# Patent

## Title
DHAD variants for butanol production

## Abstract

Dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 14/142,398, filed on Dec. 27, 2013 which claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 61/747,158, filed on Dec. 28, 2012; U.S. Provisional Patent Application No. 61/747,161, filed on Dec. 28, 2012; U.S. Provisional Patent Application No. 61/747,178, filed on Dec. 28, 2012; U.S. Provisional Patent Application No. 61/885,924, filed on Oct. 2, 2013; and U.S. Provisional Patent Application No. 61/885,939, filed on Oct. 2, 2013; all of which are incorporated herein by reference in their entireties.


REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The content of the electronically submitted sequence listing, filed herewith, is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the field of industrial microbiology and dihydroxy-acid dehydratase variants for production pathways, including isobutanol biosynthetic pathways, in microorganisms. For example, dihydroxy-acid dehydratase variants are disclosed and expressed as heterologous proteins in bacterial and yeast hosts.
BACKGROUND
Dihydroxy-acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate and of 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. DHAD-catalyzed conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is a common step in the multiple isobutanol biosynthetic pathways that are disclosed, for example, in U.S. Pat. No. 7,851,188. Disclosed therein is engineering of recombinant microorganisms for production of isobutanol. Isobutanol is useful as a fuel additive, and the availability of biologically-produced isobutanol can reduce the demand for petrochemical fuels.
BRIEF SUMMARY OF THE INVENTION
The present invention provides, for example, isolated polypeptides and fragments thereof having DHAD activity.
One aspect of the invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:547, wherein the amino acid residue at position 2 of SEQ ID NO:547 is not isoleucine or leucine, and wherein the amino acid motif comprises one or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In an embodiment of the invention, the amino acid residue at position 2 of SEQ ID NO:547 is lysine. In another embodiment, the amino acid motif comprises at least an amino acid substitution at I10V. In yet another embodiment, the amino acid motif comprises at least an amino acid substitution at L8V.
In another aspect, the invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:548, wherein the amino acid motif comprises two or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In an embodiment of the invention, the amino acid motif comprises the amino acid sequence of SEQ ID NO:553, SEQ ID NO:558, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:573, SEQ ID NO:578, SEQ ID NO:583, or SEQ ID NO:588. In another embodiment, the polypeptide or fragment thereof having DHAD activity comprises the amino acid sequence of SEQ ID NO:550, SEQ ID NO:555, SEQ ID NO:560, SEQ ID NO:565, SEQ ID NO:570, SEQ ID NO:575, SEQ ID NO:580, or SEQ ID NO:585. In another embodiment, the polypeptide or fragment thereof having DHAD activity comprises the amino acid sequence of SEQ ID NO:565. In another embodiment, the amino acid motif comprises the amino acid sequence of SEQ ID NO:573.
In certain embodiments, 9 amino acids are deleted from the C-terminal end of the amino acid sequence of SEQ ID NO:550, SEQ ID NO:555, SEQ ID NO:560, SEQ ID NO:565, SEQ ID NO:570, SEQ ID NO:575, SEQ ID NO:580, or SEQ ID NO:585.
In certain embodiments, the polypeptide or fragment thereof having DHAD activity catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate.
In other embodiments, the isolated polypeptide or fragment thereof having DHAD activity further comprises a polycysteine or polyhistidine tag. In still other embodiments, the isolated polypeptide or fragment thereof having DHAD activity further comprises the amino acid sequence of SEQ ID NO:589.
In certain embodiments, the polypeptide or fragment thereof having DHAD activity is a [2Fe-2S]2+ DHAD. In other embodiments, the polypeptide or fragment thereof having DHAD activity is a [4Fe-4S]2+ DHAD. In still other embodiments, the polypeptide or fragment thereof having DHAD activity has an amino acid sequence that matches the Profile Hidden Markov Model (HMM) of Table 6 with an E value of <10−5.
In another embodiment, the polypeptide or fragment thereof having DHAD activity comprises three conserved cysteines corresponding to positions 56, 129, and 201 in the amino acid sequences of the Streptococcus mutans DHAD corresponding to SEQ ID NO:544.
In other embodiments, the polypeptide or fragment thereof having DHAD activity is from a prokaryotic organism. In certain embodiments, the polypeptide or fragment thereof having DHAD activity is from bacteria, fungi, or plant. In a particular embodiment, the polypeptide or fragment thereof having DHAD activity is from Streptococcus mutans. Other sources of DHADs include, for example, Streptococcus downei, Oscillatoria species PCC 6506, Zea mays, Lactococcus lactis, Neurospora crassa, and Streptococcus macacae. 
Another aspect of the invention is directed to an isolated nucleic acid molecule encoding a dihydroxy-acid dehydratase (DHAD), wherein the DHAD comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:547, wherein the amino acid residue at position 2 of SEQ ID NO:547 is not isoleucine or leucine, and wherein the amino acid motif comprises one or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In an embodiment of the invention, the amino acid residue at position 2 of SEQ ID NO:547 is lysine. In another embodiment, the amino acid motif comprises at least an amino acid substitution at I10V. In yet another embodiment, the amino acid motif comprises at least an amino acid substitution at L8V.
Another aspect of the invention is directed to an isolated nucleic acid molecule encoding a DHAD, wherein the DHAD comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:548, wherein the amino acid motif comprises two or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In some embodiments, the amino acid motif comprises the amino acid sequence of SEQ ID NO:553, SEQ ID NO:558, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:573, SEQ ID NO:578, SEQ ID NO:583, or SEQ ID NO:588. In other embodiments, the DHAD encoded by the isolated nucleic acid molecule comprises the amino acid sequence of SEQ ID NO:550, SEQ ID NO:555, SEQ ID NO:560, SEQ ID NO:565, SEQ ID NO:570, SEQ ID NO:575, SEQ ID NO:580, or SEQ ID NO:565. In another embodiment, the DHAD encoded by the isolated nucleic acid molecule comprises the amino acid sequence of SEQ ID NO:11. In other embodiments, 9 amino acids are deleted from the C-terminal end of the amino acid sequence of SEQ ID NO:550, SEQ ID NO:555, SEQ ID NO:560, SEQ ID NO:565, SEQ ID NO:570, SEQ ID NO:575, SEQ ID NO:580, or SEQ ID NO:585. In another embodiment, the DHAD encoded by the isolated nucleic acid molecule comprises the amino acid motif of SEQ ID NO:573.
In some embodiments, the DHAD encoded by the isolated nucleic acid molecule catalyzes the conversion of 2,3-dihydroxy isovalerate to α-ketoisovalerate. In other embodiments, the DHAD encoded by the nucleic acid molecule further comprises a polycysteine or polyhistidine tag. In another embodiment, the DHAD encoded by the nucleic acid further comprises the amino acid sequence of SEQ ID NO:589.
In some embodiments, the DHAD encoded by the isolated nucleic acid molecule is a [2Fe-2S]2+ DHAD. In other embodiments, the DHAD encoded by the isolated nucleic acid molecule is a [4Fe-4S]2+ DHAD. In other embodiments, the DHAD encoded by the isolated nucleic acid molecule has an amino acid sequence that matches the Profile Hidden Markov Model (HMM) of Table 6 with an E value of <10−5.
In other embodiments, the DHAD encoded by the isolated nucleic acid molecule comprises three conserved cysteines corresponding to positions 56, 129, and 201 in the amino acid sequences of the Streptococcus mutans DHAD corresponding to SEQ ID NO:544.
In other embodiments, the DHAD encoded by the isolated nucleic acid molecule is from a prokaryotic organism. In other embodiments, the DHAD encoded by the isolated nucleic acid molecule is from bacteria, fungi, or plant. In a particular embodiment, the DHAD encoded by the isolated nucleic acid molecule is from Streptococcus mutans. 
In some embodiments of the invention, the isolated nucleic acid molecule is operatively linked to a promoter sequence. In other embodiments, the isolated nucleic acid molecules of the invention are comprised in a vector.
The invention also provides polypeptides encoded by the isolated nucleic acid molecules described above.
Another aspect of the invention is directed to a recombinant host cell comprising the isolated nucleic acid molecules of the invention or a vector of the invention. In certain embodiments, the DHAD encoded by the isolated nucleic acid molecule is heterologous to the recombinant host cell. In other embodiments, the DHAD encoded by the isolated nucleic acid molecule is over-expressed in the recombinant host cell.
In still other embodiments, the recombinant host cell of the invention is a bacterial cell or a yeast cell. In some embodiments, the recombinant host cell of the invention is a bacterial cell, and the bacterial cell is a member of a genus of bacteria selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. In other embodiments, the recombinant host cell of the invention is a yeast cell, and the yeast cell is a member of a genus of yeast selected from Saccharomyces, Schizosaccharomyces, Hansenula, Kluyveromyces, Candida, Pichia, and Yarrowia. In other embodiments, the recombinant host cell of the invention is Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis, or Yarrowia lipolytica. In another embodiment, the recombinant host cell of the invention is Saccharomyces cerevisiae. 
In some embodiments, the recombinant host cell of the invention is a yeast cell and the yeast cell further comprises a disruption in an endogenous ILV3 gene that encodes mitochondrial DHAD. In other embodiments, the yeast cell further comprises a disruption in one or more endogenous genes affecting iron-sulfur cluster biosynthesis selected from FRA2, GRX3, GRX4, and CCC1. In yet other embodiments, the yeast cell has been further genetically engineered to upregulate the activity of at least one gene selected from AFT1 and AFT2.
In some embodiments, the recombinant host cell of the invention is a bacterial cell, and the bacterial cell is a Lactobacillus. In other embodiments, the Lactobacillus further comprises at least one recombinant genetic expression element encoding iron-sulfur (Fe—S) cluster forming proteins. In yet other embodiments, the recombinant genetic expression element encoding iron-sulfur cluster forming proteins contains coding regions of an operon selected from Isc, Suf, and Nif operons. In some embodiments, the Suf operon comprises at least one coding region selected from SufC, SufD, SufS, SufU, SufB, SufA, and yseH. In some embodiments, the Suf operon is derived from Lactococcus lactis or Lactobacillus plantarum. In some embodiments, the Isc operon comprises at least one coding region selected from IscS, IscU, IscA, IscX, HscA, HscB, and Fdx. In some embodiments, the Isc operon is derived from Escherichia coli. In some embodiments, the Nif operon comprises at least one coding region selected from NifS and NifU. In some embodiments, the Nif operon is derived from Wolinella succinogenes. 
In some embodiments, the recombinant host cell of the invention produces butanol, for example, isobutanol. In other embodiments, the recombinant host cell of the invention comprises an isobutanol biosynthetic pathway. In some embodiments, the isobutanol biosynthetic pathway comprises genes encoding acetolactate synthase, acetohydroxy acid isomeroreductase, DHAD, α-keto acid decarboxylase, and alcohol dehydrogenase. In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: (i) pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate; (iv) α-ketoisovalerate to isobutyraldehyde; and (v) isobutyraldehyde to isobutanol.
The substrate to product conversion of pyruvate to acetolactate can be catalyzed in some embodiments by an acetolactate synthase. The substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate can be catalyzed in some embodiments by a ketol-acid reductoisomerase. The substrate to product conversion of 2,3-dihydroxy isovalerate to α-ketoisovalerate can be catalyzed in some embodiments by the DHAD. The substrate to product conversion of α-ketoisovalerate to isobutyraldehyde can be catalyzed in some embodiments by an α-keto acid decarboxylase. The substrate to product conversion of isobutyraldehyde to isobutanol can be catalyzed in some embodiments by an alcohol dehydrogenase.
In some embodiments, two or more of: acetolactate synthase, ketol-acid reductoisomerase, and α-keto acid decarboxylase are heterologous to the recombinant host cell. In other embodiments, two or more of: acetolactate synthase, ketol-acid reductoisomerase, and α-keto acid decarboxylase are over-expressed in the recombinant host cell.
In some embodiments, the recombinant host cell comprising the isolated nucleic acid molecules of the invention produces an isobutanol titer that is increased as compared to a recombinant host cell that does not contain the amino acid substitutions. In some embodiments, the recombinant host cell comprising the isolated nucleic acid molecules produces isobutanol at a rate that is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% as compared to a recombinant host cell that does not contain the amino acid substitutions.
Another aspect of the invention is directed to a method for the production of butanol, for example, isobutanol, comprising providing a recombinant host cell comprising the isolated nucleic acid molecules of the invention; culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol from pyruvate; and recovering the isobutanol. In some embodiments, the isobutanol is produced at a titer that is increased as compared to a recombinant host cell that does not contain the amino acid substitutions. In other embodiments, the isobutanol is produced at a rate that is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% as compared to a recombinant host cell that does not contain the amino acid substitutions. In another embodiment, the concentration of isobutanol in the fermentation medium is greater than or equal to about 50 mM.
Another aspect of the invention is directed to a method of converting 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate, comprising providing the isolated polypeptide or fragment thereof of the invention, wherein the isolated polypeptide or fragment thereof catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. In some embodiments of the method to convert 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate, the isolated polypeptide or fragment thereof is comprised within a recombinant host cell.
In some embodiments of the method to convert 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate, the recombinant host cell is cultured in a fermentation medium under suitable conditions to produce isobutanol or 2-methylbutanol, respectively, from pyruvate, and the isobutanol or 2-methylbutanol is recovered. In some embodiments, the isobutanol or 2-methylbutanol is recovered by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation, or combinations thereof. In some embodiments, solids are removed from the fermentation medium. In some embodiments, solids are removed from the fermentation medium by centrifugation, filtration, decantation, or combinations thereof. In other embodiments, the solids are removed before the isobutanol is recovered. In other embodiments, the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate is improved as compared to a control conversion under the same conditions with a control polypeptide having DHAD activity which does not comprise an amino acid substitution.
Another aspect of the invention is directed to a composition comprising one or more recombinant host cells of the invention, and a fermentable carbon substrate.
Another aspect of the invention is directed to a composition comprising one or more recombinant host cells of the invention, and isobutanol. In other embodiments, the composition further comprises an extractant.
The present invention is also directed to, for example, polypeptides having DHAD activity that have been altered to improve DHAD activity, for example, by the addition of a peptide tag at the C-terminal region of the DHAD. In some embodiments, the present invention is directed to a recombinant polypeptide having DHAD activity, or variant thereof, wherein the polypeptide or variant thereof comprises a C-terminal tag comprising the polypeptide sequence of CCPGCCG (SEQ ID NO:723), MCPGCCG (SEQ ID NO:724), CMPGCCG (SEQ ID NO:725), CCPGMCG (SEQ ID NO:726), CCPGCMG (SEQ ID NO:727), MCPGMCG (SEQ ID NO:728), MMPGCCG (SEQ ID NO:729), CMPGMCG (SEQ ID NO:730), CMPGCMG (SEQ ID NO:731), CCPGMMG (SEQ ID NO:732), MCPGCMG (SEQ ID NO:733), MMPGMCG (SEQ ID NO:734), CMPGMMG (SEQ ID NO:735), MCPGMMG (SEQ ID NO:736), MMPGCMG (SEQ ID NO:737), MMPGMMG (SEQ ID NO:738), CSCPGCCG (SEQ ID NO:739), CPCPGCCG (SEQ ID NO:740), CECPGCCG (SEQ ID NO:741), CCPGCSCG (SEQ ID NO:742), CCPGCPCG (SEQ ID NO:743), CCPGCECG (SEQ ID NO:744), CCPEGCCG (SEQ ID NO:745), CCPAGCCG (SEQ ID NO:746), SEQ ID NO:747, SEQ ID NO:748, or a variant thereof. In embodiments, the recombinant polypeptide having DHAD activity comprises any one of SEQ ID NOs:749-772, or a variant thereof. In embodiments, the present invention is directed to a DHAD polypeptide or variant thereof having at least about 85%, at least about 90%, or at least about 95% identity to any one of SEQ ID NOs:773-784. In embodiments, the present invention is directed to a DHAD polypeptide or variant thereof having at least about 85%, at least about 90%, or at least about 95% identity to any one of SEQ ID NOs:773-784, which further comprises a C-terminal tag comprising the polypeptide sequence of any one of SEQ ID NOs:723-772, or a variant thereof.
The present invention is also directed to a recombinant nucleic acid molecule which encodes a polypeptide or variant thereof of the present invention.
The present invention is also directed to a recombinant host cell comprising a polypeptide or variant thereof of the present invention, or a recombinant nucleic acid molecule which encodes a polypeptide or variant thereof of the present invention.
The present invention is also directed to various methods of use of the polypeptides or variants thereof of the present invention. In some embodiments, the present invention is directed to a method of converting 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate, comprising (a) providing a polypeptide having DHAD activity or variant thereof, wherein the polypeptide or variant thereof comprises a C-terminal polycysteine tag or variant thereof, or a polyhistidine tag or variant thereof, and (b) contacting the polypeptide or variant thereof with 2,3-dihydroxyisovalerate or 2,3-dihydroxymethylvalerate under conditions whereby 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate, or whereby 2,3-dihydroxymethylvalerate is converted to α-ketomethylvalerate. In other embodiments, the present invention is directed to a method of producing isobutanol, comprising (a) providing a recombinant host cell comprising a recombinant nucleic acid molecule which encodes a polypeptide or variant thereof of the present invention, and (b) contacting the host cell with a carbon substrate under conditions whereby isobutanol is produced; and (c) optionally, recovering the isobutanol.
The present invention is also directed to other isolated polypeptides and fragments thereof having dihydroxy-acid dehydratase (DHAD) activity and to isolated nucleic acids encoding such polypeptides and fragments thereof. In one aspect, the present invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO:724. In another aspect, the present invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO:773 having a deletion of 1 or more of the C-terminal amino acids of SEQ ID NO:773. In another aspect, the present invention is directed to an isolated nucleic acid molecule encoding a DHAD, wherein the DHAD comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO:724. In another aspect, the present invention is directed to an isolated nucleic acid molecule encoding a DHAD, wherein the DHAD comprises the amino acid sequence of SEQ ID NO:773 having a deletion of 1 or more of the C-terminal amino acids of SEQ ID NO:773.
The present invention is also directed to a vector comprising an isolated nucleic acid molecule of the present invention.
The present invention is also directed to a recombinant host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention.
The present invention is also directed to a method for the production of isobutanol, comprising providing a recombinant host cell of the present invention, culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol from pyruvate, and recovering the isobutanol.
The present invention is also directed to a method of converting 2,3-dihydroxyisovalerate to α-ketoisovalerate, comprising providing the isolated polypeptide or fragment thereof of the present invention, wherein the isolated polypeptide or fragment thereof catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate.
The present invention is also directed to a composition comprising a recombinant host cell of the invention and a fermentable carbon substrate.
The present invention is also directed to a composition comprising a recombinant host cell of the invention and isobutanol.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.
FIG. 1 shows biosynthetic pathways for isobutanol production.
FIG. 2 is a schematic diagram showing an 11 amino acid motif spanning positions 378 to 388 of Streptococcus mutans (S. mutans) DHAD (SEQ ID NO:547).
FIG. 3 is a graph illustrating isobutanol production in yeast strain PNY2115 transformants harboring Δ9 (Delta 9) S. mutans DHAD variants (P2A1, G2S2, L2F3, L2V4, I2V5, I2M6, L2I7, and L2M8) and the parental Δ9 S. mutans DHAD (control).
FIG. 4 is a graph illustrating the isobutanol production in yeast strain PNY2115 transformants harboring Δ9 S. mutans DHAD variants (I2V5, L2F3, L2M8, and L2V4) and the parental Δ9 S. mutans DHAD (control).
FIG. 5 is a graph illustrating the isobutanol production in yeast strain PNY2115 transformants harboring full length S. mutans DHAD variants (I2V5 clones 1 and 2 and L2V4) and the parental full length S. mutans DHAD (WT).
FIG. 6 is a graph illustrating DHAD activity in yeast strain PNY2115 harboring full length S. mutans DHAD variants (I2V5 clone 1, I2V5 clone 2, and L2V4) and the parental full length S. mutans DHAD (WT).
FIG. 7 is a graph illustrating DHIV accumulation in yeast strain PNY2115 harboring full length S. mutans DHAD variants (804-L2V4 and 804-I2V5) and the parental full length S. mutans DHAD (WT control).
FIG. 8 is a graph illustrating DHAD activity in yeast strains expressing full length S. mutans DHAD (PNY1566/pLH804 (control)) and a tagged S. mutans DHAD (PNY1566/pLH689 (tagged)).
FIG. 9 is a graph illustrating DHAD activity in yeast strains expressing full length S. mutans DHAD (pHR81 FBA-ilvD(Sm)) and a C-terminal-tagged S. mutans DHAD (pHR81 FBA-ilvD(Sm)-lum).
FIG. 10 is a graph illustrating DHAD activity in yeast strains expressing C-terminal-tagged or non-tagged DHAD enzymes, including C-terminal-tagged constructs for S. mutans DHAD, Streptococcus downei (S. downei) DHAD, and Lactococcus lactis (L. lactis) DHAD (pBP1296, pBP4579, and pBP4588, respectively).
FIG. 11 is a gel image showing tag detection for selected DHAD variants as described in the examples.
FIG. 12 is a gel image showing tag detection for selected DHAD variants as described in the examples.
FIG. 13 is a protein stained gel for selected DHAD variants as described in the examples.
FIG. 14 is a graph illustrating the specific activity of DHAD ilvD S. mutans and DHAD ilvD Streptococcus macacae (S. macacae) in a BY4741 strain background.
FIG. 15 is a graph illustrating the specific activity of DHAD ilvD S. mutans and DHAD ilvD S. macacae in a BY4741 fra2Δ strain background.
FIG. 16 is a graph illustrating DHAD activity in yeast strains expressing full length S. mutans DHAD (PNY1566/pLH804 (control)) and a C-terminal deletion S. mutans DHAD (PNY1566/pLH691(Δ9)).
FIG. 17 is a graph illustrating DHAD activity in yeast strains expressing full length S. mutans DHAD (pHR81 FBA-ilvD(Sm)) and a C-terminal deletion S. mutans DHAD (pHR81 FBA-ilvD(Sm)Δ9 amino acids).



Table 6 is a table of the Profile HAM for dihydroxy-acid dehydratases based on enzymes with assayed function prepared as described in the Examples. Table 6 is submitted herewith electronically and is incorporated herein by reference.
DETAILED DESCRIPTION OF THE INVENTION
For improved production of compounds synthesized in pathways including dihydroxy-acid dehydratase (DHAD), it is desirable to express a heterologous DHAD enzyme that provides this enzymatic activity in the production host of interest. However, there exists a need for alternative DHAD enzymes and DHAD variants that display modified activity as compared to a parental DHAD enzyme in heterologous organisms. Such enzymes may be employed for production of compounds from DHAD-requiring biosynthetic pathways.
The present invention satisfies these and other needs, and provides further related advantages, as will be made apparent by the description of the embodiments that follow.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.
Definitions
In order to further define this invention, the following terms and definitions are herein provided.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition.
As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition.
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The terms “invention” or “present invention” as used herein are non-limiting terms and are not intended to refer to any single embodiment of the particular invention but encompass all possible embodiments as described in the application.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value; in another embodiment, within 5% of the reported numerical value.
The term “alcohol” as used herein refers to any of a series of hydroxyl compounds, the simplest of which are derived from saturated hydrocarbons, having the general formula CnH2n+1OH. Examples of alcohol include ethanol and butanol.
The term “butanol” as used herein refers to n-butanol, 2-butanol, isobutanol, tert-butyl alcohol, individually or any mixtures thereof. Butanol can be from a biological source (i.e., biobutanol), for example.
The term “[2Fe-2S]2+ DHAD” refers to DHAD enzymes having a bound [2Fe-2S]2+ iron-sulfur cluster.
The term “[4Fe-4S]2+ DHAD” refers to DHAD enzymes having a bound [4Fe-4S]2+ iron-sulfur cluster.
The terms “acetohydroxy acid dehydratase” and “dihydroxy-acid dehydratase” (“DHAD”) refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Example acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. Such enzymes are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: YP_026248, NC_000913), Saccharomyces cerevisiae (GenBank Nos: NP_012550, NC_001142), Methanococcus maripaludis (GenBank Nos: CAF29874, BX957219), Bacillus subtilis (GenBank Nos: CAB14105, Z99115), Lactobacillus lactic, and Neurospora crassa. U.S. Appl. Pub. No. 2010/0081154 and U.S. Pat. No. 7,851,188, which are incorporated herein by reference, describe dihydroxy-acid dehydratases (DHADs), including a DHAD from Streptococcus mutans (Nucleic Acid: SEQ ID NO:543; Amino Acid: SEQ ID NO:544).
The term “isobutanol biosynthetic pathway” as used herein refers to an enzyme pathway to produce isobutanol from pyruvate.
The terms “acetohydroxyacid synthase,” “acetolactate synthase” and “acetolactate synthetase” (abbreviated “ALS”) are used interchangeably herein to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO2. Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos. CAB07802.1, CAB15618 and Z99122, NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae (GenBank Nos. AAA25079 and M73842), and Lactococcus lactis (GenBank Nos. AAA25161 and L16975).
The terms “ketol-acid reductoisomerase” (“KARI”), “acetohydroxy acid reductoisomerase,” and “acetohydroxy acid isomeroreductase” are used interchangeably herein to refer an enzyme that catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymes are classified as EC number 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos. NP_418222 and NC_000913), Saccharomyces cerevisiae (GenBank Nos. NP_013459 and NC_001144), Methanococcus maripaludis (GenBank Nos. CAF30210 and BX957220), and Bacillus subtilis (GenBank Nos. CAB14789 and Z99118). KARIs include, for example, Anaerostipes caccae KARI variants “K9G9”, “K9D3”, and “K9JB4P” (e.g., SEQ ID NO:697). KARI enzymes are also described in U.S. Pat. Nos. 7,910,342 and 8,129,162, U.S. Appl. Pub. No. 2010/0197519, and PCT Appl. Pub. Nos. WO2011/041415, WO2012/129555, and WO2013/176909A2, which are incorporated herein by reference. Examples of KARIs disclosed therein include those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, Pseudomonas fluorescens PF5, and Anaerostipes caccae. In some embodiments, KARI utilizes NADH (reduced nicotinamide adenine dinucleotide). In some embodiments, KARI utilizes NADPH (reduced nicotinamide adenine dinucleotide phosphate).
The terms “branched-chain α-keto acid decarboxylase,” “α-ketoacid decarboxylase,” “α-ketoisovalerate decarboxylase,” and “2-ketoisovalerate decarboxylase” (“KIVD”) are used interchangeably herein to refer to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde and CO2. Example branched-chain α-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos. AAS49166, AY548760, CAG34226, and AJ746364, Salmonella typhimurium (GenBank Nos. NP_461346 and NC_003197), Clostridium acetobutylicum (GenBank Nos. NP_149189 and NC_001988), Macrococcus caseolyticus, and Listeria grayi. Example KIVD enzymes are disclosed in U.S. Appl. Pub. US 2013/0203138, incorporated by reference.
The terms “branched-chain alcohol dehydrogenase” and “alcohol dehydrogenase” (“ADH”) are used interchangeably herein to refer to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Example branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but can also be classified under other alcohol dehydrogenases (specifically, EC numbers 1.1.1.1 or 1.1.1.2). Alcohol dehydrogenases can be, for example, NADPH dependent or NADH dependent. Such enzymes are available from a number of sources, including, but not limited to, Saccharomyces cerevisiae (GenBank Nos. NP_010656, NC_001136, NP_014051, and NC_001145), Escherichia coli (GenBank Nos. NP_417484 and NC_000913), and Clostridium acetobutylicum (GenBank Nos. NP_349892, NC_003030, NP_349891, and NC_003030). U.S. Pat. No. 8,188,250 (incorporated herein by reference) describes SadB, an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol dehydrogenases also include horse liver ADH and Beijerinkia indica ADH (as described in U.S. Appl. Publ. No. 2011/0269199, which is incorporated herein by reference).
The terms “carbon substrate” and “fermentable carbon substrate” are used interchangeably herein to refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof. Carbon substrates can include six carbon (C6) and five carbon (C5) sugars and mixtures thereof, such as, for example, glucose, sucrose or xylose.
The term “polynucleotide” as used herein encompasses a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5′ and 3′ sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. “Polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign gene” or “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
As used herein, the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
“Regulatory sequences” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, enhancers, operators, repressors, transcription termination signals, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression can also refer to translation of mRNA into a polypeptide.
The term “over-expression”, as used herein, refers to expression that is higher than endogenous expression of the same or related polynucleotide or gene. A heterologous polynucleotide or gene is also over-expressed if its expression is higher than that of a comparable endogenous gene, or if its expression is higher than that of the same polynucleotide or gene introduced by a means that does not overexpress the polynucleotide or gene. For example, a polynucleotide can be expressed in a host cell from a low copy number plasmid, which is present in only limited or few copies, and the same polynucleotide can be over-expressed in a host cell from a high copy number plasmid or a plasmid with a copy number that can be regulated, which is present in multiple copies. Any means can be used to over-express a polynucleotide, so long as it increases the copies of the polynucleotide in the host cell. In addition to using a high copy number plasmid, or a plasmid with a copy number that can be regulated, a polynucleotide can be over-expressed by multiple chromosomal integrations.
Expression or over-expression of a polypeptide of the invention in a recombinant host cell can be quantified according to any number of methods known to the skilled artisan and can be represented, e.g., by a percent of total cell protein. The percent of total protein can be an amount selected from greater than about 0.001% of total cell protein; greater than about 0.01% of total cell protein; greater than about 0.1% of total cell protein; greater than about 0.5% of total cell protein; greater than about 1.0% of total cell protein; greater than about 2.0% of total cell protein; greater than about 3.0% of total cell protein; greater than about 4.0% of total cell protein; greater than about 5.0% of total cell protein; greater than about 6.0% of total cell protein; greater than about 7.0% of total cell protein; greater than about 8.0% of total cell protein; greater than about 9.0% of total cell protein; greater than about 10% of total cell protein; or greater than about 20% of total cell protein. In one embodiment, the amount of polypeptide expressed is greater that about 0.5% of total cell protein. In another embodiment, the amount of polypeptide expressed is greater than about 1.0% of total cell protein or greater than about 2.0% of total cell protein.
As used herein, the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
A “recombinant host cell” is defined as a host cell that has been genetically manipulated to express a biosynthetic production pathway, wherein the host cell either produces a biosynthetic product in greater quantities relative to an unmodified host cell or produces a biosynthetic product that is not ordinarily produced by an unmodified host cell.
The term “engineered” as applied to a isobutanol biosynthetic pathway refers to the isobutanol biosynthetic pathway that is manipulated, such that the carbon flux from pyruvate through the engineered isobutanol biosynthetic pathway is maximized, thereby producing an increased amount of isobutanol directly from the fermentable carbon substrate. Such engineering includes expression of heterologous polynucleotides or polypeptides, overexpression of endogenous polynucleotides or polypeptides, cytosolic localization of proteins that do not naturally localize to cytosol, increased cofactor availability, decreased activity of competitive pathways, etc.
The term “codon optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.







TABLE 1







The Standard Genetic Code












T
C
A
G














T
TTT Phe (F)
TCT Ser (S)
TAT Tyr (Y)
TGT Cys (C)



TTC Phe (F)
TCC Ser (S)
TAC Tyr (Y)
TGC



TTA Leu (L)
TCA Ser (S)
TAA Stop
TGA Stop



TTG Leu (L)
TCG Ser (S)
TAG Stop
TGG Trp (W)


C
CTT Leu (L)
CCT Pro (P)
CAT His (H)
CGT Arg (R)



CTC Leu (L)
CCC Pro (P)
CAC His (H)
CGC Arg (R)



CTA Leu (L)
CCA Pro (P)
CAA Gln (Q)
CGA Arg (R)



CTG Leu (L)
CCG Pro (P)
CAG Gln (Q)
CGG Arg (R)


A
ATT Ile (I)
ACT Thr (T)
AAT Asn (N)
AGT Ser (S)



ATC Ile (I)
ACC Thr (T)
AAC Asn (N)
AGC Ser (S)



ATA Ile (I)
ACA Thr (T)
AAA Lys (K)
AGA Arg (R)



ATG Met
ACG Thr (T)
AAG Lys (K)
AGG Arg (R)



(M)





G
GTT Val (V)
GCT Ala (A)
GAT Asp (D)
GGT Gly (G)



GTC Val (V)
GCC Ala (A)
GAC Asp (D)
GGC Gly (G)



GTA Val (V)
GCA Ala (A)
GAA Glu (E)
GGA Gly (G)



GTG Val (V)
GCG Ala (A)
GAG Glu (E)
GGG Gly (G)









Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
As used herein, an “isolated nucleic acid fragment” or “isolated nucleic acid molecule” are used interchangeably herein and mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Accordingly, an “isolated” nucleic acid fragment or molecule can be, for example, “recombinant” or “engineered.” An isolated nucleic acid fragment in the form of a polymer of DNA can be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
A nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified, for example, in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (incorporated herein by reference in its entirety). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.
Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably, a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as length of the probe.
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
As used herein, the term “variant” refers to a polypeptide differing from a specifically recited polypeptide of the invention, such as DHAD, by amino acid insertions, deletions, mutations, and substitutions, created using, e.g., recombinant DNA techniques, such as mutagenesis. Guidance in determining which amino acid residues can be replaced, added, or deleted without abolishing activities of interest, can be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, e.g., yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.
Alternatively, recombinant polynucleotide variants encoding these same or similar polypeptides can be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as silent changes which produce various restriction sites, can be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence can be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.
Amino acid “substitutions” can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they can be the result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. “Conservative” amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, “non-conservative” amino acid substitutions can be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. “Insertions” or “deletions” can be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed can be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides can be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases can be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, can now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” or “sequence identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: (1. Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); (2) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); (3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); (4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and (5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally, the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992); Thompson, J. D., Higgins, D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673-4680) and found in the MegAlignTM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment are GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.
It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% is useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” can be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: (1) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); (2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); (3) DNASTAR (DNASTAR, Inc. Madison, Wis.); (4) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and (5) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein, “default values” means any set of values or parameters that originally load with the software when first initialized.
“Fermentation medium” as used herein means a mixture of water, fermentable carbon substrates, dissolved solids, fermentation product and all other constituents of the material held in the fermentation vessel in which the fermentation product is being made by the reaction of fermentable carbon substrates to fermentation products, water and carbon dioxide (CO2) by the microorganisms present. From time to time, as used herein, the terms “fermentation broth” and “fermentation mixture” can be used synonymously with “fermentation medium.”
As used herein, the term “yield” refers to the amount of product in grams per amount of carbon source in grams (g/g). The yield can be exemplified, for example, for glucose as the carbon source. It is understood, unless otherwise noted, that yield is expressed as a percentage of the theoretical yield. In reference to a microorganism or metabolic pathway, “theoretical yield” is defined as the maximum amount of product that can be generated per total amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isopropanol is 0.33 g/g. As such, a yield of isopropanol from glucose of 0.297 g/g would be expressed as 90% of theoretical or 90% theoretical yield. It is understood that, while in the present disclosure the yield is exemplified for glucose as a carbon source, the invention can be applied to other carbon sources and the yield can vary depending on the carbon source used. One skilled in the art can calculate yields on various carbon sources.
The term “titer” as used herein, refers to the total amount of butanol isomer produced by fermentation per liter of fermentation medium. The total amount of butanol isomer includes: (i) the amount of butanol in the fermentation medium; (ii) the amount of butanol isomer recovered from the organic extractant; and (iii) the amount of butanol isomer recovered from the gas phase, if gas stripping is used.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used herein are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).
DHAD Variants
As described above, dihydroxy-acid dehydratase (DHAD), also called acetohydroxy acid dehydratase, catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate and of 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. The DHAD enzyme is part of naturally occurring biosynthetic pathways producing valine, isoleucine, leucine and pantothenic acid (vitamin B5). DHAD catalyzed conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is also a step in the multiple isobutanol biosynthetic pathways that are disclosed in U.S. Pat. No. 7,851,188 and (both incorporated herein by reference). For production of compounds synthesized in pathways including DHAD, it is desirable to express a heterologous DHAD enzyme that provides DHAD enzymatic activity in a host cell. A consideration for functional expression of dihydroxy-acid dehydratases in a heterologous host is the enzyme's requirement for an iron-sulfur (Fe—S) cluster, which involves availability and proper loading of the cluster into the DHAD apo-protein.
The present invention is based on the discovery that certain variants of DHAD have DHAD activity, and, in some embodiments, improved activity compared to the parental DHAD molecule. DHAD variants are desirable for production of products produced by DHAD containing biosynthetic pathways, particularly isobutanol.
It has been discovered that alterations in the amino acid sequence of an 11-amino acid motif found in DHAD enzymes can lead to improved DHAD activity as indicated by increased isobutanol production. For the purposes of the present invention, amino acid substitutions were made in the Streptococcus mutans DHAD enzyme (SEQ ID NO:544), however, equivalent substitutions can be made in the homologous regions of DHAD enzymes from other organisms. A list of example DHAD enzymes that may be used to produce the DHAD variants of the invention is included below in Tables 3-5. Other sources of DHADs include, for example, Streptococcus downei, Oscillatoria species PCC 6506, Zea mays, Lactococcus lactis, Neurospora crassa, and Streptococcus macacae. 
An 11-amino acid motif sequence which may be found in DHAD enzymes is: P-K-X-X-X-G-X-I/L-X-I-L, wherein X represents any amino acid (SEQ ID NO:875). This motif encompasses amino acid positions 378 through 388 of the Streptococcus mutans DHAD enzyme. Throughout this description, the positions of the amino acids in the 11-amino acid motif are numbered 1 through 11, with amino acid residue 1 representing the first proline and amino acid residue 11 representing the last leucine. In addition, amino acids are described herein using either the full name of the amino acid or the 1-letter or 3-letter abbreviation of the amino acid, as indicated in Table 2.







TABLE 2







Amino Acids and their Abbreviations











Amino Acid
1-Letter Symbol
3-Letter Symbol






Alanine
A
Ala



Arginine
R
Arg



Asparagine
N
Asn



Aspartic acid
D
Asp



Cysteine
C
Cys



Glutamine
Q
Gln



Glutamic acid
E
Glu



Pyroglutamic acid
pQ
pGlu



Glycine
G
Gly



Histidine
H
His



Hydroxylysine

Hyl



Hydroxyproline, 4(R)-L-
O
Hyp



Isoleucine
I
Ile



Leucine
L
Leu



Lysine
K
Lys



Methionine
M
Met



Phenylalanine
F
Phe



Proline
P
Pro



Serine
S
Ser



Threonine
T
Thr



Tryptophan
W
Trp



Tyrosine
Y
Tyr



Valine
V
Val









The amino acid changes that were made and/or contemplated by the present invention to produce alternative, active DHAD enzymes are described herein, for example, by a three character code that begins with the 1-letter abbreviation of the native amino acid, followed by the amino acid position number, and followed by the 1-letter abbreviation of the identity of the substituted amino acid. For example, “P1A” refers to a proline to alanine substitution of the first position of the DHAD motif described, e.g., in SEQ ID NO:547. As explained above, the substitution in the first position represented by P1A (numbering
according to the DHAD motif of SEQ ID NO:547) corresponds to position 378 of the Streptococcus mutans DHAD enzyme. Accordingly, P1A corresponds to P378A when the substitution is expressed in terms of the corresponding position of Streptococcus mutans DHAD. In some embodiments, the amino acid changes that were made and/or contemplated to produce alternative, active DHAD enzymes include, for example, P1A, P1G, P1V, P1I, P1L, K2I, K2L, G6S, G6A, G6V, G6L, G6I, L8F, L8V, L8G, L8A, L8V, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11G, corresponding to the DHAD motif described, e.g., in SEQ ID NO:547. When expressed in terms of the corresponding positions of Streptococcus mutans DHAD, these substitutions are P378A, P378G, P378V, P378I, P378L, K379I, K379L, G383S, G383A, G383V, G383L, G383I, L385F, L385V, L385G, L385A, L385V, I387V, I387M, I387L, I387G, I387A, L388I, L388M, L388V, L388A, and L388G. Accordingly, examples of DHAD variants of the invention include, for example, a DHAD having a substitution of one or more of P378A, P378G, P378V, P378I, P378L, K379I, K379L, G383S, G383A, G383V, G383L, G383I, L385F, L385V, L385G, L385A, L385V, I387V, I387M, I387L, I387G, I387A, L388I, L388M, L388V, L388A, and L388G. As described in the Examples, clones containing a P378A substitution are referenced as P2A1 (for example, delta9-P2A1 and 689-P2A1). Clones containing a G383S substitution are referenced as G2S2 (for example, delta9-G2S2 and 689-G2S2). Clones containing a L385F substitution are referenced as L2F3 (for example, delta9-L2F3 and 689-L2F3). Clones containing a L385V substitution are referenced as L2V4 (for example, delta9-L2V4, 804-L2V4 and 689-L2V4). Clones containing a I387V substitution are referenced as I2V5 (for example, delta9-12V5, 804-I2V5 and 689-I2V5). Clones containing a I387M substitution are referenced as I2M6 (for example, delta9-I2M6 and 689-I2M6). Clones containing a L388I substitution are referenced as L2I7 (for example, delta9-L2I7 and 689-L2I7). Clones containing a L388M substitution are referenced as L2M8 (for example, delta9-L2M8 and 689-L2M8). However, one or more of such substitutions can be made in the corresponding residue(s) of other DHAD enzymes.
Examples of alternative, active DHAD enzymes are described herein and include, for example, Streptococcus mutans P2A1 (Nucleic Acid: SEQ ID NO:549; Amino Acid: SEQ ID NO:550), Streptococcus mutans P2A1 delta9 (A9) (Nucleic Acid: SEQ ID NO:551; Amino Acid: SEQ ID NO:552), Streptococcus mutans G2S2 (Nucleic Acid: SEQ ID NO:554; Amino Acid: SEQ ID NO:555), Streptococcus mutans G2S2 delta9 (AΔ9) (Nucleic Acid: SEQ ID NO:556; Amino Acid: SEQ ID NO:557), Streptococcus mutans L2F3 (Nucleic Acid: SEQ ID NO:559; Amino Acid: SEQ ID NO:560), Streptococcus mutans L2F3 (Δ9) (Nucleic Acid: SEQ ID NO:561; Amino Acid: SEQ ID NO:562), Streptococcus mutans L2V4 (Nucleic Acid: SEQ ID NO:564; Amino Acid: SEQ ID NO:565), Streptococcus mutans L2V4 delta9 (Δ9) (Nucleic Acid: SEQ ID NO:566; Amino Acid: SEQ ID NO:567), Streptococcus mutans I2V5 (Nucleic Acid: SEQ ID NO:569; Amino Acid: SEQ ID NO:570), Streptococcus mutans I2V5 delta9 (Δ9) (Nucleic Acid: SEQ ID NO:571; Amino Acid: SEQ ID NO:572), Streptococcus mutans I2M6 (Nucleic Acid: SEQ ID NO:574; Amino Acid: SEQ ID NO:575), Streptococcus mutans I2M6 delta9 (Δ9) (Nucleic Acid: SEQ ID NO:576; Amino Acid: SEQ ID NO:577), Streptococcus mutans L2I7 (Nucleic Acid: SEQ ID NO:579; Amino Acid: SEQ ID NO:580), Streptococcus mutans L2I7 delta9 (Δ9) (Nucleic Acid: SEQ ID NO:581; Amino Acid: SEQ ID NO:582), Streptococcus mutans L2M8 (Nucleic Acid: SEQ ID NO:584; Amino Acid: SEQ ID NO:585), Streptococcus mutans L2M8 delta9 (Δ9) (Nucleic Acid: SEQ ID NO:586; Amino Acid: SEQ ID NO:587), Streptococcus mutans 689-P2A1 (Nucleic Acid: SEQ ID NO:860; Amino Acid: SEQ ID NO:861), Streptococcus mutans 689-G2S2 (Nucleic Acid: SEQ ID NO:862; Amino Acid: SEQ ID NO:863), Streptococcus mutans 689-L2F3 (Nucleic Acid: SEQ ID NO:864; Amino Acid: SEQ ID NO:865), Streptococcus mutans 689-L2V4 (Nucleic Acid: SEQ ID NO:866; Amino Acid: SEQ ID NO:867), Streptococcus mutans 689-I2V5 (Nucleic Acid: SEQ ID NO:786; Amino Acid: SEQ ID NO:787), Streptococcus mutans 689-I2M6 (Nucleic Acid: SEQ ID NO:868; Amino Acid: SEQ ID NO:869), Streptococcus mutans 689-L2I7 (Nucleic Acid: SEQ ID NO:870; Amino Acid: SEQ ID NO:871), and Streptococcus mutans 689-L2M8 (Nucleic Acid: SEQ ID NO:872; Amino Acid: SEQ ID NO:873). Accordingly, in some embodiments, the invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises one or more amino acid substitutions selected from P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F (numbering corresponding to the conserved 11-amino acid motif sequence found in DHAD enzymes of P-K-X-X-X-G-X-I/L-X-I-L, wherein X represents any amino acid amino acid), or one or more amino acid substitutions corresponding to amino acid positions P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F of S. mutans DHAD.
In some embodiments, the invention provides an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:547, wherein the amino acid residue at position 2 of SEQ ID NO:547 is not isoleucine or leucine, and wherein the amino acid motif comprises one or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In other embodiments, the polypeptide or fragment comprises an amino acid motif which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of SEQ ID NO:547, wherein the amino acid residue at position 2 of SEQ ID NO:547 is not isoleucine or leucine, and wherein the amino acid motif comprises one or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In some embodiments, the amino acid residue at position 2 of SEQ ID NO:547 is lysine. In some embodiments, the amino acid motif comprises at least an amino acid substitution at I10V. In other embodiments, the amino acid motif comprises at least an amino acid substitution at L8V.
The invention also provides an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid motif which is at least 90% identical to the amino acid sequence of SEQ ID NO:548, wherein the amino acid motif comprises two or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In other embodiments, the amino acid motif is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identifcal to the amino acid sequence of SEQ ID NO:548, wherein the amino acid motif comprises two or more amino acid substitutions selected from: P1A, P1S, G6S, G6A, L8F, L8V, L8M, L8A, I10V, I10M, I10L, I10G, I10A, L11I, L11M, L11V, L11A, and L11F. In other embodiments, the amino acid motif comprises the amino acid sequence of SEQ ID NO:553, SEQ ID NO:558, SEQ ID NO:563, SEQ ID NO:568, SEQ ID NO:573, SEQ ID NO:578, SEQ ID NO:583, or SEQ ID NO:588. In other embodiments, the polypeptide or fragment thereof having DHAD activity comprises the amino acid sequence of SEQ ID NO:550, SEQ ID NO:555, SEQ ID NO:560, SEQ ID NO:565, SEQ ID NO:570, SEQ ID NO:575, SEQ ID NO:580, or SEQ ID NO:585. In other embodiments, the polypeptide or fragment thereof having DHAD activity comprises the amino acid sequence of SEQ ID NO:565. In other embodiments, the amino acid motif comprises the amino acid sequence of SEQ ID NO:573.
In embodiments, DHAD variant proteins display increased DHAD activity compared to DHAD proteins without the amino acid substitutions. In some embodiments, DHAD variant proteins expressed in yeast cytosol have a specific activity of greater than about 0.10 units/mg, greater than about 0.15 units/mg, greater than about 0.20 units/mg, greater than about 0.25 units/mg, greater than about 0.30 units/mg, greater than about 0.35 units/mg, or greater than about 0.40 units/mg.
Other alterations in the amino acid sequence of DHAD enzymes may lead to improved DHAD activity, as indicated, for example, by increased isobutanol production. These alterations include, for example, the addition of a peptide tag (“tag”) to the C-terminal region of a DHAD. In some embodiments, the alteration is, for example, the addition of a sequence comprising a polyhistidine tag, a polycysteine tag, a V5 epitope, a myc epitope, a LumioTM tag (Life Technologies; DNA: SEQ ID NO:785; Amino Acid: SEQ ID NO:747), or a tag such as SEQ ID NO: 748, for example, or a variant thereof, to the C-terminal end of a DHAD. In other embodiments, the alterations include, for example, the addition of a tag to the C-terminal region of a DHAD that comprises the polypeptide sequence of CCPGCCG (SEQ ID NO:723), MCPGCCG (SEQ ID NO:724), CMPGCCG (SEQ ID NO:725), CCPGMCG (SEQ ID NO:726), CCPGCMG (SEQ ID NO:727), MCPGMCG (SEQ ID NO:728), MMPGCCG (SEQ ID NO:729), CMPGMCG (SEQ ID NO:730), CMPGCMG (SEQ ID NO:731), CCPGMMG (SEQ ID NO:732), MCPGCMG (SEQ ID NO:733), MMPGMCG (SEQ ID NO:734), CMPGMMG (SEQ ID NO:735), MCPGMMG (SEQ ID NO:736), MMPGCMG (SEQ ID NO:737), MMPGMMG (SEQ ID NO:738), CSCPGCCG (SEQ ID NO:739), CPCPGCCG (SEQ ID NO:740), CECPGCCG (SEQ ID NO:741), CCPGCSCG (SEQ ID NO:742), CCPGCPCG (SEQ ID NO:743), CCPGCECG (SEQ ID NO:744), CCPEGCCG (SEQ ID NO:745), CCPAGCCG (SEQ ID NO:746), or variant thereof.
Disclosed herein are DHAD variants of a Streptococcus mutans DHAD enzyme (SEQ ID NO:773), however, equivalent alterations can be made in homologous regions of DHAD enzymes from other organisms. Examples include SEQ ID NOs:749-772. A list of other DHAD enzymes that can be used to produce the DHAD variants of the invention is included below in Tables 3-5. Other sources of DHADs include, for example, Streptococcus downei, Oscillatoria species PCC 6506, Zea mays, Lactococcus lactis, Neurospora crassa, and Streptococcus macacae. 
In some embodiments, DHAD variant proteins display increased DHAD activity compared to DHAD proteins without amino acid alterations. In some embodiments, DHAD variant proteins expressed in yeast cytosol have a specific activity of greater than 0.1 units/mg (U/mg), greater than 0.15 units/mg, greater than 0.2 units/mg, greater than 0.25 units/mg, greater than 0.3 units/mg, greater than 0.35 units/mg, greater than 0.4 units/mg, greater than 0.45 units/mg, greater than 0.5 units/mg, greater than 0.55 units/mg, greater than 0.6 units/mg, greater than 0.65 units/mg, greater than 0.7 units/mg, greater than 0.75 units/mg, greater than 0.8 units/mg, greater than 0.85 units/mg, greater than 0.9 units/mg, greater than 1 unit/mg, greater than 1.1 units/mg, greater than 1.2 units/mg, greater than 1.3 units/mg, greater than 1.4 units/mg, greater than 1.5 units/mg, greater than 1.6 units/mg, greater than 1.7 units/mg, greater than 1.8 units/mg, greater than 1.9 units/mg, greater than 2 units/mg, or any range of values thereof. In some embodiments, DHAD variant proteins expressed in yeast cytosol have a specific activity of about 0.1 units/mg, about 0.15 units/mg, about 0.2 units/mg, about 0.25 units/mg, about 0.3 units/mg, about 0.35 units/mg, about 0.4 units/mg, about 0.45 units/mg, about 0.5 units/mg, about 0.55 units/mg, about 0.6 units/mg, about 0.65 units/mg, about 0.7 units/mg, about 0.75 units/mg, about 0.8 units/mg, about 0.85 units/mg, about 0.9 units/mg, about 1 unit/mg, about 1.1 units/mg, about 1.2 units/mg, about 1.3 units/mg, about 1.4 units/mg, about 1.5 units/mg, about 1.6 units/mg, about 1.7 units/mg, about 1.8 units/mg, about 1.9 units/mg, about 2 units/mg, or more, or any range of values thereof. In some embodiments, the DHAD variant proteins have a specific activity of about 0.1 units/mg to about 2 units/mg, about 0.2 units/mg to about 2 units/mg, about 0.3 units/mg to about 2 units/mg, about 0.4 units/mg to about 2 units/mg, or about 0.5 units/mg to about 2 units/mg. In other embodiments, DHAD activity is increased in the DHAD variant by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to DHAD proteins without an amino acid alteration, or any range of values thereof. In other embodiments, DHAD activity is increased in the DHAD variant by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, compared to DHAD proteins without an amino acid alteration, or any range of values thereof. In some embodiments, DHAD activity is increased in the DHAD variant by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 50%, or about 50% to about 60% compared to DHAD proteins without an amino acid alteration.
Other alterations in the amino acid sequence of DHAD enzymes can lead to improved DHAD activity, as indicated, for example, by increased isobutanol production. These alterations include, for example, a deletion of one or more of the C-terminal amino acids of DHAD. In some embodiments, the deletion is a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 C-terminal amino acids of DHAD, or any range of values thereof. In some embodiments, the deletion is a deletion of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, or more C-terminal amino acids of DHAD, or any range of values thereof. In other embodiments, the deletion is a deletion of about 1 to about 40 C-terminal amino acids of DHAD, including, but not limited to, about 1 to about 35, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 5 to about 40, about 5 to about 35, about 5 to about 30, about 5 to about 25, about 5 to about 20, about 5 to about 15, or about 5 to about 10 C-terminal amino acids of DHAD. In some embodiments, the deletion is the 9 C-terminal amino acids of DHAD (DNA: SEQ ID NO:545; Protein: SEQ ID NO:546).
For the purposes of the present invention, amino acid deletions were made of the Streptococcus mutans DHAD enzyme (e.g., SEQ ID NO:544), however, equivalent deletions can be made in homologous regions of DHAD enzymes from other organisms. A list of other DHAD enzymes that may be used to produce the DHAD variants of the invention is included below in Tables 3-5.
In some embodiments, the invention is directed to an isolated polypeptide or fragment thereof having DHAD activity, wherein the polypeptide or fragment thereof comprises an amino acid sequence which is at least 90% identical to the amino acid sequence of SEQ ID NO:544. In other embodiments, the polypeptide or fragment comprises an amino acid sequence which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:528, or any range of values thereof. In other embodiments, the polypeptide or fragment comprises an amino acid sequence which is about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence of SEQ ID NO:544, or any range of values thereof. In other embodiments, the polypeptide or fragment comprises an amino acid sequence which is about 60% to about 99%, about 65% to about 99%, about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, or about 95% to about 99% identical to the amino acid sequence of SEQ ID NO:544.
In some embodiments, DHAD variant proteins display increased DHAD activity compared to DHAD proteins without amino acid deletions. In some embodiments, DHAD variant proteins expressed in yeast cytosol have a specific activity of greater than 0.1 units/mg (U/mg), greater than 0.15 units/mg, greater than 0.2 units/mg, greater than 0.25 units/mg, greater than 0.3 units/mg, greater than 0.35 units/mg, greater than 0.4 units/mg, greater than 0.43 units/mg, greater than 0.45 units/mg, greater than 0.5 units/mg, greater than 0.55 units/mg, greater than 0.6 units/mg, greater than 0.65 units/mg, greater than 0.7 units/mg, greater than 0.75 units/mg, greater than 0.8 units/mg, or any range of values thereof. In some embodiments, DHAD variant proteins expressed in yeast cytosol have a specific activity of about 0.1 units/mg, about 0.15 units/mg, about 0.2 units/mg, about 0.25 units/mg, about 0.3 units/mg, about 0.35 units/mg, about 0.4 units/mg, about 0.43 units/mg, about 0.45 units/mg, about 0.5 units/mg, about 0.55 units/mg, about 0.6 units/mg, about 0.65 units/mg, about 0.7 units/mg, about 0.75 units/mg, about 0.8 units/mg, or more, or any range of values thereof. In some embodiments, the DHAD variant proteins have a specific activity of about 0.10 units/mg to about 0.8 units/mg, about 0.2 units/mg to about 0.8 units/mg, about 0.3 units/mg to about 0.8 units/mg, about 0.4 units/mg to about 0.8 units/mg, or about 0.1 units/mg to about 0.5 units/mg. In other embodiments, DHAD activity is increased in the DHAD variant by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, or more, compared to DHAD proteins without an amino acid deletion, or any range of values thereof. In other embodiments, DHAD activity is increased in the DHAD variant by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, compared to DHAD proteins without an amino acid deletion, or any range of values thereof. In some embodiments, DHAD activity is increased in the DHAD variant by about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 50%, or about 50% to about 60% compared to DHAD proteins without an amino acid deletion.
DHAD Proteins
Any DHAD proteins may be used as a parental, or starting, molecule for creating a DHAD variant polypeptide of the invention. DHADs that may be used herein can be derived from bacterial, fungal, or plant sources. DHADs that may be used may have a [4Fe-4S]2+ cluster or a [2Fe-2S]2+ cluster bound by the apoprotein. Tables 3-5 list SEQ ID NOs for coding regions and proteins of representative DHADs that may be used in the present invention. Proteins with at least about 95% identity to those listed sequences have generally been omitted for simplification, but it is understood that the omitted proteins with at least about 95% sequence identity to any of the proteins listed in Tables 3-5 and having DHAD activity may be used as disclosed herein. As described herein, polynucleotide sequences encoding DHADs can be codon optimized for expression in a particular organism by methods known in the art. Examples of such DHAD sequences include, for example, Streptococcus downei (S. downei) (DNA SEQ ID NO:708; protein SEQ ID NO:709), Oscillatoria species PCC 6506 (DNA SEQ ID NO:710; protein SEQ ID NO:711), Zea mays (DNA SEQ ID NO:712; protein SEQ ID NO:713), Lactococcus lactis (DNA SEQ ID NO:714; protein SEQ ID NO:715), Neurospora crassa (DNA SEQ ID NOs:716 and 718; protein SEQ ID NOs:717 and 719) and S. mutans DHAD 689-I2V5 variant (DNA SEQ ID NO:786; protein SEQ ID NO:787). Other examples of DHADs include, for example, Streptococcus macacae DHAD (S. macacae) (DNA SEQ ID NO:700; protein SEQ ID NO:701), S. macacae DHAD L2V4 (DNA SEQ ID NO:702; protein SEQ ID NO:703), S. macacae DHAD I2V5 (DNA SEQ ID NO:704; protein SEQ ID NO:705) and S. mutans DHAD 689-I2V5 variant (DNA SEQ ID NO:706; protein SEQ ID NO:707). Additional DHAD proteins and their encoding sequences can be identified by BLAST searching of public databases, as well known to one skilled in the art. Typically BLAST (described above) searching of publicly available databases with known DHAD sequences, such as those provided herein, is used to identify DHADs and their encoding sequences that can be expressed in the present cells. For example, DHAD proteins having amino acid sequence identities of at least about 80-85%, at least about 85-90%, at least about 90-95%, or at least about 98% sequence identity to any of the DHAD proteins disclosed herein can be expressed in the present cells. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.







TABLE 3







SEQ ID NOs of Representative Bacterial [2Fe—2S]2+ DHAD Proteins


and Encoding Sequences











SEQ



SEQ ID NO:
ID NO:


Organism of derivation
Nucleic acid
Peptide












Mycobacterium sp. MCS
1
2


Mycobacterium gilvum PYR-GCK
3
4


Mycobacterium smegmatis str. MC2 155
5
6


Mycobacterium vanbaalenii PYR-1
7
8


Nocardia farcinica IFM 10152
9
10


Rhodococcus sp. RHA1
11
12


Mycobacterium ulcerans Agy99
13
14


Mycobacterium avium subsp.
15
16


paratuberculosis K-10




Mycobacterium tuberculosis H37Ra
17
18


Mycobacterium leprae TN *
19
20


Kineococcus radiotolerans SRS30216
21
22


Janibacter sp. HTCC2649
23
24


Nocardioides sp. JS614
25
26


Renibacterium salmoninarum ATCC 33209
27
28


Arthrobacter aurescens TC1
29
30


Leifsonia xyli subsp. xyli str. CTCB07
31
32


marine actinobacterium PHSC20C1
33
34


Clavibacter michiganensis subsp.
35
36


michiganensis NCPPB 382




Saccharopolyspora erythraea NRRL 2338
37
38


Acidothermus cellulolyticus 11B
39
40


Corynebacterium efficiens YS-314
41
42


Brevibacterium linens BL2
43
44


Tropheryma whipplei TW08/27
45
46


Methylobacterium extorquens PA1
47
48


Methylobacterium nodulans ORS 2060
49
50


Rhodopseudomonas palustris BisB5
51
52


Rhodopseudomonas palustris BisB18
53
54


Bradyrhizobium sp. ORS278
55
56


Bradyrhizobium japonicum USDA 110
57
58


Fulvimarina pelagi HTCC2506
59
60


Aurantimonas sp. SI85-9A1
61
62


Hoeflea phototrophica DFL-43
63
64


Mesorhizobium loti MAFF303099
65
66


Mesorhizobium sp. BNC1
67
68


Parvibaculum lavamentivorans DS-1
69
70


Loktanella vestfoldensis SKA53
71
72


Roseobacter sp. CCS2
73
74


Dinoroseobacter shibae DFL 12
75
76


Roseovarius nubinhibens ISM
77
78


Sagittula stellata E-37
79
80


Roseobacter sp. AzwK-3b
81
82


Roseovarius sp. TM1035
83
84


Oceanicola batsensis HTCC2597
85
86


Oceanicola granulosus HTCC2516
87
88


Rhodobacterales bacterium HTCC2150
89
90


Paracoccus denitrificans PD1222
91
92


Oceanibulbus indolifex HEL-45
93
94


Sulfitobacter sp. EE-36
95
96


Roseobacter denitrificans OCh 114
97
98


Jannaschia sp. CCS1
99
100


Caulobacter sp. K31
101
102


Candidatus Pelagibacter ubique HTCC1062
103
104


Erythrobacter litoralis HTCC2594
105
106


Erythrobacter sp. NAP1
107
108


Comamonas testosterone KF-1
109
110


Sphingomonas wittichii RW1
111
112


Burkholderia xenovorans LB400
113
114


Burkholderia phytofirmans PsJN
115
116


Bordetella petrii DSM 12804
117
118


Bordetella bronchiseptica RB50
119
120


Bradyrhizobium sp. ORS278
121
122


Bradyrhizobium sp. BTAi1
123
124


Bradyrhizobium japonicum
125
126


Sphingomonas wittichii RW1
127
128


Rhodobacterales bacterium HTCC2654
129
130


Solibacter usitatus Ellin6076
131
132


Roseiflexus sp. RS-1
133
134


Rubrobacter xylanophilus DSM 9941
135
136


Salinispora tropica CNB-440
137
138


Acidobacteria bacterium Ellin345
139
140


Thermus thermophilus HB27
141
142


Maricaulis maris MCS10
143
144


Parvularcula bermudensis HTCC2503
145
146


Oceanicaulis alexandrii HTCC2633
147
148


Plesiocystis pacifica SIR-I
149
150


Bacillus sp. NRRL B-14911
151
152


Oceanobacillus iheyensis HTE831
153
154


Staphylococcus saprophyticus subsp.
155
156


saprophyticus ATCC 15305




Bacillus selenitireducens MLS10
157
158


Streptococcus pneumoniae SP6-BS73
159
160


Streptococcus sanguinis SK36
161
162


Streptococcus thermophilus LMG 18311
163
164


Streptococcus suis 89/1591
165
166


Streptococcus mutans UA159
167
168


Leptospira borgpetersenii serovar Hardjo-
169
170


bovis L550




Candidatus Vesicomyosocius okutanii HA
171
172


Candidatus Ruthia magnifica str. Cm
173
174


(Calyptogena magnifica)




Methylococcus capsulatus str. Bath
175
176


uncultured marine bacterium EB80_02D08
177
178


uncultured marine gamma proteobacterium
179
180


EBAC31A08




uncultured marine gamma proteobacterium
181
182


EBAC20E09




uncultured gamma proteobacterium
183
184


eBACHOT4E07




Alcanivorax borkumensis SK2
185
186


Chromohalobacter salexigens DSM 3043
187
188


Marinobacter algicola DG893
189
190


Marinobacter aquaeolei VT8
191
192


Marinobacter sp. ELB17
193
194


Pseudoalteromonas haloplanktis TAC125
195
196


Acinetobacter sp. ADP1
197
198


Opitutaceae bacterium TAV2
199
200


Flavobacterium sp. MED217
201
202


Cellulophaga sp. MED134
203
204


Kordia algicida OT-1
205
206


Flavobacteriales bacterium ALC-1
207
208


Psychroflexus torquis ATCC 700755
209
210


Flavobacteriales bacterium HTCC2170
211
212


unidentified eubacterium SCB49
213
214


Gramella forsetii KT0803
215
216


Robiginitalea biformata HTCC2501
217
218


Tenacibaculum sp. MED152
219
220


Polaribacter irgensii 23-P
221
222


Pedobacter sp. BAL39
223
224


Flavobacteria bacterium BAL38
225
226


Flavobacterium psychrophilum JIP02/86
227
228


Flavobacterium johnsoniae UW101
229
230


Lactococcus lactis subsp. cremoris SK11
231
232


Psychromonas ingrahamii 37
233
234


Microscilla marina ATCC 23134
235
236


Cytophaga hutchinsonii ATCC 33406
237
238


Rhodopirellula baltica SH 1
239
240


Blastopirellula marina DSM 3645
241
242


Planctomyces maris DSM 8797
243
244


Algoriphagus sp. PR1
245
246


Candidatus Sulcia muelleri str. Hc
247
248


(Homalodisca coagulata)




Candidatus Carsonella ruddii PV
249
250


Synechococcus sp. RS9916
251
252


Synechococcus sp. WH 7803
253
254


Synechococcus sp. CC9311
255
256


Synechococcus sp. CC9605
257
258


Synechococcus sp. WH 8102
259
260


Synechococcus sp. BL107
261
262


Synechococcus sp. RCC307
263
264


Synechococcus sp. RS9917
265
266


Synechococcus sp. WH 5701
267
268


Prochlorococcus marinus str. MIT 9313
269
270


Prochlorococcus marinus str. NATL2A
271
272


Prochlorococcus marinus str. MIT 9215
273
274


Prochlorococcus marinus str. AS9601
275
276


Prochlorococcus marinus str. MIT 9515
277
278


Prochlorococcus marinus subsp. pastoris str.
279
280


CCMP1986




Prochlorococcus marinus str. MIT 9211
281
282


Prochlorococcus marinus subsp. marinus
283
284


str. CCMP1375




Nodularia spumigena CCY9414
285
286


Nostoc punctiforme PCC 73102
287
288


Nostoc sp. PCC 7120
289
290


Trichodesmium erythraeum IMS101
291
292


Acaryochloris marina MBIC11017
293
294


Lyngbya sp. PCC 8106
295
296


Synechocystis sp. PCC 6803
297
298


Cyanothece sp. CCY0110
299
300


Thermosynechococcus elongatus BP-1
301
302


Synechococcus sp. JA-2-3B′a(2-13)
303
304


Gloeobacter violaceus PCC 7421
305
306


Nitrosomonas eutropha C91
307
308


Nitrosomonas europaea ATCC 19718
309
310


Nitrosospira multiformis ATCC 25196
311
312


Chloroflexus aggregans DSM 9485
313
314


Leptospirillum sp. Group II UBA
315
316


Leptospirillum sp. Group II UBA
317
318


Halorhodospira halophila SL1
319
320


Nitrococcus mobilis Nb-231
321
322


Alkalilimnicola ehrlichei MLHE-1
323
324


Deinococcus geothermalis DSM 11300
325
326


Polynucleobacter sp. QLW-P1DMWA-1
327
328


Polynucleobacter necessarius STIR1
329
330


Azoarcus sp. EbN1
331
332


Burkholderia phymatum STM815
333
334


Burkholderia xenovorans LB400
335
336


Burkholderia multivorans ATCC 17616
337
338


Burkholderia cenocepacia PC184
339
340


Burkholderia mallei GB8 horse 4
341
342


Ralstonia eutropha JMP134
343
344


Ralstonia metallidurans CH34
345
346


Ralstonia solanacearum UW551
347
348


Ralstonia pickettii 12J
349
350


Limnobacter sp. MED105
351
352


Herminiimonas arsenicoxydans
353
354


Bordetella parapertussis
355
356


Bordetella petrii DSM 12804
357
358


Polaromonas sp. JS666
359
360


Polaromonas naphthalenivorans CJ2
361
362


Rhodoferax ferrireducens T118
363
364


Verminephrobacter eiseniae EF01-2
365
366


Acidovorax sp. JS42
367
368


Delftia acidovorans SPH-1
369
370


Methylibium petroleiphilum PM1
371
372


gamma proteobacterium KT 71
373
374


Tremblaya princeps
375
376


Blastopirellula marina DSM 3645
377
378


Planctomyces maris DSM 8797
379
380


Microcystis aeruginosa PCC 7806
381
382


Salinibacter ruber DSM 13855
383
384


Methylobacterium chloromethanicum
385
386
















TABLE 4







SEQ ID NOs of Representative Fungal and Plant [2Fe—2S]2+ DHAD


Proteins and Encoding Sequences










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












Schizosaccharomyces pombe ILV3
387
388


Saccharomyces cerevisiae ILV3
389
390


Kluyveromyces lactis ILV3
391
392


Candida albicans SC5314 ILV3
393
394


Pichia stipitis CBS 6054 ILV3
395
396


Yarrowia lipolytica ILV3
397
398


Candida glabrata CBS 138 ILV3
399
400


Chlamydomonas reinhardtii
401
402


Ostreococcus lucimarinus CCE9901
403
404


Vitis vinifera
405
406


(Unnamed protein product: CAO71581.1)




Vitis vinifera
407
408


(Hypothetical protein: CAN67446.1)




Arabidopsis thaliana
409
410


Oryza sativa (indica cultivar-group)
411
412


Physcomitrella patens subsp. patens
413
414


Chaetomium globosum CBS 148.51
415
416


Neurospora crassa OR74A
417
418


Magnaporthe grisea 70-15
419
420


Gibberella zeae PH-1
421
422


Aspergillus niger
423
424


Neosartorya fischeri NRRL 181
425
426


(XP_001266525.1)




Neosartorya fischeri NRRL 181
427
428


(XP_001262996.1)




Aspergillus niger
429
430


(hypothetical protein An03g04520)




Aspergillus niger
431
432


(Hypothetical protein An14g03280)




Aspergillus terreus NIH2624
433
434


Aspergillus clavatus NRRL 1
435
436


Aspergillus nidulans FGSC A4
437
438


Aspergillus oryzae
439
440


Ajellomyces capsulatus NAm1
441
442


Coccidioides immitis RS
443
444


Botryotinia fuckeliana B05.10
445
446


Phaeosphaeria nodorum SN15
447
448


Pichia guilliermondii ATCC 6260
449
450


Debaryomyces hansenii CBS767
451
452


Lodderomyces elongisporus NRRL YB-
453
454


4239




Vanderwaltozyma polyspora DSM 70294
455
456


Ashbya gossypii ATCC 10895
457
458


Laccaria bicolor S238N-H82
459
460


Coprinopsis cinerea okayama7#130
461
462


Cryptococcus neoformans var. neoformans
463
464


JEC21




Ustilago maydis 521
465
466


Malassezia globosa CBS 7966
467
468


Aspergillus clavatus NRRL 1
469
470


Neosartorya fischeri NRRL 181
471
472


(Putative)




Aspergillus oryzae
473
474


Aspergillus niger (hypothetical protein
475
476


An18g04160)




Aspergillus terreus NIH2624
477
478


Coccidioides immitis RS (hypothetical
479
480


protein CIMG_04591)




Paracoccidioides brasiliensis
481
482


Phaeosphaeria nodorum SN15
483
484


Gibberella zeae PH-1
485
486


Neurospora crassa OR74A
487
488


Coprinopsis cinerea okayama 7#130
489
490


Laccaria bicolor S238N-H82
491
492


Ustilago maydis 521
493
494
















TABLE 5







SEQ ID NOs of Representative [4Fe—4S]2+ DHAD Proteins


and Encoding Sequences










SEQ ID NO:
SEQ ID NO:


Organism
Nucleic acid
Peptide












Escherichia coli str. K-12 substr. MG1655
495
496


Bacillus subtilis subsp. subtilis str. 168
497
498


Agrobacterium tumefaciens str. C58
499
500


Burkholderia cenocepacia MC0-3
501
502


Psychrobacter cryohalolentis K5
503
504


Psychromonas sp. CNPT3
505
506


Deinococcus radiodurans R1
507
508


Wolinella succinogenes DSM 1740
509
510


Zymomonas mobilis subsp. mobilis ZM4
511
512


Clostridium acetobutylicum ATCC 824
513
514


Clostridium beijerinckii NCIMB 8052
515
516


Pseudomonas fluorescens Pf-5
517
518


Methanococcus maripaludis C7
519
520


Methanococcus aeolicus Nankai-3
521
522


Vibrio fischeri ATCC 700601 (ES114)
523
524


Shewanella oneidensis MR-1 ATCC
525
526


700550









Additional [2Fe-2S]2+ DHADs can be identified using the analysis described in co-pending U.S. Appl. Pub. No. 2010/0081154, which is herein incorporated by reference. The analysis is as follows: A Profile Hidden Markov Model (HMM) was prepared based on amino acid sequences of eight functionally verified DHADs. These DHADs are from Nitrosomonas europaea (DNA SEQ ID NO:309; protein SEQ ID NO:310), Synechocystis sp. PCC6803 (DNA SEQ ID:297; protein SEQ ID NO:298), Streptococcus mutans (DNA SEQ ID NO:167; protein SEQ ID NO:168), Streptococcus thermophilus (DNA SEQ ID NO:163; protein SEQ ID NO:164), Ralstonia metallidurans (DNA SEQ ID NO:345; protein SEQ ID NO:346), Ralstonia eutropha (DNA SEQ ID NO:343; protein SEQ ID NO:344), and Lactococcus lactis (DNA SEQ ID NO:231; protein SEQ ID NO:232). In addition, the DHAD from Flavobacterium johnsoniae (DNA SEQ ID NO:229; protein SEQ ID NO:230) was found to have DHAD activity when expressed in Escherichia coli and was used in making the Profile.
The Profile HMM was built as follows:
Step 1. Build a Sequence Alignment
The eight sequences for the functionally verified DHADs listed above were aligned using Clustal W with default parameters.
Step 2. Build a Profile HMM
The hmmbuild program was run on the set of aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new Profile HMM, and saves the Profile HMM to file. Using this program an un-calibrated profile was generated from the multiple alignment for each set of subunit sequences described above.
The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a Profile HMM. A Profile HMM is capable of modeling gapped alignments, e.g., including insertions and deletions, which lets the software describe a complete conserved domain (rather than just a small ungapped motif). Insertions and deletions are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”. These states are interconnected with arrows called state transition probabilities. M and I states are emitters, while D states are silent. The transitions are arranged so that at each node, either the M state is used (and a residue is aligned and scored) or the D state is used (and no residue is aligned, resulting in a deletion-gap character, ‘−’). Insertions occur between nodes, and I states have a self-transition, allowing one or more inserted residues to occur between consensus columns.
The scores of residues in a match state (i.e., match state emission scores), or in an insert state (i.e., insert state emission scores) are proportional to Log_2 (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the Profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24.
State transition scores are also calculated as log odds parameters and are proportional to Log_2 (t_x). Where t_x is the probability of transiting to an emitter or non-emitter state.
Step 3. Calibrate the Profile HMM
The Profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the Profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the Profile HMM.
The calibrated Profile HMM for the DHAD set of sequences is provided in Table 6. The Profile HMM is provided in a chart that gives the probability of each amino acid occurring at each position in the amino acid sequence. The highest probability is highlighted for each position. The first line for each position reports the match emission scores: probability for each amino acid to be in that state (highest score is highlighted). The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E.
For example, the DHAD Profile HMM shows that methionine has a 1757 probability of being in the first position, the highest probability which is highlighted. In the second position glutamic acid has the highest probability, which is 1356. In the third position lysine has the highest probability, which is 1569.
Step 4. Test the Specificity and Sensitivity of the Built Profile HMMs
The Profile HMM was evaluated using hmmsearch, which reads a Profile HMM from hmmfile and searches a sequence file for significantly similar sequence matches. The sequence file searched contained 976 sequences (see above). During the search, the size of the database (Z parameter) was set to 1 billion. This size setting ensures that significant E-values against the current database will remain significant in the foreseeable future. The E-value cutoff was set at 10.
A hmmer search with the Profile HMM generated from the alignment of the eight DHADs with experimentally verified function, matched all 976 sequences with an E value <10−5. This result indicates that members of the dehydratase superfamily share significant sequence similarity. A hmmer search with a cutoff of E value 10−5 was used to separate DHAD related dehydratases from other more remote but related proteins, as described above.
The Profile HMM is prepared using the HMMER software package (The theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.). The output of the HMMER software program is a Profile Hidden Markov Model (HMM) that characterizes the input sequences. The Profile HMM prepared for the eight DHAD proteins is given in Table 6.
This Profile HMM for DHADs can be used to identify DHAD related proteins. Any protein that matches the Profile HMM with an E value of <10−5 is a DHAD related protein, which includes [4Fe-4S]2+ DHADs, [2Fe-2S]2+ DHADs, aldonic acid dehydratases, and phosphogluconate dehydratases.
Sequences matching the Profile HMM given herein are then analyzed for the presence of the three conserved cysteines described above. The exact positions of the three conserved cysteines can vary, and these can be identified in the context of the surrounding sequence using multiple sequence alignments performed with the Clustal W algorithm (Thompson, J. D., Higgins, D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673 4680) employing the following parameters: 1) for pairwise alignment parameters, a Gap opening=10; Gap extend=0.1; matrix is Gonnet 250; and mode—Slow-accurate, 2) for multiple alignment parameters, Gap opening=10; Gap extension=0.2; and matrix is Gonnet series. For example, the three conserved cysteines are located at amino acid positions 56, 129, and 201 in the Streptococcus mutans (S. mutans) DHAD (SEQ ID NO:168), and at amino acid positions 61, 135, and 207 in the Lactococcus lactis (L. lactis) DHAD (SEQ ID NO:232). The exact positions of the three conserved cysteines in other protein sequences correspond to these positions in the S. mutans or the L. lactis amino acid sequence. One skilled in the art will readily be able to identify the presence or absence of each of the three conserved cysteines in the amino acid sequence of a DHAD protein using pairwise or multiple sequence alignments. In addition, other methods can be used to determine the presence of the three conserved cysteines, such as analysis by eye.
The DHAD Profile HMM matching proteins that have two but not the third (position 56) conserved cysteine include [4Fe-4S]2+ DHADs and phosphogluconate dehydratases (EDDs). Proteins having the three conserved cysteines include arabonate dehydratases and [2Fe-2S]2+ DHADs, and are members of a [2Fe-2S]2+ DHAD/aldonic acid dehydratase group. The [2Fe-2S]2+ DHADs can be distinguished from the aldonic acid dehydratases by analyzing for signature conserved amino acids found to be present in the [2Fe-2S]2+ DHADs or in the aldonic acid dehydratases at positions corresponding to the following positions in the Streptococcus mutans DHAD amino acid sequence. These signature amino acids are in [2Fe-2S]2+ DHADs or in aldonic acid dehydratases, respectively, at the following positions (with greater than 90% occurrence): 88 asparagine vs. glutamic acid; 113 not conserved vs. glutamic acid; 142 arginine or asparagine vs. not conserved; 165: not conserved vs. glycine; 208 asparagine vs. not conserved; 454 leucine vs. not conserved; 477 phenylalanine or tyrosine vs. not conserved; and 487 glycine vs. not conserved.
The disclosed methods for identification of [2Fe-2S]2+ DHAD enzymes can be carried out on a single sequence or on a group of sequences. In a preferred embodiment, one or more sequence databases are queried with a Profile HMM as described herein.
Additionally, the sequences of DHAD coding regions provided herein can be used to identify other homologs in nature. Such methods are well-known in the art, and various methods that can be used to isolate genes encoding homologous proteins are described in U.S. Appl. Pub. No. 2010/0081154, which such methods are incorporated by reference herein.
DHAD variant polypeptides provided herein may be, for example, of a size of about 10 or more, about 20 or more, about 25 or more, about 50 or more, about 75 or more, about 100 or more, about 200 or more, about 500 or more, about 1,000 or more, or about 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
Also provided are active fragments of the DHAD variant polypeptides. A “fragment” is a unique portion of a polypeptide or other enzyme used in the invention which is identical in sequence to but shorter in length than the parent full-length sequence. A fragment can comprise up to the entire length of the defined sequence, minus one amino acid residue. For example, a fragment can comprise from about 5 to about 1,000 contiguous amino acid residues. A fragment can be, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250, 500, 750, or 1,000 contiguous amino acid residues in length. Fragments can be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment can comprise a certain length of contiguous amino acids selected from the first 100, 200, 300, 400, or 500 amino acids of a polypeptide as shown in a certain defined sequence. Alternatively, a polypeptide fragment can comprise a certain length of contiguous amino acids selected from the last 100, 200, 300, 400, or 500 amino acids of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, can be encompassed by the present embodiments. An exemplary DHAD fragment encompassed by the present invention is the Streptococcus mutans DHAD enzyme (Amino Acid: SEQ ID NO:544; Nucleic Acid: SEQ ID NO:543) lacking the last 9 amino acids from the C-terminus (“the Δ9 variant”; Amino Acid: SEQ ID NO:546; Nucleic Acid: SEQ ID NO:545). In certain embodiments, the DHAD variant polypeptide fragments have DHAD activity, and thus are capable of catalyzing the conversion of 2,3-dihydroxy isovalerate to α-ketoisovalerate.
The DHAD variant polypeptides of the invention can further comprise a label (such as for detection) or peptide tag. Peptide tags can include, for example, a polyhistidine tag, a polycysteine tag, a V5 epitope, a myc epitope, or a sequence comprising a LumioTM tag (protein SEQ ID NOs:589 or 720; DNA SEQ ID NO:721). A detectable label can include, for example, an enzyme, a substrate for an enzyme, a fluorescent compound, a luminescent compound, a chemiluminescent compound, a radionuclide, a paramagnetic compound, or biotin.
DHAD Activity Assays
The presence of DHAD activity in a cell engineered to express a heterologous DHAD can be confirmed using methods known in the art and/or described herein. As one example, crude extracts from cells engineered to express a bacterial DHAD can be used in a DHAD assay as described in the Examples herein or as described by Flint and Emptage (J. Biol. Chem. (1988) 263(8): 3558-64) using dinitrophenylhydrazine. In another example, DHAD activity can be assayed by the methods disclosed in U.S. App. Pub. No. US20100081154, incorporated herein by reference, in a yeast strain that lacks endogenous DHAD activity. In such a yeast strain, if sufficient DHAD activity is present, the yeast strain will grow in the absence of branched-chain amino acids. DHAD activity can also be confirmed by more indirect methods, such as by assaying for a downstream product in a pathway requiring DHAD activity. Any product that has α-ketoisovalerate or α-ketomethylvalerate as a pathway intermediate can be measured in an assay for DHAD activity. A list of such products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol, and isobutanol.
Nucleic Acid Molecules
Provided herein are isolated nucleic acid molecules that encode for the above-described DHAD variant polypeptides. The coding region of the isolated nucleic acid encoding the DHAD variant can be codon optimized for a particular target host cell, as well known to one skilled in the art. The isolated nucleic acid molecules of the invention can be comprised in a vector. Vectors useful for the transformation of a variety of host cells are common and commercially available from companies such as EpicentreTM (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically, the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment can be inserted, to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be, for example, derived from genes that are not native to the specific species chosen as a host.
Initiation control regions or promoters, which are useful to drive expression of bacterial DHAD variant coding regions in the desired bacterial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements is suitable for the present invention including, but not limited to, lac, ara, tet, trp, IPL, IPR, T7, tac, and trc promoters (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas); the amy, apr, and npr promoters, and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans; nisA (useful for expression Gram-positive bacteria, Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). In addition, the ldhL1 and fabZ1 promoters of L. plantarum are useful for expression of chimeric genes in bacteria. The fabZ1 promoter directs transcription of an operon with the first gene, fabZ1, encoding (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase. Termination control regions can also be derived from various genes, typically from genes native to the preferred hosts. In other embodiments, a termination site is unnecessary. Optionally, a termination site can be unnecessary; however, it is most preferred if included.
Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors: pRK437, pRK442, and pRK442(H), are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid 50(1):74-79 (2003)). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmids pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for heterologous gene expression in Gram-negative bacteria. Some vectors that are useful for transformation of Bacillus subtilis and Lactobacillus include pAMβ1 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al., Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (van Kranenburg et al., Appl. Environ. Microbiol. 71(3):1223-1230 (2005)).
Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWV101 has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin et al., J. Bacteriol. 174(17):5633-5638 (1992)). Additionally, in vitro transposomes are available from commercial sources such as EpicentreTM to create random mutations in a variety of genomes.
Vectors suitable for expression and propagation in yeast cells are also well known. Methods for gene expression in yeast are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes in yeast, including, but not limited to, promoters derived from the following genes: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, CUP1, FBA, GPD, GPM, AOX1, ILV5 and TEF(M7). Suitable transcriptional terminators include, but are not limited to, FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, ADH1, and ILV5t.
Suitable promoters, transcriptional terminators, and a DHAD variant coding regions can be cloned into Escherichia coli (E. coli)-yeast shuttle vectors, and transformed into yeast cells, for example. These vectors allow strain propagation in both E. coli and yeast strains. Typically, the vector used contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, pHR81, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2μ origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Construction of expression vectors with a chimeric gene encoding the described DHAD variants can be performed, for example, by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast.
The gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast (see, e.g., Ma et al. Gene 58:201-216; 1987)). Typically, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain a 21 by sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X”, a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 by overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g., TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally, the construct can be verified by sequence analysis.
Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. Typically, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding region X-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding region X-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 base pairs (bps) of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.
Recombinant Host Cells
The isolated nucleic acid molecules and vectors of the invention can be transformed into a host cell for DHAD expression and activity. Suitable host cells include any cell capable of genetic manipulation, and include, for example, bacteria, cyanobacteria, filamentous fungi, and yeasts.
The microbial hosts selected for the production of isobutanol are preferably tolerant to isobutanol and should be able to convert carbohydrates to isobutanol. The criteria for selection of suitable microbial hosts include, for example, the following: intrinsic tolerance to isobutanol, high rate of glucose utilization, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations.
Yeast Cells
Yeast cells that can be hosts for expression of a DHAD variant of the invention are any yeast cells that are amenable to genetic manipulation and include, but are not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis, and Yarrowia lipolytica. In some embodiments, the yeast host is Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm ProTM yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMaxTM Green yeast, FerMaxTM Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.
Expression is achieved by transforming the host cell with a gene comprising a sequence encoding any of the DHAD variants of the invention. The coding region for the DHAD to be expressed can be codon optimized for the yeast cell, as well known to one skilled in the art.
In embodiments, reducing production of an endogenous iron-sulfur (Fe—S) protein in a yeast host cell may result in an improvement in activity of an expressed heterologous Fe—S cluster protein, such as the variant DHAD enzymes of the invention. For example, in the yeast Saccharomyces cerevisiae, the native DHAD is encoded by ILV3, and is a mitochondrially-localized protein. The applicants have found that a Saccharomyces cerevisiae host cell with a heterologous DHAD expressed in the cytosol had 1.5 fold comparative activity in a mitochondrial ILV3 deletion host cell. Thus, in any of the yeast hosts described herein, an endogenous ILV3 gene can be inactivated to reduce endogenous Fe—S protein expression. ILV3 encodes mitochondrial DHAD that is involved in branched chain amino acid biosynthesis. Mitochondrial DHAD is encoded by a nuclear gene, and has a mitochondrial targeting signal sequence so that it is transported to and localized in the mitochondrion. Any ILV3 gene can be inactivated in a yeast host cell of this disclosure. Examples of yeast ILV3 inactivation target genes and their encoded proteins are those from Saccharomyces cerevisiae YJM78 (coding SEQ ID NO:389; protein SEQ ID NO:390), Schizosaccharomyces pombe (coding SEQ ID NO:387; protein SEQ ID NO:388), Candida galbrata strain CBS 138 (coding SEQ ID NO:399; protein SEQ ID NO:400), Candida albicans SC5314 (coding SEQ ID NO:393; protein SEQ ID NO:394), Kluyveromyces lactis (coding SEQ ID NO:391; protein SEQ ID NO:392), Yarrowia lipolytica (coding SEQ ID NO:397; protein SEQ ID NO:398) and Pichia stipitis CBS 6054 (coding SEQ ID NO:395; protein SEQ ID NO:396).
In addition, in embodiments, overexpression of the transcriptional activator genes AFT1 and/or AFT2 or homologs thereof in a recombinant yeast microorganism improves DHAD activity. Thus, the invention also provides recombinant yeast host cells comprising the isolated nucleic acid molecules of the invention, further genetically engineered to have increased heterologous or native expression of AFT1 and/or AFT2 or homologs thereof. In general, cells that overexpress AFT1 and/or AFT2 or homologs thereof exhibit enhanced DHAD activity. The observed increases in DHAD activity resulting from the increased expression of AFT1 and/or AFT2 have broad applicability to any DHAD-requiring biosynthetic pathway, as DHAD activity is often a rate-limiting component of such pathways.
Grx3, Grx4, Fra2 and Ccc1 are proteins involved in iron-sulfur cluster biosynthesis in yeast. Grx3 and Grx4 are monothiol glutaredoxins that have been shown to be involved in cellular Fe content modulation and delivery in yeast. Glutaredoxins are glutathione-dependent thiol-disulfide oxidoreductases that function in maintaining the cellular redox homeostasis. Saccharomyces cerevisiae has two dithiol glutaredoxins (Grx1 and Grx2) and three monothiol glutaredoxins (Grx3, Grx4, and Grx5). The monothiol glutaredoxins are believed to reduce mixed disulfides formed between a protein and glutathione in a process known as deglutathionylation. Thus, the invention is also directed to a recombinant host described herein (e.g., yeast) further genetically modified to disrupt a gene encoding an endogenous Fra2, Grx3, Grx4, and/or Ccc1 or a homolog thereof. In embodiments, increases in DHAD activity may be observed in yeast cells with disruptions in FRA2, GRX3, GRX4, and/or CCC1.
In some embodiments, the invention is also directed to a recombinant host described herein (e.g., yeast) further genetically modified to disrupt (e.g., delete) a gene encoding pyruvate decarboxylase (PDC). In some embodiments, the PDC is PDC1, PDC5, PDC6, or combinations thereof.
Bacterial Cells
In some embodiments, the recombinant host cell is a prokaryotic cell. In certain embodiments, the recombinant host cell is a bacterial cell. In other embodiments, the bacterial cell is a lactic acid bacterial (LAB) cell selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. In still other embodiments, the bacterial host cell is the lactic acid bacteria Lactobacillus. In some embodiments, the bacterial host cell is Lactobacillus plantarum. 
Bacterial cells that can be hosts for expression of a heterologous bacterial [2Fe-2S]2+ DHAD include, but are not limited to, Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus. Engineering expression of a heterologous DHAD variant can increase DHAD activity in a host bacterial cell that naturally expresses a [2Fe-2S]2+ DHAD or a [4Fe-4S]2+ DHAD. Such host cells can include, for example, Escherichia coli and Bacillus subtilis. Furthermore, engineering expression of a heterologous DHAD variant provides DHAD activity in a host bacterial cell that has no endogenous DHAD activity. Such host cells can include, for example, Lactobacillus, Enterococcus, Pediococcus and Leuconostoc. 
Specific hosts include, for example, Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, and Bacillus subtilis. Bacterial cells can be genetically modified for expression of DHAD variants using methods well known to one skilled in the art. Expression of DHAD variants is generally achieved by transforming suitable bacterial host cells with a sequence encoding a DHAD variant protein. Typically, the coding sequence is part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a termination control region. The coding region can be from the host cell for transformation and combined with regulatory sequences that are not native to the natural gene encoding the variant DHAD. Alternatively, the coding region can be from another host cell.
Vectors can be introduced into LAB host cells using methods known in the art, such as electroporation (Cruz-Rodz et al., Molecular Genetics and Genomics 224:1252-154 (1990), Bringel et al., Appl. Microbiol. Biotechnol. 33: 664-670 (1990), Alegre et al., FEMS Microbiology letters 241:73-77 (2004)), and conjugation (Shrago et al., Appl. Environ. Microbiol. 52:574-576 (1986)). A chimeric DHAD gene can also be integrated into the chromosome of LAB using integration vectors (Hols et al., Appl. Environ. Microbiol. 60:1401-1403 (1990), and Jang et al., Micro. Lett. 24:191-195 (2003)).
Lactic acid bacteria are well characterized and are used commercially in a number of industrial processes. Although it is known that some lactic acid bacteria possess iron-sulfur (Fe—S) cluster requiring enzymes (Liu et al., Journal of Biological Chemistry (2000), 275(17), 12367-12373), and therefore possess the genetic machinery to produce Fe—S clusters, little is known about the ability of lactic acid bacteria to insert Fe—S clusters into heterologous enzymes, and little is known about the facility with which Fe—S cluster forming proteins can be expressed in lactic acid bacteria.
To obtain high levels of product in a lactic acid bacterium from a biosynthetic pathway including DHAD activity, high expression of DHAD activity is desired. The activity of the Fe—S requiring DHAD enzyme in a host cell can be limited, for example, by the availability of Fe—S clusters in the cell. Increasing the expression of Fe—S cluster forming proteins effectively increased the activity of DHAD in LAB cells. Thus, in certain embodiments, a lactic acid bacterial host cell is genetically engineered to express at least one recombinant genetic expression element encoding Fe—S cluster forming proteins. The genetic engineering of lactic acid bacteria to express iron-sulfur cluster forming proteins is described in U.S. Appl. Pub. No. 2010/0081182, which is herein incorporated by reference.
Expression of any set of proteins for Fe—S cluster formation can be used to increase DHAD activity in LAB cells. There are three known groups of Fe—S cluster forming proteins. These proteins are encoded by three types of operons: the Suf operon, the Isc operon, and the Nif operon. U.S. Appl. Pub. No. 2010/0081182 discloses the Suf operons of Lactobacillus plantarum (L. plantarum), Lactobacillus lactis (L. lactis), and Escherichia coli (E. coli); the Isc operon of E. coli; and the Nif operon of Wolinella succinogenes. Additional Fe—S cluster forming proteins can be readily identified by a skilled artisan, for example, by using the sequences disclosed in U.S. Appl. Pub. No. 2010/0081182 as sequence probes to identify homologous proteins in a desired organism.
Culture Conditions for Butanol Production
The invention is also directed to a method for the production of butanol (e.g., isobutanol), comprising providing a recombinant host cell comprising the isolated nucleic acid molecules of the present invention encoding a variant DHAD described herein; culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol from pyruvate; and recovering the isobutanol. In certain embodiments, the butanol (e.g., isobutanol) is produced at a titer that is increased as compared to a recombinant host cell that does not contain a variant DHAD.
In other embodiments, the isobutanol is produced at a rate that is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 15%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, or more, as compared to a recombinant host cell that does not contain a variant DHAD. In other aspects of the method to produce isobutanol, the concentration of isobutanol in the fermentation medium is greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 950 mM, or greater than or equal to about 100 mM.
The invention is also directed to methods for the production of butanol comprising providing a recombinant host cell comprising a polypeptide or variant thereof of the present invention, or a nucleic acid molecule which encodes a polypeptide or variant thereof of the present invention. In some embodiments, the method is a method for the production of isobutanol, comprising providing a recombinant host cell comprising a nucleic acid molecule of the invention encoding a variant DHAD, culturing the recombinant host cell in a fermentation medium under suitable conditions to produce isobutanol from pyruvate; and recovering the isobutanol. In some embodiments, the butanol (e.g., isobutanol) is produced at a titer that is increased as compared to a recombinant host cell that does not contain a variant DHAD. In other embodiments, the butanol (e.g., isobutanol) is produced at a rate that is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or more, as compared to a recombinant host cell that does not contain a variant DHAD, or any range of values thereof. In other embodiments, the butanol (e.g., isobutanol) is produced at a titer that is increased as compared to a recombinant host cell that does not contain a variant DHAD. In other embodiments, the butanol (e.g., isobutanol) is produced at a rate that is increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, or more, as compared to a recombinant host cell that does not contain a variant DHAD, or any range of values thereof. In other embodiments, the butanol (e.g., isobutanol) is produced at a rate that is increased by about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%, as compared to a recombinant host cell that does not contain a variant DHAD.
In other aspects of the method to produce butanol, the concentration of butanol (e.g., isobutanol) in the fermentation medium is greater than or equal to about 10 mM, greater than or equal to about 20 mM, greater than or equal to about 30 mM, greater than or equal to about 40 mM, greater than or equal to about 50 mM, greater than or equal to about 60 mM, greater than or equal to about 70 mM, greater than or equal to about 80 mM, greater than or equal to about 90 mM, greater than or equal to about 100 mM, or more, or any range of values thereof. In some embodiments, the concentration of butanol (e.g., isobutanol) in the fermentation medium is about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 30 mM to about 100 mM, about 40 mM to about 100 mM, about 50 mM to about 100 mM, about 60 mM to about 100 mM, about 70 mM to about 100 mM, about 80 mM to about 100 mM, or about 90 mM to about 100 mM.
In other embodiments, the invention is directed to a method of converting 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate, comprising providing a polypeptide or variant thereof of the invention. In some embodiments, the method comprises (a) providing a polypeptide or variant thereof of the invention, and (b) contacting the polypeptide or variant thereof with 2,3-dihydroxy isovalerate or 2,3-dihydroxymethylvalerate under conditions whereby 2,3-dihydroxyisovalerate is converted to α-ketoisovalerate, or whereby 2,3-dihydroxymethylvalerate is converted to α-ketomethylvalerate. It has been discovered that the activity of the polypeptides or variants of the invention is increased, and/or the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate is improved, as compared to a control polypeptide having DHAD activity which does not comprise a tag.
Recombinant host cells disclosed herein are grown in media which contains suitable carbon substrates. Additional carbon substrates can include, but are not limited to, monosaccharides such as fructose, oligosaccharides such as lactose, maltose, galactose, sucrose, or mixtures thereof, polysaccharides such as starch, cellulose or mixtures thereof, and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates can include, but are not limited to, ethanol, lactate, succinate, glycerol, or mixtures thereof.
Additionally, in some embodiments, the carbon substrate can also be a one carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine, and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7th (1993), 415 32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485 489 (1990)). Hence, it is contemplated that the source of carbon utilized in the present invention can encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, in some embodiments, the carbon substrates are glucose, fructose, and sucrose, or mixtures of these with five-carbon (C5) sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose can be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose can be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Pat. No. 7,932,063, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipid. Biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass can comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs, bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.
In addition to an appropriate carbon source, fermentation media contains suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art and suitable for growth of the cultures and promotion of an enzymatic pathway comprising a DHAD.
Typically, cells are grown at a temperature in the range of about 20° C. to about 40° C. in an appropriate medium. Suitable growth media for the present invention include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, Yeast Medium (YM) broth, or broth that includes yeast nitrogen base, ammonium sulfate, and dextrose (as the carbon/energy source) or Yeast Extract Peptone Dextrose (YPD) Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media can also be used, and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′ monophosphate, can also be incorporated into the fermentation medium.
Suitable pH ranges for the fermentation are from about pH 5.0 to about pH 9.0. In one embodiment, about pH 6.0 to about pH 8.0 is used for the initial condition. Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0. In one embodiment, about pH 5.0 to about pH 8.0 is used for the initial condition. Suitable pH ranges for the fermentation of other microorganisms are from about pH 3.0 to about pH 7.5. In one embodiment, about pH 4.5 to about pH 6.5 is used for the initial condition.
Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentations.
Industrial Batch and Continuous Fermentations
Isobutanol, or other products, can be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed batch system. Fed batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed batch fermentations are common and well known in the art, examples of which are found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, M.A., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.
Isobutanol, or other products, can also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
It is contemplated that the production of isobutanol, or other products, can be practiced using batch, fed batch or continuous processes and that any known mode of fermentation is suitable. Additionally, it is contemplated that cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.
Biosynthetic Pathways
Expression of a DHAD variant in bacteria or yeast, as described herein, provides the transformed, recombinant host cell with dihydroxy-acid dehydratase (DHAD) activity for conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate or 2,3-dihydroxymethylvalerate to α-ketomethylvalerate. Any product that has α-ketoisovalerate or α-ketomethylvalerate as a pathway intermediate can be produced in a bacterial or yeast strain disclosed herein having the described heterologous DHAD variants. A list of such products includes, but is not limited to, valine, isoleucine, leucine, pantothenic acid, 2-methyl-1-butanol, 3-methyl-1-butanol and isobutanol.
For example, yeast biosynthesis of valine includes steps of acetolactate conversion to 2,3-dihydroxyisovalerate by acetohydroxyacid reductoisomerase (ILV5), conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate (also called 2-keto-isovalerate) by dihydroxy-acid dehydratase, and conversion of α-ketoisovalerate to valine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). Biosynthesis of leucine includes the same steps to α-ketoisovalerate, followed by conversion of α-ketoisovalerate to α-isopropylmalate by α-isopropylmalate synthase (LEU9, LEU4), conversion of α-isopropylmalate to beta-isopropylmalate by isopropylmalate isomerase (LEU1), conversion of beta-isopropylmalate to α-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally conversion of α-ketoisocaproate to leucine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). The bacterial pathway is similar, involving differently named proteins and genes. Increased conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate will increase flow in these pathways, particularly if one or more additional enzymes of a pathway is overexpressed. Thus, it is desired for production of valine or leucine to use a strain disclosed herein.
Biosynthesis of pantothenic acid includes a step performed by DHAD, as well as steps performed by ketopantoate hydroxymethyltransferase and pantothenate synthase. Engineering of expression of these enzymes for enhanced production of pantothenic acid biosynthesis in microorganisms is described, for example, in U.S. Pat. No. 6,177,264, which is incorporated by reference herein.
The α-ketoisovalerate product of DHAD is an intermediate in the isobutanol biosynthetic pathways disclosed, for example, in U.S. Pat. No. 7,851,188, which is incorporated by reference herein. A diagram of the disclosed isobutanol biosynthetic pathways is provided in FIG. 1. Production of isobutanol in a strain disclosed herein benefits from increased DHAD activity. As disclosed herein, DHAD activity is provided by expression of a variant DHAD in a bacterial or yeast cell. As described in U.S. Pat. No. 7,851,188, steps in an example isobutanol biosynthetic pathway include conversion of: pyruvate to acetolactate as catalyzed for example by acetolactate synthase, acetolactate to 2,3-dihydroxyisovalerate as catalyzed for example by acetohydroxy acid isomeroreductase; 2,3-dihydroxy isovalerate to α-ketoisovalerate as catalyzed for example by acetohydroxy acid dehydratase, also called dihydroxy-acid dehydratase (DHAD); α-ketoisovalerate to isobutyraldehyde as catalyzed for example by branched-chain α-keto acid decarboxylase; and isobutyraldehyde to isobutanol as catalyzed for example by branched-chain alcohol dehydrogenase. The substrate to product conversions, and enzymes involved in these reactions, are described, for example, in U.S. Pat. No. 7,851,188, which is incorporated by reference herein.
Genes that can be used for expression of the pathway step enzymes named above other than the variant DHADs disclosed herein, as well as those for two additional isobutanol pathways, are described, for example, in U.S. Pat. No. 7,851,188, which is incorporated by reference herein. Additional genes that can be used can be identified by one skilled in the art through bioinformatics or experimentally as described above. Ketol-acid reductoisomerase (KARI) enzymes are also disclosed, for example, in U.S. Pat. No. 7,910,342 and PCT App. Pub. No. WO2012/129555, which are incorporated by reference herein. Examples of KARIs disclosed therein include KARIs from Vibrio cholerae (DNA: SEQ ID NO:684; protein SEQ ID NO:685), Pseudomonas aeruginosa PAO1, (DNA: SEQ ID NO:686; protein SEQ ID NO:687), Pseudomonas fluorescens PF5 (DNA: SEQ ID NO:688; protein SEQ ID NO:689) and Anaerostipes caccae (protein SEQ ID NO:697).
Additionally described in U.S. Pat. No. 7,851,188 is the construction of chimeric genes and genetic engineering of bacteria and yeast for isobutanol production using the disclosed biosynthetic pathways. In some embodiments, one or more components of the above-described biosynthetic pathways can be endogenous to the host cell of choice, or can be heterologous. Additionally, in other embodiments, one or more of the genes encoding the enzymes required in the above-described biosynthetic pathways can be overexpressed in the host cell.
Methods for Butanol Isolation from Fermentation Medium
Bioproduced butanol (e.g., isobutanol) may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the isobutanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.
Because isobutanol forms a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
The isobutanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the butanol from the solvent.
Distillation in combination with adsorption can also be used to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).
Additionally, distillation in combination with pervaporation may be used to isolate and purify the isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).
In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields. One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction. In general, with regard to butanol fermentation, for example, the fermentation medium, which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the fermentation medium form a biphasic mixture. The butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.
Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 2009/0305370, the disclosure of which is hereby incorporated in its entirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C12 to C22 fatty alcohols, C12 to C22 fatty acids, esters of C12 to C22 fatty acids, C12 to C22 fatty aldehydes, and mixtures thereof. The extractant(s) for ISPR can be non-alcohol extractants. The ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.
In some embodiments, the alcohol can be formed by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst capable of esterifying the alcohol with the organic acid. In such embodiments, the organic acid can serve as an ISPR extractant into which the alcohol esters partition. The organic acid can be supplied to the fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol. The catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock. When the catalyst is supplied to the fermentation vessel, alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel. Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant. The extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration. In addition, unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.
In situ product removal can be carried out in a batch mode or a continuous mode. In a continuous mode of in situ product removal, product is continually removed from the reactor. In a batchwise mode of in situ product removal, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. For in situ product removal, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some embodiments of the present invention, the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel. The ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved. In some embodiments, the ester-containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.
EXAMPLES
Example 1
Construction of Yeast Strain PNY2115, PNY2145 and Other Plasmids Creation of PNY2115
Yeast strain PNY2115 has the following genotype: MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66.
Saccharomyces cerevisiae strain PNY0827 is used as the starting strain for the construction of strain PNY2115. PNY0827 refers to a strain derived from Saccharomyces cerevisiae which has been deposited at the ATCC under the Budapest Treaty on Sep. 22, 2011 at the American Type Culture Collection, Patent Depository 10801 University Boulevard, Manassas, Va. 20110-2209 and has the patent deposit designation PTA-12105.
1. Deletion of URA3 and Sporulation into Haploids
In order to delete the endogenous URA3 coding region, a deletion cassette was PCR-amplified from pLA54 (SEQ ID NO:592) which contains a PTEF1-kanMX4-TEF1t cassette flanked by loxP sites to allow homologous recombination in vivo and subsequent removal of the KANMX4 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and primers BK505 (SEQ ID NO:593) and BK506 (SEQ ID NO:594). The URA3 portion of each primer was derived from the 5′ region 180 nucleotides upstream of the URA3 ATG and 3′ region 78 nucleotides downstream of the coding region such that integration of the kanMX4 cassette results in replacement of the URA3 coding region. The PCR product was transformed into PNY0827 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on Yeast Extract Peptone (YEP) medium supplemented with 2% glucose and 100 μg/ml Geneticin® (Invitrogen Life TechnologiesTM, Grand Island, N.Y.) at 30° C. Transformants were screened by colony PCR with primers LA468 (SEQ ID NO:595) and LA492 (SEQ ID NO:596) to verify presence of the integration cassette. A heterozygous diploid was obtained: NYLA98, which has the genotype MATa/α URA3/ura3::loxP-kanMX4-loxP. To obtain haploids, NYLA98 was sporulated using standard methods (Codón A. C., Gasent-Ramírez J. M., Benítez T. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeast. Appl Environ Microbiol. 1995 PMID: 7574601). Tetrads were dissected using a micromanipulator and grown on rich YEP medium supplemented with 2% glucose. Tetrads containing four viable spores were patched onto synthetic complete medium lacking uracil supplemented with 2% glucose, and the mating type was verified by multiplex colony PCR using primers AK109-1 (SEQ ID NO:597), AK109-2 (SEQ ID NO:598), and AK109-3 (SEQ ID NO:599). The resulting identified haploid strain was called NYLA103, which has the genotype: MATα ura3Δ::loxP-kanMX4-loxP, and NYLA106, which has the genotype: MATa ura3Δ::loxP-kanMX4-loxP.
2. Deletion of HIS3
To delete the endogenous HIS3 coding region, a scarless deletion cassette was used. The four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen Inc., Valencia, Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO:600) and primer oBP453 (SEQ ID NO:601), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO:602), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO:603) containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO:604), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO:605), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO:606), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO:607). PCR products were purified with a PCR Purification kit (Qiagen Inc., Valencia, Calif.). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO:600) and oBP455 (SEQ ID NO:603). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO:604) and oBP459 (SEQ ID NO:607). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen Inc., Valencia, Calif.). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO:600) and oBP459 (SEQ ID NO:607). The PCR product was purified with a PCR Purification kit (Qiagen Inc., Valencia, Calif.). Competent cells of NYLA106 were transformed with the HIS3 ABUC PCR cassette and were plated on synthetic complete medium lacking uracil supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating onto synthetic complete medium lacking histidine and supplemented with 2% glucose at 30° C. Genomic DNA preparations were made to verify the integration by PCR using primers oBP460 (SEQ ID NO:608) and LA135 (SEQ ID NO:609) for the 5′ end and primers oBP461 (SEQ ID NO:610) and LA92 (SEQ ID NO:611) for the 3′ end. The URA3 marker was recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-fluoroorotic acid (5-FOA) at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from 5-fluoroorotic Acid (5-FOA) plates onto SD-URA medium to verify the absence of growth. The resulting identified strain, called PNY2003 has the genotype: MATa ura3Δ::loxP-kanMX4-loxP his3Δ.
3. Deletion of PDC1
To delete the endogenous PDC1 coding region, a deletion cassette was PCR-amplified from pLA59 (SEQ ID NO:612), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and primers LA678 (SEQ ID NO:613) and LA679 (SEQ ID NO:614). The PDC1 portion of each primer was derived from the 5′ region 50 nucleotides downstream of the PDC1 start codon and 3′ region 50 nucleotides upstream of the stop codon such that integration of the URA3 cassette results in replacement of the PDC1 coding region but leaves the first 50 nucleotides and the last 50 nucleotides of the coding region. The PCR product was transformed into PNY2003 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by colony PCR using primers LA337 (SEQ ID NO:615), external to the 5′ coding region and LA135 (SEQ ID NO:609), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA692 (SEQ ID NO:616) and LA693 (SEQ ID NO:617), internal to the PDC1 coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 2% glucose at 30° C. Transformants were plated on rich medium supplemented with 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 2% glucose to verify absence of growth. The resulting identified strain, called PNY2008 has the genotype: MATa ura3Δ::loxP-kanMX4-loxP his3Δ pdc1Δ::loxP71/66.
4. Deletion of PDC5
To delete the endogenous PDC5 coding region, a deletion cassette was PCR-amplified from pLA59 (SEQ ID NO:612), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and primers LA722 (SEQ ID NO:619) and LA733 (SEQ ID NO:620). The PDC5 portion of each primer was derived from the 5′ region 50 nucleotides upstream of the PDC5 start codon and 3′ region 50 nucleotides downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire PDC5 coding region. The PCR product was transformed into PNY2008 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers LA453 (SEQ ID NO:621), external to the 5′ coding region and LA135 (SEQ ID NO:609), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA694 (SEQ ID NO:622) and LA695 (SEQ ID NO:623), internal to the PDC5 coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich YEP medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2009 has the genotype: MATa ura3Δ::loxP-kanMX4-loxP his3Δ pdc1Δ::loxP71/66 pdc5Δ::loxP71/66.
5. Deletion of FRA2
The FRA2 deletion was designed to delete 250 nucleotides from the 3′ end of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding sequence intact. An in-frame stop codon was present seven nucleotides downstream of the deletion. The four fragments for the PCR cassette for the scarless FRA2 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen Inc., Valencia, Calif.). FRA2 Fragment A was amplified with primer oBP594 (SEQ ID NO:624) and primer oBP595 (SEQ ID NO:625), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment B. FRA2 Fragment B was amplified with primer oBP596 (SEQ ID NO:626), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment A, and primer oBP597 (SEQ ID NO:627), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment U. FRA2 Fragment U was amplified with primer oBP598 (SEQ ID NO:628), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO:629), containing a 5′ tail with homology to the 5′ end of FRA2 Fragment C. FRA2 Fragment C was amplified with primer oBP600 (SEQ ID NO:630), containing a 5′ tail with homology to the 3′ end of FRA2 Fragment U, and primer oBP601 (SEQ ID NO:631). PCR products were purified with a PCR Purification kit (Qiagen Inc., Valencia, Calif.). FRA2 Fragment AB was created by overlapping PCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying with primers oBP594 (SEQ ID NO:624) and oBP597 (SEQ ID NO:627). FRA2 Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U and FRA2 Fragment C and amplifying with primers oBP598 (SEQ ID NO:628) and oBP601 (SEQ ID NO:631). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen Inc., Valencia, Calif.). The FRA2 ABUC cassette was created by overlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO:624) and oBP601 (SEQ ID NO:631). The PCR product was purified with a PCR Purification kit (Qiagen Inc., Valencia, Calif.).
To delete the endogenous FRA2 coding region, the scarless deletion cassette obtained above was transformed into PNY2009 using standard techniques and plated on synthetic complete medium lacking uracil and supplemented with 1% ethanol. Genomic DNA preparations were made to verify the integration by PCR using primers oBP602 (SEQ ID NO:632) and LA135 (SEQ ID NO:609) for the 5′ end, and primers oBP602 (SEQ ID NO:632) and oBP603 (SEQ ID NO:633) to amplify the whole locus. The URA3 marker was recycled by plating on synthetic complete medium supplemented with 1% ethanol and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify the absence of growth. The resulting identified strain, PNY2037, has the genotype: MATa ura3Δ::loxP-kanMX4-loxP his3Δ pdc1Δ::loxP71/66 pdc5Δ::loxP71/66 fra2Δ.
6. Addition of Native 2 Micron Plasmid
The loxP71-URA3-loxP66 marker was PCR-amplified using Phusion DNA polymerase (New England BioLabs; Ipswich, Mass.) from pLA59 (SEQ ID NO:612), and transformed along with the LA811x817 (SEQ ID NOs:634 and 635) and LA812x818 (SEQ ID NOs:636 and 637) 2-micron plasmid fragments (amplified from the native 2-micron plasmid from CEN.PK 113-7D; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre) into strain PNY2037 on SE-URA plates at 30° C. The resulting strain PNY2037 2μ::loxP71-URA3-loxP66 was transformed with pLA34 (pRS423::cre) (also called, pLA34) (SEQ ID NO:618) and selected on SE-HIS-URA plates at 30° C. Transformants were patched onto YP-1% galactose plates and allowed to grow for 48 hrs at 30° C. to induce Cre recombinase expression. Individual colonies were then patched onto SE-URA, SE-HIS, and YPE plates to confirm URA3 marker removal. The resulting identified strain, PNY2050, has the genotype: MATa ura3Δ::loxP-kanMX4-loxP, his3Δ pdc1Δ::loxP71/66 pdc5Δ::loxP71/66 fra2Δ 2-micron.
7. Construction of PNY2115 from PNY2050
Construction of PNY2115 [MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66] from PNY2050 was as follows:
a. pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66
To integrate alsS into the pdc1Δ::loxP66/71 locus of PNY2050 using the endogenous PDC1 promoter, an integration cassette was PCR-amplified from pLA71 (SEQ ID NO:643), which contains the gene acetolactate synthase from the species Bacillus subtilis with a FBA1 promoter and a CYC1 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using the KAPA HiFiTM PCR Kit (Kapabiosystems, Woburn, Mass.) and primers 895 (SEQ ID NO:663) and 679 (SEQ ID NO:664). The PDC1 portion of each primer was derived from 60 nucleotides of the upstream of the coding sequence and 50 nucleotides that are 53 nucleotides upstream of the stop codon. The PCR product was transformed into PNY2050 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers 681 (SEQ ID NO:665), external to the 3′ coding region and 92 (SEQ ID NO:666), internal to the URA3 gene. Positive transformants were then prepped for genomic DNA and screened by PCR using primers N245 (SEQ ID NO:667) and N246 (SEQ ID NO:668). The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2090 has the genotype MATa ura3Δ::loxP, his3Δ, pdc1Δ::loxP71/66, pdc5Δ::loxP71/66 fra2Δ 2-micron pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66.
b. pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66
To delete the endogenous PDC6 coding region, an integration cassette was PCR-amplified from pLA78 (SEQ ID NO:648), which contains the kivD gene from the species Listeria grayi with a hybrid FBA1 promoter and a TDH3 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using the KAPA HiFiTM PCR Kit (Kapabiosystems, Woburn, Mass.) and primers 896 (SEQ ID NO:669) and 897 (SEQ ID NO:670). The PDC6 portion of each primer was derived from 60 nucleotides upstream of the coding sequence and 59 nucleotides downstream of the coding region. The PCR product was transformed into PNY2090 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers 365 (SEQ ID NO:671) and 366 (SEQ ID NO:672), internal primers to the PDC6 gene. Transformants with an absence of product were then screened by colony PCR N638 (SEQ ID NO:673), external to the 5′ end of the gene, and 740 (SEQ ID NO:674), internal to the FBA1 promoter. Genomic DNA was prepared from positive transformants and screened by PCR with two external primers to the PDC6 coding sequence. Positive integrants would yield a 4720 nucleotide long product, while PDC6 wild type transformants would yield a 2130 nucleotide long product. The URA3 marker was recycled by transforming with pLA34 containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain is called PNY2093 and has the genotype MATa ura3Δ::loxP his3Δ pdc5Δ::loxP71/66 fra2Δ 2-micron pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66.
c. adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66
To delete the endogenous ADH1 coding region and integrate BiADH using the endogenous ADH1 promoter, an integration cassette was PCR-amplified from pLA65 (SEQ ID NO:654), which contains the alcohol dehydrogenase from the species Beijerinckii indica with an ILV5 promoter and a ADH1 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using the KAPA HiFiTM PCR Kit (Kapabiosystems, Woburn, Mass.) and primers 856 (SEQ ID NO:675) and 857 (SEQ ID NO:676). The ADH1 portion of each primer was derived from the 5′ region 50 nucleotides upstream of the ADH1 start codon and the last 50 nucleotides of the coding region. The PCR product was transformed into PNY2093 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers BK415 (SEQ ID NO:677), external to the 5′ coding region and N1092 (SEQ ID NO:678), internal to the BiADH gene. Positive transformants were then screened by colony PCR using primers 413 (SEQ ID NO:679), external to the 3′ coding region, and 92 (SEQ ID NO:666), internal to the URA3 marker. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2101 has the genotype MATa ura3Δ::loxP his3Δ pdc5Δ::loxP71/66 fra2Δ 2-micron pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66.
d. fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66
To integrate BiADH into the fra2Δ locus of PNY2101, an integration cassette was PCR-amplified from pLA65 (SEQ ID NO:654), which contains the alcohol dehydrogenase from the species Beijerinckii indica with an ILV5 promoter and an ADH1 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using the KAPA HiFiTM PCR Kit (Kapabiosystems, Woburn, Mass.) and primers 906 (SEQ ID NO:680) and 907 (SEQ ID NO:681). The FRA2 portion of each primer was derived from the first 60 nucleotides of the coding sequence starting at the ATG and 56 nucleotides downstream of the stop codon. The PCR product was transformed into PNY2101 using standard genetic techniques and transformants were selected on synthetic complete media lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers 667 (SEQ ID NO:682), external to the 5′ coding region and 749 (SEQ ID NO:683), internal to the ILV5 promoter. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete media lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich media supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete media lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2110 has the genotype MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 2-micron pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66.
e. GPD2 Deletion
To delete the endogenous GPD2 coding region, a deletion cassette was PCR amplified from pLA59 (SEQ ID NO:612), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using the KAPA HiFiTM PCR Kit (Kapabiosystems, Woburn, Mass.) and primers LA512 (SEQ ID NO:638) and LA513 (SEQ ID NO:639). The GPD2 portion of each primer was derived from the 5′ region 50 nucleotides upstream of the GPD2 start codon and 3′ region 50 nucleotides downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire GPD2 coding region. The PCR product was transformed into PNY2110 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were screened to verify correct integration by colony PCR using primers LA516 (SEQ ID NO:640) external to the 5′ coding region and LA135 (SEQ ID NO:609), internal to URA3. Positive transformants were then screened by colony PCR using primers LA514 (SEQ ID NO:641) and LA515 (SEQ ID NO:642), internal to the GPD2 coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO:618) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were plated on rich medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2115, has the genotype MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66.
pLH689
Plasmid pLH689 (SEQ ID NO:698) is a yeast-E. coli shuttle vector based on pHR81 (ATCC#87541). It contains genes for the expression of KARI variant K9JB4P (SEQ ID NO:697) and S. mutans DHAD with a C-terminal tag (SEQ ID NO:721). The positions of the relevant gene features are listed below in Table 7.







TABLE 7







Nucleotide positions of pathway gene features


of plasmid pLH689 (SEQ ID NO: 698)













Element
Description
Start
End
Strand
















promoter
ILV5p
427
1620
T



CDS
K9JB4P
1628
2659
T



terminator
ILV5t
2685
3307
T



terminator
FBAt
3320
3632
B



CDS
S. mutans ilvD-lum
3641
5377
B



promoter
TEF1(M7)p
5387
5787
B









pLH691

Plasmid pLH691 (SEQ ID NO:590) is a yeast-E. coli shuttle vector based on pHR81 (ATCC#87541). It contains genes for the expression of KARI variant K9JB4P and S. mutans DHAD with a 9 amino acid (Δ9) deletion. The positions of the relevant gene features are listed in the Table 8.







TABLE 8







Nucleotide positions of pathway gene features


of plasmid pLH691 (SEQ ID NO: 590)













Element
Description
Start
End
Strand
















promoter
ILV5p
427
1620
T



CDS
K9JB4P
1628
2659
T



terminator
ILV5t
2685
3307
T



terminator
FBAt
3320
3632
B



CDS
S. mutans ilvD-□9
3644
5335
B



promoter
TEF1(M7)p
5339
5739
B









pLH804

Plasmid pLH804 (SEQ ID NO:591) is a yeast-E. coli shuttle vector based on pHR81 (ATCC#87541). It contains genes for the expression of KARI variant K9JB4P and S. mutans DHAD. The positions of the relevant gene features are listed in the Table 9.







TABLE 9







Nucleotide positions of pathway gene features


of plasmid pLH804 (SEQ ID NO: 591)













Element
Description
Start
End
Strand
















promoter
ILV5p
427
1620
T



CDS
K9JB4P
1628
2659
T



terminator
ILV5t
2685
3307
T



terminator
FBAt
3320
3632
B



CDS
S. mutans ilvD
3641
5356
B



promoter
TEF1(M7)p
5366
5766
B









pRS413::BiADH-kivD

Plasmid pRS413::BiADH-kivD (SEQ ID NO:874) is a yeast-E. coli shuttle vector based on pRS413 (ATCC#87518). It contains genes for the expression of BiADH and kivD. The positions of the relevant gene features are listed in the Table 10.







TABLE 10







Nucleotide positions of pathway gene features in


plasmid pRS413::BiADH-kivD (SEQ ID NO: 874)













Element
Description
Start
End
Strand






Promoter
FBA1p
2293
2893
T



CDS
kivD_Lg(y)
2902
4548
T



Terminator
TDH3t
4560
5139
T



Promoter
PDC1p
5983
6852
T



CDS
adhBiy
6853
7896
T



Terminator
ADH1t
7905
8220
T









Creation of PNY2145

PNY2145 was constructed from PNY2115 (MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66; described in, for example, U.S. Provisional Appl. No. 61/842,817, filed Jul. 3, 2013, which is incorporated by reference herein) by the additional integration of a phosphoketolase gene cassette at the pdc5Δ locus and by replacing the native AMN1 gene with a codon optimized version of the ortholog from CEN.PK. Integration constructs are further described below.
pdc5Δ::FBA(L8)-xpk1-CYC1t-loxP71/66
The TEF(M4)-xpk1-CYC1t gene from pRS423::TEF(M4)-xpk1+ENO1-eutD (SEQ ID NO:829) was PCR amplified using primers N1341 and N1338 (SEQ ID NOs: 830 and 831), generating a 3.1 kb product. The loxP-flanked URA3 gene cassette from pLA59 (SEQ ID NO:832) was amplified with primers N1033c and N1342 (SEQ ID NOs: 833 and 834), generating a 1.6 kb product. The xpk1 and URA3 PCR products were fused by combining them without primers for an additional 10 cycles of PCR using Phusion DNA polymerase. The resulting reaction mix was then used as a template for a PCR reaction with KAPA Hi Fi and primers N1342 and N1364 (SEQ ID NOs:834 and 835). A 4.2 kb PCR product was recovered by purification from an electrophoresis agarose gel (Zymo kit). FBA promoter variant L8 (SEQ ID NO:836) was amplified using primers N1366 and N1368 (SEQ ID NOs:837 and 838). The xpk1::URA3 PCR product was combined with the FBA promoter by additional rounds of PCR. The resulting product was phosphorylated with polynucleotide kinase and ligated into pBR322 that had been digested with EcoRV and treated with calf intestinal phosphatase. The ligation reaction was transformed into E. coli cells (Stb13 competent cells from Invitrogen). The integration cassette was confirmed by sequencing. To prepare DNA for integration, the plasmid was used as a template in a PCR reaction with Kapa HiFi and primers N1371 and N1372 (SEQ ID NOs:839 and 840). The PCR product was isolated by phenol-chloroform extraction and ethanol precipitation (using standard methods; e.g., Maniatis et al.). Five micrograms of DNA were used to transform strain PNY2115. Transformants were selected on medium lacking uracil (synthetic complete medium minus uracil with 1% ethanol as the carbon source). Colonies were screened for the integration event using PCR (JumpStart) with primers BK93 and N1114 (SEQ ID NOs:841 and 842). Two clones were selected to carry forward. The URA3 marker was recycled by transforming with pJT254 (SEQ ID NO:843) containing the CRE recombinase under the GAL1 promoter and plating on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30° C. Transformants were grown in rich medium supplemented with 1% ethanol to derepress the recombinase. Marker removal was confirmed for single colony isolates by patching to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. Loss of the recombinase plasmid, pJT254, was confirmed by patching the colonies to synthetic complete medium lacking histidine and supplemented with 1% ethanol. Proper marker removal was confirmed by PCR (primers N160SeqF5 (SEQ ID NO:844) and BK380). One resulting clone was designated PNY2293.
amn1Δ::AMN1(y)-loxP71/66
To replace the endogenous copy of AMN1 with a codon-optimized version of the AMN1 gene from CEN.PK2, an integration cassette containing the CEN.PK AMN1 promoter, AMN1(y) gene (nucleic acid SEQ ID NO:845; amino acid SEQ ID NO:846), and CEN.PK AMN1 terminator was assembled by SOE PCR and subcloned into the shuttle vector pLA59. The AMN1(y) gene was ordered from DNA 2.0 with codon-optimization for S. cerevisiae. The completed pLA67 plasmid (SEQ ID NO:847) contained: 1) pUC19 vector backbone sequence containing an E. coli replication origin and ampicillin resistance gene; 2) URA3 selection marker flanked by loxP71 and loxP66 sites; and 3) PAMN1(CEN.PK)-AMN1(y)-termAMN1(CEN.PK) expression cassette.
PCR amplification of the AMN1(y)-loxP71-URA3-loxP66 cassette was done by using KAPA HiFi from Kapa Biosystems, Woburn, Mass. and primers LA712 (SEQ ID NO:848) and LA746 (SEQ ID NO:849). The PCR product was transformed into PNY2293 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30° C. Transformants were observed under magnification for the absence of a clumping phenotype with respect to the control (PNY2293). The URA3 marker was recycled using the pJT254 Cre recombinase plasmid as described above. After marker recycle, clones were again observed under magnification to confirm absence of the clumping phenotype. A resulting identified strain, PNY2145, has the genotype: MATa ura3Δ::loxP his3Δ pdc5Δ::P[FBA(L8)]-XPK|xpk1_Lp-CYCt-loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66 amn1Δ::AMN1(y).
Example 2
Site Directed Mutagenesis of A9 Variant of S. mutans DHAD
Amino acid replacements at positions 378, 383, 385, 387, and 388 were individually incorporated into a truncated version of Streptococcus mutans DHAD lacking the nine c-terminal amino acids (Δ9 variant) via site directed mutagenesis. Mutagenesis was performed with a yeast shuttle plasmid employing the QuikChange® Lightning Site-Directed Mutagenesis Kit (Catalog #210518; Agilent Technologies, Stratagene Products Division, La Jolla, Calif.). Mutagenesis primers listed in Table 11 were commercially synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa). Primers were combined into mixes, as indicated in Table 11 (column labeled “Mix”).







TABLE 11







Primer Mixes Employed for Site Directed Mutagenesis











SEQ ID




Mix
NO:
Primers
Sequence





P2A1mix
527
P2A1
GTTATTATGCCGCTTGAAAATGCTAAACG





TGAAGATGGTCCGCTC


 


P2A1mix
528
P2A1rev
GAG CGG ACC ATC TTC ACG TTT





AGC ATT TTC AAG CGG CAT AAT





AAC


 


G2S2mix
529
G2S2
GAAAATCCTAAACGTGAAGATTCTCCGCT





CATTATTCTCCATGG


 


G2S2mix
530
G2S2rev
TGG AGA ATA ATG AGC GGA GAA





TCT TCA CGT TTA GGA TTT TC


 


L2F3mix
531
L2F3
CTAAACGTGAAGATGGTCCGTTCATTATT





CTCCATGGTAACTTGG


 


L2F3mix
532
L2F3rew
CCA AGT TAC CAT GGA GAA TAA





TGA ACG GAC CAT CTT CAC GTT





TAG


 


L2V4mix
533
L2V4
CTAAACGTGAAGATGGTCCGGTCATTATT





CTCCATGGTAACTTGG


 


L2V4mix
534
L2V4rev
CCA AGT TAC CAT GGA GAA TAA





TGA CCG GAC CAT CTT CAC GTT





TAG


 


I2V5mix
535
I2V5
GAAGATGGTCCGCTCATTGTTCTCCATGG





TAACTTGGC


 


I2V5mix
536
I2V5rev
GCC AAG TTA CCA TGG AGA ACA





ATG AGC GGA CCA TCT TC


 


I2M6mix
537
I2M6
GAAGATGGTCCGCTCATTATGCTCCATGG





TAACTTGGC


 


I2M6mix
538
I2M6rev
GCC AAG TTA CCA TGG AGC ATA





ATG AGC GGA CCA TCT TC


 


L2I7mix
539
L2I7
GAAGATGGTCCGCTCATTATTATCCATGG





TAACTTGGCTCCAGAC


 


L2I7mix
540
L2I7rev
GTC TGG AGC CAA GTT ACC ATG





GAT AAT AAT GAG CGG ACC ATC





TTC


 


L2M8mix
541
L2M8
GAAGATGGTCCGCTCATTATTATGCATGG





TAACTTGGCTCCAGAC


 


L2M8mix
542
L2M8rev
GTC TGG AGC CAA GTT ACC ATG





CAT AAT AAT GAG CGG ACC ATC





TTC









Except for the primers, templates, and double distilled water (ddH2O), all reagents used here were supplied with the kit indicated above. A stock mixture for the eight mutagenesis reactions contained 1 μl of pLH691 (495 ng)(SEQ ID NO:590), 50 μl of 10× reaction buffer, 10 μl of dNTP mix, 15 μl of QuikSolution reagent, and 404 μl of ddH2O. Each mutagenesis reaction mixture contained 48 ul of the stock mixture, 1 μl of a primer mix (10 uM each primer), and 1 μl of QuikChange Lightning Enzyme. The following conditions were used for the reactions: The starting temperature was 95° C. for 2 min followed by 18 heating/cooling cycles. Each cycle consisted of 95° C. for 20 sec, 60° C. for 10 sec, and 68° C. for 10 min. At the completion of the temperature cycling, the samples were incubated at 68° C. for 5.0 min and then held awaiting sample recovery at 4° C. 2 μl of the Dpn I was added to each reaction and the mixtures were incubated for 1 hr at 37° C.
2 μl of each mutagenic reaction was transformed into One Shot® Stbl3TM Chemically Competent Escherichia coli (E. coli)(Invitrogen, Catalog # C7373-03) or One Shot® TOP10 Chemically Competent E. coli (Invitrogen, Catalog #C404003) according to the manufacturer's instructions. The transformants were spread on agar plates containing the LB medium and 100 μg/ml ampicillin (Catalog #L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Multiple transformants for each reaction were inoculated into LB medium containing 100 μg/ml ampicillin and incubated at 37° C. with shaking at 225 rpm. Plasmid DNA was isolated from the cells with the QIAprep® Spin Miniprep Kit (Catalog #2706; Qiagen, Valencia, Calif.) according to the protocol provided by the manufacturer. Sequencing of the complete DHAD genes were performed with primers Dseq1 (aacgcgtgaagcttttgaagatg; SEQ ID NO:690), Dseq2 (tcagttcggaacaatcacgg; SEQ. ID NO:691), Dseq3 (tgctttccctttcatcaatgattgttg, SEQ ID NO:692), Dseq4 (tccatgttagccatagcgataac SEQ ID NO:693), Dseq5 (ttgtgcttcaggagcgatatg; SEQ ID NO:694), N885 (ctgctaatgtggaattgacac, SEQ ID NO:695), and N929 (gtctgttacggctcccctag, SEQ ID NO:696). Two clones were prepared for each variant listed in Table 12, with the exception of delta9-L2I7, for which only a single clone was prepared.







TABLE 12







Prepared Streptococcus mutans DHAD delta9 (Δ9) Variants













Nucleotide
Amino Acid




Variant
SEQ ID NO:
SEQ ID NO:
Substitution






delta9-P2A1
551
552
P378A



delta9-G2S2
556
557
G383S



delta9-L2F3
561
562
L385F



delta9-L2V4
566
567
L385V



delta9-I2V5
571
572
I387V



delta9-I2M6
576
577
I387M



delta9-L2I7
581
582
L388I



delta9-L2M8
586
587
L388M









Example 3
Site Directed Mutagenesis of Full Length Streptococcus mutans (S. mutans) DHAD
Full length versions of Δ9-L2V4 (substitution L385V), Δ9-I2V5 (substitution I387V), and Δ9-L2I7 (substitution L387I) were prepared by site directed mutagenesis of the wild type S. mutans DHAD. Site directed mutagenesis was performed as described in Example 2, with modifications.
For the L385V substitution, the mutagenesis reaction contained 1 ul pLH804 (50 ng)(SEQ ID NO:591), 1 ul L2V4mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange® Lightning Enzyme, and 39.5 ul of distilled water (ddH2O).
For the I387V substitution, the mutagenesis reaction contained 1 ul pLH804 (50 ng)(SEQ ID NO:591), 1 ul I2V5 mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the L388I substitution, the mutagenesis reaction contained 1 ul pLH804 (50 ng)(SEQ ID NO:591), 1 ul L2I8 mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange® Lightning Enzyme, and 39.5 ul of ddH2O.
The following conditions were used for the reactions: The starting temperature was 95° C. for 2 min followed by 18 heating/cooling cycles. Each cycle consisted of 95° C. for 20 sec, 60° C. for 10 sec, and 68° C. for 10 min. At the completion of the temperature cycling, the samples were incubated at 68° C. for 5 min and then held awaiting sample recovery at 4° C. 2 μl of DpnI restriction enzyme was added to each reaction and the mixtures were incubated for 30 min at 37° C.
Three clones were prepared for variants L2V4 and I2V5 listed in Table 13, while two clones were prepared for variant L2I7.







TABLE 13







Prepared Variants of Full Length S. mutans DHAD













Nucleotide
Amino Acid




Variant
SEQ ID NO:
SEQ ID NO:
Substitution






L2V4
564
565
L385V



I2V5
569
570
I387V



L2I7
579
580
L388I









Example 4
Isobutanol Production of S. mutans DHAD Derivatives in PNY2115
Variants prepared in Examples 2 and Example 3 were analyzed for isobutanol production in yeast strain PNY2115, described in Example 1 (MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66).
Growth Media
Four types of media were used during the growth procedure of yeast strains: SE-ura agar plate, SAG-2-ura agar plate, an aerobic pre-culture media and an anaerobic culture media. All chemicals were obtained from Sigma-Aldrich® (St. Louis, Mo.) unless otherwise noted.
Yeast transformation recovery plate (SE-ura): 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)(pH 5.5), 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 0.2% ethanol, 0.01% weight per volume (w/v) leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Glucose adaptation plate (SAG-2-Ura): 50 mM MES (pH 5.5, 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 2% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Aerobic pre-culture media (SAG-0.2-Ura): 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 0.2% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Anaerobic culture media (SAG-3-Ura): 50 mM MES (pH 5.5, 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 3% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, 0.002% w/v tryptophan, 30 mg/L nicotinic acid, 30 mg/L thiamine and 10 mg/L ergosterol made up in 50/50 volume per volume (v/v) Tween/ethanol solution.
Transformation and Glucose Adaptation
Competent cells of the PNY2115 (MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66) were prepared and transformed with 1 μL of purified plasmid ( ̃0.4 to 0.8 μg total DNA) using a Frozen-EZ Yeast Transformation II KitTM (Zymo Research Corp.; Irvine, Calif.). Transformation mixtures were plated on SE-ura plates and incubated at 30° C. for 4 days. Three or four colonies for each transformant were selected and patched onto SE-ura plates and incubated at 30° C. for 2 days. The variants then underwent glucose adaptation by patching onto SAG-2-Ura plates and growing for 2 days at 30° C.
Deep-well Plate Growth Procedure
1.5 mL aliquots of the aerobic pre-culture media were dispensed into each well of a VWR 48 deep-well plate (#82004-674, VWR, Radnor, Pa.) and inoculated with cells grown on a SAG-2-Ura agar plate, as described above. A sterile air permeable cover (#60941-086, VWR, Radnor, Pa.) was used to seal the culture plate. The plate was placed in a 30° C. incubator and was grown for 20 to 24 hours with shaking, and an OD600 value (optical density at 600 nm) was obtained using Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). OD600 values were converted to equivalent OD600 values on a Cary 300 (Agilent Technologies, Wilmington, Del.) calibration value. A dilution 48 deep-well plate was set with a target Cary OD600 value of 0.35 for each well in a total volume of 1.5 mL. Wells with Cary OD600 values of 0.35 to 0.40 were transferred directly from the original plate to the dilution plate. For all other wells, a volume of turbid culture was transferred the volume was brought up to 1.5 mL with aerobic pre-culture media. The 48 deep-well plate was returned to the 30° C. shaking incubator and grown for an additional 20 to 24 hours. OD600 values were obtained as described above.
Serum Vial Growth Procedure
The final volume of medium in each 15 mL serum vial was 10 mL of medium. Target inoculation OD600 value of 0.1 was set for each vial. Turbid culture volume was based on the equivalent Cary 300 OD600 values obtained from each well of the dilution 48 deep-well plate described in the previous section. Anaerobic culture media was added to bring the final volume up to 10 mL. Serum vials were then capped and crimped. Vials were placed in a 30° C. shaking incubator and grown for 40-50 hours. Inoculation of the serum vials were performed under aerobic conditions. Wells that did not have and OD600 value of at least 0.3 were not inoculated into serum vials.
High-performance Liquid Chromatography (HPLC) Analysis
Samples were taken for HPLC analysis and to obtain OD600 values at the end of the anaerobic growth period. Serum vials were opened and an aliquot of turbid culture was removed and filtered in preparation for HPLC analysis. Another aliquot was removed for OD600 determination (described above). The remaining turbid culture was centrifuged. The resulting supernatant was discarded and the cell pellet was saved by freezing at −80° C. in the event additional analysis was required.
HPLC analysis was performed using a Waters 2695 separations unit, 2996 photodiode array detector, and 2414 refractive index detector (Waters, Milford, Mass.) with a Shodex Sugar SH-G pre-column and Shodex Sugar SH1011 separations column (Shodex, J M Science, Grand Island, N.Y.). Compounds were separated by isocratic elution at 0.01 N sulfuric acid with a flow rate of 0.5 mL/min. Chromatograms were analyzed using the Waters Empower Pro software. Isobutanol titer and molar yield for Streptococcus mutans (S. mutans) delta9 (Δ9) DHAD variants clones 1 and 2
PNY2115 with S. mutans variants described in Example 2 were grown and analyzed as described above. Isobutanol titer and molar yield are listed in Tables 14 and 11 below.







TABLE 14







S. mutans Δ9 DHAD variants (clone 1 set) in PNY2115












Isobutanol






mM at 46 hr

Molar Yield














Clone
Mean
SD
Mean
SD
















delta9 control
45
4.4
0.40
0.02



(pLH691)(SEQ ID







NO: 590)







delta9-P2A1 #1
0
0
0
0



delta9-G2S2 #1
0.2
0.3
0.01
0.002



delta9-L2F3 #1
10
1.4
0.31
0.02



delta9-L2V4 #1
18
21
0.3
0.2



delta9-I2V5 #1
51
1.4
0.53
0.01



delta9-I2M6 #1
0
0
0.42
0.04



delta9-L2I7 #1
25
5.3
0.44
0.02



delta9-L2M8 #1
30
1.7
0.51
0.02
















TABLE 15







S. mutans Δ9 DHAD variants (clone 2 set) in PNY2115












Isobutanol






mM at 46 hr

Molar Yield














Clone
Mean
SD
Mean
SD
















delta9 control
55
15
0.42
0.02



(pLH691)(SEQ ID







NO: 590)







delta9-L2F3 #2
13
0.5
0.24
0.02



delta9-L2V4 #2
55
15
0.20
0.09



delta9-I2V5 #2
49
9
0.40
0.03



delta9-L2M8 #2
35
7
0.36
0.04









Isobutanol Titer and Molar Yield for Streptococcus mutans (S. mutans) DHAD Variants

PNY2115 with S. mutans variants described in Example 3 were grown and analyzed as described above. Isobutanol titers and molar yield are listed in Table 16 below.







TABLE 16







S. mutans DHAD variants (full length) in PNY2115












Isobutanol






mM 50 hr

Molar Yield














Clone
Mean
SD
Mean
SD
















WT Control
51
3
0.54
0.01



(pLH804)(SEQ ID







NO: 591)







804-L2V4
82
10
0.63
0.01



Clone #1







804-I2V5
49
5
0.55
0.02



Clone#1







804-I2V5
41
14
0.51
0.04



Clone#2









FIG. 3 is a graph illustrating the isobutanol production in yeast strain PNY2115 transformants harboring Δ9 (Delta 9) S. mutans DHAD variants (P2A1, G2S2, L2F3, L2V4, I2V5, I2M6, L2I7, and L2M8) and the parental Δ9 S. mutans DHAD (control).
FIG. 4 is a graph illustrating the isobutanol production in yeast strain PNY2115 transformants harboring Δ9 S. mutans DHAD variants (I2V5, L2F3, L2M8, and L2V4) and the parental Δ9 S. mutans DHAD (control).
FIG. 5 is a graph illustrating the isobutanol production in yeast strain PNY2115 transformants harboring full length S. mutans DHAD variants (I2V5 clones 1 and 2 and L2V4) and the parental full length S. mutans DHAD (WT).
FIG. 6 is a graph illustrating DHAD activity in yeast strain PNY2115 harboring full length S. mutans DHAD variants (I2V5 clone 1, I2V5 clone 2, and L2V4) and the parental full length S. mutans DHAD (WT).
Example 5
DHAD Specific Activities for Streptococcus mutans (S. mutans) DHAD Derivatives in Crude Extracts of PNY2115
DHAD specific activities were measured in crude extracts of yeast strain PNY2115 transformed with either the wild type S. mutans DHAD or variants of the full length enzyme containing a single amino acid change at position 385, position 387, or position 388.
Following 40-50 hr of growth in serum vials as described in Example 3, yeast cells were centrifuged and resultant pellets stored at −80° C. Frozen yeast cells were later thawed, resuspended in 0.1 M K-Hepes pH 6.8 containing 10 mM MgCl2 and a protease inhibitor cocktail (Roche, Catalog #11873580001), and then broken by bead beating. The broken cells were centrifuged to remove the cell debris and generate the yeast crude extract. Protein concentrations (mg/ml) of extracts were measured with the Pierce Coomassie Plus (Bradford) Protein Assay (Catalog #23236, Thermoscientific). DHAD enzyme activities were measured spectrophotometrically in an end point assay using the 2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D. H. and M. H. Emptage, J. Biol. Chem. 263:3558-64 (1988), with modifications. The assay buffer contained 0.1 M K-Hepes pH 6.8 and 10 mM MgCl2. Yeast extracts were diluted in assay buffer. Sufficient (R)-2,3-dihydroxyisovaleric acid was added to assay buffer so that the final concentration in the assay is 10 mM. In each assay, an enzyme containing solution and sufficient substrate containing buffer are mixed so that the final volume is 300 ul. Assay mixtures were incubated at 30° C. for 20 minutes. At five minute intervals, a 60 ul aliquot of each reaction was mixed with 70 ul of a saturated solution of 2,4-DNPH in 1 N HCl. Following a 30 minute incubation at room temperature, 70 ul of 4 N KOH in ethanol was then added to the solution, followed by brief mixing. The absorbance of the mixture was read at 540 nm with a Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). A standard curve containing 0 to 1.33 mM α-ketoisovalerate was employed to calculate enzyme activities (U/ml) for the conversion of (R)-2,3-dihydroxyvalerate to α-ketoisovalerate in the assays. DHAD specific activities (U/mg) were determined from enzyme activities (U/ml) and protein concentrations (mg/ml) measured for each sample. Averages for each clone evaluated are provided in Table 17.







TABLE 17







DHAD specific activities in PNY2115 extracts










DHAD SA U/mg












Clone
Mean
SD







WT Control
0.14
0.05



(pLH804) (SEQ ID



NO: 591)



804-L2V4
0.16
0.06



Clone #1



804-I2V5
0.19
0.08



Clone #1



804-I2V5
0.08
0.08



Clone #2










Example 6
DHIV Accumulation Levels for S. mutans DHAD Derivatives in PNY2115
Wild type S. mutans DHAD and the full length variants prepared in Example 2 were transformed into yeast strain PNY2115 and analyzed for levels of DHIV accumulation and isobutanol production. Cultures were grown as described in Example 4, with the modification that samples were removed for analysis following 26 hr and 48 hr of growth. In addition to the analyses described in Example 4, samples were subjected to liquid chromatography-mass spectrometry (“LC/MS”) to measure levels of DHIV. LC/MS quantitation was performed as described in U.S. Patent Appl. Pub. No. 2012/0258873, incorporated by reference herein. As shown in Tables 18 and 19, replacement of wild type DHAD with the variants results in DHIV accumulation levels and DHIV/isobutanol ratios that are equivalent to or lower than the wild type control.







TABLE 18







Isobutanol and DHIV levels at 26 hr growth in serum vials











Isobutanol





mM
DHIV mM
DHIV/Isobutanol













Clone
Mean
SD
Mean
SD
Mean
SD
















WT Control
7
1
0.3
0.2
0.043
0.017


(pLH804)








(SEQ ID NO: 591)








804-L2V4
9
4
0.1
0.05
0.011
0.001


Clone #1








804-I2V5
4
3
0.2
0.1
0.050
0.012


Clone#1
















TABLE 19







Isobutanol and DHIV levels at 48 hr growth in serum vials











Isobutanol

DHIV/



mM
DHIV mM
Isobutanol













Clone
Mean
SD
Mean
SD
Mean
SD
















WT Control
35
2
1.3
0.5
0.038
0.011


(pLH804)








(SEQ ID NO: 591)








804-L2V4
57
18
0.8
0.4
0.012
0.005


Clone #1








804-I2V5
24
14
0.6
0.4
0.026
0.004


Clone#1









FIG. 7 is a graph illustrating DHIV accumulation in yeast strain PNY2115 harboring full length S. mutans DHAD variants (804-L2V4 and 804-I2V5) and the parental full length S. mutans DHAD (WT control).
Example 7
Isobutanol Production with I2V5 IlvD Variant







TABLE 20







Strain referenced in Example 7









Strain




Name
Genotype
Description





PNY1665
MATa ura3Δ::loxP pdc5Δ::loxP66/71
herein



fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 2-



micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-



ALS|alsS_Bs-CYC1t-loxP71/66



pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-



TDH3t-loxP71/66 adh1Δ::P[ADH1]-



ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66



amn1Δ::AMN1









Plasmid Construction
Plasmids were constructed in a 2-micron based Saccharomyces cerevisiae-Escherichia coli shuttle vector.
pBP3765—C-terminal Tagged I2V5 IlvD_Sm
pBP3765 (SEQ ID NO:699) was constructed to contain a chimeric gene having the coding region of the I2V5 mutant ilvD gene from Streptococcus mutans (nt position 5377-3665) followed by a LumioTM tag sequence (nt 3664-3647; Invitrogen, Carlsbad, Calif.; Adams et al. J. Am. Chem. Soc., 124:6063, 2002) expressed from the yeast TEF1 mutant 7 promoter (nt 5787-5387; Nevoigt et al. Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the K9JB4P mutant ilvC gene from Anaeropstipes cacae (nt 1628-2659; described in Int'l Pub. No. WO2012/12955, which is incorporated by reference herein) expressed from the yeast ILV5 promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
Strain Construction
Following conversion of PNY2115 to PNY2121 by replacing the endogenous copy of AMN1 with a codon-optimized version of the AMN1 gene from CEN.PK2 (SEQ ID NO:722), PNY2121 was restored back to a histidine prototroph. The HISS coding sequence and 500 bp upstream and downstream of the coding sequence were amplified from a haploid (PNY0865) obtained from sporulation of PNY0827. PNY2121 was transformed with the resulting PCR product and transformants were selected on agar plates containing synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. A PNY2121 HIS3+ isolate was designated PNY1665.
PNY1665 was transformed with the plasmids described above and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Three transformants were selected for each plasmid construct.
Isobutanol Production
Isobutanol production was tested for the isobutanologen strains described above. Strains were grown overnight in 10 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% w/v ethanol, 0.3% w/v glucose, and 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were sub-cultured into the same medium to an OD600 of 0.4, and glucose was added to a final concentration of 3% w/v 15 ml of culture in 125 ml VWR vent cap shake flasks were grown for 4 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. Cells were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in high glucose medium to an OD600 of 0.2. High glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.1% w/v ethanol, 3.0% w/v glucose, and 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. 10 ml of the culture in high glucose medium was transferred to a 20 ml serum vial (Kimble Chase; Vineland, N.J.). Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 44 hours.
After 44 hours, the cultures were sampled for OD600 and culture supernatants (collected using Spin-X centrifuge tube filter units, Costar Cat. No. 8169) were analyzed by HPLC (Example 4). OD600, isobutanol concentration, and isobutanol molar yield are presented in Table 21.







TABLE 21







Average OD600, isobutanol concentration, and isobutanol molar yield


and standard deviations.













Isobutanol Yield



OD600
Isobutanol (mM)
(mol/mol)














PNY1665/pLH689
2.1 ± 0.1
112.0 ± 3.0
0.69 ± 0.01


PNY1665/pBP3765
2.2 ± 0.0
 96.2 ± 5.1
0.67 ± 0.02









Example 8
Construction of Additional Yeast Strains
The following table provides the genotypes of the various yeast strains referenced in the following Examples.







TABLE 22







Strains referenced in the following Examples








Strain Name
Genotype





PNY2115
MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 2-micron



plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-



CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-



KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-



ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-



ADHt-loxP71/66 gpd2Δ::loxP71/66


PNY1566
MATa ura3Δ::loxP pdc5Δ::loxP66/71 2-micron plasmid



(CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-



loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-



TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-



loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66



gpd2Δ::loxP71/66


PNY1602 and
PNY1566 with plasmid pLH689 containing (P[ILV5]-


PNY1612
KARI|ilvC_Ll-ILV5t P[TEF1(M7)]-DHAD|ilvD_Sm-



lum-FBA1t)


PNY1604 and
PNY1566 with plasmid pLH804 containing (P[ILV5]-


PNY1614
KARI|ilvC_Ll-ILV5t P[TEF1(M7)]-DHAD|ilvD_Sm-



FBA1t)









To obtain PNY0865, PNY0827 was sporulated using standard methods (Codón A C, Gasent-Ramírez J M, Benítez T. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeast. Appl. Environ. Microbiol. 1995). After the formation of asci (as observed by microscopy), a 100 ul aliquot of cells was resuspended in 100 ul of 1 M sorbitol and treated with 5 U of zymolyase (Zymo Research, Orange Calif.) at 37° C. for 15 min. Resulting tetrads were spread on YPD medium and dissected using a micromanipulator (Singer Instruments, Somerset UK), and the single-spore isolates were grown at 30° C. for 3 d to form colonies. One tetrad with four viable spores was characterized further. The mating type of the single-spore isolates was determined by PCR as described (Huxley, C., E. D. Green and I. Dunham (1990). “Rapid assessment of S. cerevisiae mating type by PCR.” Trends Genet 6(8): 236). One spore isolate, of mating type MATα, was designated PNY0865.
PNY2115 was restored back to a histidine prototroph. The HIS3 coding sequence and 500 base pairs (bp) upstream and downstream of the coding sequence were amplified from the haploid PNY0865. PNY2115 was transformed with the resulting PCR product and transformants were selected on agar plates containing synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. A PNY2115 HIS3+ isolate was designated PNY1566.
PNY1566 was transformed with pLH804 (ilvD Sm; SEQ ID NO:591) and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Two transformants were selected and designated PNY1604 and PNY1614. PNY1566 was transformed with pLH689 (tagged ilvD Sm) and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Two transformants were selected and designated PNY1602 and PNY1612.
Example 9
DHAD Activity of Variant DHADs
PNY1604, PNY1614, PNY1602, and PNY1612 were grown overnight in 12 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% weight per volume (w/v) ethanol, 0.3% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 1 ml of high glucose medium. High glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.1% w/v ethanol, 3.0% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. 15 ml of high glucose medium in 125 ml VWR vent cap shake flasks was inoculated with cells to a final OD600 0.4 and grown for 5 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. Cells were again centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 2 ml of high glucose medium. 30 ml of high glucose medium in 60 ml serum vials (Kimble Chase; Vineland, N.J.) was inoculated with resuspended cells to a final OD600 of 0.15. Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 20 hours. Cultures were centrifuged at 3800×g for 5 minutes at 4° C. The pellets were washed with cold 50 mM HEPES pH 6.8 and then centrifuged at 3800×g for 5 minutes at 4° C. Cell pellets were frozen on dry ice and stored at −80° C. until they were assayed for DHAD activity.
For DHAD activity measurements the frozen yeast cells were thawed, resuspended in 0.1 M K-Hepes pH 6.8 containing 10 mM MgCl2 and a protease inhibitor cocktail (Roche, Catalog #11873580001), and then broken by bead beating. The broken cells were centrifuged to remove the cell debris and generate the yeast crude extract. Protein concentrations (mg/ml) of extracts were measured with the Pierce Coomassie Plus (Bradford) Protein Assay (Catalog #23236, Thermoscientific). DHAD enzyme activities were measured spectrophotometrically in an end point assay using the 2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D. H. and M. H. Emptage, J. Biol. Chem. 263:3558-64, 1988, with modifications. The assay buffer contained 0.1 M K-Hepes pH 6.8 and 10 mM MgCl2. Yeast extracts were diluted in assay buffer. Sufficient (R)-2,3-dihydroxyisovaleric acid was added to assay buffer so that the final concentration in the assay is 10 mM. In each assay, an enzyme containing solution and sufficient substrate containing buffer are mixed so that the final volume is 300 ul. Assay mixtures were incubated at 30° C. for 20 minutes. At five minute intervals, a 60 ul aliquot of each reaction was mixed with 70 ul of a saturated solution of 2,4-DNPH in 1 N HCl. Following a 30 minute incubation at room temperature, 70 ul of 4 N KOH in ethanol was then added to the solution, followed by brief mixing. The absorbance of the mixture was read at 540 nm with a Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). A standard curve containing 0 mM to 1.33 mM α-ketoisovalerate was employed to calculate enzyme activities (units per milliliter, U/ml) for the conversion of (R)-2,3-dihydroxyvalerate to α-ketoisovalerate in the assays. DHAD specific activities (units per milligram, U/mg) were determined from enzyme activities (U/ml) and protein concentrations (mg/ml) measured for each sample.
Strains PNY1604 and PNY1614 containing the control ilvD_Sm had an average DHAD activity of 0.32 U/mg. See FIG. 8. Strains PNY1602 and PNY1612 containing the tagged ilvD_Sm had an average DHAD activity of 1.25 U/mg. See FIG. 8.
Example 10
Constructs for Expressing Variant DHAD in Yeast Strain BY4741
Vector derived from pHR81 (ATCC 87541) was used for expressing the wild type and mutant DHAD from S. mutans under the control of FBA promoter. Vector pHR81 FBA-IlvD(Sm) contained wild-type (WT) DHAD. In this-vector, the FBA promoter is in the region from nucleotides (nt) 7626 to 8623. The IlvD gene is from nt 8631 to 10343 flanked by restriction sites SpeI and NotI. For the expression of the IlvD protein containing a C-terminal tag, vector pHR81 FBA-IlvD(Sm)-lum was used. In this vector, the IlvD gene was located between nt 8631 to 10343 and a C-terminal tag sequence (SEQ ID NO:785) was added in frame from 10344 to 10364.
Example 11
DHAD Activity Measurement in Yeast Strain BY4741
Vectors pHR81 FBA-IlvD(Sm) (SEQ ID NO:788) and pHR81 FBA-IlvD (Sm)-lum (SEQ ID NO:789) were transformed into yeast strain BY4741. Competent cells were prepared with a Frozen Yeast Transformation kit (Zymo Research). The transformants were selected on plates with complete synthetic yeast growth medium minus Ura (Teknova). Growth on liquid medium was carried out by adding 5 ml of an overnight culture into 100 ml medium in a 250 ml flask. Cells from 80 ml culture were harvested by centrifugation (4,000 rpm for 10 min at 4° C.) and washed with 10 ml TM8 buffer (50 mM Tris, pH 8.0, 10 mM MgSO4) stored at 4° C. The cells were resuspended in 1 ml of TM8 and transferred to a lysing matrix tubes with 0.1 mm silica spheres (MP Biomedicals, Solon, Ohio). The cells were broken with a beads beater (4× with 30 seconds each). The crude extract was obtained by centrifugation with a table top microfuge at 12,000 rpm at 4° C. for 30 min. The supernatants were removed and stored on ice until assayed as described above.
It was found that the DHADs assayed herein were stable in crude extracts kept on ice for a few hours. The activity was also preserved when samples were frozen in liquid N2 and stored at −80° C. Unexpectedly, results from enzymatic measurement (FIG. 9) showed that about 50% increase in activity with the IlvD enzyme containing a tag at the C-terminus as compared to the activity obtained with the wild-type (WT) enzyme.
Example 12
C-terminal-Tagged DHAD Expression Strains
Plasmid and Strain Construction
Plasmids were constructed in a pRS423-based 2-micron Saccharomyces cerevisiae-Escherichia coli shuttle vector. Genes encoding the DHAD sequence or the C-terminal-tagged DHAD sequence were cloned into the PmlI and NotI restriction sites of the plasmid, such that the gene was expressed from the yeast FBA1 promoter and followed by the yeast FBA1 terminator. All genes, except the Streptococcus mutans ilvD gene, were synthesized codon optimized for expression in Saccharomyces cerevisiae (Genscript, Piscataway, N.J.). The native sequence was utilized for the Streptococcus mutans ilvD gene. The predicted Zea mays chloroplast targeting peptide sequence was not included in the synthesized gene. The sequence encoding the first 33 amino acids was removed and replaced with a methionine codon. The predicted Neurospora crassa mitochondrial targeting peptide sequence was not included in the synthesized gene. Two start sites were tested. Construct Neurospora crassa DHAD(1) had the sequence encoding the first 32 amino acids removed and replaced with a methionine codon. Construct Neurospora crassa DHAD(2) had the sequence encoding the first 36 amino acids removed and replaced with a methionine codon.
pBP4582 (SEQ ID NO:790) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nt position 2260-3972) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3984-4296) for expression of the Streptococcus mutans DHAD with no C-terminal tag.
pBP1296 (SEQ ID NO:791) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nt position 2260-3972) followed by a C-terminal tag sequence (nt 3973-3993; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 4005-4317) for expression of the Streptococcus mutans C-terminal-tagged DHAD.
pBP4578 (SEQ ID NO:792) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus downei (nt position 2260-3954) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3966-4278) for expression of the Streptococcus downei DHAD with no C-terminal tag.
pBP4579 (SEQ ID NO:793) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus downei (nt position 2260-3954) followed by a C-terminal tag sequence (nt 3955-3975; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3987-4299) for expression of the Streptococcus downei C-terminal-tagged DHAD.
pBP4580 (SEQ ID NO:794) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Oscillatoria species PCC 6506 (nt position 2260-3942) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3954-4266) for expression of the Oscillatoria species PCC 6506 DHAD with no C-terminal tag.
pBP4581 (SEQ ID NO:795) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Oscillatoria species PCC 6506 (nt position 2260-3942) followed by a C-terminal tag sequence (nt 3943-3963; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3975-4287) for expression of the Oscillatoria species PCC 6506 C-terminal-tagged DHAD.
pBP4585 (SEQ ID NO:796) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Zea mays (nt position 2260-3936) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3948-4260) for expression of the Zea mays DHAD with no C-terminal tag.
pBP4586 (SEQ ID NO:797) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Zea mays (nt position 2260-3936) followed by a C-terminal tag sequence (nt 3937-3957; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3969-4281) for expression of the Zea mays C-terminal-tagged DHAD.
pBP4587 (SEQ ID NO:798) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Lactococcus lactis (nt position 2260-3969) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3981-4293) for expression of the Lactococcus lactis DHAD with no C-terminal tag.
pBP4588 (SEQ ID NO:799) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Lactococcus lactis (nt position 2260-3969) followed by a C-terminal tag sequence (nt 3970-3990; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 4002-4314) for expression of the Lactococcus lactis C-terminal-tagged DHAD.
pBP4642 (SEQ ID NO:800) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Neurospora crassa (nt position 2260-3954) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3966-4278) for expression of the Neurospora crassa DHAD(1) with no C-terminal tag.
pBP4644 (SEQ ID NO:801) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Neurospora crassa (nt position 2260-3954) followed by a C-terminal tag sequence (nt 3955-3975; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3987-4299) for expression of the Neurospora crassa C-terminal-tagged DHAD(1).
pBP4643 (SEQ ID NO:802) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Neurospora crassa (nt position 2260-3942) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3954-4266) for expression of the Neurospora crassa DHAD(2) with no C-terminal tag.
pBP4645 (SEQ ID NO:803) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Neurospora crassa (nt position 2260-3942) followed by a C-terminal tag sequence (nt 3943-3963; Adams et al., 2002. J. Am. Chem. Soc., v124 p 6063) expressed from the yeast FBA1 promoter (nt 1661-2250) and followed by the FBA1 terminator (nt 3975-4287) for expression of the Neurospora crassa C-terminal-tagged DHAD(2).
pBP4577 (SEQ ID NO:804) was constructed as a negative control (no heterologous DHAD expression). The Streptococcus mutans ilvD gene and C-terminal tag sequence in pBP1296 were removed by restriction digestion and the remaining vector was re-ligated to create pBP4577.
BY4741 fra2Δ (Thermo Scientific Open Biosystems Yeast Knock Out Clone) was transformed using a Frozen-EZ Yeast Transformation II kit (Zymo Research) and the resulting plasmids and transformants were selected on agar plates containing synthetic complete media lacking histidine supplemented with 2% glucose at 30° C. Three transformants were selected for each plasmid transformation.
DHAD Activity
Strains were grown overnight in synthetic complete media lacking histidine supplemented with 2% glucose (Teknova). Overnight cultures were sub-cultured to an optical density at a wavelength of 600 nm (OD600) of 0.25 and grown in 25 ml synthetic complete media lacking histidine supplemented with 2% glucose (Teknova) in 125 ml Erlenmeyer flat cap flasks (VWR) at 30° C. 250 revolutions per minute (RPM) in a New Brunswick I24 incubated shaker for 7.5 hours. Cultures were centrifuged at 3800×g for 5 minutes at 4° C. The pellets were washed with cold 50 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) pH 6.8 and then centrifuged at 3800×g for 5 minutes at 4° C. Cell pellets were frozen on dry ice and stored at −80° C. until they were assayed for DHAD activity. DHAD activity was measured as described above. All samples, except pBP1296 transformant #2, were assayed with three different volumes of cell extract; 20 μl, 40 μl, and 150 μl. pBP1296 transformant #2 was measured with 20 μl and 40 μl of cell extract.
DHAD activity is shown in FIG. 10 as units of enzyme activity per mg of total protein, with error bars reflecting the standard deviation. The DHAD activity for each DHAD construct is the average of the activities for the cell extract volumes tested and the average of the three transformants for each plasmid. The C-terminal-tagged constructs for the Streptococcus mutans DHAD, Streptococcus downei DHAD, and Lactococcus lactis DHAD (pBP1296, pBP4579, and pBP4588, respectively) all demonstrated higher DHAD activity than their non-C-terminal-tagged equivalents (pBP4582, pBP4578, and pBP4587, respectively). The activities for the other DHAD enzymes were too low to determine the effect of the C-terminal tag on their enzyme activities.
Example 13
Site Directed Mutagenesis to Replace Cysteines with Methionines in the C-terminal Tag of the S. mutans DHAD 689-I2V5 Variant
The four cysteines in the C-terminal tag of the 689-I2V5 variant of the S. mutans DHAD (DNA SEQ ID NO:786; protein SEQ ID NO:787) were replaced with methionines, individually and in every combination. Fifteen variants and a control were prepared via site directed mutagenesis. Mutagenesis was performed in a two-step process with the yeast shuttle plasmid pBP376. The first step entailed PCR with mutagenic primers. Primer ilvD F1 (SEQ ID NO:805; GTG AGT ATG ACT GAC AAA AAA ACT CTT AAA GAC) and the primers listed in Table 23 were commercially synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).







TABLE 23







Primers Employed for Site Directed Mutagenesis










SEQ ID



Primer
NO:
Sequence





Lum_R1
806
ATT AAT CAA CCA CAG CAA CCA GGA CAA CAT




TTT TTG CCA G


 


Lum_R2
807
ATT AAT CAA CCA CAG CAA CCA GGA CAC ATT




TTT TTG CCA G


 


Lum_R3
808
ATT AAT CAA CCA CAG CAA CCA GGC ATA CAT




TTT TTG CCA G


 


Lum_R4
809
ATT AAT CAA CCA CAC ATA CCA GGA CAA CAT




TTT TTG CCA G


 


Lum_R5
810
ATT AAT CAA CCC ATG CAA CCA GGA CAA CAT




TTT TTG CCA G


 


Lum_R6
811
ATT AAT CAA CCA CAC ATA CCA GGA CAC ATT




TTT TTG CCA G


 


Lum_R7
812
ATT AAT CAA CCA CAG CAA CCA GGC ATC ATT




TTT TTG CCA G


 


Lum_R8
813
ATT AAT CAA CCA CAC ATA CCA GGC ATA CAT




TTT TTG CCA G


 


Lum_R9
814
ATT AAT CAA CCC ATG CAA CCA GGC ATA CAT




TTT TTG CCA G


 


Lum_R10
815
ATT AAT CAA CCC ATC ATA CCA GGA CAA CAT




TIT TTG CCA G


 


Lum_R11
816
ATT AAT CAA CCC ATG CAA CCA GGA CAC ATT




TTT TTG CCA G


 


Lum_R12
817
ATT AAT CAA CCA CAC ATA CCA GGC ATC ATT




TTT TTG CCA G


 


Lum_R13
818
ATT AAT CAA CCC ATC ATA CCA GGC ATA CAT




TTT TTG CCA G


 


Lum_R14
819
ATT AAT CAA CCC ATC ATA CCA GGA CAC ATT




TTT TTG CCA G


 


Lum_R15
820
ATT AAT CAA CCC ATG CAA CCA GGC ATC ATT




TTT TTG CCA G


 


Lum_R16
821
ATT AAT CAA CCC ATC ATA CCA GGC ATC ATT




TTT TTG CCA G









Sixteen PCR reactions were performed with PFUultra polymerase (Catalog #600380; Agilent Technologies, Stratagene Products Division, La Jolla, Calif.). Each reaction consisted of 1 μl of pBP3765 (10 ng/μl), 1 μl of primer ilvD F1 (10 uM), 1 ul of a primer listed in Table 23 (10 uM), 5 ul of 10× PFUultra buffer, 1 μl of 10 mM dNTP mix, 1 μl of PFUultra DNA polymerase, and 40 μl of ddH2O. The following conditions were used for the PCR reactions: The starting temperature was 95° C. for 2.0 min followed by 30 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 30 sec, and 68° C. for 120 sec. At the completion of the temperature cycling, the sample was kept at 72° C. for 10.0 min more, and then held awaiting sample recovery at 4° C. The reaction products were separated from the template via agarose gel electrophoresis (1% agarose, 1×TBE buffer) and recovered using the illustra GFX PCR DNA and Gel Band Purification kit (Cat#28-9034-70, GE Healthcare Life Sciences, Piscataway, N.J.) as recommended by the manufacturer.
In the second step, the purified PCR products were employed as a megaprimers for reactions with the QuikChange® Lightning Site-Directed Mutagenesis Kit (Catalog #200523; Agilent Technologies, Stratagene Products Division, La Jolla, Calif.). Except for the primers, templates, and ddH2O, all reagents used here were supplied with the kit. The reaction mixtures contained 1 μl of pBP3765 (50 ng/μl), 2.5 μl of each megaprimer (100 ng/ul), 2.5 μl of 10× reaction buffer, 0.5 μl of dNTP mix, 0.75 ul QuikSolution, 0.5 ul QuikChange Lightning Enzyme, and 17.5 μl of ddH2O. The following conditions were used for the reactions: The starting temperature was 95° C. for 2 min followed by 18 heating/cooling cycles. Each cycle consisted of 95° C. for 20 sec, 60° C. for 10 sec, and 68° C. for 7 min. At the completion of the temperature cycling, the samples incubated at 68° C. for 5 min and then held awaiting sample recovery at 4° C. 1 μl of the Dpn I (10 U/μl) was added to each reaction and the mixtures were incubated for 30 min at 37° C. Reaction products were isolated and concentrated to 6 ul with the DNA Clean & ConcentratorTM-5 kit (D4013; Zymo Research; Irvine, Calif.).
2.5 μl of each mutagenic reaction was transformed into One Shot® TOP10 Chemically Competent E. coli (Invitrogen, Catalog #C404003) according to the manufacturer's instructions. The transformants were spread on agar plates containing the LB medium and 100 μg/ml ampicillin (Catalog #L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Multiple transformants for each reaction were inoculated into LB medium containing 100 μg/ml ampicillin and incubated at 37° C. with shaking at 225 rpm. Plasmid DNA was isolated from the cells with the QIAprep® Spin Miniprep Kit (Catalog #2706; Qiagen, Valencia, Calif.) according to the protocol provided by the manufacturer. Sequencing of the complete DHAD genes were performed with primers Dseq1 (aacgcgtgaagcttttgaagatg; SEQ ID NO:822), Dseq2 (tcagttcggaacaatcacgg; SEQ ID NO:823), Dseq3 (tgctttccctttcatcaatgattgttg, SEQ ID NO:824), Dseq4 (tccatgttagccatagcgataac SEQ ID NO:825), Dseq5 (ttgtgcttcaggagcgatatg; SEQ ID NO:826), and N885 (ctgctaatgtggaattgacac, SEQ ID NO:827).







TABLE 24







Prepared Versions of the C-terminal Tag


in the I2V5 Variant










Amino Acid



Variant
SEQ ID NO:
C-terminal Tag





YW1 (689-I2V5)
749
CCPGCCG


 


YW2
750
MCPGCCG


 


YW3
751
CMPGCCG


 


YW4
752
CCPGMCG


 


YW5
753
CCPGCMG


 


YW6
754
MCPGMCG


 


YW7
755
MMPGCCG


 


YW8
756
CMPGMCG


 


YW9
757
CMPGCMG


 


YW10
758
CCPGMMG


 


YW11
759
MCPGCMG


 


YW12
760
MMPGMCG


 


YW13
761
CMPGMMG


 


YW14
762
MCPGMMG


 


YW15
763
MMPGCMG


 


YW16
764
MMPGMMG









The variants listed in Table 24 together with 804-I2V5 variant lacking a C-terminal tag were transformed in yeast strain PNY2145 (MATa ura3Δ::loxP his3Δ pdc5Δ::P[FBA(L8)]-XPK|xpk1_Lp-CYCt-loxP66/71 fra2Δ 2-micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-ALS|alsS_Bs-CYC1t-loxP71/66 pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2Δ::loxP71/66 amn1Δ::AMN1(y); described, for example, in Int'l Appl. No. PCT/US2012/072186, filed Dec. 28, 2012, which is incorporated by reference herein) and analyzed for isobutanol production and DHAD activity.
Growth Media
Four types of media were used during the growth procedure of yeast strains: SE-ura agar plate, SAG-2-ura agar plate, an aerobic pre-culture media and an anaerobic culture media. All chemicals were obtained from Sigma-Aldrich® (St. Louis, Mo.) unless otherwise noted.
Yeast transformation recovery plate (SE-ura): 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)(pH 5.5), 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 0.2% ethanol, 0.01% weight per volume (w/v) leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Glucose adaptation plate (SAG-2-Ura): 50 mM MES (pH 5.5, 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 2% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Aerobic pre-culture media (SAG-0.2-Ura): 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 0.2% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, and 0.002% w/v tryptophan.
Anaerobic culture media (SAG-3-Ura): 50 mM MES (pH 5.5, 6.7 g/L yeast nitrogen base without amino acids (DifcoTM, 291940, Sparks, Md.), 1.4 g/L yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil, 3 mM sodium acetate (pH 7.0), 3% w/v glucose, 0.01% w/v leucine, 0.01% w/v histidine, 0.002% w/v tryptophan, 30 mg/L nicotinic acid, 30 mg/L thiamine and 10 mg/L ergosterol made up in 50/50 volume per volume (v/v) Tween/ethanol solution.
Transformation and Glucose Adaptation
Competent cells of the PNY2145 were prepared and transformed with 1 μL of purified plasmid ( ̃0.4 to 0.8 μg total DNA) using a Frozen-EZ Yeast Transformation II KitTM (Zymo Research Corp.; Irvine, Calif.). Transformation mixtures were plated on SE-ura plates and incubated at 30° C. for 4 days. Three or four colonies for each transformant were selected and patched onto SE-ura plates and incubated at 30° C. for 2 days. The variants then underwent glucose adaptation by patching onto SAG-2-Ura plates and growing for 2 days at 30° C.
Deep-well Plate Growth Procedure
1.5 mL aliquots of the aerobic pre-culture media were dispensed into each well of a VWR 48 deep-well plate (#82004-674, VWR, Radnor, Pa.) and inoculated with cells grown on a SAG-2-Ura agar plate, as described above. A sterile air permeable cover (#60941-086, VWR, Radnor, Pa.) was used to seal the culture plate. The plate was placed in a 30° C. incubator and was grown for 20 to 24 hours with shaking, and an OD600 value (optical density at 600 nm) was obtained using Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). OD600 values were converted to equivalent OD600 values on a Cary 300 (Agilent Technologies, Wilmington, Del.) calibration value. A dilution 48 deep-well plate was set with a target Cary OD600 value of 0.35 for each well in a total volume of 1.5 mL. Wells with Cary OD600 values of 0.35 to 0.40 were transferred directly from the original plate to the dilution plate. For all other wells, a volume of turbid culture was transferred the volume was brought up to 1.5 mL with aerobic pre-culture media. The 48 deep-well plate was returned to the 30° C. shaking incubator and grown for an additional 20 to 24 hours. OD600 values were obtained as described above.
Isobutanol Titers and DHAD Activities
PNY2145 with S. mutans DHAD variants were grown and analyzed as described above. Additionally, the resultant cells were pelleted via centrifugation and the DHAD activities were measured as described above. Isobutanol titers and DHAD specific activities are listed in Table 25 below. FIG. 11 shows gel analysis for selected variants to determine whether these proteins are detected with the LumioTM reagent. The gel was prepared was prepared as described in Example 14. Only lanes for variants YW1, YW4, and YW7 contain bands consistent with detected DHAD protein. The lane 804-I2V5 represents the negative control lacking a c-terminal tag.







TABLE 25







Isobutanol titers and DHAD specific activities for


S. mutans DHAD variants in PNY2145












Isobutanol






mM at

DHAD SA



44 hr

(U/mg)














Variant
C-terminal Tag
Mean
SD
Mean
SD


















YW1
CCPGCCG
82.1
3.9
1.06
0.09



(689-



I2V5)



YW2
MCPGCCG
73.5
5.7
0.81
0.09



YW3
CMPGCCG
78.0
4.5
0.76
0.04



YW4
CCPGMCG
76.6
5.3
0.39
0.33



YW5
CCPGCMG
74.6
4.3
0.99
0.13



YW6
MCPGMCG
59.1
5.0
0.16
0.19



YW7
MMPGCCG
65.9
6.6
0.41
0.07



YW8
CMPGMCG
57.0
4.7
0.29
0.10



YW9
CMPGCMG
50.5
3.4
0.33
0.04



YW10
CCPGMMG
57.9
1.1
0.30
0.06



YW11
MCPGCMG
70.0
3.2
0.47
0.17



YW12
MMPGMCG
39.3
2.0
0.28
0.13



YW13
CMPGMMG
43.0
3.4
0.22
0.13



YW14
MCPGMMG
44.9
4.5
0.19
0.07



YW15
MMPGCMG
44.7
3.7
0.21
0.02



YW16
MMPGMMG
39.1
1.6
0.22
0.02



804-I2V5
No tag
38.3
6.6
0.14
0.06










Example 14
Construction of DHAD-C-terminal Tag Variants
Eight C-terminal tag variants containing either amino acid changes or additions were constructed by PCR amplification of the native sequence from the plasmid pBP3765 (SEQ ID NO:828). One forward primer N1560 was used with eight reverse primers N1561, N1562, N1563, N1564, N1565, N1566, N1567 and N1568. The resulting PCR products were cloned into a TOPO vector (Invitrogen Cat. No. K288020) and sequenced to verify the engineered nucleotide changes.
Construction of IlvD-I2V5-C-terminal Tag Plasmids
The TOPO-clones containing the PCR products described above were restriction digested with AscI and PacI and ligated to the AscI and PacI digested plasmid pBP3765 thus replacing the 3′ end of the ilvD-I2V5-Lum. The ligation reaction was transformed into. E. coli Stbl3 cells (Invitrogen Cat. No. C737303) which were incubated on LB ampicillin plates to select for transformants. Successful insertion was identified by PCR colony screen using PCR primers N1576 and N1577. Colonies screening positive for an insertion were cultivated, plasmids isolated and the C-terminal tag locus sequenced for final verification of engineered nucleotide changes. The plasmids were designated, respectively: pJT386, pJT387, pJT388, pJT389, pJT390, pJT391, pJT392, and pJT39.







TABLE 26





C-terminal tag amino acid sequences and


corresponding expression plasmids

















Control
CCPGCCG (SEQ ID NO: 748)
pBP3765


 


Variant 1
CSCPGCCG (SEQ ID NO: 739)
pJT386


 


Variant 2
CPCPGCCG (SEQ ID NO: 740)
pJT387


 


Variant 3
CECPGCCG (SEQ ID NO: 741)
pJT388


 


Variant 4
CCPGCSCG (SEQ ID NO: 742)
pJT389


 


Variant 5
CCPGCPCG (SEQ ID NO: 743)
pJT390


 


Variant 6
CCPGCECG (SEQ ID NO: 744)
pJT391


 


Variant 7
CCPEGCCG (SEQ ID NO: 745)
pJT392


 


Variant 8
CCPAGCCG (SEQ ID NO: 746)
pJT393









Construction of Isobutanologen Strains with C-terminal Tag Variant DHAD Enzymes

The plasmids above (plus control) were each transformed along with pRS413::BiADH-kivD into strain PNY2145. Transformation mixtures were plated on synthetic complete medium without uracil or histidine containing 1% ethanol as carbon source. Cells from transformant colonies were patched to fresh plates. After two days, cells from patches were transferred to plates containing 2% glucose instead of ethanol as carbon source. After two days, cells from patches were used to inoculate liquid medium (synthetic complete without uracil or histidine with 0.3% glucose). The next morning, the optical density of each culture was adjusted with fresh medium. After approximately 4 hours, cultures were used to inoculate 15 mL serum vials containing synthetic complete medium minus uracil and histidine with 2% glucose as carbon source and supplemented with 1×BME vitamins (Sigma Cat. No. B6891). The starting OD for each culture was 0.1 and the final volume was 10 mL. Vials were stoppered, crimped and incubated at 30° C. in an Infors Multitron platform shaker (220 rpm). After 38.5 hours, stoppers were removed for sampling. Culture supernatant obtained from Costar Spin-X columns (3,000 rpm, 3 minutes) was analyzed by HPLC, as described in Example 4. Concentration of isobutanol was determined using a standard curve. Glucose consumption was also calculated by standard curve. A second series of serum vial cultures was set up as described above with one clone from each genotype. This time cells were collected from the cultures 24 hours after inoculation (3 minutes, 500×g). Cell pellets were then stored at −80° C. DHAD assay as described in the Examples herein or as described by Flint and Emptage (J. Biol. Chem., 263(8):3558-64, 1988) using dinitrophenylhydrazine.
Table 27 shows the glucose consumption and isobutanol concentration from 39 hour serum vial incubation. For all variants except variant #6, there were n=3 biological replicates. For variant #6, there were n=2 biological replicates and for the control, n=2 technical replicates. The DHAD specific activity from 24 hour serum vial incubation is one individual clone of each variant.







TABLE 27







Glucose consumption and isobutanol concentration


from 39 hour serum vial incubation










C-terminal

Isobutanol
Representative DHAD


Tag
Glucose Consumed
concentration
Specific Activity


Variant
(mM)
(mM)
(μmole/min/mg)













1
81.2 ± 1.8
53.7 ± 1.7
0.85


2
88.4 ± 6.0
59.4 ± 3.3
0.89


3
88.9 ± 1.9
60.0 ± 1.0
1.06


4
86.3 ± 7.8
57.2 ± 3.7
1.2


5
81.4 ± 1.6
54.2 ± 1.0
1.2


6
87.2 ± 0.4
57.9 ± 0.4
1.1


7
86.4 ± 2.3
57.2 ± 1.4
1.51


8
84.4 ± 6.9
55.8 ± 4.4
1.93


Control
95.8 ± 0.5
64.4 ± 0.7
1.47









Sequences were detected using the LumioTM Green Detection kit (LC6090, Invitrogen Life Technologies, Carlsbad, Calif.) according to the manufacturer's instructions, with the following modifications. The buffer used to lyse the cells was a HEPES buffer, pH 6.8. A master mix combining 60 μL of 4× LumioTM Gel Sample buffer and 2.4 μL of LumioTM Green Detection reagent was made; each sample received a 5.2 μL aliquot of this master mix. Cell lysates were concentrated approximately 3 to 4 fold using a YM-10 spin filter prior to the addition of the master mix.
A 10% Bis-Tris gel (NP0303, Invitrogen, Carlsbad, Calif.) was run in 1×MOPS buffer (NP000102, Invitrogen, Carlsbad, Calif.) at 185V for 50 minutes. A BioRad Gel Doc (BioRad, Hercules, Calif.) system was used to visualize and image the gel, according to the LumioTM Green Detection kit instructions (FIG. 12). The lanes of the gel in FIG. 12 are as follows: 1. LumioTM Molecular Weight Marker; 2. pJT386; 3. pJT387; 4. pJT388; 5. pJT389; 6. pJT390; 7. pJT391; 8. pJT392; 9. pJT393; and 10. PNY2312 (positive control). After the gel image was acquired, the gel was stained following the Simple Blue (LC6060, Invitrogen, Carlsbad, Calif.) protocol in order to visualize total protein (FIG. 13). The lanes of the gel in FIG. 13 are as follows: 1. LumioTM Molecular Weight Marker; 2. pJT386; 3. pJT387; 4. pJT388; 5. pJT389; 6. pJT390; 7. pJT391; 8. pJT392; 9. pJT393; 10. PNY2312 (positive control); 11. PNY2145+pLH804; and 12. PNY2145+PLH804 L2V4 #1. Analysis of gel image was performed using Image Lab 4.0 (BioRad, Hercules, Calif.) to quantitate the signal, with the arrow indicating the DHAD reference band (Table 28).







TABLE 28







Relative Quantity of signal











Relative



Lane
Quantity







1
n/d



2
n/d



3
n/d



4
0.24



5
0.38



6
0.25



7
0.28



8
0.16



C
1.00










Example 15
ilvD Sm with C-terminal Tag Variants in Isobutanologen Plasmid Construction
Plasmids were constructed in a 2-micron based Saccharomyces cerevisiae-Escherichia coli shuttle vector.







TABLE 29







Plasmids referenced in Example 15










DHAD



Plasmid
description
C-terminal Amino Acid Sequence





pBP3763
Full-length
Lys-Lys-Cterm


pBP3765
tag
Lys-Lys-Cys-Cys-Pro-Gly-Cys-Cys-Gly-Cterm


pBP3767
tag 2C-2A
Lys-Lys-Cys-Ala-Pro-Gly-Ala-Cys-Gly-Cterm


pBP3769
tag 2C-2S
Lys-Lys-Cys-Ser-Pro-Gly-Ser-Cys-Gly-Cterm


pBP3771
Tag 4C-
Lys-Lys-Ser-Ala-Pro-Gly-Ala-Ser-Gly-Cterm



2A2S









pBP3763—Native Length ilvD Sm

pBP3763 (SEQ ID NO:850) was constructed to contain a chimeric gene having the coding region of the Streptococcus mutans ilvD variant I2V5 (nucleotide (nt) position 5356-3644) expressed from the yeast TEF1 mutant 7 promoter (nt 5766-5366; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006) and followed by the FBA1terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659; described in WO2012/12955, incorporated herein by reference) expressed from the yeast ILV5 promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of ketol-acid reductoisomerase (KARI).
pBP3765 —C-terminal Tagged ilvD Sm
pBP3765 (SEQ ID NO:828) was constructed to contain a chimeric gene having the coding region of the Streptococcus mutans ilvD variant I2V5 (nt position 5377-3665) followed by a C-terminal tag sequence (nt 3664-3647; Adams et al., J. Am. Chem. Soc., 124:6063, 2002) expressed from the yeast TEF1 mutant 7 promoter (nt 5787-5387; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659; described in, for example, Int'l Publ. No. WO2012/12955, which is incorporated by reference herein) expressed from the yeast ILV5 promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
pBP3767 —2C-2A Variant C-terminal Tagged ilvD Sm
pBP3767 (SEQ ID NO:851) was constructed to contain a chimeric gene having the coding region of the Streptococcus mutans ilvD variant I2V5 (nt position 5377-3665) followed by the 2C-2A C-terminal tag sequence (nt 3664-3647) expressed from the yeast TEF1 mutant 7 promoter (nt 5787-5387; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659; described in, for example, Int'l Pub. No. WO2012/12955, which is incorporated by reference herein) expressed from the yeast ILV5promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
pBP3769 —2C-2S Variant C-terminal Tagged ilvD Sm
pBP3769 (SEQ ID NO:852) was constructed to contain a chimeric gene having the coding region of the Streptococcus mutans ilvD variant I2V5 (nt position 5377-3665) followed by the 2C-2S C-terminal tag sequence (nt 3664-3647) expressed from the yeast TEF1mutant 7 promoter (nt 5787-5387; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659; described in, for example, Int'l Pub. No. WO2012/12955, which is incorporated by reference herein) expressed from the yeast ILV5promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
pBP3771 —4C-2A2S Variant C-terminal Tagged ilvD Sm
pBP3771 (SEQ ID NO:853) was constructed to contain a chimeric gene having the coding region of the Streptococcus mutans ilvD variant I2V5 (nt position 5377-3665) followed by the 4C-2A2S C-terminal tag sequence (nt 3664-3647) expressed from the yeast TEF1 mutant 7 promoter (nt 5787-5387; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659; described in, for example, Int'l Pub. No. WO2012/12955, which is incorporated by reference herein) expressed from the yeast ILV5promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
Strain Construction
Following conversion of PNY2115 to PNY2121 by replacing the endogenous copy of AMN1 with a codon-optimized version of the AMN1 gene from CEN.PK2 (SEQ ID NO:854), PNY2121 was restored back to a histidine prototroph. The HIS3 coding sequence and 500 bp upstream and downstream of the coding sequence were amplified from a haploid (PNY0865) obtained from sporulation of PNY0827. PNY2121 was transformed with the resulting PCR product and transformants were selected on agar plates containing synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. A PNY2121 HIS3+isolate was designated PNY1665.
PNY1665 was transformed with the plasmids described above and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Three transformants were selected for each plasmid construct.
DHAD Activity
Strains were grown overnight in 12 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% w/v ethanol, 0.3% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were sub-cultured into 15 ml of high glucose medium in 125 ml VWR vent cap shake flasks to a final OD600 0.4 and grown for 4 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The high glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% w/v ethanol, 3.0% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Cells were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 2 ml of high glucose medium. 30 ml of high glucose medium in 60 ml serum vials (Kimble Chase; Vineland, N.J.) was inoculated with resuspended cells to a final OD600 of 0.2. Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 24 hours. Cultures were centrifuged at 3800×g for 5 minutes at 4° C. The pellets were washed with cold 50 mM HEPES pH 6.8 and then centrifuged at 3800×g for 5 minutes at 4° C. Cell pellets were frozen on dry ice and stored at −80° C. until they were assayed for DHAD activity. DHAD activity was measured as described above.
Strains containing the tagged ilvD Sm plasmid (pBP3765) had an average DHAD activity of 0.68 (U/mg) (Table 30). Strains containing the plasmids with the C-terminal tag variants (pBP3767, pBP3769, pBP3771) had DHAD activities similar to the strains containing the native length ilvD Sm plasmid (pBP3763); average DHAD activities ranging from of 0.11 to 0.16 (U/mg).







TABLE 30







Average DHAD activity and standard deviation for three transformants












DHAD
DHAD Activity



Plasmid
description
(U/mg)







pBP3763
Full-length
0.14 ± 0.02



pBP3765
tag
0.68 ± 0.05



pBP3767
tag 2C-2A
0.15 ± 0.04



pBP3769
tag 2C-2S
0.16 ± 0.03



pBP3771
tag 4C-2A2S
0.11 ± 0.04










Example 16
C-terminal-tagged ilvD Sm in Isobutanol Production
Isobutanol production was tested for isobutanologen strains. PNY1604, PNY1614, PNY1602, and PNY1612 were grown overnight in 10 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% w/v ethanol, 0.3% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 2 ml of high glucose medium. High glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.1% w/v ethanol, 3.0% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. 10 ml of high glucose medium in 125 ml VWR vent cap shake flasks was inoculated with cells to a final OD600 0.4 and grown for 5 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. Cells were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in high glucose medium to an OD600 0.2. 10 ml of the culture in high glucose medium was transferred to a 20 ml serum vial (Kimble Chase; Vineland, N.J.). Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 42 hours.
After 42 hours, the cultures were sampled for OD600 and culture supernatants (collected using Spin-X centrifuge tube filter units, Costar Cat. No. 8169) were analyzed by HPLC (as described in Example 4). OD600, isobutanol concentration, and isobutanol molar yield are presented in Table 31.







TABLE 31







Average OD600, isobutanol concentration,


and isobutanol molar yield and standard deviations












Isobutanol
Isobutanol yield


Strain
OD600
(mM)
(mol/mol)





PNY1604/PNY1614
1.5 ± 0.1
81.8 ± 1.9
0.60 ± 0.00


(control)


PNY1602/PNY1612
2.1 ± 0.1
102.5 ± 3.1 
0.62 ± 0.02


(tagged)









Example 17
S. mutans DHAD and S. macacae DHAD Plasmid and Strain Construction
Plasmids were constructed in the 2-micron Saccharomyces cerevisiae-Escherichia coli shuttle vector pHR81 (SEQ ID NO:855; ATCC 87541).
pBP5062 (SEQ ID NO:856) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nt position 9644-11356) expressed from the yeast FBA1 promoter (nt 8639-9636) and followed by the FBA1 terminator (nt 7-1006) for expression of the Streptococcus mutans DHAD.
pBP5063 (SEQ ID NO:857) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus macacae codon optimized for expression in Saccharomyces cerevisiae (nt position 9644-11356) expressed from the yeast FBA1 promoter (nt 8639-9636) and followed by the FBA1 terminator (nt 7-1006) for expression of the Streptococcus macacae DHAD.
BY4741 (ATCC 201388) and BY4741 fra2Δ (Thermo Scientific Open Biosystems Yeast Knock Out Clone) were transformed using a Frozen-EZ Yeast Transformation II kit (Zymo Research) with pHR81, pBP5062, and pBP5063 and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Three transformants were selected for each plasmid transformation.
DHAD Activity
Strains were grown overnight in synthetic complete media lacking uracil supplemented with 2% glucose (Teknova). Overnight cultures were sub-cultured to an OD600 0.4 and grown in 25 ml synthetic complete media lacking uracil supplemented with 2% glucose (Teknova) in 250 ml Erlenmeyer vent cap flasks (VWR) at 30° C. 250 RPM in a New Brunswick I24 incubated shaker for 5.5 hours. Cultures were centrifuged at 3800×g for 5 minutes at 4° C. The pellets were washed with cold 50 mM HEPES pH 6.8 and then centrifuged at 3800×g for 5 minutes at 4° C. Cell pellets were frozen on dry ice and stored at −80° C. until they were assayed for DHAD activity.
For DHAD activity measurements the frozen yeast cells were thawed, resuspended in 0.1 M K-Hepes pH 6.8 containing 10 mM MgCl2 and a protease inhibitor cocktail (Roche, Catalog #11873580001), and then broken by bead beating. The broken cells were centrifuged to remove the cell debris and generate the yeast crude extract. Protein concentrations (mg/ml) of extracts were measured with the Pierce Coomassie Plus (Bradford) Protein Assay (Catalog #23236, Thermoscientific). DHAD enzyme activities were measured spectrophotometrically in an end point assay using the 2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D. H. and M. H. Emptage, J. Biol. Chem. 263:3558-64, 1988, with modifications. The assay buffer contained 0.1 M Tris pH 8.0, 10 mM MgCl2, and 0.5 mM TPP. Yeast extracts were diluted in assay buffer. Sufficient (R)-2,3-dihydroxyisovaleric acid was added to assay buffer so that the final concentration in the assay was 10 mM. In each assay, an enzyme containing solution and sufficient substrate containing buffer were mixed so that the final volume was 300 ul. Assay mixtures were incubated at 37° C. for 20 minutes. At five minute intervals, a 60 ul aliquot of each reaction was mixed with 70 ul of a saturated solution of 2,4-DNPH in 1 N HCl. Following a 30 minute incubation at room temperature, 70 ul of 4 N KOH in ethanol was then added to the solution, followed by brief mixing. The absorbance of the mixture was read at 540 nm with a Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). A standard curve containing 0 mM to 1.33 mM α-ketoisovalerate was employed to calculate enzyme activities (units per milliliter, U/ml) for the conversion of (R)-2,3-dihydroxyvalerate to α-ketoisovalerate in the assays. DHAD specific activities (units per milligram, U/mg) were determined from enzyme activities (U/ml) and protein concentrations (mg/ml) measured for each sample.
DHAD activities are shown in FIG. 14 for the BY4741 strain background and in FIG. 15 for the BY4741fra2Δ strain background for control vector-transformations (“vector”), for S. mutans ilvD (“ilvD S. mutans”), and S. macacae ilvD (“ilvD S. macacae”). The DHAD activity for each construct is the average of the activities for the three transformants for each plasmid.
Example 18
Plasmid Construction for Expression of C-terminal Deletion Variants of Dehydroxy-Acid Dehydratase (DHAD)
The following table provides the genotypes of the various yeast strains referenced in the following Examples.







TABLE 32







Strain names and genotypes










Strain Name
Genotype







PNY2115
MATa ura3Δ::loxP his3Δ pdc5Δ::loxP66/71 2-




micron plasmid (CEN.PK2) pdc1Δ::P[PDC1]-




ALS|alsS_Bs-CYC1t-loxP71/66




pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-




TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-




ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-




ADHt-loxP71/66 gpd2Δ::loxP71/66



PNY1566
MATa ura3Δ::loxP pdc5Δ::loxP66/71 2-micron




plasmid (CEN.PK2) pdc1Δ::P[PDC1]-




ALS|alsS_Bs-CYC1t-loxP71/66




pdc6Δ::(UAS)PGK1-P[FBA1]-KIVD|Lg(y)-




TDH3t-loxP71/66 adh1Δ::P[ADH1]-ADH|Bi(y)-




ADHt-loxP71/66 fra2Δ::P[ILV5]-ADH|Bi(y)-




ADHt-loxP71/66 gpd2Δ::loxP71/66



PNY1603 and 
PNY1566 with plasmid pLH691 containing



PNY1613
(P[ILV5]-KARI|ilvC_Ll-ILV5t P[TEF1(M7)]-




DHAD|ilvD_Sm_Δ9-FBA1t)



PNY1604 and
PNY1566 with plasmid pLH804 containing



PNY1614
(P[ILV5]-KARI|ilvC_Ll-ILV5t P[TEF1(M7)]-




DHAD|ilvD_Sm-FBA1t)










Plasmids were constructed in a 2-micron based Saccharomyces cerevisiae-Escherichia coli shuttle vector.
pLH804—IlvD Sm
pLH804 (SEQ ID NO:591) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nucleotide (nt) position 5356-3644) expressed from the yeast TEF1 mutant 7 promoter (nt 5766-5366; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006) and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the K9JB4P mutant ilvC gene from Anaeropstipes cacae (nt 1628-2659) expressed from the yeast ILV5 promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of ketol-acid reductoisomerase (KARI), described, for example, in PCT App. Pub. No. WO2012/12955, which is incorporated by reference herein.
pLH691—IlvD Sm Δ9
pLH691 (SEQ ID NO:590) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans (nt position 5329-3644) with the C-terminal 9 amino acids deleted, expressed from the yeast TEF1 mutant 7 promoter (nt 5739-5339; Nevoigt et al., Applied and Environmental Microbiology, 72:5266, 2006), and followed by the FBA1 terminator (nt 3632-3320) for expression of DHAD, and a chimeric gene having the coding region of the Anaerostipes caccae ilvC variant K9JB4P (nt 1628-2659) expressed from the yeast ILV5 promoter (nt 434-1614) and followed by the ILV5 terminator (nt 2673-3307) for expression of KARI.
Example 19
Construction of Yeast Strains PNY1566, PNY1603, PNY1604, PNY1613, and PNY1614
To obtain PNY0865, PNY0827 was sporulated using standard methods (Codón A C, Gasent-Ramírez J M, Benítez T. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeast. Appl Environ Microbiol. 1995). After the formation of asci (as observed by microscopy), a 100 ul aliquot of cells was resuspended in 100 ul of 1 M sorbitol and treated with 5 U of zymolyase (Zymo Research, Orange Calif.) at 37° C. for 15 min. Resulting tetrads were spread on YPD medium and dissected using a micromanipulator (Singer Instruments, Somerset UK), and the single-spore isolates were grown at 30° C. for 3 d to form colonies. One tetrad with four viable spores was characterized further. The mating type of the single-spore isolates was determined by PCR as described (Huxley, C., E. D. Green and I. Dunham (1990). “Rapid assessment of S. cerevisiae mating type by PCR.” Trends Genet 6(8): 236). One spore isolate, of mating type MATα, was designated PNY0865.
PNY2115 was restored back to a histidine prototroph. The HIS3 coding sequence and 500 base pairs (bp) upstream and downstream of the coding sequence were amplified from the haploid PNY0865. PNY2115 was transformed with the resulting PCR product and transformants were selected on agar plates containing synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. A PNY2115 HIS3+ isolate was designated PNY1566.
PNY1566 was transformed with pLH804 (control ilvD_Sm) (SEQ ID NO:591) and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Two transformants were selected and designated PNY1604 and PNY1614. PNY1566 was transformed with pLH691 (Δ9 ilvD_Sm)(SEQ ID NO:590) and transformants were selected on agar plates containing synthetic complete media lacking uracil supplemented with 1% ethanol at 30° C. Two transformants were selected and designated PNY1603 and PNY1613.
Example 20
DHAD Activity of C-terminal Deletion Strains
PNY1603, PNY1613, PNY1604, and PNY1614 were grown overnight in 12 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% weight per volume (w/v) ethanol, 0.3% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 1 ml of high glucose medium. High glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.1% w/v ethanol, 3.0% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. 15 ml of high glucose medium in 125 ml VWR vent cap shake flasks was inoculated with cells to a final OD600 0.4 and grown for 5 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. Cells were again centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 2 ml of high glucose medium. 30 ml of high glucose medium in 60 ml serum vials (Kimble Chase; Vineland, N.J.) was inoculated with resuspended cells to a final OD600 of 0.15. Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 20 hours. Cultures were centrifuged at 3800×g for 5 minutes at 4° C. The pellets were washed with cold 50 mM HEPES pH 6.8 and then centrifuged at 3800×g for 5 minutes at 4° C. Cell pellets were frozen on dry ice and stored at −80° C. until they were assayed for DHAD activity.
To assay for DHAD activity, frozen yeast cells were thawed, resuspended in 0.1 M K-Hepes pH 6.8 containing 10 mM MgCl2 and a protease inhibitor cocktail (Roche, Catalog #11873580001), and then broken by bead beating. The broken cells were centrifuged to remove the cell debris and generate the yeast crude extract. Protein concentrations (mg/ml) of extracts were measured with the Pierce Coomassie Plus (Bradford) Protein Assay (Catalog #23236, Thermo Scientific). DHAD enzyme activities were measured spectrophotometrically in an end point assay using the 2,4-dinitrophenylhydrazine (2,4-DNPH) method as described in Flint, D. H. and M. H. Emptage, J. Biol. Chem. 263:3558-64, 1988, with modifications. The assay buffer contained 0.1 M K-Hepes pH 6.8 and 10 mM MgCl2. Yeast extracts were diluted in assay buffer. Sufficient (R)-2,3-dihydroxyisovaleric acid was added to assay buffer so that the final concentration in the assay is 10 mM. In each assay, an enzyme containing solution and sufficient substrate containing buffer are mixed so that the final volume is 300 ul. Assay mixtures were incubated at 30° C. for 20 minutes. At five minute intervals, a 60 ul aliquot of each reaction was mixed with 70 ul of a saturated solution of 2,4-DNPH in 1 N HCl. Following a 30 minute incubation at room temperature, 70 ul of 4 N KOH in ethanol was then added to the solution, followed by brief mixing. The absorbance of the mixture was read at 540 nm with a Spectra Max384 Plus plate reader (Molecular Devices, Sunnyvale, Calif.). A standard curve containing 0 mM to 1.33 mM α-ketoisovalerate was employed to calculate enzyme activities (units per ml, or U/ml) for the conversion of (R)-2,3-dihydroxyvalerate to α-ketoisovalerate in the assays. DHAD specific activities (units per mg, or U/mg) were determined from enzyme activities (U/ml) and protein concentrations (mg/ml) measured for each sample.
Strains PNY1604 and PNY1614 containing the control ilvD Sm had an average DHAD activity of 0.32 (U/mg). See FIG. 16. Strains PNY1603 and PNY1613 containing the Δ9 ilvD Sm had an average DHAD activity of 0.43 (U/mg). See FIG. 16.
Example 21
Isobutanol Production with ilvD Sm Δ9
Isobutanol production was tested for isobutanologen strains. PNY1604, PNY1614, PNY1603, and PNY1613 were grown overnight in 10 ml of low glucose medium in 125 ml VWR vent cap shake flasks at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. The low glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.2% w/v ethanol, 0.3% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. Overnight cultures were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in 2 ml of high glucose medium. High glucose medium consisted of: 6.7 g/L Difco Yeast Nitrogen Base without amino acids (Becton Dickinson; Sparks, Md.), 1.92 g/L Synthetic Drop-out Medium Supplement without Uracil (Sigma; St. Louis, Mo.), 0.1% w/v ethanol, 3.0% w/v glucose, 100 mM 2-Morpholinoethanesulphonic acid (MES) buffer, adjusted to pH 5.5 with KOH. 10 ml of high glucose medium in 125 ml VWR vent cap shake flasks was inoculated with cells to a final OD600 0.4 and grown for 5 hours at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker. Cells were centrifuged at 3800×g for 5 minutes at room temperature and cell pellets were resuspended in high glucose medium to an OD600 0.2. 10 ml of the culture in high glucose medium was transferred to a 20 ml serum vial (Kimble Chase; Vineland, N.J.). Serum vials were sealed and cultures were grown at 30° C. at 250 RPM in a New Brunswick I24 incubated shaker for 42 hours.
After 42 hours, the cultures were sampled for OD600 and culture supernatants (collected using Spin-X centrifuge tube filter units, Costar Cat. No. 8169) were analyzed by HPLC (as described in Example 4). OD600, isobutanol concentration, and isobutanol molar yield are presented in Table 33.







TABLE 33







Average OD600, isobutanol concentration, and isobutanol molar yield


and standard deviations












Isobutanol
Isobutanol


Strain
OD600
(mM)
yield (mol/mol)





PNY1604/PNY1614
1.5 ± 0.1
81.8 ± 1.9
0.60 ± 0.00


(control)


PNY1603/PNY1613
2.0 ± 0.1
81.9 ± 4.9
0.60 ± 0.01


(Δ9)









Example 22
Plasmid Construction for Expression of C-terminal Deletion Variants of Dehydroxy-Acid Dehydratase (DHAD)
Vector derived from pHR81 (ATCC 87541) was used for expressing wild type (WT) and mutant DHAD from S. mutans under the control of FBA promoter. Vector pHR81 FBA-IlvD(Sm) (SEQ ID NO:858) contained WT DHAD. In this vector, the FBA promoter was located in the region from nucleotides (nt) 7626 to 8623. The IlvD gene was located from nt 8631 to 10343, flanked by the restriction enzyme sites for SpeI and NotI. For the expression of the IlvD protein containing a nine amino acid deletion from the C-terminus, vector pHR81 FBA-IlvD(Sm)Δ9 was used (SEQ ID NO:859). In this vector, the IlvD gene with deletion was located in the region from nt 9644 to 11332, flanked by the restriction enzyme sites for SpeI and NotI. The FBA promoter was located from nt 8639 to 9636.
Example 23
DHAD Activity of C-terminal Deletion Strains
Vectors pHR81 FBA-IlvD(Sm) and pHR81 FBA-IlvD(Sm)Δ9 were transformed into yeast strain BY4741 (ATCC 201388). Competent cells were prepared with a Frozen Yeast Transformation kit (Zymo Research, Irvine, Calif.). The transformants were selected on plates with complete synthetic yeast growth medium minus Ura (Teknova, Hollister, Calif.). Growth on liquid medium was carried out by adding 5 ml of an overnight culture into 100 ml medium in a 250 ml flask. Cells from 80 ml culture were harvested by centrifugation (4,000 rpm for 10 min at 4° C.) and washed with 10 ml TM8 buffer (50 mM Tris, pH 8.0, 10 mM MgSO4) stored at 4° C. The cells were resuspended in 1 ml of TM8 and transferred to lysing matrix tubes with 0.1 mm silica spheres (MP Biomedicals, Solon, Ohio). The cells were broken with a beads beater (4× with 30 seconds each). The crude extract was obtained by centrifugation with a table top microfuge at 12,000 rpm at 4° C. for 30 minutes. The supernatants were removed and stored on ice until assayed for DHAD activity as described above. It was found that the DHADs assayed herein were stable in crude extracts kept on ice for a few hours. DHAD activity was also preserved when samples were frozen in liquid N2 and stored at −80° C. Results from enzymatic measurement (FIG. 17) showed an about 60% increase in DHAD activity with the IlvD enzyme having a nine amino acid deletion at the C-terminus (pHR81 FBA-IlvD(Sm)Δ9) as compared to DHAD activity obtained with the control DHAD enzyme (pHR81 FBA-IlvD(Sm)).
Example 24
Site Directed Mutagenesis of C-terminal Tagged S. mutans DHAD
C-terminal-tagged versions of variants in Example 2 were prepared by site directed mutagenesis of C-terminal tagged S. mutans DHAD. Site directed mutagenesis of performed as described in Example 2, with modifications.
For the P378A substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul P2A1 mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the G383S substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul G2S2mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the L385F substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul L2F3mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the L385V substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul L2V4mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the I387V substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul I2V5mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the I387M substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul I2M6 mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the L388I substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul L2I7mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
For the L388M substitution, the mutagenesis reaction contained 1 ul pLH689 (50 ng), 1 ul L2M8mix (10 uM each primer), 1 ul dNTP mix, 1.5 ul Quiksolution, 5 ul of 10× buffer, 1 ul QuikChange Lightning Enzyme, and 39.5 ul of ddH2O.
The following conditions were used for the reactions: The starting temperature was 95° C. for 2 min followed by 18 heating/cooling cycles. Each cycle consisted of 95° C. for 20 sec, 60° C. for 10 sec, and 68° C. for 10 min. At the completion of the temperature cycling, the samples were incubated at 68° C. for 5.0 min and then held awaiting sample recovery at 4° C. 2 μl of the Dpn I was added to each reaction and the mixtures were incubated for 30 min at 37° C.







TABLE 34







Prepared Variants of C-terminal Tagged S. mutans DHAD












Variant
Nuc Seq ID
AA Seq ID
Substitution







689-P2A1
860
861
P378A



689-G2S2
862
863
G383S



689-L2F3
864
865
L385F



689-L2V4
866
867
L385V



689-I2V5
786
787
I387V



689-I2M6
868
869
I387M



689-L2I7
870
871
L388I



689-L2M8
872
873
L388M










Example 25
Comparison of DHAD Variant L2V4 (L385V) and L2I7 (L388I) with and without C-terminal Tag
Isobutanol-producing strains containing DHAD variant L2V4 (L385V) with and without a C-terminal tag were constructed and evaluated for isobutanol production using the serum vial procedure described in Example 14. These strains were designated PNY2318 (with tag) and PNY2310 (without tag) and are further described below.
PNY2310 was generated by transforming strain PNY2145 (Example 1) with plasmids pLH804-L2V4 (Example 3) and pRS413::BiADH-kivD (Example 1). Plasmid transformants were selected by plating on synthetic complete medium lacking uracil and histidine with 1% (v/v) ethanol as the carbon source. Colonies were transferred to fresh plates by patching. After two days, cells from the patches were transferred to plates containing synthetic complete medium (minus uracil and histidine) with 2% (w/v) glucose as the carbon source. The resulting strain was designated PNY2310. PNY2318 was constructed analogously to PNY2310 except that it was transformed with pLH689-L2V4 (Example 24) instead of pLH804-L2V4. Isobutanol titer (mM) and yield (mole/mole glucose) from 38 hour samples are indicated in Table 35.







TABLE 35







Comparison of isobutanol production by strains containing


DHAD variant L2V4 with (PNY2318) and without


(PNY2310) a C-terminal tag. For each strain, n = 2.












38 h serum vial
38 h serum vial molar




titer (mM) +/−
yield +/−



Strain
standard deviation
standard deviation







PNY2318 (with
68.37 +/− 0.07
0.695 +/− 0.004



C-terminal Tag)



PNY2310 (without
62 +/− 2
0.674 +/− 0.003



C-terminal Tag)










Isobutanol-producing strains containing DHAD variant L2I7 (L388I) with and without the C-terminal tag were also constructed and evaluated for isobutanol production using the serum vial procedure described in Example 14. Strains were prepared by transforming PNY2145 (Example 1) with plasmid pRS413::BiADH-kivD (Example 1) and either pLH689-L2I7 (Example 24) or pLH804-L2I7 (Example 3). Transformants were obtained as described above and three independent clones of each were tested. Isobutanol titer (mM) and yield (mole/mole glucose) from 40 hour samples are indicated in Table 36.







TABLE 36







Comparison of isobutanol production by strains containing


DHAD variant L2I7 with and without a C-terminal tag.


For each genotype, n = 3.










40 h serum vial
40 h serum vial



titer (mM) +/−
molar yield +/−


Strain Genotype
standard deviation
standard deviation





PNY2145/pRS413::BiADH-
60 +/− 2
0.693 +/− 0.005


kivD/pLH689-L2I7 (i.e. with C-


terminal Tag)


PNY2145/pRS413::BiADH-
51 +/− 2
0.637 +/− 0.007


kivD/pLH804-L2I7 (i.e. without


C-terminal Tag)









While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.
All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.








TABLE 6







HMMER2.0 [2.2 g]
Program name and version


NAME dhad_for_hmm
Name of the input sequence alignment file


LENG 564
Length of the alignment: include indels


ALPH Amino
Type of residues


MAP yes
Map of the match states to the columns of the alignment


COM /app/public/hmmer/current/bin/hmmbuild -F dhad-exp_hmm dhad_for_hmm.aln
Commands used to generate the file: this one means that hmmbuild (default patrameters) was applied to the alignment file


COM /app/public/hmmer/current/bin/hmmcalibrate dhad-exp_hmm
Commands used to generate the file: this one means that hmmcalibrate (default parametrs) was applied to the hmm profile


NSEQ 8
Number of sequences in the alignment file


DATE Tue Jun 3 10:48:24 2008
When was the file generated


XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4



NULT −4 −8455
The transition probability distribution for the null model (single G state).


NULE 595 −1558 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45 531 201
The symbol emission probability distribution for the null model (G state); consists of K (e.g. 4 or 20) integers.


384 −1998 −644
The null probability used to convert these back to model probabilities is 1/K.


EVD −499.650970 0.086142
The extreme value distribution parameters μ and lambda respectively; both floating point values. Lambda is positive and



nonzero. These values are set when the model is calibrated with hmmcalibrate.
































A
C
D
E
F
G
H
I
K











Position in



m->m
m->i
m->d
i->m
i->i
d->m
d->d
b->m
m->e
L
M
N
P
Q
R
S
T
V
W
Y
alignment






−538
*
−1684




















 1(M)
−233
−1296
99
1223
−1477
−1132
89
−1122
420
−1248
1757
1553
−1296
464
−24
−190
−188
−838
−1578
−985
6


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
−538
*














 2(E)
−220
−1288
232
1356
−1807
1016
−70
−1474
190
−1584
−775
132
−1298
300
−282
−183
1140
−1092
−1872
−1262
7


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*














 3(K)
−448
−1932
1558
658
−2220
−1048
40
−1983
1569
−1938
−1091
1558
−1319
450
−193
−278
−419
−1552
−2121
−1397
8


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*














 4(V)
−404
−498
−1497
−939
−588
−1810
−640
1591
914
−127
335
−962
−1866
−562
−767
−868
−357
1720
−1169
−763
9


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*














 5(E)
−265
−1340
−52
1376
−1572
−1189
113
−1125
1345
−1287
−496
99
−1321
505
198
−218
−205
597
−1598
−1032
10


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*














 6(S)
256
−397
−1014
−830
−1841
−646
−862
−1443
−767
−1740
−963
−568
−1249
−651
−1007
2267
1586
−862
−2080
−1672
11


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−29
−6203
−7245
−894
−1115
−701
−1378
*
*














 7(M)
−990
−889
−2630
157
−513
−2514
−1346
1309
−1767
820
3683
−1898
−2491
−1496
−1799
−1589
−925
150
−1336
−1041
12


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 8(E)
588
−1875
−194
1536
−2188
−1373
−59
−1931
957
−1890
−977
904
292
393
−162
483
−372
−1495
−2070
−1391
13


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 9(N)
−514
−1116
1207
−315
447
−1650
−304
−778
−224
825
−277
1457
−1738
−123
−618
−627
−454
−603
−1186
763
14


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 10(N)
−815
−1190
−1360
−922
−904
−1967
−797
−442
−670
381
1700
3009
−2099
−654
−934
−1051
−791
−445
−1490
−979
15


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 11(K)
−1530
−2498
−1722
−855
−3141
−2246
−428
−2627
2828
−2404
−1656
−927
662
−2
2047
−1421
−1337
−2324
−2357
−2081
16


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 12(Y)
−872
−1887
−861
−290
−1369
−1801
1662
−1797
325
−1793
−1031
893
−1876
56
2219
−812
−780
−1514
−1565
2287
17


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 13(S)
−830
−1586
−1471
−1099
−2717
−1642
−1010
−2479
−266
−2518
−1746
−1065
−2069
−676
1822
2748
−1000
−1950
−2597
−2189
18


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 14(Q)
−851
−2131
−775
−153
−2554
−1735
−211
−2205
1908
−2094
−1244
−386
−1802
2254
974
1001
−747
−1819
−2181
−1667
19


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 15(T)
−405
−1258
−618
−100
−1490
−1466
1158
−1121
1
−1299
−514
578
−1607
65
−433
960
1849
343
−1677
−1143
20


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 16(I)
−1772
−1325
−4307
−3877
−1405
−3993
−3383
2935
−3705
820
−217
−3632
−3761
−3400
−3682
−3260
−1742
2033
−2838
−2525
21


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 17(T)
−1018
−1329
−2004
−1771
−409
−1993
−1000
−1256
−1512
−1464
−966
−1543
−2367
−1428
−1638
−1257
3050
−1090
−1012
2448
22


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 18(Q)
−1509
−3056
1970
44
−3310
−1666
−896
−3242
−877
−3158
−2439
−322
−2123
3562
−1493
−1259
−1550
−2779
−3260
−2446
23


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 19(D)
−1006
−2199
2178
−88
−3159
1997
−936
−2974
−948
−2977
−2174
−382
−1960
−589
−1571
1295
−1157
−2369
−3178
−2430
24


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 20(M)
445
−796
−1082
−521
−841
−1643
−412
−403
−370
−692
2213
−646
536
1166
−698
−630
660
831
−1204
−767
25


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 21(Q)
741
−990
−1025
−507
−1249
−1551
−519
−720
−357
−1062
−345
−635
−1739
1770
−713
−589
1576
1129
−1559
−1097
26


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 22(R)
−1753
−2648
−2072
−1047
−3365
−2405
−452
−2782
1989
−2495
−1773
−1062
−2379
2402
2643
−1629
−1506
−2504
−2397
−2190
27


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 23(S)
−330
−1010
−1820
−1628
−2778
−1229
−1652
−2481
−1592
−2691
−1841
−1273
2130
−1426
−1834
2449
1034
−1716
−2961
−2594
28


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 24(P)
1882
−1119
−2231
−2302
−3062
−1360
−2209
−2710
−2339
−3013
−2243
−1676
3304
−2117
−2409
−742
−918
−1916
−3263
−3022
29


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 25(N)
969
−1230
−1066
−915
−2593
−1313
−1196
−2242
−1033
−2447
−1626
3197
−1850
−898
−1392
−582
1155
−1644
−2736
−2256
30


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 26(R)
−1847
−2640
−2014
−1161
−3282
−2428
−579
−2818
687
−2553
−1869
−1165
−2462
2447
3181
−1746
−1630
−2555
−2447
−2228
31


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 27(A)
3048
−932
−2480
−2533
−3075
−1200
−2274
−2765
−2501
−3071
−2221
−1658
−1948
−2205
−2512
1225
−739
−1842
−3322
−3078
32


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 28(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
33


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 29(Y)
−1674
−1506
−2863
−2464
596
−2872
2251
−972
−2024
2197
−552
−1986
−2876
−1739
−1988
−1987
−1601
−1002
−95
2332
34


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 30(Y)
−2013
−2305
−2428
−1781
−328
−2709
−654
−2240
−258
−2064
−1626
−1631
−2788
−899
2789
−2017
−1896
−2130
−857
3434
35


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 31(A)
2822
−1031
−2418
−2539
−3226
1898
−2364
−2941
−2626
−3229
−2379
−1722
−2026
−2302
−2634
−654
−848
−1983
−3415
−3226
36


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 32(I)
−1247
−941
−3569
−3039
−1082
−3101
−2185
2227
−2763
766
−76
−2700
−3050
−2469
−2697
−2253
1322
1974
−1988
−1633
37


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 33(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
38


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 34(F)
−1511
−1236
−3511
−3017
2747
−2982
−1069
−260
−2651
992
2737
−2407
−2904
−2088
−2418
−2099
−1434
−489
−537
2056
39


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 35(Q)
−576
−1869
−401
92
−2232
831
−173
−1930
1505
−1913
−1042
−186
−1620
1653
−51
−482
1346
−1534
−2098
−1490
40


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 36(D)
−1352
−3066
3028
1349
−3303
−1566
−724
−3141
1155
−3043
−2267
−165
−1991
−354
−1350
−1086
−1368
−2659
−3221
−2356
41


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 37(E)
−1507
−3288
2042
2762
−3520
515
−853
−3401
−981
−3296
−2566
−182
−2064
−503
−1753
−1209
−1553
−2895
−3486
−2547
42


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 38(D)
−1445
−2778
3529
−53
−3524
−1590
−1129
−3476
−1367
−3459
−2774
−396
−2156
−825
−2122
554
−1609
−2880
−3582
−2717
43


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 39(F)
−2658
−2176
−4213
−4000
3815
−3933
−1352
−531
−3638
1121
−19
−3184
−3709
−2820
−3296
−3219
−2579
−1037
−601
403
44


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 40(D)
−684
−2193
1738
1460
−2494
−1437
−249
−2257
1694
−2199
−1308
−62
−1637
185
−450
−531
633
−1808
−2374
−1657
45


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 41(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
46


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 42(P)
1882
−1119
−2231
−2302
−3062
−1360
−2209
−2710
−2339
−3013
−2243
−1676
3304
−2117
−2409
−742
−918
−1916
−3263
−3022
47


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 43(I)
−1006
−992
−2347
−1784
−650
−2452
−1256
2372
−1386
77
2213
−1720
−2455
2030
−1490
−1528
−946
106
−1441
−1111
48


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 44(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
49


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 45(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
50


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 46(I)
−1759
−1303
−4330
−3968
−1751
−4051
−3743
3027
−3837
−597
−528
−3729
−3875
−3688
−3910
−3369
−1751
2438
−3259
−2819
51


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 47(V)
1736
−1012
−3546
−3078
−1377
−3073
−2434
2052
−2843
−608
−331
−2754
−3122
−2619
−2855
−2270
−1277
2193
−2333
−1941
52


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 48(N)
−686
−1511
−702
−806
−2927
−1386
−1339
−2841
−1264
−2950
−2137
2702
−1979
−1062
−1648
2444
−971
−2105
−3054
−2475
53


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 49(M)
−411
−857
−1800
−1434
−1528
1914
−1202
−1029
−1247
−1347
2989
−1217
−1912
−1119
−1444
−676
1550
−767
−1922
−1539
54


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 50(W)
−782
−1258
793
−683
1193
346
2051
−932
−556
−1092
−441
−798
−1993
−426
−909
−904
−720
−779
3163
1546
55


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 51(W)
1009
−798
−1470
−935
−463
−1773
−545
−460
−751
−736
−66
−943
−1904
−606
−1002
1604
−507
−322
2535
1521
56


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 52(D)
−1137
−2711
2125
1647
−2995
−1523
−617
−2786
−528
−2743
−1933
−150
−1897
−234
−1165
−924
2117
−2331
−2948
−2141
57


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 53(I)
−599
−1102
−1031
−829
−1522
1429
−927
2119
−880
−1369
−699
1692
−1938
−759
−1188
−799
−698
−689
−1887
−1419
58


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 54(T)
−666
−1412
−954
−984
−2702
−1428
−1357
−2418
−1208
−2650
−1886
2293
−2000
−1101
−1519
−787
2967
−1835
−2866
−2360
59


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 55(P)
−632
−1230
−2074
−2144
−2996
−1453
−2116
−2631
−2128
−2928
−2213
−1658
3610
−2006
−2221
−852
1302
−1931
−3185
−2917
60


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 56(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
61


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 57(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
62


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 58(M)
672
−918
−3119
−2578
−742
−2668
−1734
1807
−2263
16
3713
−2271
−2704
−1960
−2216
−1806
−1058
493
−1612
−1306
63


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 59(H)
−1525
−2164
−1235
−1346
−2509
2296
4235
−3172
−1516
−3178
−2523
−1448
−2541
−1520
−1760
−1591
−1741
−2656
−2681
−2065
64


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 60(L)
−2478
−2009
−4717
−4196
−568
−4424
−3262
1334
−3887
2824
604
−4085
−3872
−3088
−3590
−3717
−2380
−199
−2217
−2207
65


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 61(H)
−682
−2191
1015
275
−2485
396
2379
−2251
62
−2197
−1307
1826
−1636
1527
−480
−529
−641
−1803
−2375
−1654
66


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 62(D)
−575
−1920
1979
184
−2299
94
−242
−2029
114
−2023
−1144
−120
−1608
186
1063
−469
1413
−1605
−2229
−1561
67


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 63(L)
−2618
−2139
−4597
−4163
2144
−4285
−2334
−83
−3854
2690
538
−3771
−3806
−2950
−3488
−3563
−2505
−751
−1442
−808
68


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 64(A)
2657
−1033
−2408
−2532
−3233
2193
−2364
−2950
−2626
−3237
−2386
−1719
−2027
−2301
−2635
−655
−850
−1988
−3420
−3231
69


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 65(K)
−443
−1857
958
270
−2158
−1393
−66
−1890
1839
−442
−957
−36
−1499
1204
−132
616
−382
−1469
−2048
−1383
70


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 66(C)
605
1553
739
−17
−1374
−1488
−182
260
969
−203
−397
−263
−1573
159
691
−426
−331
−761
−1567
−1032
71


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 67(A)
2327
−956
−3193
−2728
−1289
−2677
−2114
1664
−2485
−601
−288
−2403
−2839
−2263
−2523
−1871
−1126
1617
−2143
−1765
72


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 68(K)
−532
−1656
−490
1321
−1891
−1527
−172
−124
2206
−1591
−782
−223
−1619
237
−106
−482
−464
−98
−1904
−1326
73


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 69(H)
384
−1854
936
889
−2165
−1363
1498
−1909
1111
−1866
−948
1091
−1464
421
−131
−284
−342
−69
−2043
−1364
74


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 70(G)
1823
−932
−2330
−2313
−3120
2511
−2158
−2865
−2331
−3098
−2209
−1563
−1912
−2032
−2419
1138
−706
−1883
−3328
−3077
75


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 71(V)
−1760
−1333
−4244
−3789
−1262
−3902
−3190
1495
−3588
1270
−96
−3536
−3677
−3238
−3534
−3148
−1725
2865
−2654
−2373
76


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 72(W)
−1054
−2172
−1112
−403
−2566
−1917
−286
−2196
2516
−2095
−1292
1183
−1958
140
1333
−959
−922
−1867
2591
−1720
77


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 73(D)
611
−1995
1525
937
−2295
−1400
−148
−2043
211
−2006
−1106
−37
−1553
1420
−312
−408
1235
−1609
−2193
−1499
78


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 74(A)
2716
−902
−2380
−2205
−2799
−1197
−1975
−2459
−2081
−2736
−1895
−1520
−1895
−1844
−2201
1191
1299
−1669
−3045
−2758
79


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 75(G)
−1709
−2833
2424
−409
−3781
2819
−1457
−3777
−1728
−3733
−3076
−739
−2389
−1180
−2441
−1557
−1893
−3158
−3660
−3038
80


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−212
−2909
−8150
−273
−2534
−701
−1378
*
*














 76(A)
2529
−1119
−2614
−2330
−1245
−1983
−1829
−377
−2042
1435
−341
−1937
−2411
−1873
−2088
−1266
−1059
−397
−2063
−1713
82


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 77(W)
−472
−361
−2421
−1812
−298
−1979
−826
1164
−1486
−143
2485
873
−2028
−1185
−1426
−1048
−412
1116
2999
−454
83


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 78(P)
−1198
−1737
−2187
−2394
−3665
2006
−2550
−3630
−2743
−3756
−3008
−2052
3474
−2495
−2835
−1401
−1593
−2736
−3511
−3519
84


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 79(Q)
−999
−1075
−2106
−1568
−726
−2370
−1175
83
−1185
1373
218
−1566
−2400
2445
−1340
−1445
−946
1441
−1501
−1146
85


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 80(Q)
−885
−779
−2609
−2018
−481
−2414
−1253
1645
−1736
799
1924
−1827
−2405
2262
−1752
−1484
−821
802
−1240
−935
86


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 81(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
87


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 82(G)
−998
−2100
−120
−175
−2567
2528
2174
−2558
−587
−2583
−1806
1422
−1966
−461
−1038
−925
−1088
−2095
−2657
−1948
88


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 83(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
89


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 84(I)
−1286
−1279
−2907
−2683
−1446
−2549
−2198
3290
−2407
−726
−534
−2386
1172
−2299
−2437
−1895
−1392
283
−2302
−1913
90


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 85(T)
−493
−1105
−2189
−2267
−3101
1880
−2196
−2791
−2334
−3081
−2269
−1649
−2058
−2099
−2410
−719
3135
−1948
−3282
−3046
91


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 86(V)
−1750
−1296
−4319
−3957
−1765
−4038
−3733
2364
−3826
−619
−543
−3716
−3869
−3685
−3902
−3354
−1743
3012
−3265
−2817
92


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 87(S)
923
−962
−2348
−2422
−3132
−1207
−2248
−2850
−2440
−3140
−2285
−1624
−1954
−2158
−2477
3171
−758
−1896
−3362
−3103
93


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 88(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
94


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 89(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
95


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 90(I)
−1880
−1493
−4193
−3724
−953
−3837
−2980
3251
−3420
257
2372
−3485
−3608
−3005
−3310
−3087
−1840
617
−2373
−2155
96


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 91(S)
2150
−939
−2407
−2415
−3075
−1197
−2205
−2781
−2384
−3065
−2205
−1613
−1936
−2105
−2436
2652
−729
−1850
−3306
−3049
97


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 92(M)
−979
−1455
−1242
−1122
−1434
−1860
−1131
−1171
−974
−1285
4091
2176
−2226
−1017
−1187
−1166
−1086
−1063
−1929
−1345
98


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 93(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
99


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 94(T)
−959
−1691
−1249
−949
−2563
−1747
−929
−2093
1282
−2263
−1554
−995
−2115
−600
−354
−1037
3152
−1726
−2494
−2098
100


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 95(E)
−572
−1860
−208
2213
−2107
−1461
−191
−1808
199
−116
−983
−127
318
1199
−269
−475
−517
−1448
−2078
−1441
101


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 96(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
102


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 97(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
103


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 98(R)
−2097
−2786
−2688
−1415
−3622
−2625
−555
−2964
2585
−2627
−1957
−1318
−2577
−137
3015
−1979
−1791
−2732
−2469
−2363
104


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














 99(Y)
−3615
−2706
−4169
−4413
2626
−4044
−396
−2535
−3993
−1939
−1985
−2747
−3930
−2852
−3446
−3296
−3494
−2686
347
4252
105


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














100(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
106


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














101(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
107


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














102(V)
−1381
−1065
−3714
−3252
−1453
−3300
−2646
1872
−3023
−615
−373
−2949
−3287
−2816
−3039
−2506
1346
2750
−2489
−2087
108


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














103(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
109


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














104(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
110


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














105(E)
−1719
−3572
2596
2779
−3767
−1632
−993
−3700
−1241
−3578
−2920
−234
−2167
−666
−2090
−1380
−1789
−3182
−3742
−2756
111


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














106(V)
−1746
−1296
−4308
−3946
−1757
−4020
−3712
2190
−3811
−614
−539
−3702
−3858
−3667
−3884
−3336
−1740
3098
−3250
−2803
112


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














107(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
113


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














108(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
114


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














109(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
115


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














110(S)
−352
2942
−2955
−2957
−2876
−1254
−2382
−2573
−2692
−2927
−2128
−1827
−2001
−2405
−2607
3103
−778
−1757
−3171
−2911
116


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














111(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
117


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














112(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
118


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














113(T)
1556
−936
−2493
−2457
−2805
−1256
−2159
−2210
−2319
−2681
−1932
−1656
−1974
−2089
−2352
−598
3235
−1547
−3111
−2847
119


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














114(C)
1784
2119
−2013
−1532
−1093
−1580
−1089
−436
−1322
−937
−273
1093
−1932
−1127
−1472
−748
−515
1585
−1536
−1163
120


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














115(M)
1831
2019
−2596
−2038
−605
−1979
−1126
244
−1727
−359
2501
−1655
−2145
−1435
−1683
−1106
−557
1087
−1153
−804
121


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














116(Q)
−987
−2211
−43
−62
−2833
2229
−691
−2616
−407
−2604
−1797
1197
−1917
2260
−858
−880
−1045
−2139
−2772
−2099
122


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














117(G)
2313
−1042
−2391
−2526
−3250
2601
−2372
−2972
−2637
−3257
−2407
−1721
−2032
−2310
−2646
−662
−859
−2003
−3434
−3247
123


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














118(Q)
−914
−2350
−48
1661
−2621
−1571
2504
−2400
68
−2331
−1486
−201
−1796
2646
−351
−754
−865
−1984
−2463
−1787
124


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














119(W)
−517
−1294
−733
−183
−1062
−1605
−234
−1037
19
−1207
−456
1435
−1690
33
756
411
−454
−819
3340
1286
125


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














120(M)
410
−469
−2417
−1828
−341
−2041
−897
195
−1513
−156
3130
−1534
−2102
−1230
−1484
−1117
−507
954
−894
2253
126


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














121(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
127


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














122(G)
2142
−930
−2334
−2298
−3100
2237
−2139
−2842
−2302
−3074
−2187
−1557
−1909
−2010
−2397
1136
−701
−1871
−3308
−3053
128


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














123(V)
−1514
−1144
−3950
−3459
1821
−3487
−2577
2274
−3208
−209
−87
−3112
−3362
−2864
−3118
−2680
−1476
2426
−2194
−1786
129


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














124(V)
−1743
−1294
−4292
−3873
−1511
−3988
−3433
2287
−3712
598
−319
−3626
−3774
−3456
−3716
−3260
−1717
2790
−2931
−2577
130


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














125(A)
2911
−954
−2808
−2665
−2115
−1577
−2196
−575
−2445
−1646
−1202
−1906
−2208
−2218
−2451
−901
−876
1294
−2727
−2394
131


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














126(I)
−1764
−1323
−4298
−3936
−1668
−3994
−3655
3337
−3783
−508
−462
−3689
−3838
−3608
−3835
−3311
−1759
1847
−3164
−2747
132


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














127(G)
−1157
−1705
−2169
−2375
−3654
3021
−2534
−3611
−2730
−3741
−2984
−2024
2418
−2475
−2826
−1361
−1555
−2705
−3513
−3509
133


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














128(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
134


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














129(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
135


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














130(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
136


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














131(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
137


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














132(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
138


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














133(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
139


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














134(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
140


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














135(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
141


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














136(A)
2180
−935
−2286
−2196
−3057
1098
−2058
−2796
−2174
−3021
−2134
−1516
−1898
−1906
−2302
2146
−689
−1849
−3256
−2983
142


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














137(M)
−1799
−1433
−4142
−3579
−669
−3668
−2608
1558
−3293
1235
3799
−3296
−3401
−2717
−3088
−2843
−1726
1156
−2002
−1868
143


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














138(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
144


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














139(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
145


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














140(M)
−2325
−1891
−4598
−4012
−498
−4222
−3013
1242
−3722
1864
3929
−3855
−3711
−2910
−3414
−3439
−2215
−299
−2076
−2098
146


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














141(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
147


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














142(R)
−1588
−2442
−1399
−953
−3069
−2171
−708
−2795
373
−2625
−1916
1858
−2357
−324
3294
−1520
−1505
−2453
−2523
−2186
148


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














143(M)
−1448
−1256
−3396
−2819
−474
−3024
−1923
175
−2473
2225
2756
−2574
−2922
−2063
−2375
−2153
952
−151
−1599
−1410
149


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














144(N)
−1662
−3306
2055
78
−3621
−1643
−1040
−3622
−1272
−3531
−2870
3477
−2182
−724
−2071
−1371
−1757
−3092
−3633
−2700
150


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














145(I)
−1066
−921
−2828
−2239
−1041
−2675
−1601
2235
−1668
−455
−92
−2067
−2692
−1688
1701
−1795
−1024
1960
−1771
−1396
151


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














146(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
152


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














147(S)
1568
−940
−2267
−2192
−3082
1101
−2068
−2826
−2185
−3049
−2159
−1515
−1901
−1915
−2313
2603
−694
−1866
−3279
−3006
153


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














148(I)
−1880
−1492
−4195
−3728
−963
−3841
−2991
3272
−3425
246
2277
−3490
−3613
−3014
−3317
−3092
−1841
628
−2385
−2163
154


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














149(F)
−2204
−1797
−3724
−3473
3206
−3383
−628
−1077
−3092
−746
3167
−2502
−3309
−2372
−2792
−2535
−2120
−1245
28
2460
155


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














150(V)
1265
−1028
−3200
−2994
−1833
−2150
−2480
417
−2771
−1122
−818
−2349
−2640
−2559
−2766
−1464
−1118
3028
−2700
−2325
156


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














151(Y)
−3482
−2868
−3701
−3919
238
−3552
−1112
−3000
−3638
−2516
−2526
−3027
−3772
−3101
−3341
−3418
−3527
−3071
−441
4711
157


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














152(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
158


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














153(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
159


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














154(T)
−359
−976
−2225
−2229
−2900
−1242
−2074
−2560
−2170
−2875
−2064
−1561
−1958
−1969
−2247
1110
3375
−1760
−3152
−2850
160


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














155(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
161


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














156(H)
861
−1924
−384
1010
−2260
−1477
1787
−1974
1769
−1918
−1022
−120
−1566
362
697
−417
−459
−1557
−2073
−1446
162


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














157(P)
−655
−1502
−711
−557
−2204
−1463
2143
−2122
−586
−2233
−1445
−688
2941
−560
−941
855
−805
−1657
−2369
−1763
163


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














158(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
164


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














159(H)
−744
−2193
−114
1118
−2513
−1512
2486
−2252
1178
−2183
−1308
2230
−1689
180
−233
−598
−687
−1823
−2335
−1670
165


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














160(W)
−2672
−2139
−3850
−3748
941
−3611
−469
−1691
−3306
1047
−1217
−2551
−3534
−2514
−2960
−2788
−2577
−1799
4205
3466
166


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














161(K)
386
−1981
779
279
−2295
−1403
−114
−2043
2059
−1991
−1082
941
−1536
1263
−211
−384
−457
−1602
−2161
−1476
167


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














162(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
168


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














163(K)
−1144
−2365
−912
2048
−2856
−1912
−326
−2459
2267
−2295
−1482
−556
−1989
108
1334
−1013
−1014
−2093
−2324
−1881
169


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














164(D)
−1091
−2610
2941
174
−2957
−1527
−595
−2750
1084
−2696
−1877
−176
−1885
−206
−1006
740
−1098
−2288
−2880
−2105
170


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














165(L)
−2387
−1922
−4674
−4155
−617
−4366
−3250
1889
−3865
2650
558
−4023
−3847
−3098
−3586
−3647
−2296
−38
−2247
−2224
171


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














166(N)
−1021
−2427
1806
133
−2870
−1499
−635
−2647
−521
−2640
−1825
2171
−1874
−255
−1124
−860
2122
−2184
−2853
−2090
172


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














167(I)
−1830
−1390
−4327
−3873
−1210
−3994
−3274
2967
−3678
1259
−30
−3633
−3730
−3283
−3604
−3249
−1791
1570
−2661
−2417
173


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














168(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
174


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














169(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
175


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














170(A)
2440
−824
−2371
−2082
−1993
−1344
−1704
−1264
−1899
−1832
−1137
−1517
−1946
−1674
−2005
1075
−641
1474
−2390
−2055
176


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














171(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
177


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














172(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
178


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














173(A)
2966
−1031
−2429
−2551
−3222
1544
−2368
−2934
−2633
−3225
−2377
−1727
−2028
−2309
−2637
−656
−850
−1980
−3412
−3224
179


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














174(V)
−1769
−1342
−4255
−3793
−1216
−3901
−3162
1633
−3589
1486
−51
−3537
−3667
−3214
−3518
−3143
−1731
2692
−2609
−2345
180


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














175(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
181


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














176(Q)
−729
−2116
−413
1096
−2484
−1587
1599
−2186
1695
−2094
−1219
−223
−1698
2418
90
−599
−649
−1770
−2213
−1615
182


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














177(W)
−1652
−1707
−2340
−1879
1996
−2733
2013
−1398
1758
−1386
−938
−1641
−2751
−1364
−1762
−1780
−1577
−1325
3577
2136
183


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














178(T)
−421
−753
−1251
−704
−846
−1670
−535
894
−548
−690
−1
1376
−1791
−421
−846
373
1461
858
−1236
−812
184


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














179(H)
1498
−1593
−504
15
−1895
−1484
2279
−1559
1119
−1640
−810
−242
−1611
194
−171
−462
815
−1231
−1914
−1340
185


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














180(G)
−1515
−2130
−1298
−1450
−2658
3285
2212
−3276
−1691
−3291
−2638
−1524
−2562
−1662
−1925
−1600
−1764
−2713
−2804
−2234
186


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














181(K)
−528
−2010
1346
1082
−2329
−1408
−118
−2080
1475
−2018
−1108
1161
−1543
331
1052
−394
−471
−1632
−2181
−1494
187


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














182(M)
−1894
−1521
−4170
−3679
−840
−3793
−2866
2827
−3360
375
3445
−3437
−3555
−2902
−3223
−3028
−1846
470
−2249
−2059
188


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














183(T)
−670
−1758
1731
−141
−2591
−1399
−691
−2319
−499
−2384
−1543
−387
−1786
−316
−1016
1576
2044
−1811
−2624
−1981
189


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














184(E)
345
−2074
925
1994
−2378
−1408
−177
−2135
922
−2084
−1183
−38
641
264
−356
−444
−536
−1690
−2261
−1556
190


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














185(E)
−1493
−2900
93
3174
−2903
−1743
1987
−3042
−646
−2957
−2238
−411
−2146
−506
−1121
−1272
−1503
−2629
−2905
−2134
191


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














186(D)
−1293
−2959
2673
2121
−3219
−1546
−713
−3043
−707
−2974
−2191
−158
−1967
−342
−1394
−1043
701
−2567
−3172
−2311
192


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














187(F)
−1137
−905
−3250
−2707
2365
−2647
−1016
−34
−2336
1239
267
−2150
−2626
−1861
−2133
−1752
−1069
1461
−599
1844
193


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














188(K)
−479
−1713
−409
1031
−1925
−1467
1755
−1650
1844
−349
−827
−140
−1556
319
−75
−403
−411
−1301
−1900
843
194


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














189(G)
433
−2144
52
1047
−2717
2303
−615
−2467
−442
−2482
−1655
1123
−1828
−233
−995
−763
−923
−2000
−2710
−2005
195


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














190(V)
−1752
−1320
−4254
−3806
−1311
−3916
−3232
1701
−3614
1188
−140
−3551
−3693
−3280
−3568
−3166
−1718
2833
−2703
−2409
196


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














191(E)
−1199
−1750
−734
2668
−1820
−2038
−1068
1892
−867
−1273
−897
−922
−2295
−797
−1238
−1340
−1197
−426
−2325
−1789
197


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














192(C)
−1182
3528
−1398
−620
−2541
−2038
−358
−2093
1181
−2037
−1272
−747
−2070
1553
2213
−1123
−1038
−1817
−2142
−1774
198


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














193(N)
−1478
−2527
−261
−403
−2011
−1837
2032
−2925
−735
−2845
−2195
3635
−2259
−721
−1085
−1352
−1546
−2522
−2307
−1431
199


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














194(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
200


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














195(C)
−1220
4911
−3609
−3314
−1440
−2525
−2482
1565
−2922
−706
−544
−2678
−2896
−2710
−2836
−1869
−1375
379
−2371
−1957
201


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














196(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
202


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














197(G)
−477
−1115
−1983
−2189
−3315
3154
−2272
−3172
−2506
−3387
−2522
−1599
−2042
−2177
−2583
1217
−905
−2130
−3477
−3225
203


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














198(A)
1653
−1347
−705
−249
−1969
−1385
−477
−1629
−159
−1759
−935
−434
1285
1404
−586
−450
1019
−1243
−2070
−1522
204


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














199(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
205


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














200(S)
1870
−938
−2270
−2183
−3068
1488
−2056
−2810
−2168
−3032
−2144
−1511
−1898
−1901
−2300
2236
−690
−1857
−3265
−2990
206


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














201(C)
−2476
5735
−4102
−4358
−3712
−2763
−3545
−3518
−4167
−3859
−3569
−3631
−3363
−4030
−3832
−2793
−2860
−3158
−3464
−3718
207


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














202(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
208


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














203(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
209


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














204(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
210


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














205(Y)
−3590
−2700
−4146
−4379
2092
−4028
−404
−2517
−3963
−1928
−1973
−2744
−3921
−2845
−3431
−3284
−3474
−2669
336
4423
211


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














206(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
212


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














207(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
213


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














208(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
214


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














209(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
215


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














210(M)
−2355
−1988
−4343
−3834
−504
−4051
−2868
105
−3385
1451
4460
−3680
−3671
−2806
−3171
−3327
−2274
−474
−2039
−1925
216


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














211(S)
2150
−939
−2407
−2415
−3075
−1197
−2205
−2781
−2384
−3065
−2205
−1613
−1936
−2105
−2436
2652
−729
−1850
−3306
−3049
217


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














212(S)
−344
−979
−2190
−2162
−2959
−1227
−2042
−2651
−2116
−2934
−2100
−1526
−1941
−1909
−2222
2940
1775
−1804
−3187
−2882
218


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














213(A)
3048
−932
−2480
−2533
−3075
−1200
−2274
−2765
−2501
−3071
−2221
−1658
−1948
−2205
−2512
1225
−739
−1842
−3322
−3078
219


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














214(I)
−1924
−1546
−4067
−3658
2312
−3663
−2081
3030
−3367
150
99
−3197
−3492
−2821
−3179
−2894
−1877
293
−1445
−692
220


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














215(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
221


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














216(A)
2389
−814
−2506
−2162
−1696
−1545
−1698
−499
−1942
−1398
−813
−1640
−2076
−1723
−2027
−806
1148
1559
−2200
−1856
222


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














217(M)
−2576
−2118
−4725
−4165
−461
−4430
−3165
99
−3811
2513
3454
−4075
−3839
−2978
−3488
−3704
−2457
−591
−2111
−2145
223


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














218(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
224


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














219(M)
−2313
−1968
−4258
−3765
−518
−3966
−2806
98
−3289
1292
4523
−3599
−3636
−2769
−3097
−3249
−2243
−457
−2026
−1874
225


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














220(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
226


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














221(L)
−2631
−2159
−4786
−4228
−462
−4506
−3231
96
−3878
2828
2482
−4157
−3880
−3016
−3541
−3793
−2509
−608
−2134
−2182
227


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














222(P)
−1501
−1778
−2473
−2371
−1710
−2311
−2045
−1321
−2060
827
−1068
−2173
3594
−2082
−2130
−1799
−1699
−1373
−2373
−1942
228


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














223(Y)
−1068
−1670
−865
−836
−631
1198
−767
−1828
−1059
−1914
−1304
692
−2203
−906
−1387
−1136
−1163
−1566
−1185
3670
229


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














224(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
230


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














225(S)
1172
−954
−2367
−2422
−3120
−1204
−2237
−2835
−2426
−3122
−2265
−1621
−1948
−2145
−2467
3107
−749
−1884
−3349
−3092
231


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














226(S)
−342
−975
−2176
−2124
−2912
−1229
−2003
−2594
−2067
−2878
−2048
−1510
−1936
−1866
−2184
2553
2492
−1773
−3143
−2833
232


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














227(M)
−720
−1440
−710
−343
−1228
−1693
2436
−1209
−132
−1364
3099
1904
−1852
−183
−458
−776
−680
−1004
−1540
−890
233


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














228(P)
2240
−1100
−2241
−2293
−3037
−1346
−2188
−2683
−2317
−2986
−2210
−1663
3041
−2093
−2391
−722
−895
−1893
−3243
−2998
234


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














229(A)
2958
−1235
−1299
−1377
−2868
−1345
−1673
−2580
−1661
−2843
−2054
1555
−1995
−1468
−1921
−715
−888
−1871
−3064
−2630
235


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














230(E)
−509
−1046
−884
1564
−1116
−1669
−441
−485
−283
250
−206
−577
689
−200
−656
−670
−459
1290
−1467
−995
236


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














231(D)
−1203
−2412
2595
−117
−3286
−1536
−1057
−3176
−1165
−3186
−2436
−428
−2068
−736
−1824
2377
−1366
−2578
−3334
−2552
237


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














232(Q)
954
−1983
−100
971
−2337
177
−267
−2067
81
−2060
−1189
−125
−1637
2600
−418
−514
−597
−1649
−2268
−1597
238


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














233(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
239


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














234(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
240


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














235(R)
377
−1802
−415
988
−2095
−1474
−95
−1786
1452
−1785
−911
−135
−1560
343
1555
−409
−431
376
−1986
−1375
241


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














236(D)
1083
−1565
2662
−244
−1941
−1573
−679
612
−527
−1651
−980
−490
−1869
−358
−1003
−771
−766
−903
−2208
−1633
242


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














237(E)
−1225
−2868
1894
1948
−3149
−1532
−671
−2975
−630
−2902
−2101
−150
−1935
−293
−1299
1884
−1241
−2496
−3093
−2248
243


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














238(C)
1375
3262
−2620
−2108
−827
−1866
−1267
1631
−1811
−599
−10
−1674
−2137
−1531
−1786
−1034
790
249
−1361
−1010
244


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














239(E)
635
−1796
1055
1761
−2018
−1464
−263
1191
28
−1767
−946
−148
−1637
135
−481
−520
−553
−1300
−2077
−1441
245


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














240(E)
593
−2044
−252
2548
−2437
−1542
−329
−2133
151
−2120
−1274
−244
−1738
89
946
−646
−717
−1734
−2305
−1686
246


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














241(S)
1884
−835
−1962
−1576
−1634
−1436
−1320
1041
−1409
−1453
−781
−1293
−1922
−1241
−1606
1973
−597
−669
−2036
−1656
247


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














242(G)
2267
−1043
−2388
−2526
−3253
2642
−2373
−2975
−2639
−3260
−2410
−1722
−2033
−2311
−2648
−663
−860
−2005
−3436
−3250
248


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














243(R)
−876
−2087
−829
1490
−2474
−1766
−229
−2106
1269
−44
−1198
−424
−1829
205
2225
−775
−768
−1753
−2143
−1647
249


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














244(V)
2339
−967
−2970
−2766
−1878
−1847
−2252
32
−2541
−1299
−918
−2087
−2399
−2316
−2545
−1157
−971
2345
−2605
−2251
250


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














245(I)
−1827
−1398
−4307
−3831
−1099
−3939
−3142
2286
−3619
1835
69
−3579
−3671
−3177
−3511
−3178
−1781
1918
−2524
−2310
251


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














246(V)
−1178
−1448
−1943
−1452
−1776
−2261
−1140
−227
1866
−1260
−816
−1444
−2448
−902
−540
−1496
−1176
2697
−2161
−1764
252


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














247(E)
−508
−1976
840
1547
−2280
−1393
−117
−2029
1400
−1984
−1077
1158
−1531
330
−253
−378
−454
262
−2163
−1471
253


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














248(M)
1703
−991
−2901
−2342
−528
−2567
−1550
166
−2031
1544
2668
−2104
−2591
−1715
−2010
−1685
−1052
−12
−1442
−1177
254


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














249(I)
−1947
−1516
−4385
−3885
−916
−4013
−3118
2193
−3656
2186
257
−3656
−3687
−3109
−3494
−3250
−1889
1383
−2397
−2258
255


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














250(E)
−1322
−2647
−272
2491
−3071
−1811
−576
−2759
2306
−2633
−1854
−464
−2066
−175
−177
−1144
−1256
−2368
−2692
−2140
256


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














251(K)
−1395
−2059
−1711
−1014
−2215
−2218
−641
−1709
3021
−1652
2578
−1075
−2303
−282
287
−1423
−1283
−1603
−2159
−1803
257


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














252(D)
−1285
−2888
2677
176
−3210
1189
−737
−3047
−715
−2977
−2195
−190
−1979
2106
−1379
−1050
−1315
−2564
−3161
−2320
258


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














253(I)
−2073
−1632
−4434
−3975
−911
−4130
−3238
3164
−3706
1451
244
−3779
−3785
−3187
−3557
−3413
−2021
546
−2449
−2273
259


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














254(K)
−1570
−2144
−1887
−1191
−2098
−2363
−750
−1603
3034
938
−1112
−1231
−2436
−408
215
−1616
−1443
−1580
−2166
−1804
260


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














255(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
261


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














256(R)
−928
−1705
−1507
−1055
−2761
−1730
−896
−2490
−44
−2489
−1723
−1042
−2102
−543
2614
2258
−1053
−1998
−2546
−2158
262


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














257(D)
−1280
−2865
3154
175
−3194
−1547
−743
−3034
−728
−2971
−2194
−190
−1979
1342
−1391
553
−1316
−2552
−3161
−2317
263


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














258(I)
−1997
−1562
−4355
−3927
−1042
−4066
−3261
3343
−3654
937
97
−3718
−3783
−3239
−3555
−3364
−1959
702
−2549
−2295
264


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














259(M)
−2252
−1821
−4572
−3991
−530
−4164
−2990
2068
−3709
1993
3197
−3808
−3685
−2916
−3406
−3378
−2149
−172
−2084
−2091
265


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














260(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
266


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














261(R)
−2131
−2786
−2704
−1460
−3618
−2638
−587
−2976
1735
−2645
−1985
−1353
−2603
−173
3492
−2020
−1828
−2748
−2484
−2384
267


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














262(K)
−1349
−2635
−381
2083
−3083
−1857
−565
−2750
2690
−2612
−1837
−514
−2090
−161
−61
−1178
−1271
−2369
−2655
−2138
268


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














263(A)
2821
−932
−2451
−2472
−3065
−1198
−2233
−2763
−2434
−3056
−2201
−1633
−1940
−2147
−2468
1831
−730
−1840
−3305
−3055
269


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














264(F)
−2063
−1686
−4037
−3677
3437
−3644
−1706
2063
−3359
135
67
−3095
−3486
−2739
−3127
−2876
−2012
−83
−1038
−158
270


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














265(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
271


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














266(N)
−1662
−3306
2055
78
−3621
−1643
−1040
−3622
−1272
−3531
−2870
3477
−2182
−724
−2071
−1371
−1757
−3092
−3633
−2700
272


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














267(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
273


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














268(I)
−1760
−1307
−4325
−3962
−1735
−4042
−3726
3135
−3828
−579
−515
−3722
−3869
−3673
−3896
−3359
−1752
2276
−3240
−2806
274


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














269(T)
1428
−904
−2334
−2158
−2747
−1206
−1940
−2392
−2037
−2678
−1846
−1504
−1896
−1809
−2163
902
3001
−1635
−2999
−2705
275


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














270(V)
−1745
−1300
−4286
−3858
−1446
−3967
−3370
2358
−3688
852
−261
−3606
−3749
−3403
−3673
−3232
−1717
2643
−2856
−2524
276


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














271(V)
−1404
−1072
−3766
−3305
−1464
−3356
−2696
2276
−3080
−616
−379
−3001
−3325
−2870
−3091
−2563
1344
2521
−2516
−2113
277


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














272(M)
866
−1113
−2656
−2412
−1322
−1920
−1883
−487
−2061
−587
4451
−1950
−2387
−1928
−2078
−1220
−1053
−498
−2134
−1803
278


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














273(A)
2601
−957
−2898
−2711
−1943
−1740
−2211
−165
−2487
−1406
−1001
−2008
−2320
−2260
−2494
−1053
−929
1990
−2626
−2279
279


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














274(L)
−1171
−983
−3266
−2733
−796
−2795
−1888
590
−2418
2001
198
−2418
−2816
−2106
−2362
−1944
965
1777
−1724
−1426
280


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














275(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
281


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














276(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
282


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














277(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
283


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














278(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
284


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














279(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
285


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














280(A)
3134
−934
−2491
−2567
−3083
−1203
−2300
−2766
−2540
−3082
−2237
−1672
−1954
−2240
−2537
874
−747
−1844
−3333
−3093
286


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














281(V)
−984
−1045
−3169
−2909
−1709
−2304
−2404
531
−2643
−988
−697
−2378
−2722
−2480
−2661
−1601
1504
3014
−2588
−2201
287


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














282(L)
−2631
−2159
−4786
−4228
−462
−4506
−3231
96
−3878
2828
2482
−4157
−3880
−3016
−3541
−3793
−2509
−608
−2134
−2182
288


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














283(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
289


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














284(L)
−1623
−1338
−3726
−3164
−251
−3255
−1820
1373
−2808
2371
514
−2785
−3086
−2281
−2613
−2389
−1543
−161
−1311
1782
290


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














285(L)
−2333
−1873
−4640
−4127
−650
−4326
−3241
2176
−3843
2519
523
−3982
−3833
−3105
−3579
−3604
−2247
56
−2268
−2230
291


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














286(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
292


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














287(M)
−1886
−1507
−4178
−3693
−877
−3806
−2901
3008
−3380
335
3109
−3451
−3570
−2934
−3251
−3044
−1840
524
−2288
−2089
293


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














288(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
294


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














289(H)
−1490
−2484
−362
−476
−1816
−1880
4320
−2854
−684
−2770
−2133
2185
−2285
−728
−1000
−1377
−1550
−2475
−2146
−1255
295


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














290(A)
2439
−911
−2326
−2131
−2811
−1197
−1934
−2480
−2011
−2745
−1898
−1490
−1888
−1785
−2153
1898
1073
−1682
−3044
−2749
296


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














291(I)
2038
−985
−3388
−2919
−1320
−2893
−2277
2155
−2677
−587
−297
−2593
−2992
−2450
−2697
−2087
−1208
1681
−2229
−1846
297


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














292(G)
−1243
−2769
311
1902
−3172
1980
−744
−2992
−697
−2936
−2152
1923
−1974
−377
−1331
−1030
−1284
−2506
−3125
−2308
298


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














293(V)
−1738
−1298
−4281
−3921
−1737
−3979
−3665
1917
−3774
−601
−528
−3671
−3834
−3628
−3843
−3293
−1735
3205
−3215
−2770
299


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














294(E)
−833
−2344
1092
2412
−2643
−1464
−386
−2413
−146
−2369
−1505
−96
562
29
−717
−666
862
−1966
−2562
−1818
300


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














295(W)
−1380
−1116
−3614
−3026
1322
−2981
−1582
1966
−2661
1775
556
−2562
−2865
−2117
−2424
−2098
−1302
−187
2908
−629
301


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














296(T)
−350
−973
−2204
−2178
−2893
−1236
−2035
−2561
−2117
−2862
−2043
−1536
−1946
−1916
−2214
1618
3198
−1758
−3137
−2831
302


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














297(L)
−1443
−1269
−3144
−2576
−528
−3014
−1816
1945
−2155
2102
508
−2422
−2899
1193
−2133
−2129
−1369
−50
−1616
−1384
303


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














298(D)
−1826
−3682
3559
1199
−3883
−1662
−1073
−3846
−1391
−3720
−3110
−272
−2222
−760
−2283
−1471
−1913
−3321
−3864
−2864
304


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














299(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
305


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














300(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
306


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














301(Q)
−1048
−2608
205
2170
−2893
−1535
−505
−2680
−255
−2604
−1769
1814
−1849
2272
−789
−848
−1028
−2228
−2770
−2013
307


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














302(R)
1083
−1687
691
135
−2058
−1406
−178
−1755
214
−1793
−924
−145
−1553
247
1670
−383
1217
−1367
−2031
−1404
308


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














303(I)
−1915
−1536
−4077
−3667
2027
−3678
−2155
3137
−3381
144
94
−3225
−3506
−2848
−3202
−2914
−1871
345
−1522
−791
309


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














304(R)
−689
−2015
−494
24
−2395
−1582
−184
−2087
444
−2020
−1151
1161
−1687
1832
2131
626
−614
−1684
−2156
−1573
310


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














305(D)
387
−1967
1600
1359
−2275
−1391
1561
−2025
282
−1976
−1067
−25
−1525
342
1024
−369
−443
−1584
−2152
−1462
311


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














306(R)
−1460
−2315
−1793
−887
−2832
−2237
−431
−2288
2193
−2199
−1473
−946
−2245
−20
2706
−1394
−1275
591
−2248
−1961
312


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














307(V)
−941
−1027
−3099
−2832
−1692
−2234
−2324
470
−2565
−1003
−695
−2305
−2663
−2399
−2587
−1527
1858
2876
−2536
−2152
313


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














308(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
314


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














309(V)
−1090
−1215
−2097
−1824
−819
−2221
2699
−287
−1392
−1027
−591
−1674
−2482
−1446
−1482
−1482
−1143
2879
−1420
−707
315


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














310(L)
−2439
−1972
−4702
−4181
−588
−4401
−3258
1582
−3881
2757
587
−4061
−3862
−3093
−3590
−3689
−2344
−130
−2230
−2217
316


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














311(C)
2157
4166
−3012
−2973
−2780
1022
−2337
−2398
−2724
−2744
−1930
−1786
−1943
−2372
−2623
−540
−692
−1624
−3091
−2881
317


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














312(D)
−1732
−3453
3468
99
−3733
−1645
−1066
−3747
−1356
−3641
−3008
1690
−2201
−755
−2209
−1416
−1833
−3208
−3752
−2776
318


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














313(L)
−2477
−2023
−4713
−4122
1592
−4329
−2920
72
−3835
2593
2472
−3948
−3754
−2914
−3466
−3550
−2350
−634
−1927
−1830
319


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














314(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
320


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














315(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
321


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














316(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
322


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














317(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
323


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














318(K)
2
−2257
−1073
−374
−2740
−1908
−278
−2339
2328
−2192
−1373
−562
−1953
2273
1344
−952
−933
−1980
−2234
−1799
324


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














319(Y)
−3482
−2868
−3701
−3919
238
−3552
−1112
−3000
−3638
−2516
−2526
−3027
−3772
−3101
−3341
−3418
−3527
−3071
−441
4711
325


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














320(M)
−1559
−1267
−3829
−3380
−1103
−3357
−2655
805
−3067
−64
3046
−3065
−3326
−2779
−3011
−2591
−1556
2855
−2312
−1998
326


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














321(M)
1225
−469
−2256
−1679
1656
−1926
−870
90
−1396
−210
2763
−1424
−2028
−1129
−1411
−1008
712
154
−951
−586
327


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














322(T)
−738
−2094
−84
1704
−2416
−1495
−317
−2135
61
−2127
−1275
−163
−1704
1857
−405
−613
1930
−1734
−2331
−1668
328


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














323(D)
−1746
−3458
3540
90
−3744
−1650
−1081
−3767
−1381
−3662
−3036
1386
−2211
−772
−2239
−1429
−1850
−3226
−3765
−2789
329


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














324(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
330


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














325(H)
−2923
−2573
−2959
−2926
826
−3449
4553
−2508
−2463
−2054
−1948
−2279
−3499
−2191
−2397
−2761
−2855
−2540
123
2920
331


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














326(K)
373
−1957
−342
1025
−2297
−1472
−98
−2018
2111
−1954
−1056
906
−1570
352
685
−424
−473
−1592
−2105
−1469
332


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














327(V)
1739
−1008
−3509
−3043
−1376
−3028
−2406
1765
−2807
−615
−334
−2718
−3093
−2585
−2823
−2226
−1263
2376
−2322
−1931
333


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














328(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
334


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














329(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
335


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














330(I)
−1758
−1302
−4331
−3970
−1756
−4054
−3748
2976
−3840
−603
−533
−3731
−3877
−3693
−3914
−3372
−1750
2505
−3265
−2824
336


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














331(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
337


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














332(Q)
1795
−1440
−730
−492
−2453
682
−812
−2151
−508
−2256
−1426
−624
−1796
2666
−901
−590
−689
−1636
−2510
−1971
338


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














333(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
339


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














334(M)
−2355
−1988
−4343
−3834
−504
−4051
−2868
105
−3385
1451
4460
−3680
−3671
−2806
−3171
−3327
−2274
−474
−2039
−1925
340


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














335(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
341


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














336(Y)
−1187
−974
−3186
−2638
−117
−2732
−1255
1905
−2270
73
1977
−2217
−2699
−1882
−2144
−1841
−1124
71
−907
3254
342


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














337(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
343


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














338(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
344


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














339(K)
−864
−1785
−860
−366
−2128
−1763
−407
−1612
2624
−1800
−1045
629
−1900
−28
62
−851
−805
1127
−2064
−1581
345


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














340(N)
602
−1686
−275
1008
−1926
−1415
1528
−1618
244
−1673
−815
1897
−1530
299
−244
−371
−391
322
−1934
−1306
346


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














341(G)
−1709
−2639
1362
−690
−3785
3257
−1671
−3805
−1946
−3792
−3137
−980
−2480
−1424
−2576
−1630
−1936
−3150
−3628
−3155
347


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














342(F)
−942
−799
−2828
−2226
1797
−2476
−1269
1109
581
1793
516
−1952
−2453
−1557
−1815
−1558
−875
52
−1138
−794
348


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














343(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
349


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














344(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
350


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














345(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
351


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














346(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
352


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














347(C)
774
4452
−2162
−1688
−1962
−1478
−1302
−1474
−944
−1796
−1088
−1351
−1979
−1147
1684
−732
−719
−1116
−2225
−1881
353


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














348(L)
−2387
−1922
−4674
−4155
−617
−4366
−3250
1889
−3865
2650
558
−4023
−3847
−3098
−3586
−3647
−2296
−38
−2247
−2224
354


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














349(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
355


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














350(C)
−1489
2972
−4007
−3563
−1524
−3541
−2939
2612
−3350
−617
−413
−3224
−3470
−3129
−3335
−2770
−1475
2269
−2657
−2248
356


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














351(T)
−364
−979
−2232
−2250
−2904
−1245
−2090
−2559
−2191
−2881
−2075
−1571
−1964
−1991
−2260
905
3428
−1762
−3159
−2858
357


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














352(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
358


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














353(K)
−1716
−2632
−2004
−1008
−3336
−2379
−444
−2764
2775
−2484
−1756
−1035
−2357
2151
1811
−1592
−1477
−2481
−2391
−2172
359


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














354(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
360


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














355(V)
−1771
−1339
−4275
−3816
−1235
−3919
−3194
2139
−3617
1520
−66
−3558
−3681
−3244
−3547
−3164
−1733
2390
−2634
−2369
361


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














356(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
362


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














357(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
363


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














358(N)
−823
−1917
−96
1188
−2187
−1547
−506
−1711
−265
−1955
−1191
2711
−1815
−144
−747
−757
−815
1140
−2297
−1666
364


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














359(L)
−2153
−1779
−4360
−3884
−675
−3965
−3012
392
−3561
2726
467
−3673
−3662
−2955
−3355
−3239
−2102
1281
−2207
−2099
365


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














360(E)
1136
−2084
−175
2027
−2436
−1510
−274
−2147
1525
−2118
−1254
−175
−1692
152
−251
−593
−670
−1736
−2296
−1650
366


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














361(H)
893
−1761
1357
214
−2092
−1387
1862
−1810
229
−1825
−942
−83
−1527
293
−273
640
793
−1409
−2050
−1397
367


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














362(I)
608
−458
−2776
−2176
1666
−2202
−1113
1712
−1836
−222
338
−1782
−2245
−1512
−1731
−1292
867
1366
−1036
−684
368


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














363(P)
−922
−1912
1681
−141
−2123
−1604
−687
−1787
−550
187
−1245
−427
2677
−363
−1049
−882
−947
−1524
−2338
−1711
369


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














364(D)
−1692
−3605
3364
1256
−3770
−1599
−957
−3700
−1216
−3569
−2909
1025
−2138
−628
−2083
−1346
−1761
−3174
−3765
−2738
370


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














365(Q)
−877
−1646
−633
499
−1610
−1781
−505
−1210
−63
1648
−649
−558
−1931
2241
−360
−907
−814
−1097
−1882
−1385
371


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














366(P)
−648
−2019
1139
203
−2354
−1436
−285
−2089
29
−2086
−1217
−114
1965
1445
−492
−529
1244
−1672
−2300
−1616
372


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−571
−7108
−1646
−894
−1115
−701
−1378
*
*














367(R)
−422
−1009
−851
−304
1406
−1496
−183
−740
147
−894
−230
−440
775
21
2009
−539
−381
−568
−1136
−521
373


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−23
−6560
−7602
−894
−1115
−341
−2249
*
*














368(D)
1472
−1668
1835
−70
−2356
−1385
−511
−2062
−246
−2128
−1275
−318
1353
−118
−746
−526
425
−1602
−2380
−1752
374


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














369(G)
−1044
−2230
2141
−100
−3222
2291
−982
−3045
−1033
−3050
−2258
−395
−1985
−644
−1669
858
−1207
−2428
−3250
−2493
375


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














370(Q)
−2562
−2904
−1886
−1971
−3251
−2661
−2079
−3690
−1565
−3469
−3081
−2107
−3091
4371
−1665
−2585
−2674
−3411
−3077
−2821
376


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














371(D)
−1275
−2955
2862
1330
−3205
−1556
−670
−3029
1509
−2936
−2141
−158
−1955
−290
−1213
−1025
−1281
−2554
−3111
−2272
377


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














372(V)
−1738
−1298
−4281
−3921
−1737
−3979
−3665
1917
−3774
−601
−528
−3671
−3834
−3628
−3843
−3293
−1735
3205
−3215
−2770
378


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














373(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
379


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














374(M)
−584
−1354
−847
−246
−1467
−1659
2505
−1087
212
−374
2571
−449
−1729
1171
1074
−634
−507
−876
−1617
−1128
380


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














375(P)
−910
−2031
−73
1195
−2792
−1488
−794
−2539
−629
−2588
−1788
−401
3005
−439
−1131
612
−1014
−2050
−2815
−2151
381


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














376(W)
−1588
−1300
−3783
−3197
−329
−3245
−1926
2071
−2827
1901
558
−2822
−3072
−2297
−2616
−2381
−1508
−111
3483
−1042
382


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














377(E)
−1024
−2640
1844
2310
−2908
−1498
−505
−2711
−344
−2636
−1791
−107
−1824
1521
−957
207
−1011
−2243
−2817
−2021
383


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














378(N)
−826
−2349
1089
227
−2651
−1487
−341
−2416
1494
−2346
−1475
2601
−1724
1005
−522
−657
−787
−1968
−2511
−1791
384


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














379(P)
1932
−1116
−2232
−2301
−3058
−1358
−2206
−2706
−2336
−3009
−2238
−1674
3274
−2114
−2406
−739
−914
−1913
−3260
−3019
385


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














380(V)
−914
−773
−2713
−2129
−712
−2505
−1388
1452
1084
1324
204
−1926
−2507
−1580
−1808
−1591
−859
1713
−1424
−1081
386


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














381(Y)
−1484
−2331
−1762
−887
−2436
−2254
−420
−2325
2137
−2195
−1475
−949
−2258
−39
1983
−1411
−1295
−2075
−2087
2868
387


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














382(E)
1256
−1890
−206
1353
−2196
−1401
−89
−1930
812
−1898
−996
−45
547
1252
−162
−356
−414
−1507
−2083
−1416
388


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














383(Q)
−752
−2272
1586
1407
−2561
−1448
−308
−2329
−23
−2276
−1396
−71
−1677
1749
−577
−590
1569
−1881
−2459
−1727
389


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














384(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
390


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














385(H)
−964
−2089
−200
−136
−2264
−1600
3833
−2320
−296
−2338
−1558
1362
1479
−276
−699
−881
−992
−1924
−2364
−1652
391


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














386(L)
−2451
−1983
−4707
−4186
−582
−4409
−3259
1510
−3884
2778
592
−4069
−3865
−3091
−3590
−3698
−2355
−150
−2226
−2214
392


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














387(Q)
1643
−1017
−1196
−721
−1189
−1714
−668
1336
−497
−907
−297
−823
−1893
2044
−794
−784
−569
−339
−1579
−1135
393


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














388(I)
−1760
−1308
−4323
−3961
−1730
−4039
−3721
3156
−3825
−575
−512
−3720
−3867
−3669
−3893
−3356
−1753
2241
−3236
−2802
394


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














389(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
395


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














390(K)
−1259
−2115
−1267
−676
−970
−2105
1794
−2040
2549
−1955
−1282
−808
−2165
−167
114
−1192
−1140
−1801
−1301
2517
396


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














391(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
397


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














392(N)
−2171
−2655
−1458
−1748
−3334
−2364
−2267
−3943
−2365
−3936
−3437
4205
−2932
−2205
−2608
−2224
−2439
−3392
−3253
−2909
398


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














393(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
399


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














394(A)
3121
−934
−2489
−2561
−3081
−1203
−2295
−2766
−2533
−3080
−2234
−1669
−1953
−2234
−2533
936
−746
−1844
−3331
−3090
400


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














395(E)
−522
−1773
−240
1676
−2248
−1396
−289
−1968
50
−1989
−1115
−174
1198
131
−448
1226
677
−1538
−2214
−1565
401


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














396(E)
−1481
−3230
1425
2936
−3481
751
−843
−3354
−954
−3256
−2520
−187
−2057
−492
−1711
−1193
−1527
−2852
−3445
−2523
402


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














397(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
403


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














398(A)
2847
−932
−2454
−2477
−3066
−1198
−2236
−2763
−2439
−3057
−2202
−1635
−1940
−2152
−2471
1777
−731
−1840
−3306
−3056
404


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














399(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
405


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














400(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
406


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














401(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
407


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














402(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
408


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














403(S)
−348
−981
−2200
−2194
−2989
−1227
−2073
−2686
−2157
−2970
−2136
−1541
−1946
−1946
−2253
3060
1398
−1824
−3217
−2916
409


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














404(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
410


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














405(V)
−917
−809
−2556
−1976
−827
−2491
−1367
1339
1455
721
94
−1841
−2501
−1487
−1710
−1570
−863
2038
−1514
−1151
411


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














406(K)
−1386
−2643
−447
1824
−3108
−1893
−570
−2762
2860
−2616
−1848
−552
−2117
−166
−3
−1217
−1300
−2388
−2647
−2154
412


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














407(N)
−537
−1563
−449
−36
−1889
1143
−307
−1529
932
−1655
−844
1794
−1658
73
−356
−518
−516
924
−1962
−1392
413


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














408(P)
−894
−2181
−369
1705
−2576
−1650
−357
−2268
243
−2210
−1375
−330
2093
63
1619
−774
−835
−1876
−2347
−1769
414


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














409(V)
−419
−634
−1376
−807
1053
−1737
−499
−198
−623
−505
178
600
−1807
−475
475
313
−360
1389
−1016
1303
415


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














410(I)
−1282
−1082
−3022
−2555
2426
−2683
1767
2555
−2191
−443
−88
−2038
−2692
−1794
−2075
−1793
−1220
−317
−361
552
416


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














411(T)
−499
−1595
−431
966
−1830
−1487
−185
−1449
1092
−1574
−754
−207
−1601
213
−206
−458
2067
159
−1877
−1296
417


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














412(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
418


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














413(P)
−632
−1230
−2074
−2144
−2996
−1453
−2116
−2631
−2128
−2928
−2213
−1658
3610
−2006
−2221
−852
1302
−1931
−3185
−2917
419


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














414(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
420


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














415(R)
−1454
−2316
−1780
−878
−2834
−2232
−428
−2292
2281
−2200
−1473
−940
−2240
−17
2627
−1386
−1270
588
−2249
−1960
421


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














416(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
422


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














417(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
423


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














418(D)
−1572
−3426
2573
2447
−3613
−1583
−879
−3513
−1050
−3393
−2684
1292
−2085
−535
−1855
−1253
−1623
−3000
−3585
−2609
424


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














419(S)
−879
−1989
1498
−177
−3045
1600
−939
−2843
−904
−2867
−2046
−438
−1922
−591
−1483
2171
−1044
−2226
−3072
−2372
425


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














420(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
426


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














421(Q)
−705
−1925
−199
2112
917
−1534
−288
−1824
42
−1842
−1054
−210
−1709
2163
−420
−611
−656
−1502
−1997
−1291
427


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














422(H)
−569
−2048
1450
1526
−2349
−1405
1830
−2103
181
−2058
−1157
−37
−1569
272
−349
713
620
−1662
−2240
−1537
428


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














423(C)
1626
2878
−2671
−2107
1264
−1968
−1091
233
−1777
−334
250
−1672
−2128
−1459
−1691
−1096
−529
1209
−1066
−704
429


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














424(M)
−2042
−1634
−4379
−3826
−659
−3976
−2899
2765
−3546
1204
3085
−3605
−3604
−2896
−3318
−3183
−1961
195
−2135
−2058
430


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














425(E)
412
−2447
1356
2379
−2747
−1477
−445
−2527
−243
−2477
−1622
−107
855
−36
−831
−730
−894
−2073
−2668
−1906
431


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














426(A)
2822
−1031
−2418
−2539
−3226
1898
−2364
−2941
−2626
−3229
−2379
−1722
−2026
−2302
−2634
−654
−848
−1983
−3415
−3226
432


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














427(I)
−1772
−1325
−4307
−3877
−1405
−3993
−3383
2935
−3705
820
−217
−3632
−3761
−3400
−3682
−3260
−1742
2033
−2838
−2525
433


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














428(L)
−875
−1634
−575
959
−1581
−1769
−525
−1179
−135
1884
−625
−547
−1931
1405
−450
−909
−816
−1074
−1883
−1383
434


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














429(A)
1705
−1826
−180
949
−2318
−1410
−359
−2041
−53
−2067
−1204
1001
−1652
52
−561
1232
−595
−1609
−2298
−1643
435


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














430(D)
−1074
−2458
2381
60
−2921
1927
−658
−2710
−463
−2675
−1860
−271
−1918
−276
866
−915
−1100
−2245
−2845
−2124
436


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














431(K)
−688
−2117
785
888
−2469
−1529
−187
−2189
2380
−2106
−1221
−162
−1661
256
1134
−553
−619
−1760
−2240
−1607
437


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














432(I)
−2019
−1582
−4380
−3941
−1000
−4086
−3253
3295
−3671
1100
145
−3736
−3783
−3222
−3556
−3378
−1976
657
−2517
−2289
438


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














433(Q)
−490
−1797
−369
171
−2078
−1457
1762
−1779
1157
−1780
−905
1165
−1550
1798
−48
−396
−422
725
−1986
−1366
439


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














434(A)
1954
−1836
1733
−180
−2714
−1429
−806
−2438
−679
−2518
−1698
−430
1775
−448
−1211
−736
−894
−1923
−2765
−2117
440


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














435(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
441


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














436(D)
−1736
−3455
3490
97
−3737
−1646
−1070
−3753
−1363
−3647
−3016
1602
−2204
−760
−2218
−1420
−1838
−3213
−3756
−2780
442


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














437(V)
−1721
−1302
−4229
−3874
−1705
−3894
−3582
1607
−3706
−582
−513
−3610
−3786
−3559
−3767
−3209
−1725
3294
−3158
−2712
443


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














438(V)
594
−988
−3391
−2911
−1164
−2888
−2187
845
−2637
765
−154
−2576
−2962
−2387
−2622
−2074
−1205
2800
−2084
−1724
444


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














439(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
445


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














440(I)
−1754
−1308
−4295
−3867
−1434
−3978
−3377
2661
−3697
862
−247
−3617
−3754
−3406
−3679
−3243
−1725
2373
−2852
−2526
446


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














441(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
447


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














442(Y)
−1321
−1438
−1994
−1608
2186
527
−450
−1117
−1481
−1211
−693
1178
−2522
−1217
−1665
−1518
−1275
−1021
−198
3178
448


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














443(C)
−675
2205
−2544
972
−572
−2236
−1121
1373
−1671
679
261
−1700
−2270
−1403
−1668
−1311
−621
1601
−1150
−790
449


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














444(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
450


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














445(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
451


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














446(K)
−1060
−2058
−1088
−460
−2432
−1917
−357
−1970
2801
−1978
−1220
−632
−1990
1339
367
−999
−946
536
−2145
−1717
452


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














447(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
453


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














448(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
454


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














449(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
455


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














450(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
456


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














451(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
457


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














452(P)
−1659
−2241
−2022
−1646
−3185
−2242
−1373
−3000
−450
−2936
−2274
−1624
3435
−1065
2095
−1730
−1750
−2593
−2816
−2613
458


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














453(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
459


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














454(M)
−2406
−2296
−3638
−3594
−1525
−3105
−2824
−1047
−3121
−596
5043
−3293
−3425
−3046
−2996
−2911
−2552
−1398
−2513
−2207
460


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














455(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
461


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














456(K)
1368
−1491
−763
−332
−2319
−1417
−551
−1998
1786
−2068
−1221
−500
−1721
−160
−470
1631
−587
−1532
−2299
−1754
462


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














457(P)
−1500
−1738
−2514
−2380
−1555
−2358
−2022
−1126
−2063
1224
−841
−2189
3436
−2061
−2129
−1822
−1674
−1231
−2290
−1878
463


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














458(T)
−351
−974
−2208
−2185
−2894
−1237
−2041
−2561
−2125
−2863
−2046
−1539
−1948
−1923
−2218
1543
3230
−1758
−3139
−2834
464


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














459(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
465


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














460(M)
2706
−986
−2433
−2144
−1502
−1684
−1706
−700
−1858
−968
2744
−1705
−2188
−1713
−1932
−963
−862
−592
−2145
−1794
466


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














461(I)
−2103
−1659
−4461
−3992
−869
−4152
−3233
3082
−3723
1619
290
−3801
−3788
−3171
−3557
−3432
−2046
487
−2418
−2265
467


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














462(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
468


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














463(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
469


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














464(K)
1641
−2033
−323
914
−2415
−1565
−296
−2097
2052
−2080
−1233
−257
−1736
125
−133
−646
−702
−1707
−2258
−1657
470


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














465(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
471


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














466(L)
−1699
−1807
−2268
−1925
−830
−2795
−1551
−455
−1225
2510
90
−1958
−2845
1927
−1308
−2067
−1651
−846
−1841
−1454
472


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














467(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
473


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














468(D)
−853
−2415
2115
1717
−2702
−1468
−378
−2484
1085
−2417
−1546
−84
−1732
41
−699
696
−824
−2025
−2594
−1839
474


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














469(S)
−892
−1780
−931
−688
−2757
−1643
−830
−2472
1671
−2492
−1708
−799
−2018
−468
−365
2676
−1004
−1981
−2598
−2130
475


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














470(C)
−1135
3503
−3700
−3406
−1670
−2549
−2675
653
−3101
−916
−667
−2727
−2925
−2870
−3030
−1868
−1288
2927
−2619
−2222
476


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














471(A)
2590
−1035
−2404
−2530
−3236
2290
−2365
−2954
−2627
−3240
−2389
−1719
−2027
−2302
−2637
−656
−851
−1991
−3423
−3234
477


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














472(L)
−2632
−2152
−4630
−4185
1767
−4324
−2442
−61
−3879
2789
563
−3833
−3823
−2970
−3513
−3609
−2518
−738
−1527
−945
478


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














473(I)
−2073
−1632
−4434
−3975
−911
−4130
−3238
3164
−3706
1451
244
−3779
−3785
−3187
−3557
−3413
−2021
546
−2449
−2273
479


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














474(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
480


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














475(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
481


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














476(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
482


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














477(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
483


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














478(F)
−3342
−2776
−4026
−4232
4354
−3545
−1431
−2315
−4038
−1801
−1900
−3299
−3780
−3350
−3645
−3490
−3420
−2566
−739
349
484


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














479(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
485


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














480(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
486


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














481(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
487


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














482(T)
−359
−976
−2225
−2229
−2900
−1242
−2074
−2560
−2170
−2875
−2064
−1561
−1958
−1969
−2247
1110
3375
−1760
−3152
−2850
488


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














483(Y)
−3402
−2632
−3941
−4011
1064
−3924
3388
−2526
−3541
−1996
−1973
−2625
−3821
−2664
−3170
−3135
−3280
−2619
3420
3756
489


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














484(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
490


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














485(M)
−2322
−1904
−4536
−3951
2387
−4112
−2676
67
−3649
2034
3156
−3710
−3633
−2803
−3311
−3309
−2204
−588
−1794
−1586
491


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














486(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
492


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














487(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
493


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














488(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
494


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














489(H)
−3205
−3079
−2723
−2890
−2110
−3046
5295
−4135
−2617
−3813
−3561
−2886
−3482
−2833
−2620
−3291
−3356
−3895
−2397
−1681
495


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














490(V)
−1754
−1297
−4329
−3968
−1770
−4053
−3752
2604
−3840
−621
−545
−3728
−3878
−3699
−3917
−3370
−1746
2859
−3276
−2829
496


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














491(A)
2587
−828
−2477
−2155
−1837
−1468
−1728
−743
−1941
−1564
−954
−1607
−2033
−1725
−2034
−738
1178
1108
−2310
−1972
497


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














492(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
498


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














493(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
499


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














494(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
500


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














495(Y)
−866
−976
−1863
−1331
1353
−2145
1318
−556
−1116
−777
−173
−1242
−2197
1714
−1301
−1173
−802
888
−445
2749
501


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














496(D)
417
−1831
1647
1094
−2065
−1488
−353
−1618
−107
−1820
−1019
−189
−1698
30
−623
−603
−643
1629
−2154
−1520
502


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














497(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
503


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














498(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
504


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














499(T)
492
−1190
−706
−181
−1475
311
−333
−1099
−81
71
−509
570
1113
−6
−509
−450
1123
−835
−1680
−1161
505


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














500(I)
−2091
−1746
−3971
−3840
−1676
−3532
−3289
3684
−3581
−659
−693
−3562
−3674
−3445
−3521
−3194
−2146
449
−2877
−2493
506


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














501(A)
3103
−1036
−2445
−2572
−3222
1051
−2380
−2930
−2650
−3226
−2381
−1739
−2034
−2327
−2648
−664
−857
−1981
−3412
−3228
507


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














502(L)
−2239
−1892
−3711
−3400
301
−3520
−1210
−542
−2948
2564
−35
−2786
−3395
−2438
−2750
−2747
−2165
−945
−573
2562
508


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














503(V)
−1757
−1387
−4101
−3681
−1174
−3714
−3031
880
−3410
1254
−60
−3407
−3585
−3094
−3354
−2984
−1743
3014
−2536
−2219
509


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














504(Q)
−982
−2251
−866
971
−2711
−1822
−252
−2340
1444
−2194
−1356
−464
−1885
2646
1632
−858
−863
−1958
−2245
−1765
510


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














505(E)
−1162
−2771
2137
2239
−3046
−1526
−626
−2849
−546
−2792
−1983
−145
−1905
−242
−1192
−940
1396
−2385
−2990
−2169
511


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














506(G)
−1707
−2684
1591
−614
−3783
3190
−1613
−3795
−1887
−3775
−3119
−915
−2456
−1358
−2539
−1610
−1924
−3150
−3636
−3124
512


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














507(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
513


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














508(M)
−473
−522
−1819
−1236
−468
−1879
−687
1519
−996
566
1677
−1154
−1937
836
−1131
1079
−413
102
−957
−585
514


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














509(I)
−1761
−1312
−4317
−3954
−1713
−4027
−3703
3225
−3814
−556
−498
−3712
−3859
−3653
−3877
−3344
−1754
2110
−3216
−2787
515


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














510(T)
782
−1467
−550
1029
−2202
−1425
−709
−1791
−472
−1993
−1203
−528
−1787
−368
−902
−617
2685
−1400
−2333
−1783
516


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














511(I)
−1766
−1333
−4283
−3923
−1635
−3967
−3619
3388
−3759
−473
−437
−3672
−3822
−3576
−3804
−3285
−1764
1695
−3126
−2717
517


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














512(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
518


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














513(A)
2705
−1451
−1036
−913
−2506
−1504
−1143
−2174
−794
−2337
−1613
−946
−1993
2040
−1061
−809
−910
−1703
−2633
−2156
519


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














514(H)
−615
−1680
1444
66
−1883
168
2650
−1558
−86
−1691
−891
−223
−1680
31
−577
−571
−585
1267
−2007
−1397
520


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














515(K)
−654
−2006
−546
42
−2376
−1581
−133
−2066
1935
−1987
−1107
1132
−1658
1043
1058
−540
1180
−1660
−2113
−1532
521


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














516(N)
−933
−2085
−946
−284
−2472
−1822
−253
−2090
1711
76
−1204
1918
−1876
175
1799
−841
−817
−1755
−2132
−1663
522


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














517(E)
−416
−987
−843
1107
−1070
−1583
−338
−623
−183
879
−172
−489
−1679
−94
−565
544
813
265
−1379
−905
523


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














518(I)
−2258
−1804
−4588
−4084
−706
−4269
−3231
2527
−3807
2292
465
−3923
−3814
−3118
−3570
−3544
−2181
190
−2303
−2237
524


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














519(Q)
−477
−1909
958
282
−2211
−1389
1484
−1953
285
−1921
−1018
−32
−1517
2318
−225
630
559
−1525
−2110
−1430
525


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














520(L)
−2127
−1743
−4402
−3796
1257
−3918
−2674
149
−3492
2527
2164
−3553
−3509
−2714
−3181
−3095
−2019
570
−1870
−1818
526


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














521(N)
−723
−2217
958
236
−2518
−1466
1611
−2279
1719
−2217
−1334
2285
−1666
166
−401
−570
−677
−1837
−2382
−1678
527


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














522(V)
−1754
−1297
−4330
−3968
−1770
−4053
−3752
2623
−3841
−620
−545
−3729
−3878
−3699
−3918
−3371
−1746
2846
−3277
−2830
528


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














523(S)
1545
−974
−2003
−1825
−2867
−1206
−1790
−2580
−1788
−2795
−1932
−1362
1826
−1586
−1999
2362
−672
−1755
−3057
−2721
529


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














524(D)
−1776
−3649
3326
1869
−3838
−1642
−1031
−3788
−1322
−3660
−3029
−245
−2192
−711
−2201
−1425
−1855
−3264
−3821
−2816
530


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














525(E)
423
−2950
1944
2696
−3223
−1545
−718
−3047
−715
−2979
−2196
−161
−1968
−347
−1403
−1043
−1314
−2569
−3177
−2316
531


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














526(E)
−2641
−3308
−896
3732
−3966
−2458
−2043
−4105
−2128
−4016
−3555
−1531
−2959
−1842
−2560
−2479
−2750
−3722
−3563
−3385
532


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














527(L)
−2339
−1899
−4618
−4042
1570
−4204
−2849
1440
−3758
2558
676
−3825
−3700
−2902
−3418
−3418
−2226
−382
−1924
−1778
533


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














528(A)
2338
−1990
−241
938
−2395
−1557
−423
−2061
954
−2103
−1286
−301
−1791
−26
−375
−717
−784
−1691
−2330
−1728
534


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














529(R)
524
−2098
−789
−146
−2504
−1729
1632
−2153
1229
−2054
−1204
−379
−1789
1328
2313
−719
−724
−1774
−2150
−1637
535


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














530(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
536


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














531(R)
−1895
−2713
−2327
−1192
−3484
−2502
−481
−2856
2144
−2544
−1842
−1161
−2458
1393
3023
−1770
−1619
−2599
−2421
−2259
537


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














532(A)
2935
−1714
−553
857
−2769
−1546
−1218
−2333
−1106
−2591
−1873
−809
−2065
−934
−1502
−954
−1103
−1872
−2898
−2374
538


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














533(A)
1291
−1874
−176
1227
−2177
−1392
−109
−1909
277
−1891
−995
1134
−1522
1248
−228
−361
562
−1492
−2090
−1419
539


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














534(W)
−805
−687
−2581
−2028
138
−2236
−697
897
−1681
−421
141
−1645
−2282
−1369
−1627
−1315
636
−90
4479
1809
540


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














535(H)
−408
−1801
−274
1284
−2096
−1385
1500
−1822
1168
−1802
−899
−33
−1479
1381
−102
−303
595
221
−1996
−1339
541


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














536(Q)
−650
−1737
−627
−72
−1981
−1615
−209
−1625
1223
392
−866
−318
1222
2120
50
−598
−572
−1326
−1932
−1394
542


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














537(P)
−2931
−2878
−3420
−3706
−4181
−2925
−3468
−4621
−3859
−4490
−4165
−3491
4225
−3781
−3695
−3182
−3279
−4087
−3594
−4064
543


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−324
−7108
−2368
−894
−1115
−701
−1378
*
*














538(A)
2195
−924
−968
−546
−1397
−1356
−583
−812
−365
−1167
−487
−618
−1660
1324
−684
−483
−404
462
−1703
−1242
544


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−19
−6804
−7846
−894
−1115
−428
−1961
*
*














539(P)
411
−1017
−1886
−1616
−1600
−1588
−1411
−962
−1408
495
−755
−1384
3156
−1323
−1577
−847
−785
−783
−2111
−1716
545


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














540(R)
−1612
−2397
−2037
−1033
−2897
−2352
−458
−2365
2184
665
−1520
−1051
−2334
−51
2602
−1545
−1395
−2143
−2262
−2014
546


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














541(Y)
712
−796
−2334
−1883
−370
−2028
−986
−143
−1607
−663
−131
−1587
−2243
−1383
−1656
−1178
−771
1114
−965
3479
547


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














542(T)
−527
−1669
1091
−27
−2315
−1379
−443
−2033
−151
−2081
−1218
−282
557
−41
−650
1128
2077
−1576
−2321
−1690
548


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














543(R)
−2957
−3022
−3318
−2735
−3796
−2998
−1968
−3912
−846
−3631
−3157
−2611
−3280
−1724
4056
−3026
−2913
−3650
−3096
−3185
549


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














544(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
550


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














545(V)
−1747
−1296
−4310
−3948
−1758
−4023
−3716
2215
−3813
−615
−540
−3705
−3860
−3670
−3887
−3339
−1741
3087
−3252
−2806
551


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














546(L)
−2871
−2457
−4231
−4103
−1033
−3803
−3165
−541
−3734
3130
−31
−3935
−3797
−3286
−3484
−3713
−2869
−1136
−2394
−2220
552


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














547(A)
2404
−890
−1926
−1629
−1803
1275
−1415
−1282
−1490
392
−963
−1316
−1930
−1328
−1674
−654
−644
−952
−2187
−1810
553


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














548(K)
−2620
−2961
−2461
−2046
−3743
−2791
−1570
−3603
3784
−3387
−2839
−2048
−3039
−1260
−465
−2604
−2536
−3331
−3001
−2988
554


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














549(Y)
−3621
−2707
−4176
−4424
2950
−4049
−394
−2539
−4002
−1942
−1987
−2749
−3933
−2854
−3451
−3299
−3499
−2690
349
4094
555


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














550(A)
3438
−1472
−2846
−3040
−3287
−1726
−2735
−2840
−3028
−3257
−2662
−2236
−2447
−2798
−2944
−1216
−1387
−2183
−3405
−3320
556


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














551(H)
−1741
−2627
−2070
−1046
−3303
−2401
2713
−2751
2478
−2476
−1755
−1061
−2375
−27
2379
−1621
−1497
−2477
−2379
−2161
557


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














552(L)
−1014
−876
−2956
−2408
−582
−2550
−1529
1721
−2079
2042
345
−2114
−2581
−1775
−2028
454
−980
286
−1414
−1096
558


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














553(V)
933
−842
−2818
−2467
−1542
−1870
−1890
154
−2226
−1095
−617
−1932
−2326
−1995
−2259
−1126
1070
2769
−2180
−1826
559


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














554(S)
−787
−1522
−1486
−1172
−2714
−1599
−1112
−2500
−433
−2563
−1791
−1110
−2067
−796
1351
2916
−989
−1943
−2648
−2234
560


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














555(S)
−326
−1010
−1779
−1541
−2691
−1234
−1566
−2386
−1486
−2594
−1749
−1228
1196
−1330
−1747
2396
1967
−1662
−2876
−2496
561


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














556(A)
3121
−934
−2489
−2561
−3081
−1203
−2295
−2766
−2533
−3080
−2234
−1669
−1953
−2234
−2533
936
−746
−1844
−3331
−3090
562


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














557(S)
−897
−1462
−2333
−2543
−3185
−1640
−2474
−3294
−2686
−3497
−2780
−1973
−2360
−2483
−2703
3465
−1316
−2413
−3310
−3025
563


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














558(R)
−586
−1873
−516
979
−2188
−1543
−123
−1869
1290
−353
−980
−202
−1622
314
1886
−491
782
−1495
−2024
−1439
564


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














559(G)
−2594
−2690
−3304
−3623
−4328
3747
−3462
−4761
−3953
−4671
−4212
−3320
−3352
−3748
−3779
−2839
−2981
−4004
−3668
−4222
565


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














560(C)
2804
3772
−3185
−3198
−2739
−1303
−2462
−2065
−2882
−2628
−1924
−1927
−2044
−2547
−2727
−661
−799
−1463
−3099
−2886
566


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














561(V)
−1771
−1603
−3750
−3689
−2037
−3050
−3231
403
−3479
−1154
−1076
−3246
−3399
−3383
−3437
−2628
−1917
3536
−3074
−2677
567


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














562(T)
−1213
−1674
−2755
−2906
−3163
−1922
−2659
−2698
−2788
−3105
−2612
−2311
−2600
−2708
−2753
−1463
3819
−2197
−3286
−3156
568


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−16
−7108
−8150
−894
−1115
−701
−1378
*
*














563(D)
−2784
−3432
4016
−1200
−4140
−2466
−2197
−4505
−2621
−4365
−3956
−1551
−3014
−2039
−3232
−2593
−2938
−4046
−3710
−3552
569


—
−149
−500
233
43
−381
399
106
−626
210
−466
−720
275
394
45
96
359
117
−369
−294
−249



—
−21
−6715
−7757
−894
−1115
−701
−1378
*
*














564(F)
−525
−445
−2202
−1627
1946
−2001
−744
1247
−1346
952
561
1079
−2030
−1067
−1362
−1067
−465
338
−714
−230
570


—
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*



—
*
*
*
*
*
*
*
*
0

## Claims

1. A method for producing isobutanol comprising:
(a) providing a recombinant host cell comprising a polypeptide or fragment thereof having dihydroxy-acid dehydratase (DHAD) activity, wherein the polypeptide or fragment thereof comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 565, and wherein the amino acid sequence comprises a deletion of about 5 to about 20 amino acids from the C-terminal end of the amino acid sequence;
(b) contacting the recombinant host cell with a fermentation medium under conditions whereby isobutanol is produced; and
(c) optionally, recovering the isobutanol.

2. The method of claim 1, wherein the amino acid sequence further comprises one or more amino acid substitutions selected from P378A, P378G, P378V, P378I, P378L, G383S, G383A, G383V, G383L, G383I, I387V, I387M, I387L, I387G, I387A, L388I, L388V, L388A, and L388M.

3. The method of claim 1, wherein the polypeptide or fragment further comprises a polycysteine or polyhistidine tag.

4. The method of claim 1, wherein the polypeptide or fragment further comprises the amino acid sequence of SEQ ID NO: 589.

5. The method of claim 2, wherein the amino acid comprises the amino acid sequence of SEQ ID NO: 573.

6. The method of claim 1, wherein the polypeptide or fragment further comprises a polypeptide sequence selected from SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725, SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ ID NO: 730, SEQ ID NO: 731, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734, SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ ID NO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743, SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, and SEQ ID NO: 748.

7. The method of claim 1, wherein the amino acid sequence comprises a deletion of about 5 to about 15 amino acids from the C-terminal end of the amino acid sequence.

8. The method of claim 1, wherein the amino acid sequence comprises a deletion of 9 C-terminal amino acids.

9. The method of claim 1, wherein the recombinant host cell comprises an isobutanol biosynthetic pathway.

10. The method of claim 9, wherein the recombinant host cell is genetically modified to disrupt a gene encoding pyruvate decarboxylase (PDC).

11. The method of claim 9, wherein the recombinant host cell is genetically modified to disrupt a gene encoding glycerol-3-phosphate dehydrogenase (GPD2).

12. The method of claim 1, wherein the recombinant host cell is a yeast cell.

13. The method of claim 12, wherein the yeast cell comprises a disruption in one or more endogenous genes affecting iron-sulfur cluster biosynthesis selected from FRA2, GRX3, GRX4, and CCC1.

14. The method of claim 1, wherein the isobutanol is recovered by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation, or combinations thereof.

15. The method of claim 1, wherein the isobutanol is recovered by contacting the fermentation medium with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase.

16. The method of claim 15, wherein the extractant is selected from the group consisting of C12 to C22 fatty alcohols, C12 to C22 fatty acids, esters of C12 to C22 fatty acids, C12 to C22 fatty aldehydes, and mixtures thereof.

17. The method of claim 1, wherein the isobutanol is recovered by contacting the isobutanol with an organic acid and a catalyst capable of esterifying the isobutanol with the organic acid.

18. The method of claim 1, wherein solids are removed from the fermentation medium.

19. The method of claim 18, wherein the solids are removed from the fermentation medium by centrifugation, filtration, decantation, or combinations thereof.

20. The method of claim 18, wherein the solids are removed before the isobutanol is recovered.