# Patent

## Title
Ketol-acid reductoisomerase using NADH

## Abstract

Methods for the evolution of NADPH specific ketol-add reductoisomerase enzymes to acquire NADH specificity are provided. Specific mutant ketol-acid reductoisomerase enzymes isolated from Pseudomonas that have undergone co-factor switching to utilize NADH are described.

## Background
This application is a continuation of U.S. Ser. No. 12/637,905, filed Dec. 15, 2009, which is a continuation-in-part of U.S. Ser. No. 12/337,736, filed Dec. 18, 2008, now U.S. Pat. No. 8,129,162, and claims the benefit of the U.S. Provisional Applications, 61/015,346, filed Dec. 20, 2007, and 61/109,297, filed Oct. 29, 2008.


FIELD OF THE INVENTION
The invention relates to protein evolution. Specifically, ketol-acid reductoisomerase enzymes have been evolved to use the cofactor NADH instead of NADPH.
BACKGROUND OF THE INVENTION
Ketol-acid reductoisomerase enzymes are ubiquitous in nature and are involved in the production of valine and isoleucine, pathways that may affect the biological synthesis of isobutanol. Isobutanol is specifically produced from catabolism of L-valine as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of incomplete metabolism of amino acids by yeasts. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the Ehrlich pathway (Dickinson, et al., J. Biol. Chem., 273: 25752-25756, 1998).
Addition of exogenous L-valine to the fermentation increases the yield of isobutanol, as described by Dickinson et al., supra, wherein it is reported that a yield of isobutanol of 3 g/l is obtained by providing L-valine at a concentration of 20 g/L in the fermentation. In addition, production of n-propanol, isobutanol and isoamylalcohol has been shown by calcium alginate immobilized cells of Zymomonas mobilis (Oaxaca, et al., Acta Biotechnol., 11: 523-532, 1991).
An increase in the yield of C3-C5 alcohols from carbohydrates was shown when amino acids leucine, isoleucine, and/or valine were added to the growth medium as the nitrogen source (WO 2005040392).
While methods described above indicate the potential of isobutanol production via biological means these methods are cost prohibitive for industrial scale isobutanol production. The biosynthesis of isobutanol directly from sugars would be economically viable and would represent an advance in the art. However, to date the only ketol-acid reductoisomerase (KARI) enzymes known are those that bind NADPH in its native form, reducing the energy efficiency of the pathway. A KARI that would bind NADH would be beneficial and enhance the productivity of the isobutanol biosynthetic pathway by capitalizing on the NADH produced by the existing glycolytic and other metabolic pathways in most commonly used microbial cells. The discovery of a KARI enzyme that can use NADH as a cofactor as opposed to NADPH would be an advance in the art.
The evolution of enzymes having specificity for the NADH cofactor as opposed to NADPH is known for some enzymes and is commonly referred to as “cofactor switching”. See for example Eppink, et al. (J. Mol. Biol., 292: 87-96, 1999), describing the switching of the cofactor specificity of strictly NADPH-dependent p-Hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens by site-directed mutagenesis; and Nakanishi, et al., (J. Biol. Chem., 272: 2218-2222, 1997), describing the use of site-directed mutagenesis on a mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D) resulting in an enzyme having a 200-fold increase in the KM values for NADP(H) and a corresponding decrease of more than 7-fold in those for NAD(H). Co-factor switching has been applied to a variety of enzymes including monooxygenases, (Kamerbeek, et al., Eur. J. Biochem., 271: 2107-2116, 2004); dehydrogenases; Nishiyama, et al., J. Biol. Chem., 268: 4656-4660, 1993; Ferredoxin-NADP reductase, Martinez-Julvez, et al., Biophys. Chem., 115: 219-224, 2005); and oxidoreductases (US2004/0248250).
Rane et al., (Arch. Biochem. Biophys., 338: 83-89, 1997) discuss cofactor switching of a ketol acid reductoisomerase isolated from E. coli by targeting four residues in the enzyme for mutagenesis, (R68, K69, K75, and R76,); however the effectiveness of this method is in doubt.
Although the above cited methods suggest that it is generally possible to switch the cofactor specificity between NADH and NADPH, the methods are enzyme specific and the outcomes unpredictable. The development of a ketol-acid reductoisomerase having a high specificity for NADH with decreased specificity for NADPH would greatly enhance this enzyme's effectiveness in the isobutanol biosynthetic pathway and hence increase isobutanol production. However, no such KARI enzyme has been reported.
SUMMARY OF THE INVENTION
Applicants have solved the stated problem by identifying a number of mutant ketol-acid reductoisomerase enzymes that either have a preference for specificity for NADH as opposed to NADPH or use NADH exclusively in their reaction. The method involves mutagenesis of certain specific residues in the KARI enzyme to produce the co-factor switching.
Accordingly the invention provides A mutant ketol-acid reductoisomerase enzyme comprising the amino acid sequence as set forth in SEQ ID NO: 29; a nucleic acid molecule encoding a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence as set forth in SEQ ID NO:19; a mutant ketol-acid reductoisomerase enzyme as set for in SEQ ID NO:19; a mutant ketol-acid reductoisomerase enzyme having the amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27, 28, 67, 68, 70, 75, 79, 80, 81 and 82; and a mutant ketol-acid reductoisomerase enzyme as set forth in SEQ ID NO:17 comprising at least one mutation at a residue selected from the group consisting of 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170.
In another embodiment the invention provides a method for the evolution of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:

    
    
        a) providing a ketol-acid reductoisomerase enzyme which uses NADPH having a specific native amino acid sequence;
        b) identifying the cofactor switching residues in the enzyme of (a) based on the amino acid sequence of the Pseudomonas fluorescens ketol-acid reductoisomerase enzyme as set for the in SEQ ID NO:17 wherein the cofactor switching residues are at positions selected from the group consisting of: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170; and
        c) creating mutations in at least one of the cofactor switching residues of (b) to create a mutant enzyme wherein said mutant enzyme binds NADH.
    
    


In another embodiment the invention provides a method for the production of isobutanol comprising:

    
    
        a) providing a recombinant microbial host cell comprising the following genetic constructs:
        
            i) at least one genetic construct encoding an acetolactate synthase enzyme for the conversion of pyruvate to acetolactate;
            ii) at least one genetic construct encoding a ketol-acid reductoisomerase enzyme of either of claim 1 or 6;
            iii) at least one genetic construct encoding an acetohydroxy acid dehydratase for the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c);
            iv) at least one genetic construct encoding a branched-chain keto acid decarboxylase, of the conversion of α-ketoisovalerate to isobutyraldehyde, (pathway step d);
            v) at least one genetic construct encoding a branched-chain alcohol dehydrogenase for the conversion of isobutyraldehyde to isobutanol (pathway step e); and
        
        
        b) growing the host cell of (a) under conditions where iso-butanol is produced.
    
    


In another embodiment the invention provides a method for the evolution and identification of an NADPH binding ketol-acid reductoisomerase enzyme to an NADH using form comprising:

    
    
        a) providing a ketol-acid reductoisomerase enzyme which uses NADPH having a specific native amino acid sequence;
        b) identifying the amino acid residues in the native amino acid sequence whose side chains are in close proximity to the adenosyl 2′-phosphate of NADPH as mutagenesis targets;
        c) creating a library of mutant ketol-acid reductoisomerase enzymes from the class I ketol-acid reductoisomerase enzyme of step (a), having at least one mutation in at least one of the mutagenesis target sites of step (b); and
        d) screening the library of mutant ketol-acid reductoisomerase enzymes of step (c) to identify NADH binding mutant of ketol-acid reductoisomerase enzyme.
    
    


Alternatively the invention provides a method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:

    
    
        a) providing a mutant enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, and 84;
        b) constructing a site-saturation library targeting amino acid positions 47, 50, 52 and 53 of the mutant enzyme of (a); and
        c) screening the site-saturation library of (b) to identify mutants which accept NADH instead of NADPH as cofactor.
    
    


Similarly the invention provides a method for evolution of an NADPH specific ketol-acid reductoisomerase enzyme to an NADH using form comprising:

    
    
        a) providing a DNA fragment encoding a mutant enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, and 98 containing mutations in cofactor specificity domain;
        b) producing a DNA fragment cofactor specificity domain of (a);
        c) providing a DNA fragment encoding a mutant enzyme having mutations in cofactor binding affinity domain selected from the group consisting of SEQ ID NOs: 28, 67, 68, 69, 70, 84 and 86;
        d) incorporating mutations of step (b) into mutants of step (c); and
        e) screening mutants of step (d) for mutant enzymes having a ratio of NADH/NADPH utilization is greater than one.
    
    





BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS
The invention can be more fully understood from the following detailed description, the Figures, and the accompanying sequence descriptions, which form part of this application.
FIGS. 1A and 1B—Show four different isobutanol biosynthetic pathways. The steps labeled “a”, “b”, “c”, “d”, “f”, “g”, “h”, “i”, “j” and “k” represent the substrate to product conversions described below.
FIGS. 2A and 2B—Multiple sequence alignment (MSA) of KARI enzymes from different recourses; FIG. 2A—MSA among three NADPH-requiring KARI enzymes; FIG. 2B—MSA among PF5-KARI and other KARI enzymes, with promiscuous nucleotide specificity, where, MMC5—is from Methanococcus maripaludis C5; MMS2—is from Methanococcus maripaiudis S2; MNSB—is from Methanococcus vanniellii SB; ilv5—is from Saccharomyces corevisiae ilv5; KARI-D1—is from Sulfolobus solfataricus P2 ilvC; KARI-D2—is from Pyrobaculum aerophilum P2ilvC; and KARI S1—is from Raistonia solanacearum GMI1000 ivlC.
FIG. 3—Interaction of phosphate binding loop with NADPH based on homology modeling.
FIG. 4—KARI activities of top performers from library C using cofactor NADH versus NADPH. Activity and standard deviation were derived from triple experiments. The mutation information is as follows: C3A7=R47Y/S50A/T52D/V53W; C3A10=R47Y/S50A/T52G/V53W; C3B11=R47F/S50A/T52DA/53W; C3C8=R47G/S50M/T52D/V53W; and C4D12=R47C/S50MT52D/V53W
FIGS. 5A and 5B—FIG. 5A-Comparison of KARI activities of top performers from libraries E, F and G using cofactors NADH and NADPH. FIG. 5B—KARI activities of positive control versus wild type Pf5-ilvC using cofactors NADH. Activity and standard deviation were derived from at least three parallel experiments. “Wt” represents the wild type of Pf5-ilvC and “Neg” means negative control. Experiments for NADH and NADPH reactions in FIG. 5A were 30 min; in FIG. 5B were 10 min.
FIG. 6—Activities of top performers from library H using cofactors NADH versus NADPH. Activity and standard deviation were derived from triple experiments. Mutation information is as follows: 24F9=R47P/S50G/T52D; 68F10=R47P/T52S; 83G10=R47P/S50D/T52S; 39G4=R47P/S50C/T52D; 91A9=R47P/S50CT52D; and C3B11=R47F/S50A/T52D/V53W and Wt is wild type.
FIG. 7—Thermostability of wild type PF5-ilvC. The remaining activity of the enzyme after heating at certain temperatures for 10 min was the average number of triple experiments and normalized to the activity measured at room temperature.
FIG. 8—Multiple DNA sequence alignment among 5 naturally existing KARI molecules. The positions both bolded and boxed were identified by error prone PCR and the positions only boxed were targeted for mutagenesis.
FIGS. 9A through 9k—Alignment of the twenty-four functionally verified KARI sequences. The GxGXX(G/A) motif involved in the binding of NAD(P)H is indicated below the alignment.
FIGS. 10A and 10B—An example of the alignment of Pseudomonas fluorescens Pf-5 KARI to the profile HIM of KARI. The eleven positions that are responsible for co-factor switching are boxed.
FIG. 11A is a linear depiction of the KARI amino acid sequence with specific amino acids numbered. The cofactor specificity domain residues are shown in shaded rectangles. The cofactor binding domain is shown in dotted ovals. FIG. 11B is a table that shows changed amino acids, using single letter code, at numbered positions in four KART mutants.
FIG. 12A is a linear depiction of the KARI amino acid sequence. The cofactor specificity domain residues are shown in shaded rectangles. FIG. 12B is a linear depiction of the KARI amino acid sequence with specific amino acids of the cofactor binding domain shown in dotted ovals. FIG. 12C depicts incorporation of the domain swapping library into the mutants containing KM improving mutations. FIG. 12D is a table summarizing the KM values for NADH for mutations resulting from combining mutations in the cofactor binding affinity domain with mutations in the cofactor specificity determining domain.
Table 9—is a table of the Profile HMI of the KARI enzymes described in Example 3. The eleven positions in the profile HMM representing the columns in the alignment which correspond to the eleven cofactor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170. The lines corresponding to these positions in the model file are highlighted in yellow. Table 9 is submitted herewith electronically and is incorporated herein by reference.
The following sequences conform with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with the World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.







TABLE 1







Oligonucleotide Primers Used In This Invention









SEQUENCE ID




No.
SEQUENCE
Description












1
TGATGAACATCTTCGCGTATTCGCCGTCCT
Reverse Primer for




pBAD vector


 


2
GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNN
Forward primer



GGCTNNCTGGGCCAAGGCT GAAGCCCACGGCTTG
library C


 


3
GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCTCG
Forward primer for



GCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG
library E


 


4
GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCNNT
Forward primer for



GCTACCGTTGCCAAGGCTGAAGCCCACGGCTTG
library F


 


5
GCGTAGACGTGACTGTTGGCCTGCGTAAAGGCTCG
Forward primer for



GCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG
library G


 


6
GCGTAGACGTGACTGTTGGCCTGNNTAAAGGCNNT
Forward primer for



GCTNNTGTTGCCAAGGCTGAAGCCCACGGCTTG
library H


 


7
AAGATTAGCGGATCCTACCT
Sequencing primer




(forward)


 


8
AACAGCCAAGCTTTTAGTTC
Sequencing primer




(reverse)


 


20
CTCTCTACTGTTTCTCCATACCCG
pBAD_266-021308f


 


21
CAAGCCGTGGGCTTCAGCCTTGGCKNN
PF5_53Mt022908r


 


22
CGGTTTCAGTCTCGTCCTTGAAG
pBAD_866-021308


 


49
GCTCAAGCANNKAACCTGAAGG
pBAD-405-




C33_090808f


 


50
CCTTCAGGTTKNNTGCTTGAGC
pBAD-427-




C33_090808r


 


51
GTAGACGTGNNKGTTGGCCTG
pBAD-435-




T43_090808f


 


52
CAGGCCAACKNNCACGTCTAC
pBAD-456-




T43_090808r


 


53
CTGAAGCCNNKGGCNNKAAAGTGAC
pBAD-484-




H59L61_090808f


 


54
GTCACTTTKNNGCCKNNGGCTTCAG
pBAD-509-




H59L61_090808r


 


55
GCAGCCGTTNNKGGTGCCGACT
pBAD-519-




A71_090808f


 


56
AGTCGGCACCKNNAACGGCTGC
pBAD-541-




A71_090808r


 


57
CATGATCCTGNNKCCGGACGAG
pBAD-545-




T80_090808f


 


58
CTCGTCCGGKNNCAGGATCATG
pBAD-567-




T80_090808r


 


59
CAAGAAGGGCNNKACTCTGGCCT
pBAD-608-




A101_090808f


 


60
AGGCCAGAGTKNNGCCCTTCTTG
pBAD-631-




A101_090808r


 


61
GTTGTGCCTNNKGCCGACCTCG
pBAD-663-




R119_090808f


 


62
CGAGGTCGGCKNNAGGCACAAC
pBAD-685-




R119_090808r


 


71
GTAGACGTGACTGTTGGCCTGNNKAAAGGCNNKGC
PF5_4Mt111008.f



TNNKNNKGCCAAGGCTGAAGCCCACGG



 


72
CCGTGGGCTTCAGCCTTGGCKNNKNNAGCKNNGC
PF5_4Mt111008.r



CTTTKNNCAGGCCAACAGTCACGTCTAC



 


73
AAGATTAGCGGATCCTACCT
pBAD_230.f


 


74
GAGTGGCGCCCTTCTTGATGTTCG
pBAD_601_021308r









Additional sequences used in the application are listed below. The abbreviated gene names in bracket are used in this disclosure.

    SEQ ID NO: 9—Methanococcus maripaludis C5-ilvC (MMC5)—GenBank Accession Number NC—009135.1 Region: 901034.902026
    SEQ ID NO: 10 is the Methanococcus maripaludis S2-ilvC (MMS2)—GenBank Accession Number NC—005791.1 Region: 645729.646721
    SEQ ID NO: 11 is the Methanococcus vannielii SB-ilv5 (MVSB)—GenBank Accession Number NZ_AAWX01000002.1 Region: 302214.303206
    SEQ ID NO: 12 is the Saccharomyces cerevisiae 11v5 (ilv5)—GenBank Accession Number NC—001144.4 Region: 838065.839252
    SEQ ID NO: 13 is the Sulfolobus solfalaricus P2 ilvC (KARI-D1)—GenBank Accession Number NC—002754.1 Region: 506253.507260
    SEQ ID NO: 14 is the Pyrobaculum aerophilum str. IM2 ilvC (KARI-D2)—GenBank Accession Number NC—003364.1 Region: 1976281.1977267
    SEQ ID NO: 15 is the Raistonia solanacearum GM11000 ilvC (KARI-S1)—GenBank Accession Number NC—003295.1 Region: 2248264.2249280
    SEQ ID NO: 16 is the Pseudomonas aeruginosa PAO1 ilvC —GenBank Accession Number NC—002516 Region: 5272455.5273471
    SEQ ID NO: 17 is the Pseudomonas fluorescens PF5 ilvC —GenBank Accession Number NC—004129 Region: 6017379.6018395
    SEQ ID NO: 18 is the Spinacia oleracea ilvC (Spinach-KARI)—GenBank Accession Number NC—002516 Region: 1.2050.
    SEQ ID NO: 19 is the amino acid sequence of the mutant (Y24F/R47Y/S50A/T52D/V53A/L61F/G170A) of the ilvC native protein of Pseudomonas fluorescens. 
    SEQ ID NO: 23 is the DNA SEQ of the mutant (Y24F/R47Y/S50A/T52D/V53A/L61F/G170A) of the ilvC native protein of Pseudomonas fluorescens. 
    SEQ ID NO: 24 is the amino acid SEQ of the mutant ZB1 (Y24F/R47Y/S50A/T52D/V53A/L61F/A156V)
    SEQ ID NO: 25 is the amino acid SEQ of the mutant ZF3 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F)
    SEQ ID NO: 26 is the amino acid SEQ of the mutant ZF2 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V)
    SEQ ID NO: 27 is the amino acid SEQ of the mutant ZB3 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A)
    SEQ ID NO: 28 is the amino acid SEQ of the mutant Z4B8 (C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V/G170A)
    SEQ ID NO: 29 is a consensus amino acid sequence comprising all experimentally verified KARI point mutations as based on SEQ ID NO:17.
    SEQ ID NO: 30 is the amino acid sequence for KARI from Natronomonas pharaonis DSM 2160
    SEQ ID NO: 31 is the amino acid sequence for KARI from Bacillus subtilis subsp. subtilis str. 168
    SEQ ID NO: 32 is the amino acid sequence for KARI from Corynebacterium glutamicurn ATCC13032
    SEQ ID NO: 33 is the amino acid sequence for KARI from Phaeospirilum molischianum 
    SEQ ID NO: 34 is the amino acid sequence for KARI from Zymomonas mobilis subsp. mobilis ZM4
    SEQ ID NO: 35 is the amino acid sequence for KARI Alkalilimnicola ehrlichei MLHE-1
    SEQ ID NO: 36 is the amino acid sequence for KARI from Campylobacter lari RM2100
    SEQ ID NO: 37 is the amino acid sequence for KARI from Marinobacter aquaeolei VT8
    SEQ ID NO: 38 is the amino acid sequence for KARI Psychrobacter arcticus 273-4
    SEQ ID NO: 39 is the amino acid sequence for KARI from Hahella chejuensis KCTC2396
    SEQ ID NO: 40 is the amino acid sequence for KARI from Thiobacillus denitrificans ATCC25259
    SEQ ID NO: 41 is the amino acid sequence for KARI from Azotobacter vinelandii AvOP
    SEQ ID NO: 42 is the amino acid sequence for KARI from Pseudomonas syringae pv, syringae B728a
    SEQ ID NO: 43 is the amino acid sequence for KARI from Pseudomonas syringae pv. tomato str. DC3000
    SEQ ID NO: 44 is the amino acid sequence for KARI from Pseudomonas putida KT2440
    SEQ ID NO: 45 is the amino acid sequence for KARI from Pseudomonas entomophila L48
    SEQ ID NO: 46 is the amino acid sequence for KARI from Pseudomonas mendocina ymp
    SEQ ID NO: 47 is the amino acid sequence for KARI from Bacillus cereus ATCC 10987 NP—977840.1
    SEQ ID NO: 48 is the amino acid sequence for KARI from Bacillus cereus ATCC10987 NP—978252.1
    SEQ ID NO: 63 is the amino acid sequence for KARI from Escherichia coli—GenBank Accession Number P05793
    SEQ ID NO: 64 is the amino acid sequence for KARI from Marine Gamma Proteobacterium HTCC2207 —GenBank Accession Number ZP—01224863.1
    SEQ ID NO: 65 is the amino acid sequence for KARI from Desulfuromonas acetoxidans—GenBank Accession Number ZP—01313517i
    SEQ ID NO: 66 is the amino acid sequence for KARI from Pisum sativum (Pea)—GenBank Accession Number O82043
    SEQ ID NO: 67 is the amino acid sequence for mutant 3361G8 (C33L/R47Y/S50A/T52D/V53A/L61F/T80I)
    SEQ ID NO: 68 is the amino acid sequence for mutant 2H10 (Y24F/C33L/R47Y/S50A/T52D/V531I/L61F/T80I/A156V)
    SEQ ID NO: 69 is the amino acid sequence for mutant 1D2 (Y24F/R47Y/S50A/T52D/V53A/L61F/T80I/A156V.
    SEQ ID NO: 70 is the amino acid sequence for mutant 3F12 (Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V).
    SEQ ID NO: 75 is the amino acid sequence for mutant JB1C6 (Y24F/C33L/R47H/S50D/T52Y/V53Y/L61F/T80I/A156V)
    SEQ ID NO: 76 is the amino acid sequence for mutant 16445E4 (C33L/R47P/S50V/T52D/V53G/L61F/T80I/A156V)
    SEQ ID NO: 77 is the amino acid sequence for mutant 16468D7 (Y24F/C33L/R47T/S50I/T52D/V53R/L61F/T80I/A156V)
    SEQ ID NO: 78 is the amino acid sequence for mutant 16469F3 (C33L/R47E/S50A/T52D/V53A/L61F/T80I)
    SEQ ID NO: 79 is the amino acid sequence for mutant JEA1 (Y24F/C33L/R47R/S50F/T52D/L61F/T80I/A156V)
    SEQ ID NO: 80 is the amino acid sequence for mutant JEG2 (Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/A156V)
    SEQ ID NO: 81 is the amino acid sequence for mutant JEG4 (Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/A56V)
    SEQ ID NO: 82 is the amino acid sequence for mutant JEA7 (Y24 F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/A156V)
    SEQ ID NO: 83 is the amino acid sequence for mutant JED1 (C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)
    SEQ ID NO: 84 is the amino acid sequence for mutant 3361E1
    SEQ ID NO: 85 is the amino acid sequence for mutant C2F6
    SEQ ID NO: 86 is the amino acid sequence for mutant C3B11
    SEQ ID NO: 87 is the amino acid sequence for mutant C4D12
    SEQ ID NO: 88 is the amino acid sequence for mutant SE1
    SEQ ID NO: 89 is the amino acid sequence for mutant SE2
    SEQ ID NO: 90 is the amino acid sequence for mutant SB3
    SEQ ID NO: 91 is the amino acid sequence for mutant SD3
    SEQ ID NO: 92 is the amino acid sequence for mutant 9650E5
    SEQ ID NO: 93 is the amino acid sequence for mutant 9667A11
    SEQ ID NO: 94 is the amino acid sequence for mutant 9862B9
    SEQ ID NO: 95 is the amino acid sequence for mutant 9875B9
    SEQ ID NO: 96 is the amino acid sequence for mutant 11461D8
    SEQ ID NO: 97 is the amino acid sequence for mutant 11463
    SEQ ID NO: 98 is the amino acid sequence for mutant 11518B4





DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the generation of mutated KARI enzymes to use NADH as opposed to NADPH. Such co-factor switched enzymes function more effectively in microbial systems designed to produce Isobutanol. Isobutanol is an important industrial commodity chemical with a variety of applications, where its potential as a fuel or fuel additive is particularly significant. Although only a four-carbon alcohol, butanol has the energy content similar to that of gasoline and can be blended with any fossil fuel. Isobutanol is favored as a fuel or fuel additive as it yields only CO2 and little or no SOx or NOx when burned in the standard internal combustion engine. Additionally butanol is less corrosive than ethanol, the most preferred fuel additive to date.
The following definitions and abbreviations are to be use for the interpretation of the claims and the specification.
The term “invention” or “present invention” as used herein is meant to apply generally to all embodiments of the invention as described in the claims as presented or as later amended and supplemented, or in the specification.
The term “isobutanol biosynthetic pathway” refers to the enzymatic pathway to produce isobutanol. Preferred isobutanol biosynthetic pathways are illustrated in FIG. 1 and described herein.
The term “NADPH consumption assay” refers to an enzyme assay for the determination of the specific activity of the KARI enzyme, involving measuring the disappearance of the KARI cofactor, NADPH, from the enzyme reaction.
“KARI” is the abbreviation for the enzyme ketol-acid reductoisomerase.
The term “close proximity” when referring to the position of various amino acid residues of a KARI enzyme with respect to the adenosyl 2′-phosphate of NADPH means amino acids in the three-dimensional model for the structure of the enzyme that are within about 4.5 Å of the phosphorus atom of the adenosyl 2′-phosphate of NADPH bound to the enzyme.
The term “ketol-acid reductoisomerase” (abbreviated “KARI”), and “acetohydroxy acid isomeroreductase” will be used interchangeably and refer to the enzyme having the EC number, EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego). Ketol-acid reductoisomerase catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate, as more fully described below. These enzymes are available from a number of sources, including, but not limited to E. coli GenBank Accession Number NC-000913 REGION: 3955993.3957468, Vibrio cholerae GenBank Accession Number NC-002505 REGION: 157441.158925, Pseudomonas aeruginosa, GenBank Accession Number NC-002516. (SEQ ID NO: 16) REGION: 5272455.5273471, and Pseudomonas fluorescens GenBank Accession Number NC-004129 (SEQ ID NO: 17) REGION: 6017379.6018395. As used herein the term “Class I ketol-acid reductoisomerase enzyme” means the short form that typically has between 330 and 340 amino acid residues, and is distinct from the long form, called class II, that typically has approximately 490 residues.
The term “acetolactate synthase” refers to an enzyme that catalyzes the conversion of pyruvate to acetolactate and CO2.
Acetolactate has two stereoisomers ((R) and (S)); the enzyme prefers the (S)-isomer, which is made by biological systems. Preferred acetolactate synthases are known by the EC number 2.2.1.6 9 (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: CAB15618, Z99122, NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), klebsiella pneumoniae (Gen Bank Nos: AAA25079, M73842 and Lactococcus lactic (GenBank Nos: AAA25161, L16975).
The term “acetohydroxy acid dehydratase” refers to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. Preferred acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. These enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP—026248, NC—000913, S. cerevisiae (GenBank Nos: NP—012550, NC—001142), M. maripaludis (GenBank Nos: CAF29874, BX957219), and B. subtilis (GenBank Nos: CAB14105, Z99115).
The term “branched-chain α-keto acid decarboxylase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde and CO2. Preferred 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 lactic (GenBank Nos: AAS49166, AY548760; CAG34226, AJ746364, Salmonella typhimurium (GenBank Nos: NP-461346, NC-003197), and Clostridium acetobutylicum (GenBank Nos: NP-149189, NC-001988).
The term “branched-chain alcohol dehydrogenase” refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol. Preferred branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH as electron donor and are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP-010656, NC-001136; NP-014051, NC-001145), E. coli (GenBank Nos: NP-417484, and C. acetobutylicum (GenBank Nos: NP-349892, NC—003030).
The term “branched-chain keto acid dehydrogenase” refers to an enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA (isobutyryl-cofactor A), using NAD+ (nicotinamide adenine dinucleotide) as electron acceptor. Preferred branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. These branched-chain keto acid dehydrogenases comprise four subunits, and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B. subtilis (GenBank Nos: CAB14336, Z99116; CAB14335, Z99116; CAB14334, Z99116; and CAB14337, Z99116) and Pseudomonas putida (GenBank Nos: AAA65614, M57613; AAA65615, M57613; AAA65617, M57613; and AAA65618, M57613).
The terms “kcat” and “KM” are known to those skilled in the art and are described in Enzyme Structure and Mechanism, 2nd ed. (Ferst; W. H. Freeman Press, NY, 1985; pp 98-120). The term “kcat”, often called the “turnover number”, is defined as the maximum number of substrate molecules converted to products per active site per unit time, or the number of times the enzyme turns over per unit time. Kcat=Vmax/[E], where [E] is the enzyme concentration (Ferst, supra). The terms “total turnover” and “total turnover number” are used herein to refer to the amount of product formed by the reaction of a KARI enzyme with substrate.
The term “catalytic efficiency” is defined as the Kcat/Km of an enzyme. Catalytic efficiency is used to quantify the specificity of an enzyme for a substrate.
The term “isolated nucleic acid molecule”, “isolated nucleic acid fragment” and “genetic construct” will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
The term “amino acid” refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:
















Three-Letter
One-Letter



Amino Acid
Abbreviation
Abbreviation









Alanine
Ala
A



Arginine
Arg
R



Asparagine
Asn
N



Aspartic acid
Asp
D



Cysteine
Cys
C



Glutamine
Gln
Q



Glutamic acid
Glu
E



Glycine
Gly
G



Histidine
His
H



Leucine
Leu
L



Lysine
Lys
K



Methionine
Met
M



Phenylalanine
Phe
F



Proline
Pro
P



Serine
Ser
S



Threonine
Thr
T



Tryptophan
Trp
W



Tyrosine
Tyr
Y



Valine
Val
V










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 may 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 a microorganism. A “foreign” gene refers to a gene not normally found in the host microorganism, but that is introduced into the host microorganism by gene transfer. Foreign genes can comprise native genes inserted into a non-native microorganism, 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 sequence” refers to a DNA sequence that encodes for a specific amino acid sequence. “Suitable regulatory sequences” refer 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 may include promoters, 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 may be derived in their entirety from a native gene, or be 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 may 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 may 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 may also refer to translation of mRNA into a polypeptide.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host microorganism, resulting in genetically stable inheritance. Host microorganisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” microorganisms.
The terms “plasmid”, “vector” and “cassette” 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 fragments. Such elements may 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. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host,
The term “site-saturation library” refers to a library which contains random substitutions at a specific amino acid position with all 20 possible amino acids at once.
The term “error-prone PCR” refers to adding random copying errors by imposing imperfect or ‘sloppy’ PCR reaction conditions which generate randomized libraries of mutations in a specific nucleotide sequence.
As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
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 microorganism without altering the polypeptide encoded by the DNA.
Molecular Techniques
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook et al. (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) (hereinafter “Maniatis”); and by Silhavy et al. (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).
The present invention addresses a need that arises in the microbial production of isobutanol where the ketol-acid reductoisomerase enzyme performs a vital role. Wild type ketol-acid reductoisomerase enzymes typically use NADPH as their cofactor. However, in the formation of isobutanol an excess of NADH is produced by ancillary metabolic pathways. The invention provides mutant Class I KARI enzymes that have been evolved to utilize NADH as a cofactor, overcoming the cofactor problem and increasing the efficiency of the isobutanol biosynthetic pathway.
Production of isobutanol utilizes the glycolysis pathway present in the host microorganism. During the production of two molecules of pyruvate from glucose during glycolysis, there is net production of two molecules of NADH from NAD+ by the glyceraldehyde-3-phosphate dehydrogenase reaction. During the further production of one molecule of isobutanol from two molecules of pyruvate, there is net consumption of one molecule of NADPH, by the KARI reaction, and one molecule of NADH by the isobutanol dehydrogenase reaction. The overall reaction of glucose to isobutanol thus leads to net production of one molecule of NADH and net consumption of one molecule of NADPH. The interconversion of NADH with NADPH is generally slow and inefficient; thus, the NADPH consumed is generated by metabolism (for example, by the pentose phosphate pathway) consuming substrate in the process. Meanwhile, the cell strives to maintain homeostasis in the NAD+/NADH ratio, leading to the excess NADH produced in isobutanol production being consumed in wasteful reduction of other metabolic intermediates; e.g., by the production of lactate from pyruvate. Thus, the imbalance between NADH produced and NADPH consumed by the isobutanol pathway leads to a reduction in the molar yield of isobutanol produced from glucose in two ways: 1) unnecessary operation of metabolism to produce NADPH, and 2) wasteful reaction of metabolic intermediates to maintain NAD′′/NADH homeostasis. The solution to this problem is to invent a KARI that is specific for NADH as its cofactor, so that both molecules of NADH produced in glycolysis are consumed in the synthesis of isobutanol from pyruvate.
Keto Acid Reductoisomerase (KARI) Enzymes
Acetohydroxy acid isomeroreductase or ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino adds and is a key enzyme in their biosynthesis. KARI is found in a variety of microorganisms and amino acid sequence comparisons across species have revealed that there are 2 types of this enzyme: a short form (class I) found in fungi and most bacteria, and a long form (class II) typical of plants.
Class I KARIs typically have between 330-340 amino acid residues. The long form KARI enzymes have about 490 amino acid residues. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. KARI is encoded by the ilvC gene and is an essential enzyme for growth of E. coli and other bacteria in a minimal medium. Typically KARI uses NADPH as cofactor and requires a divalent cation such as Mg++ for its activity. In addition to utilizing acetolactate in the valine pathway, KARI also converts acetohydroxybutanoate to dihydroxymethylpentanoate in the isoleucine production pathway.
Class II KARIs generally consist of a 225-residue N-terminal domain and a 287-residue C-terminal domain. The N-terminal domain, which contains the NADPH-binding site, has an α/β structure and resembles domains found in other pyridine nucleotide-dependent oxidoreductases. The C-terminal domain consists almost entirely of α helices and is of a previously unknown topology.
The crystal structure of the E. coli KARI enzyme at 2.6 Å resolution has been solved (Tyagi, et al., Protein Sci., 14: 3089-3100, 2005). This enzyme consists of two domains, one with mixed α/β structure which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly α-helical and shows strong evidence of internal duplication. Comparison of the active sites of KARI of E. coli. Pseudomonas aeruginosa, and spinach showed that most residues in the active site of the enzyme occupy conserved positions. While the E. coli KARI was crystallized as a tetramer, which is probably the likely biologically active unit, the P. aeruginosa KARI (Ahn, et al., J. Mol. Biol., 328: 505-515, 2003) formed a dodecamer, and the enzyme from spinach formed a dimer. Known KARIs are slow enzymes with a reported turnover number (kcat) of 2 s−1 (Aulabaugh et al.; Biochemistry, 29: 2824-2830, 1990) or 0.12 s−1 (Rene et al., Arch. Biochem. Biophys. 338: 83-89, 1997) for acetolactate. Studies have shown that genetic control of isoleucine-valine biosynthesis in E. coli is different than that in Ps. aeruginosa (Marinus, et al., Genetics, 63: 547-56, 1969).
Identification of Amino Acid Target Sites for Cofactor Switching
It was reported that phosphate p2′′ oxygen atoms of NADPH form hydrogen bonds with side chains of Arg162, Ser165 and Ser167 of spinach KARI (Bion V., et al. The EMBO Journal, 16: 3405-3415, 1997). Multiple sequence alignments were performed, using vector NTI (Invitrogen Corp. Carlsbad, Calif.), with KARI enzymes from spinach, Pseudomonas aeruginosa (PAO-KARI) and Pseudomonas fluorescens (PF5-KARI). The NADPH binding sites are shown in FIG. 2A. The amino acids, argenine, threonine and serine appear to play similar roles in forming hydrogen bonds with phosphate p2′ oxygen atoms of NADPH in KARI enzymes. Studies by Ahn et al., (J. Mol. Biol., 328: 505-515, 2003) had identified three NADPH phosphate binding sites (Arg47, Ser50 and Thr52) for Pseudomonas aeruginosa (PAO-KARI) following comparing its structure with that of the spinach KARI. Hypothesizing that these three NADPH phosphate binding sites of the three KARI enzymes used in the disclosure were conserved, Arg47, Ser50 and Thr52 of PF5-KARI were targeted as the phosphate binding sites for this enzyme. This hypothesis was further confirmed through homology modeling.
Multiple sequence alignment among PF5-ilvC and several other KARI enzymes with promiscuous nucleotide specificity was also performed. As shown in FIG. 2B, the amino acids of glycine (G50) and tryptophan (W53), in other KARI enzymes in FIG. 2B, always appear together as a pair in the sequences of those enzymes. It was therefore assumed that the tryptophan 53 bulky residue was important in determining nucleotide specificity and by reducing the size of nucleotide binding pocket one could favor binding of the smaller nucleotide, NADH. Position 53 of PF5-ilvC was therefore chosen as a target for mutagenesis.
Several site-saturation gene libraries were prepared containing genes encoding KARI enzymes by commercially available kits for the generation of mutants. Clones from each library were screened for improved KARI activity using the NADH consumption assay described herein. Screening resulted in the identification of a number of genes having mutations that can be correlated to KARI activity. The location of the mutations were identified using the amino acid sequence of the Pseudomonas fluorescens PF5/NC protein (SEQ ID NO:17). Mutants with improved KARI activity had mutations at one or more positions at amino acids: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, 165, and 170. More specifically desirable mutations included the following substitutions:

    
    
        a) the residue at position 47 has an amino acid substitution selected from the group consisting of A, C, D, F, G, I, L, N, P, and Y;
        b) the residue at position 50 has an amino acid substitution selected from the group consisting of A, C, D, E, F, G, M, N, V, W;
        c) the residue at position 52 has an amino acid substitution selected from the group consisting of A, C, D, G, H, N, W;
        d) the residue at position 53 has an amino acid substitution selected from the group consisting of A, H, I, W;
    
    


In another embodiment, additional mutagenesis, using error prone PCR, performed on the mutants listed above identified suitable mutation positions as: 156, 165, 61, 170, 115 and 24. More specifically the desirable mutants with lower KM for NADH contained the following substitutions:

    
    
        e) the residue at position 156 has an amino acid substitution of V;
        f) the residue at position 165 has an amino acid substitution of M;
        g) the residue at position 61 has an amino acid substitution of F;
        h) the residue at position 170 has an amino acid substitution of A;
        i) the residue at position 24 has an amino acid substitution of F; and
        j) the residue at position 115 has an amino acid substitution of L.
    
    


In another embodiment, multiple sequence alignment of Pseudomonas fluorescens PF5-ilvC and Bacillus cereus ilvC1 and livC2 and spinach KARI was performed which allowed identification of positions 24, 33, 47, 50, 52, 53, 61, 80, 156 and 170 for further mutagenesis. More specifically mutants with much lower Km for NADH were obtained. These mutations are also based on the Pseudomonas fluorescens, KARI enzyme (SEQ ID NO:17) as a reference sequence wherein the reference sequence comprises at least one amino acid substitution selected from the group consisting of:

    
    
        k) the residue at position 24 has an amino acid substitution of phenylalanine;
        l) the residue at position 50 has an amino acid substitution of alanine;
        m) the residue at position 52 has an amino acid substitution of aspartic acid;
        n) the residue at position 53 has an amino acid substitution of alanine;
        o) the residue at position 61 has an amino acid substitution of phenylalanine;
        p) the residue at position 156 has an amino acid substitution of valine;
        q) the residue at position 33 has an amino acid substitution of leucine;
        r) the residue at position 47 has an amino acid substitution of tyrosine;
        s) the residue at position 80 has an amino acid substitution of isoleucine;
        and
        t) the residue at position 170 has an amino acid substitution of alanine.
    
    


The present invention includes a mutant polypeptide having KARI activity, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 24, 25, 26, 27 and 28.
A consensus sequence for the mutant ilvC was generated from the multiple sequence alignment and is provided as SEQ ID NO: 29 which represents all experimentally verified mutations of the KARI enzyme based on the amino acid sequence of the KARI enzyme isolated from Pseudomonas fluorescens, (SEQ ID NO:17)
Additionally the present invention describes mutation positions identified using a profile Hidden Markov Model (HMM) built based on sequences of 25 functionally verified Class I and Class II KARI enzymes. Profile HMM identified mutation positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 (the numbering is based on the sequences of Pseudomonas fluorescens PF5 KARI). Thus, it will be appreciated by the skilled person that mutations at these positions, as well as those discussed above that have been experimentally verified will also give rise to KARI enzymes having the ability to bind NADH.
Furthermore, applicants have discovered that the ketol-acid reductoisomerase enzyme has two functionally related domains: one domain affecting nucleotide specificity and the other domain impacting the KM for the cofactor (FIGS. 11 and 12). To examine whether this characteristic could be exploited to engineer the desired KARI mutants (i.e., mutants with high NADH activity (KM<20 μM) and substantially decreased NADPH activity (KM>100 μM)), two libraries were created.
One library was a four-site saturation library targeting the NADH or NADPH binding positions, i.e., amino acids at positions 47, 50, 52 and 53 (FIG. 11). To build this library, mutants which possessed both NADH and NADPH activities and KM ̃10-20 μM for NADH, were selected from a group consisting of SEQ ID NOs: 28, 67, 68, 69, 70 and 84, as templates. Further saturation mutagenesis generated new mutants (i.e., mutants with SEQ ID NOs: 75-78) that possessed mainly NADH activity with very low NADPH activity.
The desirable mutants with higher NADH activity, following site saturation mutagenesis, comprised the following substitutions:

    
    
        u) the residue at position 24 has an amino acid substitution of phenylalanine;
        v) the residue at position 50 has an amino acid substitution of aspartic acid or valine or isoleucine or phenylalanine;
        w) the residue at position 52 has an amino acid substitution of tyrosine or aspartic acid;
        x) the residue at position 53 has an amino acid substitution of tyrosine or glycine, or argenine, or alanine;
        y) the residue at position 61 has an amino acid substitution of phenylalanine;
        z) the residue at position 156 has an amino acid substitution of valine;
        aa) the residue at position 33 has an amino acid substitution of leucine;
        bb) the residue at position 47 has an amino acid substitution of histidine, or proline, or threonine, or glutamic acid; and
        cc) the residue at position 80 has an amino acid substitution of isoleucine.
    
    


The KM for NADH in the above mutants was still slightly high (e.g., JB1C6, SEQ ID NO: 74, has KM of 22 μM for NADH). To further improve the NADH KM of the mutant KARIs, a “domain swapping library”, which combined the nucleotide switching mutations and mutations with improved KM for NADH, was created (FIG. 12). More specifically, the beneficial mutations at positions 47, 50, 52 and 53 obtained in the site saturation experiment (see Tables 3 and 4), were transferred into mutants that possessed KM ̃4-40 μM for NADH (SEQ ID NOs:24-28 and 67-70 and 84, see Tables 6 and 7). The resultant new mutants accepted NADH as cofactor with very low KM ̃10 μM and greatly reduced NADPH activity. Examples of these mutants include: JEA1 (SEQ ID NO: 79), JEG2 (SEQ ID NO: 80), JEG4 (SEQ ID NO: 81), JEA7 (SEQ ID NO: 82) and JED1 (SEQ ID NO: 83).
Following domain swapping experiments, the mutants that possessed very low KM for NADH had the following substitutions:

    
    
        dd) the residue at position 24 has an amino acid substitution of phenylalanine;
        ee) the residue at position 50 has an amino acid substitution of alanine, asparagine, or phenylalanine;
        ff) the residue at position 52 has an amino acid substitution of aspartic acid;
        gg) the residue at position 53 has an amino acid substitution of alanine;
        hh) the residue at position 61 has an amino acid substitution of phenylalanine;
        ii) the residue at position 156 has an amino acid substitution of valine;
        jj) the residue at position 33 has an amino acid substitution of leucine;
        kk) the residue at position 47 has an amino acid substitution of asparagine, proline; and phenylalanine;
        ll) the residue at position 80 has an amino acid substitution of isoleucine.
    
    


In one embodiment the present method includes a mutant polypeptide having KARI activity, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 24,-28, 67-70, and 75-98,
In another embodiment the method provides an NADH KARI mutant with a KM for NADH <15 μM.
In a preferred embodiment, the mutant KARI JEA1 (SEQ ID NO: 79) has the following substitutions:

    Y24F/C33L/R47P/S50F/T52D/L61F/T80I/A156V


In another preferred embodiment, the mutant KARI JEG2 (SEQ ID NO: 80) has the following substitutions:

    (Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/A156V)


In another preferred embodiment, the mutant KARI JEG4 (SEQ ID NO: 81), has the following substitutions:

    (Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)


In another preferred embodiment, the mutant KARI JEA7 (SEQ ID NO: 82), has the following substitutions:

    (Y24F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/A156V)


In another preferred embodiment, the mutant KARI JED1 (SEQ ID NO: 83) has the following substitutions:

    (C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V)


In another embodiment the method provides an NADH accepting KARI mutant wherein the ratio of NADH/NADPH activity is greater than one. A consensus sequence for the mutant ilvC was generated from the multiple sequence alignment and is provided as SEQ ID NO: 29 which represents all experimentally verified mutations of the KARI enzyme based on the amino acid sequence of the KARI enzyme isolated from Pseudomonas fluorescens (SEQ ID NO:17).
The Host Strains for KARI Engineering
Two host strains, E. coli TOP10 from Invitrogen and E. coli Bw25113 (ΔilvC, an ilvC gene-knockout), were used for making constructs over-expressing the KARI enzyme in this disclosure. In the Bw25113 strain, the entire ilvC gene of the E. coli chromosome was replaced by a Kanamycin cassette using the Lambda red homology recombination technology described by Kirill et al., (Kirill A. Datsenko and Barry L. Wanner, Proc. Natl. Acad. Sci. USA, 97: 6640-6645, 2000).
Homology Modeling of PF5 KARI with Bound Substrates
The structure of PF5-KARI with bound NADPH, acetolactate and magnesium ions was built based on the crystal structure of P. aeruginosa PAO1-KARI (PDB ID 1NP3, Ahn H. J. et al., J. Mol. Biol., 328: 505-515, 2003) which has 92% amino acid sequence homology to PF5 KARI. PAO1-KARI structure is a homo-dodecamer and each dodecamer consists of six homo-dimers with extensive dimer interface. The active site of KARI is located in this dimer interface. The biological assembly is formed by six homo-dimers positioned on the edges of a tetrahedron resulting in a highly symmetrical dodecamer of 23 point group symmetry. For simplicity, only the dimeric unit (monomer A and monomer B) was built for the homology model of PF5-KARI in this study because the active site is in the homo-dimer interface.
The model of PF5-KARI dimer was built based on the coordinates of monomer A and monomer B of PAO1-KARI and sequence of PF5-KARI using DeepView/Swiss PDB viewer (Guex, N. and Peitsch, M. C., Electrophoresis, 18: 2714-2723, 1997). This model was then imported to program O (Jones, T. A. et al, Acta Crystallogr. A 47: 110-119, 1991) on a Silicon Graphics system for further modification.
The structure of PAO1-KARI has no NADPH, substrate or inhibitor or magnesium in the active site. Therefore, the spinach KARI structure (PDB ID 1yve, Biou V. et al., The EMBO Journal, 16: 3405-3415, 1997.), which has magnesium ions, NADPH and inhibitor (N-Hydroxy-N-isopropyloxamate) in the acetolacate binding site, was used to model these molecules in the active site. The plant KARI has very little sequence homology to either PF5— or PAO1 KARI (<20% amino acid identity), however the structures in the active site region of these two KARI enzymes are very similar. To overlay the active site of these two KARI structures, commands LSQ_ext, LSQ_improve, LSQ_mol in the program O were used to line up the active site of monomer A of spinach KARI to the monomer A of PF5 KARI model. The coordinates of NADPH, two magnesium ions and the inhibitor bound in the active site of spinach KARI were extracted and incorporated to molecule A of PF5 KARI. A set of the coordinates of these molecules were generated for monomer B of PF5 KARI by applying the transformation operator from monomer A to monomer B calculated by the program.
Because there is no NADPH in the active site of PAO1 KARI crystal structure, the structures of the phosphate binding loop region in the NADPH binding site (residues 44-45 in PAO1 KARI, 157-170 in spinach KARI) are very different between the two. To model the NADPH bound form, the model of the PF5-KARI phosphate binding loop (44-55) was replaced by that of 1yve (157-170). Any discrepancy of side chains between these two was converted to those in the PF5-KARI sequence using the mutate_replace command in program O, and the conformations of the replaced side-chains were manually adjusted. The entire NADPH/Mg/inhibitor bound dimeric PF5-KARI model went through one round of energy minimization using program CNX (ACCELRYS San Diego Calif., Bumger, A. T. and Warren, G. L., Acta Crystallogr., D 54: 905-921, 1998) after which the inhibitor was replaced by the substrate, acetolactate (AL), in the model. The conformation of AL was manually adjusted to favor hydride transfer of C4 of the nicotinamine of NADPH and the substrate. No further energy minimization was performed on this model (coordinates of the model created for this study are attached in a separate word file). The residues in the phosphate binding loop and their interactions with NADPH are illustrated in FIG. 3.
Application of a “Profile Hidden Markov Model” for Identification of Residue Positions Involved in Cofactor Switching in KARI Enzymes
Applicants have developed a method for identifying KARI enzymes and the residue positions that are involved in cofactor switching from NADPH to NADH. To structurally characterize KARI enzymes, a Profile Hidden Markov Model (HMM) was prepared as described in Example 5 using amino acid sequences of 25 KARI proteins with experimentally verified function as outlined in Table 6. These KARIs were from [Pseudomonas fluorescens Pf-5 (SEQ ID NO: 17), Sulfolobus solfataricus P2 (SEQ ID NO: 13), Pyrobaculum aerophilum str. IM2 (SEQ ID NO: 14), Natronomonas pharaonis DSM 2160 (SEQ ID NO: 30), Bacillus subtilis subsp. subtilis str. 168 (SEQ ID NO: 31), Corynebacterium glutamicum ATCC 13032 (SEQ ID NO: 32), Phaeospririlum molischianum (SEQ ID NO: 33), Ralstonia solanacearum GMI1000 (SEQ ID NO: 15), Zymomonas mobilis subsp. mobilis ZM4 (SEQ ID NO: 34), Alkalilimnicola ehrlichei MLHE-1 (SEQ ID NO: 35), Campylobacter lani RM2100 (SEQ ID NO: 36), Marinobacter aquaeolei VT8 (SEQ ID NO: 37), Psychrobacter arcticus 273-4 (SEQ ID NO: 38), Hahella chejuensis KCTC 2396 (SEQ ID NO: 39), Thiobacillus denitrificans ATCC 25259 (SEQ ID NO: 40), Azotobacter vinelandii AvOP (SEQ ID NO: 41), Pseudomonas syringae pv. syringae B728a (SEQ ID NO: 42), Pseudomonas syringae pv. tomato str. DC3000 (SEQ ID NO: 43), Pseudomonas putida KT2440 (Protein SEQ ID NO: 44), Pseudomonas entomophila L48 (SEQ ID NO: 45), Pseudomonas mendocina ymp (SEQ ID NO: 46), Pseudomonas aeruginosa PAO1 (SEQ ID NO: 16), Bacillus cereus ATCC 10987 (SEQ ID NO: 47), Bacillus cereus ATCC 10987 (SEQ ID NO: 48), and Spinacia oleracea (SEQ ID NO: 18).
In addition using methods disclosed in this application, sequences of Class II KARI enzymes such as E. coli (SEQ ID NO: 63—GenBank Accession Number P05793), marine gamma Proteobacterium HTCC2207 (SEQ ID NO: 64—GenBank Accession Number ZP—01224863.1), Desulfuromonas acetoxidans (SEQ ID NO: 65—GenBank Accession Number ZP—01313517.1) and Pisum sativum (pea) (SEQ ID NO: 66—GenBank Accession Number O82043) could be mentioned.
This Profile HMM for KARIs may be used to identify any KARI related proteins. Any protein that matches the Profile HMM with an E value of <10−3 using hmmsearch program in the HMMER package is expected to be a functional KARI, which can be either a Class I and Class II KARI. Sequences matching the Profile HMM given herein are then analyzed for the location of the 12 positions in Pseudomonas fluorescens Pf-5 that switches the cofactor from NADPH to NADH. The eleven nodes, as defined in the section of Profile HMM buiding, in the profile HMM representing the columns in the alignment which correspond to the eleven co-factor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as node 24, 33, 47, 50, 52, 53, 61, 80, 115, 156 and 170. The lines corresponding to these nodes in the model file are identified in Table 9. One skilled in the art will readily be able to identify these 12 positions in the amino acid sequence of a KARI protein from the alignment of the sequence to the profile HMM using hmm search program in HMMER package.
The KARI enzymes identified by this method, include both Class I and Class II KARI enzymes from either microbial or plant natural sources. Any KARI identified by this method may be used for heterologous expression in microbial cells.
For example each of the KARI encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1) methods of nucleic acid hybridization; 2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR) (Mullis et al., U.S. Pat. No. 4,683,202); ligase chain reaction (LCR) (Tabor, S. et al., Proc. Acad. Sci. USA 82:1074, 1985); or strand displacement amplification (SDA) (Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89: 392, 1992); and 3) methods of library construction and screening by complementation.
Although the sequence homology between Class I and Class II KARI enzymes is low, the three dimensional structure of both Classes of the enzymes, particularly around the active site and nucleotide binding domains is highly conserved (Tygai, R., et al., Protein Science, 34: 399-408, 2001). The key amino acid residues that make up the substrate binding pocket are highly conserved between these two Classes even though they may not align well in a simple sequence comparison. It can therefore be concluded that the residues affecting cofactor specificity identified in Class I KARI (e.g., positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 of PF5 KARI) can be extended to Class II KARI enzymes.
Isobutanol Biosynthetic Pathways
Carbohydrate utilizing microorganisms employ the Embden-Meyerhof-Pamas (EMP) pathway, the Entner and Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP) as the central, metabolic routes to provide energy and cellular precursors for growth and maintenance. These pathways have in common the intermediate glyceraldehyde-3-phosphate and, ultimately, pyruvate is formed directly or in combination with the EMP pathway. Subsequently, pyruvate is transformed to acetyl-cofactor A (acetyl-CoA) via a variety of means. Acetyl-CoA serves as a key intermediate, for example, in generating fatty acids, amino acids and secondary metabolites. The combined reactions of sugar conversion to pyruvate produce energy (e.g., adenosine-5′-triphosphate, ATP) and reducing equivalents (e.g., reduced nicotinamide adenine dinucleotide, NADH, and reduced nicotinamide adenine dinucleotide phosphate, NADPH). NADH and NADPH must be recycled to their oxidized forms (NAD+ and NADP+, respectively). In the presence of inorganic electron acceptors (e.g. O2, NO3− and SO42−), the reducing equivalents may be used to augment the energy pool; alternatively, a reduced carbon byproduct may be formed.
There are four potential pathways for production of isobutanol from carbohydrate sources with recombinant microorganisms as shown in FIG. 1. All potential pathways for conversion of carbohydrates to isobutanol have been described in the commonly owned U.S. patent application Ser. No. 11/586,315, which is incorporated herein by reference.
The preferred pathway for conversion of pyruvate to isobutanol consists of enzymatic steps “a”, “b”, “c”, “d”, and “e” (FIGS. 1A and 1B) and includes the following substrate to product conversions:

    
    
        a) pyruvate to acetolactate, as catalyzed for example by acetolactate synthase,
        b) (S)-acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for example by acetohydroxy acid isomeroreductase,
        c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed for example by acetohydroxy acid dehydratase,
        d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for example by a branched-chain keto acid decarboxylase, and
        e) isobutyraldehyde to isobutanol, as catalyzed for example by, a branched-chain alcohol dehydrogenase.
    
    


This pathway combines enzymes involved in well-characterized pathways for valine biosynthesis (pyruvate to α-ketoisovalerate) and valine catabolism (α-ketoisovalerate to isobutanol). Since many valine biosynthetic enzymes also catalyze analogous reactions in the isoleucine biosynthetic pathway, substrate specificity is a major consideration in selecting the gene sources. For this reason, the primary genes of interest for the acetolactate synthase enzyme are those from Bacillus (alsS) and Klebsiella (budB). These particular acetolactate synthases are known to participate in butanediol fermentation in these microorganisms and show increased affinity for pyruvate over ketobutyrate (Gollop et al., J. Bacteriol., 172: 3444-3449, 1990); and (Holtzclaw et al., J. Bacteriol., 121: 917-922, 1975). The second and third pathway steps are catalyzed by acetohydroxy acid reductoisomerase and dehydratase, respectively. These enzymes have been characterized from a number of sources, such as for example, E. coli (Chunduru et al., Biochemistry, 28: 486-493, 1989); and (Flint et al., J. Biol. Chem., 268: 14732-14742, 1993). The final two steps of the preferred isobutanol pathway are known to occur in yeast, which can use valine as a nitrogen source and, in the process, secrete isobutanol. α-Ketoiso-valerate can be converted to isobutyraldehyde by a number of keto acid decarboxylase enzymes, such as for example pyruvate decarboxylase. To prevent misdirection of pyruvate away from isobutanol production, a decarboxylase with decreased affinity for pyruvate is desired. So far, there are two such enzymes known in the art (Smit et al., Appl. Environ. Microbiol., 71: 303-311, 2005); and (de la Plaza et al., FEMS Microbiol. Lett., 238: 367-374, 2004). Both enzymes are from strains of Lactococcus lactis and have a 50-200-fold preference for ketoisovalerate over pyruvate. Finally, a number of aldehyde reductases have been identified in yeast, many with overlapping substrate specificity. Those known to prefer branched-chain substrates over acetaldehyde include, but are not limited to, alcohol dehydrogenase VI (ADH6) and Ypr1p (Larroy et al., Biochem. J., 361: 163-172, 2002); and (Ford et al., Yeast, 19: 1087-1096, 2002), both of which use NADPH as electron donor. An NADPH-dependent reductase, YqhD, active with branched-chain substrates has also been recently identified in E. coli (Sulzenbacher et al., J. Mol. Biol., 342: 489-502, 2004).
Two of the other potential pathways for isobutanol production also contain the initial three steps of “a”, “b” and “c” (FIG. 1A). One pathway consists of enzymatic steps “a”, “b”, “c”, “f”, “g”, “e” (FIGS. 1A and 1B). Step “f” containing a “branched-chain keto acid dehydrogenase (EC1.2.4.4). Step “g” containing an “acylating aldehyde dehydrogenase” (EC1.2.1.10) and 1.2.1.57 in addition to step “e” containing the “branched chain alcohol dehydrogenase”. The other potential pathway consists of steps “a”, “b”, “c”, “h”, “i”, “j”, “e” (FIGS. 1A and 1B). The term “transaminase” (step “h”) EC numbers 2.6.1.42 and 2.6.1.66. Step “h” consists of either a “valine dehydrogenase” (EC1.4.1.8 and EC1.4.1.9) or step “i”, a “valine decarboxylase” with an EC number 4.1.1.14. Finally step “j” will use an “omega transaminase” (EC2.6.1.18) to generate isobutyraldehyde which will be reduced by step “e” to produce isobutanol. All potential pathways for conversion of pyruvate to isobutanol are depicted in FIGS. 1A and 1B.
Additionally, a number of microorganisms are known to produce butyrate and/or butanol via a butyryl-CoA intermediate (Dürre, et al., FEMS Microbiol. Rev., 17: 251-262, 1995); and (Abbad-Andaloussi et al., Microbiology, 142: 1149-1158, 1996). Therefore isobutanol production in these microorganisms will take place using steps “k”, “g” and “e” shown in FIG. 1B. Step “k” will use an “isobutyryl-CoA mutase” (EC5.4.99.13). The nest step will involve using the “acylating aldehyde dehydrogenase” (EC 1.2.1.10 and EC1.2.1.57) to produce isobutyraldehyde followed by enzymatic step “e” to produce isobutanol. All these pathways are fully described in the commonly owned patent application Ser. No. 11/586,315, herein incorporated by reference.
Thus, in providing multiple recombinant pathways from pyruvate to isobutanol, there exist a number of choices to fulfill the individual conversion steps, and the person of skill in the art will be able to use publicly available sequences to construct the relevant pathways.
Microbial Hosts for Isobutanol Production
Microbial hosts for isobutanol production may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial host used for isobutanol production should be tolerant to isobutanol so that the yield is not limited by butanol toxicity. Microbes that are metabolically active at high titer levels of isobutanol are not well known in the art. Although butanol-tolerant mutants have been isolated from solventogenic Clostridia, little information is available concerning the butanol tolerance of other potentially useful bacterial strains. Most of the studies on the comparison of alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho, et al., Microsc. Res. Tech., 64: 215-22, 2004) and (Kabelitz, et al., FEMS Microbiol. Lett., 220: 223-227, 2003, Tomas, et al., J. Bacteriol., 186: 2006-2018, 2004) report that the yield of 1-butanol during fermentation in Clostridium acetobutylicum may be limited by 1-butanol toxicity. The primary effect of 1-butanol on Clostridium acetobutylicum is disruption of membrane functions (Hermann et al., Appl. Environ. Microbiol., 50: 1238-1243, 1985).
The microbial hosts selected for the production of isobutanol should be tolerant to isobutanol and should be able to convert carbohydrates to isobutanol. The criteria for selection of suitable microbial hosts include 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.
Suitable host strains with a tolerance for isobutanol may be identified by screening based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to isobutanol may be measured by determining the concentration of isobutanol that is responsible for 50% inhibition of the growth rate (IC50) when grown in a minimal medium. The IC50 values may be determined using methods known in the art. For example, the microbes of interest may be grown in the presence of various amounts of isobutanol and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of isobutanol that produces 50% inhibition of growth may be determined from a graph of the percent inhibition of growth versus the isobutanol concentration. Preferably, the host strain should have an IC50 for isobutanol of greater than about 0.5%.
The microbial host for isobutanol production should also utilize glucose at a high rate. Most microbes are capable of metabolizing carbohydrates. However, certain environmental microbes cannot metabolize carbohydrates to high efficiency, and therefore would not be suitable hosts.
The ability to genetically modify the host is essential for the production of any recombinant microorganism. The mode of gene transfer technology may be by electroporation, conjugation, transduction or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host microorganisms based on the nature of antibiotic resistance markers that can function in that host.
The microbial host also has to be manipulated in order to inactivate competing pathways for carbon flow by deleting various genes. This requires the availability of either transposons to direct inactivation or chromosomal integration vectors. Additionally, the production host should be amenable to chemical mutagenesis so that mutations to improve intrinsic isobutanol tolerance may be obtained.
Based on the criteria described above, suitable microbial hosts for the production of isobutanol include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae. 
Construction of Production Host
Recombinant microorganisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to isobutanol may be constructed using techniques well known in the art. In the present invention, genes encoding the enzymes of one of the isobutanol biosynthetic pathways of the invention, for example, acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain α-keto acid decarboxylase, and branched-chain alcohol dehydrogenase, may be isolated from various sources, as described above.
Methods of obtaining desired genes from a bacterial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available. Some tools for codon optimization are available based on the GC content of the host microorganism.
Once the relevant pathway genes are identified and isolated they may be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, Wis.), Invitrogen Corp. (Carlsbad, Calif.), Stratagene (La Jolla, Calif.), and New England Biolabs, Inc. (Beverly, Mass.). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.
Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired 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, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, tip, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans. 
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may 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: 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. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.
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: 5633-5638, 1992). Additionally, in vitro transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®.
The expression of an isobutanol biosynthetic pathway in various preferred microbial hosts is described in more detail below.
Expression of an Isobutanol Biosynthetic Pathway in E. Coli 
Vectors or cassettes useful for the transformation of E. coli are common and commercially available from the companies listed above. For example, the genes of an isobutanol biosynthetic pathway may be isolated from various sources, cloned into a modified pUC19 vector and transformed into E. coli NM522.
Expression of an Isobutanol Biosynthetic Pathway in Rhodococcus erythropolis 
A series of E. coli-Rhodococcus shuttle vectors are available for expression in R. erythropolis, including, but not limited to, pRhBR17 and pDA71 (Kostichka et al., Appl. Microbiol. Biotechnol., 62: 61-68, 2003). Additionally, a series of promoters are available for heterologous gene expression in R. erythropolis (Nakashima et al., Appl. Environ. Microbiol., 70: 5557-5568, 2004 and Tao et al., Appl. Microbiol. Biotechnol., 68: 346-354, 2005). Targeted gene disruption of chromosomal genes in R. erythropolis may be created using the method described by Tao et al., supra, and Brans et al. (Appl. Environ. Microbiol., 66: 2029-2036, 2000).
The heterologous genes required for the production of isobutanol, as described above, may be cloned initially in pDA71 or pRhBR71 and transformed into E. coli. The vectors may then be transformed into R. erythropolis by electroporation, as described by Kostichka et al., supra. The recombinants may be grown in synthetic medium containing glucose and the production of isobutanol can be followed using methods known in the art.
Expression of an Isobutanol Biosynthetic Dathway in B. Subtilis 
Methods for gene expression and creation of mutations in B. subtilis are also well known in the art. For example, the genes of an isobutanol biosynthetic pathway may be isolated from various sources, cloned into a modified pUC19 vector and transformed into Bacillus subtilis BE1010. Additionally, the five genes of an isobutanol biosynthetic pathway can be split into two operons for expression. The three genes of the pathway (bubB, ilvD, and kivD) can be integrated into the chromosome of Bacillus subtilis BE1010 (Payne, et al., J. Bacteriol., 173, 2278-2282, 1991). The remaining two genes (ilvC and bdhB) can be cloned into an expression vector and transformed into the Bacillus strain carrying the integrated isobutanol genes
Expression of an Isobutanol Biosynthetic Dathway in B. Licheniformis 
Most of the plasmids and shuttle vectors that replicate in B. subtilis may be used to transform B. licheniformis by either protoplast transformation or electroporation. The genes required for the production of isobutanol may be cloned in plasmids pBE20 or pBE60 derivatives (Nagarajan et al., Gene, 114: 121-126, 1992). Methods to transform B. licheniformis are known in the art (Fleming et al. Appl. Environ. Microbiol., 61: 3775-3780, 1995). The plasmids constructed for expression in B. subtilis may be transformed into B. licheniformis to produce a recombinant microbial host that produces isobutanol.
Expression of an Isobutanol Biosynthetic Pathway in Paenibacillus macerans 
Plasmids may be constructed as described above for expression in B. subtilis and used to transform Paenibacillus macerans by protoplast transformation to produce a recombinant microbial host that produces isobutanol.
Expression of the Isobutanol Biosynthetic Pathway in Alcalienes (Ralstonia) eutrophus 
Methods for gene expression and creation of mutations in Alcaligenes eutrophus are known in the art (Taghavi et al., Appl. Environ. Microbiol., 60: 3585-3591, 1994). The genes for an isobutanol biosynthetic pathway may be cloned in any of the broad host range vectors described above, and electroporated to generate recombinants that produce isobutanol. The poly(hydroxybutyrate) pathway in Alcaligenes has been described in detail, a variety of genetic techniques to modify the Alcaligenes eutrophus genome is known, and those tools can be applied for engineering an isobutanol biosynthetic pathway.
Expression of an Isobutanol Biosynthetic Pathway in Pseudomonas putida 
Methods for gene expression in Pseudomonas putida are known in the art (see for example Ben-Bassat et al., U.S. Pat. No. 6,586,229, which is incorporated herein by reference). The butanol pathway genes may be inserted into pPCU18 and this ligated DNA may be electroporated into electrocompetent Pseudomonas putida DOT-T1 C5aAR1 cells to generate recombinants that produce isobutanol.
Expression of an Isobutanol Biosynthetic Pathway in Saccharomyces cerevisiae 
Methods for gene expression in Saccharomyces cerevisiae are known in the art (e.g., 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, followed by the gene of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding an isobutanol biosynthetic pathway, including, but not limited to constitutive promoters FBA, GPD, ADH1, and GPM, and the inducible promoters GAL1, GAL10, and CUP1. Suitable transcriptional terminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1, and ADH1. For example, suitable promoters, transcriptional terminators, and the genes of an isobutanol biosynthetic pathway may be cloned into E. coli-yeast shuttle vectors.
Expression of an Isobutanol Biosynthetic Pathway in Lactobacillus plantarum 
The Lactobacillus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used for lactobacillus. Non-limiting examples of suitable vectors 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 R, et al. Appl. Environ. Microbiol., 71: 1223-1230, 2005).
Expression of an Isobutanol Biosynthetic Pathway in Various Enterococcus species (E. faecium, E. gallinarium, and E. faecalis)
The Enterococcus genus belongs to the Lactobacillales family and many plasmids and vectors used in the transformation of Lactobacilli, Bacilli and Streptococci species may be used for Enterococcus species. Non-limiting examples of suitable vectors 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). Expression vectors for E. faecalis using the nisA gene from Lactococcus may also be used (Eichenbaum et al., Appl. Environ. Microbiol., 64: 2763-2769, 1998). Additionally, vectors for gene replacement in the E. faecium chromosome may be used (Nallaapareddy et al., Appl. Environ. Microbiol., 72: 334-345, 2006).
Fermentation Media
Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, comsteep liquor, sugar beet molasses, and barley malt. Additionally the carbon substrate may also be one-carbon substrates 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 microorganisms 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 yeast are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. (eds): 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 may encompass a wide variety of carbon containing substrates and will only be limited by the choice of microorganism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for growth of the cultures and promotion of the enzymatic pathway necessary for isobutanol production.
Culture Conditions
Typically cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may 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 (cAMP), may also be incorporated into the fermentation medium.
Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred for the initial condition.
Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.
Industrial Batch and Continuous Fermentations
The present process employs 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. Thus, at the beginning of the fermentation the medium is inoculated with the desired microorganism or microorganisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
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 medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund (Appl. Biochem. Biotechnol., 36: 227, 1992), herein incorporated by reference.
Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to 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 medium 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 modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth may be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. 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 present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for isobutanol production.
Methods for Isobutanol Isolation from the Fermentation Medium
The biologically produced isobutanol may be isolated from the fermentation medium using methods known in the art for Acetone-butanol-ethanol (ABE) fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49: 639-648, 1998), and (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 and 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.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
General Methods:
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology. Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C., 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.
The meaning of abbreviations used is as follows: “Å” means Angstrom, “min” means minute(s), “h” means hour(s), “μl” means microliter(s), “ng/μl” means nano gram per microliter, “pmol/μl” means pico mole per microliter, “ml” means milliliter(s), “L” means liter(s), “g/L” mean gram per liter, “ng” means nano gram, “sec” means second(s), “ml/min” means milliliter per minute(s), “w/v” means weight per volume, “v/v” means volume per volume, “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “g” means the gravitation constant, “rpm” means revolutions per minute, “HPLC” means high performance liquid chromatography, “MS” means mass spectrometry, “HPLC/MS” means high performance liquid chromatography/mass spectrometry, “EDTA” means ethylendiamine-tetraacetic acid, “dNTP” means deoxynucleotide triphosphate, “° C.” means degrees Celsius, and “V” means voltage.
The oligonucleotide primers used in the following Examples have been described herein (see Table 1).
High Throughput Screening Assay of Gene Libraries
High throughput screening of the gene libraries of mutant KARI enzymes was performed as described herein: 10× freezing medium containing 554.4 g/L glycerol, 68 mM of (NH4)2SO4, 4 mM MgSO4, 17 mM sodium citrate, 132 mM KH2PO4, 36 mM K2HPO4 was prepared with molecular pure water and filter-sterilized. Freezing medium was prepared by diluting the 10× freezing medium with the LB medium. An aliquot (200 μl) of the freezing medium was used for each well of the 96-well archive plates (cat #3370, Corning Inc. Corning, N.Y.).
Clones from the LB agar plates were selected and inoculated into the 96-well archive plates containing the freezing medium and grown overnight at 37° C. without shaking. The archive plates were then stored at −80° C. E. coli strain Bw25113 transformed with pBAD-H isB (Invitrogen) was always used as the negative control. For libraries C, E, F and G, mutant T52D of (PF5-ilvC) was used as the positive control. The mutant T52D was a mutant of PF5-ilvC in which the threonine at position 52 was changed to aspartic acid. For library H, mutant C3B11 (R47F/S50A/T52D/v53W of PF5-ilvC) was used as the positive control.
Clones from archive plates were inoculated into the 96-deep well plates. Each well contained 3.0 μl of cells from thawed archive plates, 300 μl of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose as the inducer. Cells were the grown overnight at 37° C. with 80% humidity while shaking (900 rpm), harvested by centrifugation (4000 rpm, 5 min at 25° C.). (Eppendorf centrifuge, Brinkmann Instruments, Inc. Westbury, N.Y.) and the cell pellet was stored at −20° C. for later analysis.
The assay substrate, (R,S)-acetolactate, was synthesized as described by Aulabaugh and Schloss (Aulabaugh and Schloss, Biochemistry, 29: 2824-2830, 1990): 1.0 g of 2-acetoxy-2-methyl-3-oxobutyric acid ethyl ester (Aldrich, Milwaukee, Wis.) was mixed with 10 ml NaOH (1.0 M) and stirred at room temperature. When the solution's pH became neutral, additional NaOH was slowly added until pH  ̃8.0 was maintained. All other chemicals used in the assay were purchased from Sigma.
The enzymatic conversion of acetolactate to α,β-dihydroxyisovalerate by KARI was followed by measuring the disappearance of the cofactor, NADPH or NADH, from the reaction at 340 nm using a plate reader (Molecular Device, Sunnyvale, Calif.). The activity was calculated using the molar extinction coefficient of 6220 M−1 cm−1 for either NADPH or NADH. The stock solutions used were: K2HPO4 (0.2 M); KH2PO4 (0.2 M); EDTA (0.5 M); MgCl2 (1.0 M); NADPH (2.0 mM); NADH (2.0 mM) and acetolactate (45 mM). The 100 ml reaction buffer mix stock containing: 4.8 ml K2HPO4, 0.2 ml KH2PO4, 4.0 ml MgCl2, 0.1 ml EDTA and 90.9 ml water was prepared.
Frozen cell pellet in deep-well plates and BugBuster were warmed up at room temperature for 30 min at the same time. Each well of 96-well assay plates was filled with 120 μl of the reaction buffer and 20 μl of NADH (2.0 mM), 150 μof BugBuster was added to each well after 30 min warm-up and cells were suspended using Genmate (Tecan Systems Inc. San Jose, Calif.) by pipetting the cell suspension up and down (×5). The plates were incubated at room temperature for 20 min and then heated at 60° C. for 10 min. The cell debris and protein precipitates were removed by centrifugation at 4,000 rpm for 5 min at 25° C. An aliquot (50 μl) of the supernatant was transferred into each well of 96-well assay plates, the solution was mixed and the bubbles were removed by centrifugation at 4,000 rpm at 25° C. for 1 min. Absorbance at 340 nm was recorded as background, 20 μl of acetolactate (4.5 mM, diluted with the reaction buffer) was added to each well and mixed with shaking by the plate reader. Absorbance at 340 nm was recoded at 0, and 60 minutes after substrate addition. The difference in absorbance (before and after substrate addition) was used to determine the activity of the mutants. Mutants with higher KARI activity compared to the wild type were selected for re-screening.
About 5,000 clones were screened for library C and 360 top performers were selected for re-screen. About 92 clones were screened for library E and 16 top performers were selected for re-screening. About 92 clones were screened for library F and 8 top performers were selected for re-screening. About 92 clones were screened for library G and 20 top performers were selected for re-screening. About 8,000 clones were screened for library H and 62 top performers were selected for re-screening. The re-screening was described below as secondary assay.
Secondary Assay of Active Mutants
Cells containing pBad-ilvC and its mutants identified by high throughput screening were grown overnight, at 37° C., in 3.0 ml of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose as the inducer while shaking at 250 rpm. The cells were then harvested by centrifugation at 18,000×g for 1 min at room temperature (Sigma micro-centrifuge model 1-15, Laurel, Md.). The cell pellets were re-suspended in 300 μof BugBuster Master Mix (EMD Chemicals). The reaction mixture was first incubated at room temperature for 20 min and then heated at 60° C. for 10 min. The cell debris and protein precipitate were removed by centrifugation at 18,000×g for 5 min at room temperature.
The reaction buffer (120 μl) prepared as described above was mixed with either NADH or NADPH (20 μl) stock and cell extract (20 μl) in each well of a 96-well assay plate. The absorbance at 340 nm at 25° C. was recorded as background. Then 20 μl of acetolactate (4.5 mM, diluted with reaction buffer) was added each well and mixed with shaking by the plate reader. The absorbance at 340 nm at 0 min, 2 min and 5 min after adding acetolactate was recorded. The absorbance difference before and after adding substrate was used to determine the activity of the mutants. The mutants with high activity were selected for sequencing.
Five top performers from “Library C” were identified and sequenced (FIG. 4). The best performer was mutant R47F/S50A/T52D/V53W, which completely reversed the nucleotide specificity. The best performers from “Libraries E, F and G” were R47P, S50D and T52D respectively (FIG. 5). For “Library H”, 5 top performers were identified and sequenced (FIG. 6) and the best performer was R47P/S50G/T52D, which also completely reversed the nucleotide specificity. Enzymes containing activities higher than the background were considered positive.
KARI Enzyme Assay
KARI enzyme activity can be routinely measured by NADH or NADPH oxidation as described above, however to measure formation of the 2,3-dihydroxyisovalerate product directly, analysis of the reaction was performed using HPLC/MS.
Protein concentration of crude cell extract from Bugbuster lysed cells (as described above) was measured using the BioRad protein assay reagent (BioRad Laboratories, Inc., Hercules, Calif. 94547). A total of 0.5 micrograms of crude extract protein was added to a reaction buffer consisting of 100 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 1 mM glucose-6-phosphate (Sigma-Aldrich), 0.2 Units of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (Sigma-Aldrich), and various concentrations of NADH or NADPH, to a volume of 96 μL. The reaction was initiated by the addition of 4 μL of acetolactate to a final concentration of 4 mM and a final volume of 100 μL. After timed incubations at 30° C., typically between 2 and 15 min, the reaction was quenched by the addition of 10 μL of 0.5 M EDTA, pH 8.0 (Life Technologies, Grand Island, N.Y. 14072). To measure the KM of NADH, the concentrations used were 0.03, 0.1, 0.3, 1, 3, and 10 mM.
To analyze for 2,3-dihydroxyisovalerate, the sample was diluted 10× with water, and 8.0 μl was injected into a Waters Acquity HPLC equipped with Waters SQD mass spectrometer (Waters Corporation, Milford, Mass.). The chromatography conditions were: flow rate (0.5 ml/min), on a Waters Acquity HSS T3 column (2.1 mm diameter, 100 mm length). Buffer A consisted of 0.1% (v/v) in water, Buffer B was 0.1% formic acid in acetonitrile. The sample was analyzed using 1% buffer B (in buffer A) for 1 min, followed by a linear gradient from 1% buffer B at 1 min to 75% buffer B at 1.5 min. The reaction product, 2,3-dihydroxyisovalerate, was detected by ionization at m/z=133, using the electrospray ionization devise at −30 V cone voltage. The amount of product 2,3-dihydroxyisovalerate was calculated by comparison to an authentic standard.
To calculate the KM for NADH, the rate data for DHIV formation was plotted in Kaleidagraph (Synergy Software, Reading, Pa.) and fitted to the single substrate Michaelis-Menton equation, assuming saturating acetolactate concentration.
Example 1
Construction of Site-Saturation Gene Libraries to Identify Mutants Accepting NADH as Cofactor
Seven gene libraries were constructed (Table 2) using two steps: 1) synthesis of Megaprimers using commercially synthesized oligonucleotidies described in Table 1; and 2) construction of mutated genes using the Megaprimers obtained in step 1. These primers were prepared using high fidelity pfu-ultra polymerase (Stratagene, La Jolla, Calif.) for one pair of primer containing one forward and one reverse primer. The templates for libraries C, E, F, G and H were the wild type of PF5_ilvc. The DNA templates for library N were those mutants having detectable NADH activity from library C while those for library O were those mutants having detectable NADH activity from library H. A 50 μl reaction mixture contained: 5.0 μl of 10× reaction buffer supplied with the pfu-ultra polymerase (Stratagene), 1.0 μl of 50 ng/μl template, 1.0 μl each of 10 pmol/μl forward and reverse primers, 1.0 μl of 40 mM dNTP mix (Promega, Madison, Wis.), 1.0 μl pfu-ultra DNA polymerase (Stratagene) and 39 μl water. The mixture was placed in a thin well 200 μl tube for the PCR reaction in a Mastercycler gradient equipment (Brinkmann Instruments, Inc. Westbury, N.Y.). The following conditions were used for the PCR reaction: The starting temperature was 95° C. for 30 sec followed by 30 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 54° C. for 1 min, and 70° C. for 2 min. At the completion of the temperature cycling, the samples were kept at 70° C. for 4 min more, and then held awaiting sample recovery at 4° C. The PCR product was cleaned up using a DNA cleaning kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer.







TABLE 2







Gene Libraries












Targeted



Library

position(s)


name
Templates
of Pf5_ilvC
Primers used





C
PF5_ilvc
47, 50, 52 and 53
SEQ ID No: 1 and 2


E
PF5_ilvc
47
SEQ ID No: 1 and 3


F
PF5_ilvc
50
SEQ ID No: 1 and 4


G
PF5_ilvc
52
SEQ ID No: 1 and 5


H
PF5_ilvc
47, 50, and 52
SEQ ID No: 1 and 6


N
Good mutants
53
SEQ ID NO: 20 and 21



from library C


O
Good mutants
53
SEQ ID NO: 20 and 21



from library H









The Megaprimers were then used to generate gene libraries using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.). A 50 μl reaction mixture contained: 5.0 μl of 10× reaction buffer, 1.0 μl of 50 ng/μl template, 42 μl Megaprimer, 1.0 μl of 40 mM dNTP mix, 1.0 μl pfu-ultra DNA polymerase. Except for the Megaprimer and the templates, all reagents used here were supplied with the kit indicated above. This reaction mixture was placed in a thin well 200 μl-capacity PCR tube and the following reactions were used for the PCR: The starting temperature was 95° C. for 30 sec followed by 25 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 1 min, and 68° C. for 6 min. At the completion of the temperature cycling, the samples were kept at 68° C. for 8 min more, and then held at 4° C. for later processing. Dpn I restriction enzyme (1.0 μl) (supplied with the kit above) was directly added to the finished reaction mixture, enzyme digestion was performed at 37° C. for 1 h and the PCR product was cleaned up using a DNA cleaning kit (Zymo Research). The cleaned PCR product (10 μl) contained mutated genes for a gene library.
The cleaned PCR product was transformed into an electrocompetent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on agar plates containing the LB medium and 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library.







TABLE 3







List of some mutants having NADH activity


identified from saturation libraries











Mutant
Position 47
Position 50
Position 52
Position 53





SD2
R47Y
S50A
T52H
V53W


SB1
R47Y
S50A
T52G
V53W


SE1
R47A
S50W
T52G
V53W


SH2
R47N
S50W
T52N
V53W


SB2
R47I

T52G
V53W


SG1
R47Y

T52G
V53W


SB3
R47G
S50W
T52G
V53W


SE2
R47P
S50E
T52A
V53W


SD3
R47L
S50W
T52G
V53W


C2A6
R47I
S50G
T52D
V53W


C3E11
R47A
S50M
T52D
V53W


C3A7
R47Y
S50A
T52D
V53W


C3B11
R47F
S50A
T52D
V53W


C4A5
R47Y
S50A
T52S
V53W


C3B12
R47I

T52D
V53W


C4H7
R47I

T52S
V53W


C1D3
R47G
S50M
T52D
V53W


C4D12
R47C
S50W
T52G
V53W


C1G7
R47P
S50G
T52D
V53W


C2F6
R47P
S50V
T52D
V53W


C1C4
R47P
S50E
T52S
V53W


6924F9
R47P
S50G
T52D


6881E11
R47P
S50N
T52C


6868F10
R47P

T52S


6883G10
R47P
S50D
T52S


6939G4
R47P
S50C
T52D


11463D8
R47P
S50F
T52D


9667A11
R47N
S50N
T52D
V53A


9675C8
R47Y
S50A
T52D
V53A


9650E5
R47N
S50W
T52G
V53H


9875B9
R47N
S50N
T52D
V53W


9862B9
R47D
S50W
T52G
V53W


9728G11
R47N
S50W
T52G
V53W


11461D8
R47F
S50A
T52D
V53A


11461A2
R47P
S50F
T52D
V53I









Example 2
Construction of Error Prone PCR Librar
Mutants obtained in Example 1, with mutations in their cofactor binding sites which exhibited relatively good NADH activities, were used as the DNA template to prepare the error prone (ePCR) libraries using the GeneMorph II kit (Stratagene) as recommended by the manufacturer. All the epPCR libraries target the N-terminal (which contains the NADPH binding site) of PF5_KARI. The forward primer (SED ID No: 20) and the reverse primer (SED ID No: 22) were used for all ePCR libraries.
The DNA templates for the nth epPCR library were mutants having good NADH activity from the (n−1)th epPCR library. The templates of the first epPCR library were mutants having relatively good NADH activity from libraries N and O. The mutations rate of library made by this kit was controlled by the amount of template added in the reaction mixture and the number of amplification cycles. Typically, 1.0 ng of each DNA template was used in 100 μL of reaction mixture. The number of amplification cycles was 70. The following conditions were used for the PCR reaction: The starting temperature was 95° C. for 30 sec followed by 70 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 30 min, and 70° C. for 2 min. After the first 35 heating/cooling cycles finished, more dNTP and Mutazyme II DNA polymerase were added. The PCR product was cleaned up using a DNA cleaning kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer. The cleaned PCR product was treated as Megaprimer and introduced into the vector using the Quickchange kit as described in Example 1. Table 4 below lists the KARI mutants obtained and the significant improvement observed in their NADH binding ability. The KM was reduced from 1100 μM for mutant C3B11 to 50 μM for mutant 12957G9.







TABLE 4







List of some mutants with their measured KM values











NADH


Mutant
Mutation Locations
KM (μM)












C3B11
R47F/S50A/T52D/V53W
1100


SB3
R47G/S50W/T52G/V53W
500


11518B4
R47N/S50N/T52D/V53A/A156V
141


11281G2
R47N/S50N/T52D/V53A/A156V/L165M
130


12985F6
R47Y/S50A/T52D/V53A/L61F/A156V
100


13002D8
R47Y/S50A/T52D/V53A/L61F/A156V/G170A
68


12957G9
Y24F/R47Y/S50A/T52D/V53A/L61F/G170A
50


12978D9
R47Y/S50A/T52D/V53A/L61F/Q115L/A156V
114









Example 3
Identification of Amino Acids for Cofactor Specificity Switching Using Bioinformatic Tools
To discover if naturally existing KARI sequences could provide clues for amino acid positions that should be targeted for mutagenesis, multiple sequence alignment (MSA) using PF5_KARI, its close homolog PAO1_KARI and three KARI sequences with measureable NADH activity, i.e., B. Cereus ilvC1 and ilvC2 and spinach KARI were performed (FIG. 8). Based on the multiple sequence alignment, positions 33, 43, 59, 61, 71, 80, 101, and 119 were chosen for saturation mutagenesis. Saturation mutagenesis on all of these positions was performed simultaneously using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.) with the manufacturer's suggested protocol. Starting material for this mutagenesis was a mixed template consisting of the mutants already identified in Example 2, Table 4. The primers used are listed in Table 5. The library of mutants thus obtained were named “library Z”. Mutants with good NADH activity from this library were identified using high throughput screening and their KARI activity and the KM for NADH were measured as described above. These mutants (Table 6) possess much lower KM s for NADH compared to the parent templates (Table 4). A Megaprimer, using primers (SEQ ID Nos. 20 and 58), was created and mutations at positions 156 and 170 were eliminated. Further screening of this set of mutants identified mutant 3361G8 (SEQ ID NO: 67) (Table 7). The hits from library Z were further subjected to saturation mutagenesis at position 53 using primers (SEQ ID Nos. 20 and 21), and subsequent screening identified the remaining mutants in Table 7. As shown in Table 7 the new mutants possessed much lower KM for NADH (e.g., 4.0 to 5.5 μM) compared to mutants listed in Table 6 (e.g., 14-40 μM).







TABLE 5







Primers for Example 5








Targeted position(s)



of Pf5_ilvC
Primers





 33
pBAD-405-C33_090808f: GCTCAAGCANNKAACCTGAAGG



(SEQ ID NO: 49)



pBAD-427-C33_090808r: CCTTCAGGTTKNNTGCTTGAGC



(SEQ ID NO: 50)


 


 43
pBAD-435-T43_090808f: GTAGACGTGNNKGTTGGCCTG



(SEQ ID NO: 51)



pBAD-456-T43_090808r: CAGGCCAACKNNCACGTCTAC



(SEQ ID NO: 52)


 


59 and 61
pBAD-484-H59L61_090808f: CTGAAGCCNNKGGCNNKAAAGTGAC



(SEQ ID NO: 53)



pBAD-509-H59L61_090808r: GTCACTTTKNNGCCKNNGGCTTCAG



(SEQ ID NO: 54)


 


 71
pBAD-519-A71_090808f: GCAGCCGTTNNKGGTGCCGACT



(SEQ ID NO: 55)



pBAD-541-A71_090808r: AGTCGGCACCKNNAACGGCTGC



(SEQ ID NO: 56)


 


 80
pBAD-545-T80_090808f: CATGATCCTGNNKCCGGACGAG



(SEQ ID NO: 57)



pBAD-567-T80_090808r: CTCGTCCGGKNNCAGGATCATG



(SEQ ID NO: 58)


 


101
pBAD-608-A101_090808f: CAAGAAGGGCNNKACTCTGGCCT



(SEQ ID NO: 59)



pBAD-631-A101_090808r: AGGCCAGAGTKNNGCCCTTCTTG



(SEQ ID NO: 60)


 


119
pBAD-663-R119_090808f: GTTGTGCCTNNKGCCGACCTCG



(SEQ ID NO: 61)



pBAD-685-R119_090808r: CGAGGTCGGCKNNAGGCACAAC



(SEQ ID NO: 62)
















TABLE 6







List of some mutants with their measured KM values (positions to be


mutated in this library were indentified by bioinformatic tools)











NADH


Mutant
Mutation Locations
KM (μM)





ZB1
Y24F/R47Y/S50A/T52D/V53A/L61F/A156V
40



(SEQ ID NO: 24)


ZF3
Y24F/C33L/R47Y/S50A/T52D/V53A/L61F
21



(SEQ ID NO: 25)


ZF2
Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/A156V
17



(SEQ ID NO: 26)


ZB3
Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/G170A
17



(SEQ ID NO: 27)


Z4B8
C33L/R47Y/S50A/T52D/V53A/L61F/T80I/A156V
14



(SEQ ID NO: 28)
















TABLE 7







Mutants further optimized for improved KM (for NADH)











NADH


Mutant
Mutation Locations
Km (μM)





3361G8
C33L/R47Y/S50A/T52D/V53A/L61F/T80I
5.5



(SEQ ID NO: 67)


2H10
Y24F/C33L/R47Y/S50A/T52D/V53I/L61F/T80I/
5.3



A156V (SEQ ID NO: 68)


1D2
Y24F/R47Y/S50A/T52D/V53A/L61F/T80I/
4.1



A156V (SEQ ID NO: 69)


3F12
Y24F/C33L/R47Y/S50A/T52D/V53A/L61F/T80I/
4.0



A156V (SEQ ID NO: 70)


3361E1
Y24F/R47Y/S50A/T52D/V53I/L61F
4.5



(SEQ ID NO: 84)









Further analyses using bioinformatic tools were therefore performed to expand the mutational sites to other KARI sequences as described below.
Sequence Analysis
Members of the protein family of ketol-acid reducoisomorase (KARI) were identified through BlastP searches of publicly available databases using amino acid sequence of Pseudomonas fluorescens PF5 KARI (SEQ ID NO:17) with the following search parameters: E value=10, word size=3, Matrix=Blosum62, and Gap opening=11 and gap extension=1, E value cutoff of 10−3. Identical sequences and sequences that were shorter than 260 amino acids were removed. In addition, sequences that lack the typical GxGXX(G/A) motif involved in the binding of NAD(P)H in the N-terminal domain were also removed. These analyses resulted in a set of 692 KARI sequences.
A profile HMM was generated from the set of the experimentally verified Class I and Class II KARI enzymes from various sources as described in Table 8. Details on building, calibrating, and searching with this profile HMM are provided below. Any sequence that can be retrieved by HMM search using the profile HMM for KARI at E-value above 1E−3 is considered a member of the KARI family. Positions in a KARI sequence aligned to the following in the profile HMM nodes (defined below in the section of profile HMM building) are claimed to be responsible for NADH utilization: 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170 (the numbering is based on the sequences of Pseudomonas fluorescens PF5 KARI).
Preparation of Profile HMM
A group of KARI sequences were expressed in E. coli and have been verified to have KARI activity These KARIs are listed in Table 6. The amino acid sequences of these experimentally verified functional KARIs were analyzed 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., J. Mol. Biol. 235:1501-1531, 1994), 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 (profile HMM) that characterizes the input sequences. As stated in the user guide, profile HMMs are statistical descriptions of the consensus of a multiple sequence alignment. They use position-specific scores for amino acids (or nucleotides) and position specific scores for opening and extending an insertion or deletion. Compared to other profile based methods, HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashbum, Va.).
The profile HMM was built as follows:
Step 1. Build a Sequence Alignment
The 25 sequences for the functionally verified KARIs listed above were aligned using Clustal W (Thompson, J. D., Higgins, D. G., and Gibson T. J., Nuc. Acid Res. 22: 4673 4680, 1994) with default parameters. The alignment is shown in FIG. 9.







TABLE 8







25 Experimentally verified KARI enzymes










GI

SEQ



Number
Accession
ID NO:
Microorganism













70732562
YP_262325.1
17
Pseudomonas fluorescens Pf-5


15897495
NP_342100.1
13
Sulfolobus solfataricus P2


18313972
NP_560639.1
14
Pyrobaculum aerophilum str.





IM2


76801743
YP_326751.1
30
Natronomonas pharaonis





DSM 2160


16079881
NP_390707.1
31
Bacillus subtilis subsp.





subtilis str. 168


19552493
NP_600495.1
32
Corynebacterium glutamicum





ATCC 13032


6225553
O32414
33
Phaeospririlum molischianum


17546794
NP_520196.1
15
Ralstonia solanacearum





GMI1000


56552037
YP_162876.1
34
Zymomonas mobilis subsp.





mobilis ZM4


114319705
YP_741388.1
35
Alkalilimnicola ehrlichei





MLHE-1


57240359
ZP_00368308.1
36
Campylobacter lari RM2100


120553816
YP_958167.1
37
Marinobacter aquaeolei VT8


71065099
YP_263826.1
38
Psychrobacter arcticus 273-4


83648555
YP_436990.1
39
Hahella chejuensis KCTC





2396


74318007
YP_315747.1
40
Thiobacillus denitrificans





ATCC 25259


67159493
ZP_00420011.1
41
Azotobacter vinelandii AvOP


66044103
YP_233944.1
42
Pseudomonas syringae pv.





syringae B728a


28868203
NP_790822.1
43
Pseudomonas syringae pv.





tomato str. DC3000


26991362
NP_746787.1
44
Pseudomonas putida KT2440


104783656
YP_610154.1
45
Pseudomonas entomophila





L48


146306044
YP_001186509.1
46
Pseudomonas mendocina ymp


15599888
NP_253382.1
16
Pseudomonas aeruginosa





PAO1


42780593
NP_977840.1
47
Bacillus cereus ATCC 10987


42781005
NP_978252.1
48
Bacillus cereus ATCC 10987


266346
Q01292
18
Spinacia oleracea









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 sequence alignment for twenty-four experimentally verified KARIs as 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 a linear state machine consisting of a series of nodes, each of which corresponds roughly to a position (column) in the multiple sequence alignment from which it is built. If gaps are ignored, the correspondence is exact, i.e., the profile HMM has a node for each column in the alignment, and each node can exist in one state, a match state. The word “match” here implies that there is a position in the model for every position in the sequence to be aligned to the model. Gaps 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”.
A profile HMM has several types of probabilities associated with it. One type is the transition probability—the probability of transitioning from one state to another. There are also emissions probabilities associated with each match state, based on the probability of a given residue existing at that position in the alignment. For example, for a fairly well-conserved column in an alignment, the emissions probability for the most common amino acid may be 0.81, while for each of the other 19 amino acids it may be 0.01.
A profile HMM is completely described in a HMMER2 profile save file, which contains all the probabilities that are used to parameterize the HMM. The emission probabilities of a match state or an insert state are stored as log-odds ratio relative to a null model: log2 (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 stored as log odds parameters and are proportional to log2(t_x). Where t_x is the transition probability of transiting from one state to another 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 set of KARI sequences is provided appended hereto as a profile HMM Excel chart (Table 9). In the main model section starting from the HMM flag line, the model has three lines per node, for M nodes (where M is the number of match states, as given by the LENG line). The first line reports the match emission log-odds scores: the log-odds ratio of emitting each amino acid from that state and from the Null model. The first number if the node number (1 . . . M). The next K numbers for match emission scores, one per amino acid. The highest scoring amino acid is indicated in the parenthesis after the node number. These log-odds scores can be converted back to HMM probabilities using the null model probability. The last number on the line represents the alignment column index for this match state. 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.
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 692 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.
An hmmersearch, using hmmsearch, with the profile HMM generated from the alignment of the twenty-five KARIs with experimentally verified function, matched all 692 sequences with an E value<10−3. This result indicates that members of the KARI family share significant sequence similarity. A hmmersearch with a cutoff of E value 10−3 was used to separate KARIs from other proteins.
Step 5. Identify Positions that are Relevant for NAD(P)H Utilization.
Eleven positions have been identified in KARI of Pseudomonas fluorescens Pf-5 that switches the cofactor from NADPH to NADH. Since the KARI sequences share significant sequence similarity (as described above), it can be reasoned that the homologous positions in the alignment of KARI sequences should contribute to the same functional specificity. The profile HMM for KARI enzymes has been generated from the multiple sequence alignment which contains the sequence of Pseudomonas fluorescens Pf-5 KARI. The eleven positions in the profile HMM representing the columns in the alignment which correspond to the eleven cofactor switching positions in Pseudomonas fluorescens Pf-5 KARI are identified as positions 24, 33, 47, 50, 52, 53, 61, 80, 115, 156, and 170. The lines corresponding to these positions in the model file are highlighted in yellow in Table 9.
For any query sequence, hmm search is used to search the profile HMM for KARI against the query sequence and the alignment of the query to the HMM is recorded in the output file. In the alignment section of the output, the top line is the HMM consensus. The amino acid shown for the consensus is the highest probability amino acid at that position according to the HMM (not necessarily the highest scoring amino acid). The center line shows letters for “exact” matches to the highest probability residue in the HMM, or a “+” when the match has a positive score. The third line shows the sequence itself. The positions in the query sequence that are deemed as relevant for cofactor switching are identified as those that are aligned to these eleven nodes in the profile HMM as described above. An example of the alignment of Pseudomonas fluorescens Pf-5 KARI to the profile HMM of KARI is shown in FIG. 10 and the eleven positions that are responsible for cofactor switching are shaded in grey.
Example 4
Construction of a Site-Saturation Gene Library for Complete Cofactor Switching to Nadh
To construct the site-saturation gene library for KARI mutants, mutants 3361E1, 3361G8, 1D2, 2H10, 3F12, & Z4B8 (see Example 3, Tables 6 and 7) were used as templates. The library was constructed using QuickChange kit (Cat#200524, Stratagene, La Jolla, Calif.). The concentration of each mutant in the template mixture was 5.0 ng/μl. The two primers (2.5 nM) introducing saturation mutagenesis at positions 47, 50, 52 and 53, were PF5—4Mt111008.f (SEQ ID NO: 71) and PF5—4Mt111008.r (SEQ ID NO: 72).
The PCR Reaction Mixture Contained:
















10 × reaction buffer
5.0 μl



PF5_4Mt111008.f
2.0 μl



PF5_4Mt111008.r
2.0 μl



50 × dNTP
1.0 μl



DNA Template
1.0 μl



PfuUltra
1.0 μl



Water
 38 μl










The PCR Reaction Program was:


















1) 95° C.
30
sec



2) 95° C.
30
sec



3) 55° C.
1.0
min



4) 68° C.
6.0
min










5) Go to step (2)
Repeat 35 times











6) 68° C.
8.0
min










7) 4° C.
press Enter










The mixture was placed in a thin well 200 μl tube for the PCR reaction in a Mastercycler gradient equipment (Brinkmann Instruments, Inc. Westbury, N.Y.). After the PCR reaction, 1.0 μl Dpn I restriction enzyme (supplied with the kit above) was directly added into the PCR reaction mixture, which was then incubated at 37° C. for 1 h to remove the DNA templates. The Dpn I digested PCR product was cleaned up by the Zymo DNA clearance kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer.
The cleaned PCR product was transformed into an electro-competent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on agar plates containing the LB medium and 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library. Several mutants identified in this library (Table 10 and FIG. 11) had very low NADPH activity while they had good NADH activity. Their cofactor consumption is listed in Table 11 (The data was based on three parallel measurements). “Negative” in the following Tables refers to an empty pBAD vector without the KARI gene.







TABLE 10







List of some of the mutants identified in Example 1








Mutant
Mutation Locations





JB1C6
Y24F/C33L/R47H/S50D/T52Y/V53Y/L61F/T80I/A156V


16445E4
C33L/R47P/S50V/T52D/V53G/L61F/T80I/A156V


16468D7
Y24F/C33L/R47T/S50I/T52D/V53R/L61F/T80I/A156V


16469F3
C33L/R47E/S50A/T52D/V53A/L61F/T80I
















TABLE 11







The cofactor consumption of some mutants following a 5 min reaction


(decrease in OD340 nm)












0.2 mM NADH

0.2 mM NADPH














Mutants
average
stdev
average
stdev







JB1C6
−0.232
0.127
−0.019
0.009



16445E4
−0.152
0.057
−0.013
0.001



16468D7
−0.153
0.012
−0.039
0.020



16469F3
−0.054
0.069
−0.025
0.016



Z4B8
−0.178
0.042
−0.170
0.013



PF5_WT
−0.078
0.014
−0.320
0.024



Negative
−0.061
0.029
−0.015
0.014










Example 5
Construction of a Domain Swapping Library
In this Example the beneficial mutations outside the cofactor binding sites and the beneficial mutations within the cofactor binding sites were combined to create a domain swapping library.
Mutants, which had mutations in the cofactor binding site and exhibited only NADH activity (SE1, SB3, SE2, SD3, C2F6, C3B11, C4D12, 9650E5, 9667A11, 9862B9, 9875B9, 11461D8, 11463D8, 11518B4, SEQ ID NOs: 85-98), were used to obtain additional beneficial mutations in the cofactor binding site. Two primers, pBAD—230f (SEQ ID NO: 73) and pBAD—601—021308r (SEQ ID NO: 74), were used to amplify the mutants listed in Table 12. PCR reagents used were from Invitrogen (Cat#10572-014, Invitrogen, Carlsbad, Calif.).
The PR Reaction Mixture Contained:
















PCR SuperMix
180 μl 



pBAD_230.f (18 nM)
5.0 μl



pBAD_601_021308r (10 nM)
9.0 μl



Template mix (5.0 ng/μl)
6.0 μl










The PCR Reaction Program was:


















(1) 95° C.
30
sec



(2) 95° C.
20
sec



(3) 55° C.
20
sec



(4) 72° C.
60
sec










(5) Go to step (2)
repeat 35 times











(6) 72° C.
4
min










(7) 4° C.
press enter










After the PCR reaction, 1.0 μl Dpn I restriction enzyme (supplied with the kit above) was directly added into the PCR reaction mixture, which was then incubated at 37° C. for 1 h to remove the DNA templates. The Dpn I digested PCR product was cleaned up by the Zymo DNA clearance kit (Cat#D4003, Zymo Research, Orange, Calif.) as recommended by the manufacturer and 42 μl cleaned DNA product containing beneficial mutations in the cofactor binding sites obtained was designated as Megaprimer.
The Megaprimers thus obtained were then used to generate the domain swapping library using the QuickChange II XL site directed mutagenesis kit (Catalog #200524, Stratagene, La Jolla Calif.). The templates used in Example 4 were also used in this experiment. A 50 μl reaction mixture containing: 5.0 μl of 10× reaction buffer, 1.0 μl of 5.0 ng/μl template, 42 μl Megaprimer, 1.0 μl of 40 mM dNTP mix, 1.0 μl pfu-ultra DNA polymerase was prepared. Except for the Megaprimer and the templates, all reagents used here were supplied with the purchased kit. This reaction mixture was placed in a thin well 200 μl capacity PCR tube and the following reactions were used for the PCR. The starting temperature was 95° C. for 30 sec followed by 30 heating/cooling cycles. Each cycle consisted of 95° C. for 30 sec, 55° C. for 1 min, and 68° C. for 6 min. At the completion of the temperature cycling, the samples were kept at 68° C. for 8 min, and then stored at 4° C. for later processing. Dpn I restriction enzyme (1.0 μl) (supplied with the kit above) was directly added to the finished reaction mixture, enzyme digestion was performed at 37° C. for 1 h and the PCR product was cleaned up using a DNA cleaning kit (Zymo Research). The cleaned PCR product (10 μl) contained mutated genes for a gene library.
The mutated genes were transformed into an electro-competent strain of E. coli Bw25113 (ΔilvC) using a BioRad Gene Pulser II (Bio-Rad Laboratories Inc., Hercules, Calif.). The transformed clones were streaked on LB agar plates containing 100 μg/ml ampicillin (Cat#L1004, Teknova Inc. Hollister, Calif.) and incubated at 37° C. overnight. Dozens of clones were randomly chosen for DNA sequencing to confirm the quality of the library.
This library yielded many mutants with high NADH activity (low KM for NADH), which also had very low NADPH activity. (Table 12 and FIG. 12). Their cofactor consumption is also shown in Table 13 (The data was based on three parallel measurements).







TABLE 12







Mutants with improved KM (for NADH) obtained


from the domain swapping library











NADH


Mu-

KM


tant
Mutation Locations
(μM)












JEA1
Y24F/C33L/R47P/S50F/T52D/L61F/T80I/A156V
9.1


JEG2
Y24F/C33L/R47F/S50A/T52D/V53A/L61F/T80I/A156V
9.4


JEG4
Y24F/C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V
9.6


JEA7
Y24F/C33L/R47P/S50N/T52D/V53A/L61F/T80I/A156V
10.6


JED1
C33L/R47N/S50N/T52D/V53A/L61F/T80I/A156V
11.0
















TABLE 13







The cofactor consumption of some mutants after 5 min reaction


(decrease in OD340 nm)












0.2 mM NADH

0.2 mM NADPH














Mutants
average
stdev
average
stdev







JEA1
−0.285
0.030
−0.110
0.025



JED1
−0.287
0.032
−0.074
0.014



JEG2
−0.261
0.009
−0.078
0.009



JEG4
−0.227
0.016
−0.050
0.016



JEA7
−0.205
0.079
−0.038
0.009



Z4B8
−0.178
0.042
−0.170
0.013



PF5_WT
−0.078
0.014
−0.320
0.024



Negative
−0.061
0.029
−0.015
0.014










Example 6
Thermostability of Pf5-Ilvc and its Mutants
The wildtype PF5-ILVC and various cells containing mutated pBad-ilvC were grown overnight at 37° C. in 25 ml of the LB medium containing 100 μg/ml ampicillin and 0.02% (w/v) arabinose inducer while shaking at 250 rpm. The cells were then harvested by centrifugation at 18,000×g for 1 min at room temperature and the cell pellets were re-suspended in 300 μl of BugBuster Master Mix (EMD Chemicals). The reaction mixture was first incubated at room temperature for 20 min and aliquots of this cell mixture (e.g. 50 μl) were incubated at different temperatures (from room temperature to 75° C.) for 10 min. The precipitate was removed by centrifugation at 18,000×g for 5 min at room temperature. The remaining activity of the supernatant was analyzed as described above. As shown in FIG. 7, pBad-ilvC was very stable with T50 at 68° C. (T50 is the temperature, at which 50% of protein lost its activity after 10 min incubation).
The thermostability of PF5-ilvC allowed destruction of most of the other non-KARI NADH oxidation activity within these cells, reducing the NADH background consumption and thus facilitating the KARI activity assays. This heat treatment protocol was used in all screening and re-screening assays. The mutants thus obtained were all thermostable which allowed easier selection of the desirable mutants.
Example 7
Stoichiometric Production of 2,3-Dihydroxyisovalerate By Kari During Consumption of NADH or NADPH as Cofactors
Screening and routine assays of KARI activity rely on the 340 nm absorption decrease associated with oxidation of the pyridine nucleotides NADPH or NADH. To insure that this metric was coupled to the formation of the reaction product (i.e., 2,3-dihydroxyisovalerate), oxidation of both pyridine nucleotide and formation of 2,3-dihydroxyisovalerate were measured in the same samples.
The oxidation of NADH or NADPH was measured at 340 nm in a 1 cm path length cuvette on a Agilent model 8453 spectrophotometer (Agilent Technologies, Wilmington Del.). Crude cell extract (0.1 ml) prepared as described above containing either wild type PF5 KARI or the C3B11 mutant, was added to 0.9 ml of K-phosphate buffer (10 mM, pH 7.6), containing 10 mM MgCl2, and 0.2 mM of either NADPH or NADH. The reaction was initiated by the addition of acetolactate to a final concentration of 0.4 mM. After 10-20% decrease in the absorption (about 5 min), 50 μl of the reaction mixture was rapidly withdrawn and added to a 1.5 ml Eppendorf tube containing 10 μl 0.5 mM EDTA to stop the reaction and the actual absorption decrease for each sample was accurately recorded. Production of 2,3-dihydroxyisovalerate was measured and quantitated by HPLC/MS as described above.
The coupling ratio is defined by the ratio between the amount of 2,3-dihydroxyisovalerate (DHIV) produced and the amount of either NADH or NADPH consumed during the experiment. The coupling ratio for the wild type enzyme (PF5-ilvC), using NADPH, was 0.98 DHIV/NADPH, while that for the mutant (C3B11), using NADH, was on average around 1.10 DHIVINADPH underlining the high activity of the mutant enzyme to consume NADH and produce DHIV.













HMMER2.0 [2.2g]
File format version: a unique identifier for this save file format.


NAME Functionally Verified KARIs
Name of me profile HMM 


LENG 354
Model length: the number of match states in the model. 


ALPH Amino
Symbol alphabet: This determines the symbol alphabet and the size of the symbol emission probabilty distributions.


MAP yes
IAmino, the alphabet size is set to 20 and the symbol alphabet to “ACDEFGHIKLMNPQRSTVWY” (alphabetic order). 


COM hmmbuild-n Functionally Verified KARIs exp-KARI.hmm exp-KARI_mod.aln
Map annotation flag: If set to yes, each line of data for the match state/consensus column in the main section of the


COM hmmcalibrate exp-KARI.hmm
file is followed by an extra number. This number gives the Index of the alignment column that the match state was


NSEQ 25
made from. This information provides a “map” of the match states (1..M) onto the columns of the alignment (1.alen).


DATE Mon Dec 8 17:34:51 2008
It is used for quickly aligning the model back to the original alignment e.g. when using hmmalign-mapali.


XT −8455 −4 −1000 −1000 −8455 −4 −8455 −4
Command line for every HMMER command that modifies the save file: This one means that hmmbuild


NULT −4 −8455
(default patrameters) was applied to generate the save file. 


NULE 595 −1558 85 338 −294 453 −1158 197 249 902 −1085 −142 −21 −313 45 531 201 384 −1998 −644
Command line for every HMMER command that modifies the save file: This one means that hmmcalibrate (default 


EVD −333.712708 0.110102
parameters) was applied to the save profile. 



Sequence number: the number of sequences the HMM was trained on 



Creation date: When was the save file was generated. 



Eight “special” transitions for controlling parts of the algorithm-specific parts of the Plan7 model.



The null probabilty used to convert these back to model probabilities is 1.0. The order of the eight fields 



is N->B, N->N, E->C, E->J, C->T, C->C, J->B, J->J. 



The transition probabilty distribution for the null model (single G state). 



The extreme value distribution parameters μ and lambda respectively; both floating point values. These values are set 



when the model is calibrated with hmmcalibrate. They are used to determine E-values of bit scores.
































A
C
D
E
F
G
H
I
K











Position in


HMM
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






−650
*
−1463




















1(Q)
−648
−1356
−136
−44
−1453
−1166
−219
−1455
321
−1477
−911
−227
−1496
3263
122
−643
−684
−1239
−1542
−1030
 7100%


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



—
−38
−5840
−6882
−894
−1115
−701
−1378
−650
*














2(M)
−4231
−3929
−5216
−5402
−3438
−4370
−4528
−3232
−5113
−2613
5320
−5052
−4790
−4977
−4823
−4692
−4459
−3629
−4103
−4017
 7200%


—
−147
−501
232
42
−382
397
104
−625
209
−467
−722
276
396
44
95
361
121
−368
296
−251



—
−3303
−3318
−325
−3473
−136
−701
−1378
*
*














3(F)
−1308
−1104
−2227
−2120
3516
−2093
−244
−196
−1891
64
66
−1626
−2278
−1503
−1798
−1617
−1350
−389
305
1355
 8600%


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



—
−38
−5840
−6882
−894
−1115
−943
−1060
*
*














4(A)
1616
−1744
1125
33
−2015
−1540
−262
−1686
937
−1765
−911
−252
−1658
154
−383
−488
640
−3
−2038
−1421
 8700%


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



—
−901
−7402
−1125
−894
−1115
−2352
−314
*
*














5(C)
−346
2578
1084
−712
2092
−1540
−384
−167
−624
−482
125
−731
−1705
−451
−883
−631
−338
−50
−774
−133
 8800%


—
−149
−500
235
43
−381
398
106
−626
210
−466
−721
275
394
45
96
359
118
−369
−295
−249



—
−1009
−1006
−7567
−131
−3597
−1916
−444
*
*














6(S)
800
−586
−1937
−1415
−821
−1740
−954
1279
−1204
−584
19
−1258
−1964
−1013
−1358
1715
−476
1117
−1320
−938
 9000%


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



—
−17
−6953
−7995
−894
−1115
−146
−3378
*
*














7(K)
−956
−2411
−803
501
−2743
−1919
−558
−2483
2435
−2420
−1520
57
−2010
1146
458
829
224
−2040
−2577
−1913
 9100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














8(V)
−2472
−2010
−5089
−4702
−2534
−4789
−4391
2241
−4574
−151
−1318
−4442
−4600
−4417
−4628
−4080
−82
3023
−3952
−3510
 9200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














9(Y)
−4673
−3685
−5210
−5505
2423
−5069
−1332
−3424
−5065
−392
−2838
−3726
−4920
−3835
−4458
−4313
−4533
−3643
−581
4349
 9300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














10(Y)
−2170
−2625
−2489
−2097
−1555
−2986
−1481
−2628
906
−2674
−2098
−2051
−3206
−1513
−1078
−2258
1039
−2435
−2009
4185
 9400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














11(D)
−2498
−4412
3500
1042
−4581
−2437
−1765
−4500
733
−4361
−3682
515
−2961
−1429
−2799
−2158
−2558
−3974
−4550
−3541
 9500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














12(K)
11
−2371
348
819
−2692
−535
−527
−2443
2294
−2387
−1461
590
−1960
−68
904
−67
−837
−1993
−2554
−1871
 9600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














13(D)
−2663
−4633
3700
580
−4789
−2487
−1872
−4738
731
−4578
−3963
−1073
−3046
−1551
−2987
−2292
−2742
−4201
−4759
−3709
 9700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














14(C)
2503
3193
−4266
−3818
−2010
−3276
−2896
762
−3517
−1437
−1051
−3233
−3509
−3212
−3411
−2499
−1792
1507
−2796
−2431
 9800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














15(D)
−1363
−2905
2748
542
−3202
−2072
−920
−2977
290
−2912
−2023
1270
−2294
−489
−1189
53
1116
−2518
−3086
−2349
 9900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














16(L)
−1268
−1113
−3338
−540
−1057
−2827
−1716
569
−2409
2299
−236
−2381
−2862
−2089
−2316
−232
−1213
1306
−1645
−1304
10000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














17(S)
−1350
−2877
588
1045
−3189
−496
−920
−2963
−628
−2901
−2011
1860
−2289
−489
−1184
2139
190
−2503
−3077
−2343
10100%


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



—
−2336
−8139
−325
−894
−1115
−701
−1378
*
*














18(G)
−454
−832
−968
−1110
−2112
3143
−1211
−2091
−1317
−2264
−1691
−978
−1499
−1202
−1421
−646
−774
−1550
−1916
−1919
10200%


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



—
−38
−5840
−6881
−894
−1115
−3098
−179
*
*














19(H)
−898
−1313
−545
−482
−320
−1336
4297
−1552
−160
−1493
−1035
−579
−1675
−363
−322
−934
−951
−1354
−725
107
10300%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














20(D)
−872
−1812
3234
432
−2215
−967
−433
−2172
−569
−2269
−1704
99
−1453
−184
−1141
−728
−973
−1814
−2146
−1646
10400%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














21(E)
−766
−1695
521
2831
−2050
−1029
−293
−1804
−118
−1919
−1331
69
−1441
−4
−257
−653
−814
−1512
−1968
−1505
10500%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














22(Y)
−1337
−1229
−1681
−1596
1268
−1957
121
−918
−1294
−769
−585
−1229
−2163
−1111
−1301
−1443
−1359
−932
592
3932
10600%


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



—
−38
−5840
−6882
−894
−1115
−109
−3775
*
*














23(I)
−2294
−1931
−4749
−4227
−1724
−4227
−3320
2306
−3952
1990
−684
−3878
94
−3538
−3812
−3411
−2247
1576
−2891
−2629
10700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














24(I)
−2801
−2299
−5406
−5003
−2108
−5164
−4649
3051
−4846
1593
−869
−4829
−4788
−4454
−4829
−4493
−2764
1435
−3781
−3585
10800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














25(K)
−234
−2632
306
−500
−3007
−2141
−719
−2712
2540
−2619
−1730
−778
−2231
2257
968
−1109
−1152
−2288
−2738
−2136
10900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














26(G)
−2184
−3900
796
392
−4174
2903
−1580
−4030
−1636
−3937
−3173
−967
−2810
−1
−2362
1069
−2220
−3530
−4130
−3229
11000%


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



—
−155
−3318
−9181
−3674
−118
−701
−1378
*
*














27(K)
−3243
−3775
−4129
−2558
−4750
−3647
−1490
−4021
3681
−3617
−2982
−2368
−3580
−1076
1318
−3119
−2876
−3817
−3395
−3374
12600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














28(K)
−1684
−2925
−1665
−979
−3407
−2535
−923
−3021
2737
−2865
−2032
202
−2582
1301
804
−1564
1681
−2645
−2905
−2448
12700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














29(V)
−2623
−2122
−5300
−4990
−2769
−5101
−5131
2388
−4945
−1532
−1474
−4790
−4868
−4890
−5101
−4482
−2619
3219
−4505
−3990
12800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














30(A)
3309
−1828
−4057
−4294
−4382
656
−3657
−4147
−4169
−4428
−3497
−2821
−2904
−3694
−3937
−1470
59
−2957
−4610
−4522
12900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














31(V)
−2625
−2122
−5304
−4993
−2772
−5111
−5142
2881
−4950
−1532
−1474
−4796
−4873
−4896
−5108
−4492
−2621
2896
−4512
−3997
13000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














32(I)
−2790
−2287
−5403
−5009
−2155
−5170
−4698
3324
−4899
1175
−912
−4835
−4802
−4495
−4860
−4506
−2757
1192
−3838
−3622
13100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














33(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
13200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














34(Y)
−4838
−3766
−5229
−5579
1502
−5108
−1300
−3726
−5134
−3040
−3131
−3723
−4963
−3861
−4500
−4356
−4689
−3881
2986
4507
13300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














35(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
13400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














36(S)
−1473
−2007
−3647
−3780
−3430
−2363
−3314
228
−3616
−3373
−2876
−2840
−3093
−3395
−3541
3475
−1885
−2307
−3927
−3474
13500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














37(Q)
−4589
−4392
−3927
−4146
−5099
−4221
−4099
−5973
−3840
−5564
−5304
−4230
−4693
4575
−3826
−4704
−4772
−5612
−4577
−4751
13600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














38(G)
677
−2128
−3838
−4171
−4647
3536
−3816
−4506
−4340
−4749
−3857
−3009
−3149
−3871
−4137
−1784
−2005
−3297
−4725
−4735
13700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














39(H)
−2667
−3375
−2682
−2114
−3744
−3201
−4738
−3782
−445
−3553
−2886
−2112
866
−1265
1506
−2614
−2557
−3469
−3282
−2908
13800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














40(A)
3631
−2768
−4492
−4815
−4888
−2992
−4271
−4781
−4818
−5025
−4365
−3727
−3728
−4477
−4545
−2567
−2762
−3852
−4724
−4942
13900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














41(H)
−3103
−3404
−2950
−2573
−783
−3679
4549
−3407
−1372
−3071
−2715
−2454
−3764
2546
−1428
−2990
−2976
−3308
2269
−295
14000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














42(A)
3357
−1795
−4134
−4277
−4057
−2118
−3548
−3549
−4035
−4024
−3192
−2817
−2900
−3608
−3823
217
−1660
−276
−4363
−4211
14100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














43(Q)
−1061
1950
−2044
−1475
−1236
−2372
−1154
−789
−1218
−1062
−1123
−743
−2446
2895
−1441
−1392
−1005
−693
−1678
−1278
14200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














44(N)
−4000
−4117
−3389
−3749
−5073
−3911
−4123
−6022
−4503
−5797
−5419
4397
−4479
−4255
−4592
−4115
−4312
−5371
−4650
−4731
14300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














45(L)
−4414
−3800
−5638
−5628
−2290
−4980
−4628
−1886
−5423
3316
−1236
−5514
−4997
−4750
−5002
−5379
−4399
−2629
−3665
−3690
14400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














46(R)
−1731
−3051
275
−931
−3487
−2518
−973
−3116
2321
−2955
−2123
−224
−2603
256
2808
−1596
−1613
−2730
−2995
−2515
14500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














47(D)
−2896
−4843
3855
944
−5037
−2600
−2082
−5082
−2528
−4903
−4373
−1209
−3196
−1786
−3536
−2501
−3007
−4517
−5004
−3956
14600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














48(S)
−1538
−2212
−2363
−2679
−4293
−2279
−3082
−4365
−3331
−4524
−3676
288
−3026
−2967
−3497
3508
−1962
−3259
−4477
−4066
14700%


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



—
−155
−3318
−9181
−2405
−302
−701
−1378
*
*














49(G)
−2521
−3968
1232
−911
−4849
3373
−2126
−4854
−2535
−4752
−4136
−53
−3115
−1836
−3440
−2284
−2716
−4157
−4880
−3914
15400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














50(V)
−2767
−2324
−5232
−4770
396
−4827
−3784
−36
−4546
848
−611
−4472
−4518
−3980
−4367
−4081
−2716
3323
−3037
−2660
15500%


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



—
−148
−3381
−9181
−203
−2928
−701
−1378
*
*














51(D)
−1684
−3285
2735
2014
−3554
−2196
−1177
−3350
92
−3279
−2427
692
−2505
−770
−1595
−1483
−1666
332
−3460
−2676
15700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














52(V)
−3122
−2888
−5092
−5160
−3522
−4180
−4687
−905
−5060
−2626
−2570
−4662
−4579
−4940
−4923
−4013
−3297
3796
−4414
−4190
15800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














53(V)
369
366
−3075
−2462
−883
−2257
−1420
1415
378
−757
−117
−2098
−2610
−1809
−2037
−1630
1166
2145
−1365
−343
15900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














54(V)
−2624
−2122
−5302
−4991
−2772
−5108
−5139
2623
−4948
−1533
−1475
−4794
−4871
−4894
−5106
−4488
−2620
3088
−4511
−3996
16000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














55(G)
929
−2107
−3852
−4182
−4633
3492
−3809
−4486
−4335
−4732
−3835
−2997
−3132
−3863
−4127
−1761
−1982
−3275
−4720
−4725
16100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














56(L)
−3427
−2938
−5791
−5325
−1449
−5374
−4410
−543
−5063
3041
−255
−5207
−4820
−4126
−4691
−4757
−3351
883
−3184
−3234
16200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














57(R)
−3040
−3724
−3266
82
−4620
−3470
−1396
−3905
804
−3529
−2874
−2133
−3439
−978
3800
−2894
−2709
−3682
−3353
−3267
16300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














58(K)
31
−2412
−803
1532
−2743
−1920
−559
−2483
1772
−2421
−1503
−556
1229
727
1079
−566
−893
−2041
−2579
−1915
16400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














59(G)
−2671
−4661
1614
587
−4832
3103
−1901
−4803
−2269
−4648
−4047
421
−3049
−1587
−3230
2297
−2766
−4245
−4850
−3752
16500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














60(S)
−1499
−2308
−1932
−1859
−4006
1604
−2121
−3754
1362
−3793
−2945
−1833
−2827
−1794
−1902
2738
−1771
−2970
−3910
−3479
16600%


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



—
−247
−8139
−2699
−894
−1115
−701
−1378
*
*














61(K)
1362
−2232
−619
−98
−2567
−427
−435
−2309
1599
−2265
−1349
1101
−1861
886
−512
833
−740
−1868
−2441
−1767
16700%


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



—
−9
−7900
−8943
−894
−1115
−344
−2238
*
*














62(S)
−1288
−1904
−3742
−4011
−4384
−2155
−3593
−4209
−3996
−4479
−3573
−2789
−2948
−3606
−3832
3517
228
−3028
−4600
−4451
16800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














63(W)
726
−873
−3261
−2634
1926
−2567
−1425
660
−2252
−701
−68
−2174
−2617
−1898
18
−1648
−972
983
4091
−958
16900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














64(E)
1527
−244
212
1636
−2722
−1878
−556
−2474
1241
−2419
−1497
350
−1985
−100
−659
96
70
−2025
−2589
−1903
17000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














65(K)
−8
−2242
−895
770
−2502
−1963
−609
−2192
2589
22
−1353
−631
−2052
692
−617
−889
−906
−361
−2455
−1836
17100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














66(A)
3631
−2768
−4492
−4815
−4888
−2992
−4271
−4781
−4818
−5025
−4365
−3727
−3728
−4477
−4545
−2567
−2762
−3852
−4724
−4942
17200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














67(Q)
−1006
−2441
−869
1767
−2780
−1965
−586
−2510
1702
−2445
−1534
−603
−2052
1923
873
−888
236
−630
−2596
1949
17300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














68(A)
1489
−2393
167
1234
−2711
−1873
−547
−2462
895
−2408
−1485
1161
−1977
−90
−648
666
141
−2014
−2577
−1892
17400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














69(D)
2104
−2898
2124
985
−3163
2096
1397
−2935
−693
−2897
−2025
−723
−2329
−543
−1250
−1245
−1368
−2501
−3087
−530
17500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














70(G)
−2294
−2898
−2521
−2885
−4852
3641
−3456
−5042
−3796
−5094
−4356
−365
−3545
−3376
−4005
−2451
−2700
−3996
−4706
−4575
17600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














71(F)
−2596
−2266
−4685
−4188
3199
−4136
1018
505
−3812
1986
−405
−3595
−3961
−3157
−3524
−3277
−2509
−1337
−1621
−840
17700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














72(K)
47
−2348
338
950
−2668
−1854
−512
−507
1721
−2364
−1438
−490
−1947
672
436
806
687
−1970
−2533
−1851
17800%


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



—
−155
−3318
−9181
−2159
−366
−701
−1378
*
*














73(V)
−1810
−1639
−4149
−3689
−1869
−3417
−2822
29
−3369
320
−897
−3230
112
−3099
−3291
−2619
−767
3269
−2708
−2354
18400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














74(K)
847
−1093
−2131
−1554
304
127
−1127
−637
1445
645
1186
−1534
−2401
−1174
−1547
−764
−172
−528
−1519
1413
18500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














75(T)
−1284
−2794
1526
1290
−3096
−2041
1289
−2863
−548
−2808
−1914
−668
−2242
−427
−1095
1451
1827
−2411
−2986
−2264
18600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














76(V)
−1089
−957
−3143
−2535
−943
−2618
−1496
1052
−2198
−792
1859
−146
686
−1884
−2111
−1695
945
2346
−1458
−1106
18700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














77(W)
1606
−2321
−752
612
−2628
−323
−527
−2366
1480
−2331
−1416
−510
−789
−73
421
23
−829
−1936
2212
−1843
18800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














78(E)
−1509
−3540
1372
3127
−3861
−120
−1391
−3685
−1319
−3605
−2787
−900
−2659
−1005
−1976
−400
−655
−3194
−3790
−2957
18900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














79(A)
3390
−1868
−4092
−4341
−4332
−2153
−3680
−3942
−4157
−4333
−3471
−2869
−2948
−3730
−3919
−1525
931
−2894
−4580
−4483
19000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














80(V)
2003
−1721
−4449
−3995
−2160
−3763
−3240
−1342
−3745
−1435
−1124
−3561
−3855
−3494
−3700
−2979
−58
2574
−3091
−2698
19100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














81(K)
1714
−2501
−959
446
−2858
−2043
−654
−2574
1964
−2506
−1609
−689
−2135
−203
1088
423
−1032
−2148
−2652
−2027
19200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














82(W)
265
−2347
815
432
−2663
634
−519
−210
619
−2361
−1438
−495
−1952
1955
−609
−382
147
−1966
2858
−1853
19300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














83(A)
3391
−1860
−3998
−4279
−4411
−2128
−3684
−4207
−4197
−4490
−3585
−2837
−2929
−3729
−3959
706
−1718
−3007
−4636
−4534
19400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














84(D)
−2747
−4795
3813
396
−4912
−2496
−1935
−4905
−2324
−4735
−4166
−1079
−3082
603
−3296
−2353
−2844
−4347
−4929
−3809
19500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














85(V)
−2717
−2220
−5338
−4951
−2254
−5099
−4670
1963
−4844
1553
−1011
−4759
−4771
−4509
−4836
−4427
−2688
2741
−3899
−3628
19600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














86(V)
−2635
−2129
−5306
−4970
−2652
−5125
−5011
2554
−4915
−354
−1368
−4781
−4852
−4798
−5038
−4487
−2622
3019
−4355
−3902
19700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














87(M)
−1340
−1208
−3317
−2708
−968
−2860
−1708
577
−2346
932
4131
−2382
−2878
250
−2265
−228
−1278
−506
−1629
−1313
19800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














88(I)
−2566
−2177
−5017
−4470
669
−4496
−3487
2791
−4191
1116
1394
−4156
−4228
−3615
−3972
−3687
−2499
1692
−2860
−2711
19900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














89(L)
−4414
−3800
−5638
−5628
−2290
−4980
−4628
−1886
−5423
3316
−1236
−5514
−4997
−4750
−5002
−5379
−4399
−2629
−3665
−3690
20000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














90(I)
1212
−1286
−3846
−3262
−1360
−3195
−2166
1616
−2918
1031
−493
−2824
−3211
−2583
−2782
−2308
1598
1299
−2020
−1668
20100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














91(P)
−1614
−2214
−3396
−3710
−4516
−2407
−2618
−4516
−3976
−4705
−3849
−2890
3993
−3625
−3900
666
−2068
−3354
−4610
−4474
20200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














92(D)
−4580
−4701
4174
−3014
−5700
−3967
−3905
−6376
−4478
−6024
−5744
−3355
−4501
−3870
−4926
−4440
−4750
−5894
−4922
−5231
20300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














93(E)
−1123
−2199
−983
2715
−2589
−2046
−942
−2250
−625
−2356
1979
−870
−2250
−554
−1093
463
932
−1902
−2660
−2064
20400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














94(H)
399
−1137
−2012
−14
1582
−2306
1600
246
−1252
190
−325
−1456
−2374
1474
−1479
−94
−905
896
−1557
−1158
20500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














95(Q)
−2742
−3142
−2766
−2681
−2790
−3344
−2460
−160
−1802
−2456
−2353
−2682
−3710
4317
−1866
−2894
−2844
−2559
−3295
−2711
20600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














96(A)
1981
−2315
−809
−268
−2645
−531
−579
−2374
232
−2350
−1445
−567
1217
711
445
447
−874
−1951
−2540
−1883
20700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














97(D)
491
−2351
1394
1381
−2671
−1854
1062
−2421
1010
−2367
−1440
−489
−1947
1017
362
−760
250
−623
−2535
−1852
20800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














98(V)
−2039
−1706
−4456
−3939
−1846
−3939
−3049
1986
−3656
1460
−826
804
−3870
−3351
−3565
−3105
−2000
2330
−2796
−2442
20900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














99(Y)
−4840
−3766
−5230
−5581
1898
−5109
−1300
−3727
−5135
−3041
−3132
−3723
−4964
−3861
−4501
−4357
−4690
−3883
3325
4377
21000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














100(E)
−163
−2353
−734
1681
−2674
−1859
888
−2422
1668
−792
−1443
777
−1952
890
286
−766
238
−1975
−2536
−1856
21100%


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



—
−257
−8139
−2649
−894
−1115
−701
−1378
*
*














101(E)
1017
−2763
862
2042
−3060
−1913
−775
−2836
−495
−2773
−1886
1956
−2143
−136
−1056
265
−1185
−2377
−2948
−2207
21200%


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



—
−9
−7891
−8933
−894
−1115
−338
−2261
*
*














102(E)
−944
−2422
863
2138
−2740
−436
−567
−2493
894
−2437
−1515
−518
−1994
1767
−673
109
−885
−1023
−2605
−1917
21300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














103(I)
−2660
−2156
−5316
−4965
−2520
−5119
−4900
3165
−4894
297
−1251
−4775
−4828
−4705
−4975
−4470
−2642
2240
−4202
−3814
21400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














104(E)
1068
−2341
−760
2003
628
−1887
876
−2380
1240
−2347
−1436
−529
−1983
881
−618
−804
−855
−1954
−2530
−1862
21500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














105(P)
−343
−3144
1561
442
−3538
−489
−1216
−3329
−1038
−3274
−2420
−848
2974
−812
−1635
469
−1644
−2849
−3462
−2693
21600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














106(N)
−1173
−2375
−814
827
−2376
−2071
1767
−2279
−479
−2338
−1509
3151
−2218
−415
−957
−1093
−1120
−198
−2486
647
21700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














107(M)
−3415
−2890
−5826
−5252
−1352
−5488
−4282
1361
−5022
2621
2728
−5181
−4778
−4005
−4613
−4776
−3292
69
−3071
−3194
21800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














108(K)
−1941
−3098
−1997
−1232
−3650
−2740
−1025
−3210
3059
−3010
−2208
499
−2766
1457
1261
−1817
−90
−2858
−3002
−2622
21900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














109(P)
1129
−2426
−740
964
−2747
−1913
−589
−2491
1139
−2440
−1525
−552
1941
1446
−655
−480
−913
−2050
−2610
−1935
22000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














110(G)
−2276
−2907
−2347
−2709
−4832
3554
−3349
−5005
−3678
−5053
−4315
1193
−3507
−3243
−3937
−2418
−2674
−3974
−4703
−4521
22100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














111(A)
1730
−2349
958
−198
−2661
−1868
−535
−2405
927
−2362
−1444
414
−1966
788
−630
790
−840
−303
−2540
−1863
22200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














112(T)
1350
−1149
−14
−1461
−1155
−2314
−1111
758
−1275
−1024
1167
−1475
−2388
−1111
−1501
334
1843
354
−1581
−1182
22300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














113(L)
−333
−2796
−5806
−5293
−1506
−5535
−4502
1096
−5103
2935
−282
−5232
−4857
−4172
−4762
−4864
−3236
506
−3264
−3351
22400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














114(A)
1769
−1525
158
−857
−1603
148
−891
−1181
−752
187
−712
−1040
−2228
660
−1135
−1111
−913
1305
−1913
−1442
22500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














115(F)
−4110
−3437
−5436
−5431
4216
−5143
−2159
−1742
−5074
563
−1124
−4290
−4871
−3987
−4561
−4547
−4016
−2374
−1356
−292
22600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














116(A)
3091
−1829
−3998
−4219
−4413
119
−3637
−4216
−4134
−4469
−3523
−2798
−2896
−3656
−3927
1514
−1679
−2983
−4632
−4539
22700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














117(H)
−5197
−4539
−4720
−5009
−4036
−4506
5435
−6314
−4911
−5786
−5667
−4954
−4960
−5011
−4732
−5391
−5395
−6022
−4063
−3641
22800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














118(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
22900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














119(F)
−4044
−3387
−5534
−5444
4093
−5246
−2370
−1514
−5107
1089
−868
−4443
−4880
−3998
−4592
−4639
−3934
−2200
−1536
−523
23000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














120(N)
885
−1899
−2020
−1781
−2956
−2135
−1925
−2602
−1809
3
−2052
3468
−2633
−1676
−2141
413
−1437
−2139
−3194
−2737
23100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














121(I)
−2673
−2169
−5324
−4969
−2477
−5123
−4876
3293
−4893
358
−1211
−4780
−4824
−4681
−4961
−4427
−2653
1969
−4158
−3791
23200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














122(H)
−3381
−3705
−3197
−3491
−4166
638
5216
−5496
−3798
−5304
−4811
−3481
−4185
−3770
−3879
−3508
−3702
−4793
−4170
−3759
23300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














123(Y)
−4816
−3757
−5210
−5549
3410
−5097
2153
−3719
−5105
−3041
−3127
−3715
−4955
−3851
−4483
−4344
−4669
−3870
−547
3677
23400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














124(G)
−1065
−2519
948
−272
−2820
1844
998
−2566
972
−284
−1622
1553
−2090
−229
−802
−938
−1011
−2133
2708
−2021
23500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














125(Q)
−412
−2285
−2466
−2186
−2068
−2877
−2019
−1589
−1588
1526
−1121
−2187
−3153
3585
−1718
−2137
−1964
−1719
−2789
−2414
23600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














126(I)
−2254
−1916
−4813
−4439
−2466
−4221
−3932
3248
−4248
−1515
−1324
−4044
−4259
−4063
−4255
230
−2280
2003
−3673
−3237
23700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














127(K)
−888
−2234
334
1172
−2504
−1881
−546
−93
1370
−300
−1337
465
−1974
655
−646
−794
−827
1255
−2448
−1798
23800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














128(P)
715
−1925
−3618
−3897
−4464
653
−3594
−4274
−4053
−4520
−3596
−2770
3775
−3593
−3911
−1550
−1770
−3067
−4647
−4548
23900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














129(P)
479
−2398
−1173
−637
−2915
−2106
−848
−2610
−289
−2586
−1713
−884
2238
1247
2195
51
−1147
−2184
−2757
−2174
24000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














130(A)
1787
−2663
1377
529
−2976
−1992
−762
−2736
1785
−2680
−1776
−623
−2161
−319
−936
297
−1120
−2285
−2853
−2146
24100%


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



—
−2336
−8139
−325
−894
−1115
−701
−1378
*
*














131(F)
−1308
−1104
−2227
−2120
3516
−2093
−244
−196
−1891
64
66
−1626
−2278
−1503
−1798
−1617
−1350
−389
305
1335
24200%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














132(P)
−603
−937
−997
−1058
−1832
−1041
−1092
−1737
−1074
−1874
−1416
−992
3539
−1065
−1192
−789
−866
−1383
−1765
−1661
24300%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














133(K)
−804
−1483
−564
−230
−1920
−1335
−101
−1605
2889
−1630
−1021
−349
−1569
232
698
−786
−759
−1358
−1637
−1317
24400%


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



—
−38
−5840
−6882
−894
−1115
−109
−3775
*
*














134(D)
−2405
−4159
3349
−651
−4260
−261
−1744
−4307
−1947
−4207
−3514
2151
−2936
−1416
−2754
−2102
−2471
−3802
−4324
637
24500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














135(I)
−2047
−1713
−4504
−3983
−1821
−3943
−3061
2461
−3697
1581
−797
−3593
−3873
−3371
−3587
−342
−2009
1904
−2784
−2441
24600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














136(D)
−2024
−3444
3495
−680
−3868
−2331
−1596
−44
−1632
−3675
−2915
685
−2782
−1248
−2305
478
−2088
−3196
−3911
−3096
24700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














137(V)
−3122
−2888
−5092
−5160
−3522
−4180
−4687
−905
−5060
−2626
−2570
−4662
−4579
−4940
−4923
−4013
−3297
3796
−4414
−4190
24800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














138(I)
−53
−875
−3230
−2609
1867
−393
−1422
2613
−2236
−723
−81
−2157
−2608
−1885
−2086
1633
276
−271
−1325
844
24900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














139(M)
315
−2345
−4754
−4279
−1396
−4001
−3301
−697
−3877
816
4676
−3879
−3994
−3361
−3676
−3242
−2531
−1114
−2746
−2629
25000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














140(V)
−2623
−2122
−5301
−4990
−2770
−5102
−5132
2415
−4945
−1532
−1474
−4791
−4869
−4890
−5102
−4483
−2619
3206
−4506
−3991
25100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














141(A)
3405
2528
−4529
−4796
−4340
−2257
−3851
−3901
−4447
−4351
−3532
−3057
−3052
−3976
−4112
−1643
−1844
−2929
−4572
−4519
25200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














142(P)
−4853
−4392
−5213
−5573
−5853
−4408
−5077
−6679
−5780
−6281
−6067
−5357
4310
−5648
−5396
−5166
−5194
−6092
−4900
−5786
25300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














143(K)
−4484
−4357
−4380
−3992
−5413
−4236
−3307
−5555
3994
−5171
−4707
−3921
−4535
−3079
−2169
−4529
−4408
−5264
−4403
−4729
25400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














144(G)
2167
−1833
−3963
−4199
−4430
2715
−3642
−4236
−4146
−4489
−3540
−2795
−2898
−3661
−3939
910
−1682
−2994
−4647
−4556
25500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














145(P)
−2604
−2948
−4094
−4235
−3544
−3269
−3767
−3353
−3912
−3066
2095
−3659
4036
−3912
−3822
−2883
−2963
−3249
−4027
−3787
25600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














146(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
25700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














147(H)
−2569
−3440
−1867
−1702
−3820
−2996
4731
−3830
634
−3639
−2963
−1838
1551
−1305
−748
−2470
−2510
−3482
−3434
−2990
25800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














148(T)
194
−1498
−3255
−2899
−2240
−2226
−2291
−1754
−2652
1634
−1430
−2330
−2747
−2399
−2684
567
2687
−1484
−2682
−2351
25900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














149(V)
−3122
−2888
−5092
−5160
−3522
−4180
−4687
−905
−5060
−2626
−2570
−4662
−4579
−4940
−4923
−4013
−3297
3796
−4414
−4190
26000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














150(R)
−4845
−4446
−5107
−4682
−5507
−4412
−3791
−5946
−2789
−5502
−5118
−4521
−4754
−3672
4219
−4989
−4832
−5644
−4538
−4993
26100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














151(R)
−962
−2395
−777
1012
−2721
76
1031
−2459
−142
−2413
−1501
−560
−2018
−128
2038
1224
−66
−2023
−2585
−1919
26200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














152(E)
−902
−2032
−899
2078
−2228
−1934
−611
−1897
−259
−221
−1156
520
−2024
816
−736
−858
1303
−287
−2295
537
26300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














153(Y)
−4823
−3765
−5219
−5565
3303
−5093
−1317
−3703
−5127
−3017
−3111
−3732
−4959
−3868
−4500
−4356
−4679
−3867
−565
4052
26400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














154(V)
129
−1901
−989
821
−2060
−1969
−654
−1704
498
−52
−1037
−703
−2057
695
−796
443
−344
1871
−2192
−1626
26500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














155(Q)
576
−2355
344
1156
−2675
−1856
−515
−508
1502
−2370
−1444
571
−1949
1878
419
−764
−822
−1976
−2538
−1856
26600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














156(G)
−3239
−3889
516
−2361
−5355
3646
−3337
−5629
−3818
−5498
−4951
−2619
−3905
−3187
−4377
−3211
−3532
−4837
−4895
−4826
26700%


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



—
−155
−3318
−9181
−2159
−367
−701
−1378
*
*














157(G)
753
−2516
−789
488
−2848
2300
−672
−2582
596
−2529
−1627
481
−2112
−224
471
−962
−1024
−2149
−2694
−2033
27300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














158(G)
−52
−2212
−3792
−4133
−4698
3627
−2843
−4580
−4356
−4812
−3937
−3058
−3216
−3901
−4170
−1874
−2095
−3384
−4734
−4766
27400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














159(V)
−2485
−2030
−5123
−4769
−2667
−4797
−4593
2349
−4661
−1545
−1424
−4502
−4648
−4554
−4752
−4115
825
2986
−4159
−3678
27500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














160(P)
−2541
−3139
−2413
−2753
−4726
−2991
−3342
−5055
−3527
−5058
−4393
1199
4031
−3244
−3757
−2665
−2911
−4148
−4583
−4362
27600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














161(C)
1577
3078
1357
−656
−2664
−219
891
−2359
−617
−2434
−1576
−891
−2199
−545
−1093
872
−1043
−1946
−2701
−2102
27700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














162(L)
−2140
−2404
−3995
−3997
−2053
−3121
−3283
−1687
−3689
3041
−1200
−3360
−3626
−3433
−3567
480
−2414
−1973
−3145
−2755
27800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














163(I)
−2527
−2092
−5072
−4613
2047
−4674
−3911
2668
−4413
117
−841
−4323
−4439
−4023
−4320
−3916
−2488
2176
−3342
−3040
27900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














164(A)
3631
−2766
−4492
−4815
−4888
−2992
−4271
−4781
−4818
−5025
−4365
−3727
−3728
−4477
−4545
−2567
−2762
−3852
−4724
−4942
28000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














165(V)
−2623
−2122
−5301
−4990
−2770
−5102
−5132
2426
−4946
−1532
−1474
−4791
−4869
−4891
−5102
−4483
−2619
3200
−4506
−3991
28100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














166(H)
−495
−2631
903
−2051
722
−3242
3753
−2386
−2056
−2342
−1863
−2074
−3330
−1815
−2362
−2318
−2233
−2272
−981
3315
28200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














167(Q)
−4589
−4392
−3927
−4148
−5090
−4221
−4099
−5973
−3840
−5564
−5304
−4230
−4693
4575
−3826
−4704
−4772
−5612
−4577
−4751
28300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














168(D)
−2873
−4605
3943
−902
−4948
−2633
−2157
−5087
−2604
−4922
−4387
428
−3235
−1872
−3575
−2522
−3009
−4491
−4932
−3946
28400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














169(A)
1776
−1612
−1274
138
−1698
−2092
−816
−1295
150
−1526
−780
−943
1212
−538
−1013
−1056
−894
1275
−1968
635
28500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














170(S)
−1545
−2420
1001
−1518
−4049
−2206
−2206
−3839
−2264
−3938
−3103
−1627
−2814
−1909
−2758
2666
2312
−3045
−4143
−3560
28600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














171(G)
−2999
−3461
−2978
−3207
−5161
3669
−3454
−5283
15
−5188
−4565
−3174
−3946
−3312
−3140
−3128
−3317
−4492
−4622
−4749
28700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














172(N)
−887
−2349
−736
911
−2668
−1860
−518
−2415
711
−2362
−1440
1880
−1954
436
799
671
1230
−539
−2534
−1855
28800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














173(A)
3631
−2768
−4492
−4815
−4888
−2992
−4271
−4781
−4818
−5025
−4365
−3727
−3728
−4477
−4545
−2567
−2762
−3852
−4724
−4942
28900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














174(K)
−1368
−2597
−1358
−720
−2953
−2298
1933
−2603
1975
851
−1706
−958
−2360
859
1012
−1273
1094
−2245
−2690
−2165
29000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














175(D)
−27
−2613
2320
1049
−2923
−1962
1973
−2684
544
−2624
−1712
1408
−2115
345
−869
−969
−1058
−2231
−2793
−2087
29100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














176(V)
−1096
−938
−3279
−2658
−899
−2643
−1513
1265
1006
1388
−124
−2232
−2688
−1950
−2151
−1725
−311
1669
−1425
827
29200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














177(A)
3342
−1826
−4064
−4295
−4368
111
−3651
−4129
−4159
−4412
−3484
−2820
−2903
−3689
−3929
−1469
409
−2949
−4599
−4508
29300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














178(L)
−4414
−3800
−5638
−5628
−2290
−4980
−4628
−1886
−5423
3316
−1236
−5514
−4997
−4750
−5002
−5379
−4399
−2629
−3665
−3690
29400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














179(S)
2216
−1831
−3961
−4157
−4409
656
−3608
−4213
−4076
−4462
−3514
−2782
−2892
−3613
−3895
2686
−1675
−2982
−4623
−4523
29500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














180(Y)
−3634
−3050
−4918
−4872
36
−4597
−1405
223
−4437
−250
−1998
−3545
−4494
−3522
−4004
−3782
−3536
−2621
2928
4349
29600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














181(A)
3391
−1860
−3998
−4279
−4411
−2128
−3684
−4207
−4197
−4490
−3585
−2837
−2929
−3729
−3959
706
−1718
−3001
−4636
−4534
29700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














182(K)
370
307
113
−351
−2280
−1925
−615
324
1888
−2040
−1194
−632
−2022
−205
−736
1541
14
−1628
−2330
−1726
29800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














183(G)
2572
−2028
−3934
−4246
−4575
2752
−3783
−4406
−4316
−4661
−3751
−2958
−3070
−3837
−4092
−1679
−1898
−3191
−4701
−4686
29900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














184(I)
−2178
−1808
−4630
−4153
−2094
−4190
−3417
3121
−3909
311
−1023
698
−4099
−3656
−3864
−3388
−2148
1742
−3147
−2761
30000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














185(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
30100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














186(G)
1392
2751
−4353
−4536
−4308
2864
−3681
−4084
−4233
−4354
−3425
−2859
−2890
−3744
−3957
712
−1656
−2914
−4553
−4470
30200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














187(G)
855
−1822
−3738
−3769
−4188
2507
−3358
−3950
−3667
−4196
−3283
−2667
986
−3302
−3621
−1441
2334
−2867
−4408
−4236
30300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














188(R)
−3706
−3692
−4490
−3846
1391
−4057
−2273
−3795
−1906
−3355
−3181
−3458
−4263
−2675
3948
−3768
−3671
−3813
−2293
−1328
30400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














189(A)
2844
−1670
−3814
−3873
−4048
−1958
−3316
−3787
−3686
−4061
−3156
−2598
−2734
−3301
−3572
1088
1907
−2713
−4286
−4136
30500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














190(G)
−4176
−3995
−4855
−5222
−5686
3828
−4823
−6386
−5533
−6087
−5741
−4896
−4606
−5312
−5178
−4461
−4560
−5613
−4754
−5635
30600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














191 (V)
−2496
−2036
−5139
−4788
−2680
−4825
−4637
2522
−4686
−1579
−1431
−4527
−4668
−4584
−4783
−4148
629
2919
−4191
−3706
30700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














192(I)
−2760
−2307
−5270
−4785
1172
−4884
−3992
3346
−4576
662
−572
−4539
−4535
−4008
−4400
−4134
−2703
757
−3223
−3041
30800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














193(E)
454
−3086
−801
3279
−4125
−2346
−1868
−3919
−1868
−3932
−3156
−1291
1208
−1533
−2450
−1842
−2097
−3305
−4121
−3395
30900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














194(T)
−2738
−3109
−4509
−4810
−4918
−3305
−4346
−4865
−4769
−5072
−4551
−3987
−3998
−4580
−4545
−2999
4033
−4113
−4684
−4915
31000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














195(T)
−1323
−1975
−2766
−2978
−4207
−2152
−3105
−4004
−3295
−4229
−3354
134
−2901
−2988
−3387
−113
3742
−2969
−4405
−4119
31100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














196(F)
−4110
−3437
−5436
−5431
4126
−5143
−2159
−1742
−5074
563
−1124
−4290
−4871
−3987
−4561
−4547
−4016
−2374
−1356
−292
31200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














197(K)
−111
−2844
−1470
1220
−3294
−2415
−860
−2939
2448
−2798
−1945
−1056
−2475
767
2054
−1421
−1432
−2544
−2858
−2356
31300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














198(E)
545
−3735
1715
2880
−2308
−3981
−1442
−3818
−1408
−3725
2924
−909
−2713
1281
−2087
−1777
−2041
−3331
−3909
−3046
31400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














199(E)
−4574
−4665
−2714
3919
−5655
−3995
−3886
−6219
−4238
−5898
−5604
−3415
−4513
−3838
−4570
−4456
−4726
−5786
−4878
−5197
31500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














200(T)
−1211
1331
−3446
−2962
−1399
−2495
−1930
−869
−2610
−1374
−769
−2403
−2815
−2309
−2526
−1685
−3305
195
−1933
1430
31600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














201(E)
−1941
−3222
−921
3293
−3618
−2473
−1316
−3186
−916
−3225
−2465
−1147
−2749
−923
−1064
−1790
−1902
−171
−3398
−2802
31700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














202(T)
−1286
−1898
−3764
−4016
−4329
−2157
−3572
−4120
−3959
−4408
−3517
−2789
−2947
−3588
−3797
697
3756
−2989
−4554
−4399
31800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














203(D)
−4580
−4701
4174
−3014
−5700
−3967
−3905
−6376
−4478
−6024
−5744
−3355
−4501
−3870
−4926
−4440
−4750
−5894
−4922
−5231
31900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














204(L)
−3705
−3122
−6060
−5527
−1359
−5814
−4569
1065
−5292
3069
−146
−5564
−4963
−4163
−4828
−5215
−3571
−1279
−3159
−3298
32000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














205(F)
−3777
−3220
−5271
−5259
4268
−4892
−2120
417
−4916
−1143
−1314
−4142
−4753
−3956
−4473
−4270
−3740
−1899
−1349
−269
32100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














206(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
32200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














207(E)
−4574
−4665
−2714
3919
−5655
−3995
−3886
−6219
−4238
−5898
−5604
−3415
−4513
−3838
−4570
−4456
−4726
−5786
−4878
−5197
32300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














208(Q)
−3157
−3746
−3170
−2450
−4497
−3515
−1763
−4161
−443
−3809
−3189
−2392
−3620
4200
1284
−3063
−2944
−3900
−3556
−3420
32400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














209(A)
2672
−1334
−3318
−2853
−1740
371
−2072
483
−2577
−1549
−928
−2359
−2798
−2295
−2567
−1629
191
932
−2245
−1899
32500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














210(V)
−2620
−2125
−5293
−4983
−2756
−5076
−5100
1877
−4932
−1522
−1466
−4777
−4855
−4870
−5082
−4456
−2619
3416
−4480
−3969
32600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














211(L)
−4414
−3800
−5638
−5628
−2290
−4980
−4628
−1886
−5423
3316
−1236
−5514
−4997
−4750
−5002
−5379
−4399
−2629
−3665
−3690
32700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














212(C)
−2243
5044
−4840
−4445
−1998
−3905
−3598
−31
−4138
449
−930
−3902
−4040
−3778
−4010
−3184
−2306
1347
−3209
−2883
32800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














213(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
32900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














214(G)
677
−2128
−3838
−4171
−4647
3536
−3816
−4506
−4340
−4749
−3857
−3009
−3149
−3871
−4137
−1784
−2005
−3297
−4725
−4735
33000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














215(V)
378
724
−3707
−3104
−1180
−2986
−1919
1210
−2734
1302
−359
−2627
−3014
−2382
−2566
−2089
1123
1949
−1773
−1423
33100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














216(M)
−948
−1407
−1515
156
−1452
−2164
1677
−1030
−821
−1302
1976
−1113
−2245
−718
−1173
773
1715
1332
−1794
−1343
33200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














217(E)
−1397
−2528
−725
2286
−2392
240
−791
−2681
328
−2645
−1744
−674
−2162
−351
−939
545
−1095
−2227
−2828
−2143
33300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














218(L)
−3705
−3122
−6060
−5527
−1359
−5814
−4589
1065
−5292
3069
−146
−5564
−4963
−4163
−4828
−5212
−3571
−1279
−3159
−3298
33400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














219(V)
−2600
−2108
−5251
−4894
−2568
−5025
−4783
2479
−4810
−1354
1358
−4683
−4772
−4654
−4895
−4362
−2584
3018
−4181
−3758
33500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














220(K)
−1633
−2905
−1573
706
−3375
−2487
−900
−3003
2925
−2849
−2008
−1128
−2541
1714
784
−1509
−105
−2617
−2894
−2418
33600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














221(A)
2352
2066
−2593
−2000
−947
−2434
−1271
−486
−32
−832
714
−1817
−2498
−1493
−1792
−1483
274
453
−1419
501
33700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














222(G)
224
−1905
−3562
−3696
−3684
3361
−3297
−3220
−3625
81
−2886
−2733
−2977
−3326
−3545
−1606
−1763
−2574
−4068
−3810
33800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














223(F)
−4781
−3756
−5207
−5542
4341
−5070
−1342
−3653
−5111
−2971
−3065
−3743
−4949
−3874
−4496
−4351
−4650
−3829
−591
1725
33900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














224(E)
−2413
−4114
221
3465
−4392
−2485
−1689
−4248
−1608
−4112
−3396
−1094
−2951
−1336
871
−2119
−2441
−3763
−4239
−3395
34000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














225(T)
−1461
−1864
−3139
−2645
−2659
−2483
−2136
−1734
−1646
−2359
−1761
−2298
−2936
−1995
920
−1748
3354
967
−2989
−2654
34100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














226(L)
−3831
−3266
−5314
−5148
−673
−5068
−2476
−1443
−4706
3059
−789
−4359
−4756
−3864
−4314
−4462
−3729
−2115
−1672
1736
34200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














227(V)
−1819
−1960
−4426
−4359
−2977
−3037
−3800
−439
−4098
−2145
−1909
−3451
−3600
−3879
−4011
−2397
2510
2999
−3974
−3608
34300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














228(E)
−2863
−4790
1397
3563
−4990
−2594
−2061
−5021
−2476
−4848
4298
−1204
−3182
−1761
−3454
−2476
−2968
−4462
−4951
−3920
34400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














229(A)
2686
−1916
−1440
275
−2529
−292
−1240
−2176
−998
−2345
1183
−1184
−2355
−905
−1425
512
−1158
−1817
−2697
−2174
34500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














230(G)
−4435
−4203
−5092
−5462
−5893
−3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
34600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














231(Y)
−4099
−3483
−4921
−5048
−109
−4705
−1565
−2914
−4494
−2334
2010
−3723
−4707
−3735
−4111
−4065
−4068
−3172
−847
4618
34700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














232(Q)
1711
−2410
−772
934
−2739
−1925
−604
−2477
−171
−2433
−1524
−574
−2035
2086
345
902
−923
−2042
−2608
−1941
34800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














233(P)
−3403
−4071
−1922
817
−5220
−3359
−3173
−5423
−3337
−5281
−4771
−2564
4045
−2989
−3760
−3320
−3624
−4817
−4763
−4636
34900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














234(E)
−2870
−4786
1265
3587
−4993
−2600
−2068
−5026
−2483
−4852
−4303
−1212
−3188
−1768
−3458
−2484
−2974
−4467
−4950
−3925
35000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














235(M)
−3089
−2618
−5523
−4976
−1443
−5128
−4045
653
−4735
1429
4269
−4803
−4610
−2915
−4423
−4378
−2995
1140
−3054
−3074
35100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














236(A)
3631
−2768
−4492
−4815
−4888
−2992
−4271
−4781
−4818
−5025
−4365
−3727
−3728
−4477
−4545
−2567
−2762
−3852
−4724
−4942
35200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














237(Y)
−4797
−3764
−5203
−5543
1114
−5069
−1339
−3694
−5111
−3013
−3107
−3741
−4351
−3876
−4497
−4354
−4666
−3859
−588
4723
35300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














238(F)
−3828
−3605
−4146
−4086
4292
−4207
−2060
−3492
774
−3071
−3005
−3556
−4434
−3287
−2952
−3868
−3858
−3571
−1593
−494
35400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














239(E)
−2775
−4471
−511
3582
−4815
−2610
−2057
−4863
−2317
−4711
−4124
1234
−3182
−1755
−3103
−2442
−2884
−4306
−4753
−3820
35500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














240(C)
−1407
5023
−4397
−4323
−3016
−2468
−3398
−1251
−3952
−2540
−2082
−3044
−3133
−3588
−3744
−1803
1473
1125
−3677
−3390
35600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














241(L)
−3370
−2847
−5795
−5233
−1386
−5465
−4298
708
−5010
2859
1349
−5155
−4777
−4026
−4622
−4755
−3254
814
−3103
−3213
35700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














242(H)
−2519
−4224
−445
−46
−4287
−2505
4583
−4377
−1764
−4237
−3571
2007 
−3009
−1439
−2361
−2209
−2569
−3893
−4267
−3353
35800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














243(E)
−3177
2571
−2701
3711
−4581
−3438
−3479
−4765
−3558
−4932
−4406
−3081
−4005
−3370
−3802
−3269
−3451
−4260
−4554
−4524
35900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














244(L)
−85
−1333
−3893
−3280
−1111
−3185
−2083
1066
−2910
2310
1961
−2823
−3170
−2501
−2721
−2289
−113
436
−1859
−1558
36000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














245(K)
−2513
−3173
−2941
−2370
−4402
−3094
−1824
−3895
3666
−3734
−3068
−2271
−3377
−1447
−616
−2537
751
−3485
−3625
−3471
36100%


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



—
−155
−3318
−9181
−196
−2974
−701
−1378
*
*














246(L)
−3571
−3023
−5954
−5375
−1321
−5646
−4390
−632
−5138
2962
1671
−5358
−4852
−4044
−4689
−4963
−3436
742
−3082
−3239
36300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














247(I)
−2980
−2484
−5473
−5109
−1958
−5196
−4587
3728
−4915
267
−781
−4933
−4833
−4427
−4799
−4598
−2949
−64
−3627
−3397
36400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














248(V)
−1685
−1668
−4095
−3732
−2081
−3082
−2893
−277
−3402
−1488
1383
−3123
−3419
−3145
−3320
367
−1807
3332
−2874
−2504
36500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














249(D)
−2963
−4569
3864
−1039
−4953
−2751
−2187
−4998
767
−4822
−4260
−1424
−3314
−1891
−3072
−2624
−3060
−4467
−4770
−3962
36600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














250(L)
−2768
−2715
−4842
−4633
−1675
−3998
−3790
−1038
−4207
3056
−562
−4150
−4179
−3740
−3989
−3399
699
−1545
−3154
−3367
36700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














251(M)
−2822
−2356
−5342
−4861
−1759
−4985
−4151
2587
−4663
173
4005
−4649
−4601
−4076
−4487
−4251
−2764
766
−3321
−3210
36800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














252(Y)
−4562
−3630
−5142
−5401
1516
−4992
−1300
−3544
−4968
−2963
−2986
−3671
−4868
−3786
−4393
381
−4432
−3662
2413
4375
36900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














253(E)
−1959
−3457
−568
3135
−3841
−2347
−1410
−3622
−1165
−3547
−2751
−1000
−2716
1879
−1666
−1757
692
−3162
−3709
−2960
37000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














254(G)
−347
−2818
−1215
201
−3253
2635
−921
−2921
1474
−2822
−1972
−1002
−2459
−486
658
−1397
−1435
−2521
−2923
−2378
37100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














255(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
37200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














256(I)
−2042
−1769
−4321
−3740
−1316
−3753
−2668
3134
−3389
1017
1999
194
−3653
−2958
−3221
−2884
−1980
−344
−2325
−2073
37300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














257(A)
1914
−1640
−1237
128
−1748
−661
−793
−1355
−577
−49
−817
−905
−2155
−498
−993
1037
162
−1149
−2002
624
37400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














258(N)
365
−3809
1001
557
−4083
1196
−1518
−3930
−1535
−3838
−3055
3219
−2763
−1148
−2243
−1845
−2131
−3433
−4027
−3144
37500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














259(M)
−3656
−3159
−5816
−5350
−1349
−5421
−4248
−822
−4928
948
4920
−5248
−4838
−4039
−4539
−4860
−3558
−1557
−3044
−3030
37600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














260(R)
−1614
−1949
−2260
−1663
−886
−2765
−1089
−1596
−1089
360
−1133
2239
−2814
−1215
2408
−1789
−1535
−1468
1995
1546
37700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














261(Y)
−1548
−2973
568
−509
−2846
−2207
1172
−2986
441
−2941
−2110
1645
−2446
−659
−1264
−1389
−1509
−2582
−2924
3695
37800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














262(S)
279
−1844
−3877
−4131
−4448
136
−3634
−4260
−4132
−4511
−3561
−2782
−2903
−3648
−3936
3391
−1692
−3009
−4661
−4566
37900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














263(I)
−2653
−2419
−5311
−4961
−2538
−5114
−4905
2957
−4891
332
−1267
−4770
−4827
−4713
−4978
−4466
−2636
2521
−4218
−3821
38000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














264(S)
−2212
−2711
−4019
−4348
−4697
−2899
−4045
−4988
−4527
−5102
−4364
−3492
−3638
−4203
−4355
3681
−2664
−3902
−4616
−4605
38100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














265(N)
−2725
−4778
2906
990
−4896
−2486
−1922
−4885
−2320
−4718
−4140
3045
−3069
−1612
−3311
−2334
−2821
−4325
−4919
−3794
38200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














266(T)
−2061
−3396
−596
903
−4071
−2367
−1713
−3874
−1685
−3859
−3103
2125
−2850
−1369
−2277
−1897
3157
−3351
−4055
−3271
38300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














267(A)
3410
−2035
−3979
−4290
−4573
659
−3798
−4400
−4332
−4660
−3754
−2978
−3080
−3858
−4099
−1690
−1908
−3194
−4697
−4687
38400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














268(E)
−2118
−3486
−1036
2964
−3935
−2588
−1284
−3596
1878
−3417
−2636
−1209
−2827
1323
−773
−1923
−2032
−3199
−3455
−2917
38500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














269(Y)
−4524
−3618
−5100
−5310
1910
−4972
−1299
−3522
634
−2951
−2965
−3649
−4847
−3741
−4299
−4199
−4391
−3637
2997
4211
38600%


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



—
−155
−8139
−3345
−894
−1115
−701
−1378
*
*














270(G)
−4176
−3995
−4855
−5222
−5686
3828
−4823
−6386
−5533
−6087
−5741
−4896
−4606
−5312
−5178
−4461
−4560
−5613
−4754
−5635
38700%


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



—
−8
−7992
−9034
−894
−1115
−422
−1980
*
*














271(D)
−2710
−4705
3025
1828
−4880
1758
−1932
−4863
−2320
−4703
−4115
−1084
−3073
−1621
−3297
−2330
−2809
−4301
−4894
−3793
38800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














272(Y)
−2497
−2175
−4651
−4137
2447
−4046
−2215
255
−3766
892
1558
−3537
−3886
−3109
−3468
−3181
−2410
−1282
−1615
3508
38900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














273(V)
−1425
−1250
−3480
−2894
−1283
−3035
−1955
691
−2570
−1
−429
−2568
−3060
942
−2514
−2129
1701
2578
−1907
−1553
39000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














274(T)
516
−1643
−1918
−1401
−2170
−2112
−1387
−1759
−1234
−2016
−1265
−1442
−2421
−1149
877
1341
2345
822
−2454
−2006
39100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














275(G)
677
−2128
−3838
−4171
−4647
3536
−3816
−4506
−4340
−4749
−3857
−3009
−3149
−3871
−4137
−1784
−2005
−3297
−4725
−4735
39200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














276(P)
−992
−2210
343
−359
−2447
−1960
−675
−2143
533
−2204
−1351
−651
2813
−260
−802
465
−939
−873
−2467
1093
39300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














277(R)
−1214
−2448
−1097
1072
175
−2145
−716
−2587
848
−2528
−1653
−795
−2228
−273
2862
417
−1133
−2191
−2671
−2084
39400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














278(V)
289
−2035
−5133
−4789
−2692
−4777
−4639
2142
−4689
−1561
−1443
−4511
−4649
−4585
−4784
−4102
−2487
3125
−4202
−3717
39500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














279(I)
−2286
−1919
−4828
−4452
−2473
−4254
−3954
3155
−4265
−1516
−1326
−4066
−4279
−4082
−4274
226
−2288
−2182
−3686
−3250
39600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














280(D)
−1731
−3162
2329
−550
−3318
−2239
−1273
−3221
−1145
−3214
−2406
2295
−2573
−899
−1742
−1561
1851
−2804
−3366
1327
39700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














281(E)
1097
−2699
1227
−2368
−2994
−2011
−796
−2753
291
−2704
−1808
−640
−2190
−358
−992
−1065
−1162
−2310
−2885
1152
39800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














282(E)
−166
−2372
859
1835
−2692
−1861
1182
−2444
490
−2388
−1462
590
478
1356
−620
116
−837
−1994
−2555
−1871
39900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














283(T)
228
−1688
−3655
−3444
−3179
−2145
−2891
−2611
−3215
−3076
−2328
−2557
−2821
−2916
−3214
2251
2366
1397
−3549
−3270
40000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














284(K)
−2991
−3623
−3848
−2331
−4472
−3512
−1356
−3763
2942
−3413
1860
−2188
−3430
−937
2705
−2874
−2650
−3556
−3267
−3189
40100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














285(E)
443
−2370
732
1690
−2691
−1863
−526
−2442
1639
−2385
−1460
−498
−1960
1120
437
106
−836
−1992
−2552
−1869
40200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














286(A)
1871
−2286
−843
814
−2570
−1928
−578
269
1096
−2279
−1391
−586
−2019
−136
1056
−62
−881
−1889
−2488
−1849
40300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














287(M)
−3656
−3159
−5816
−5350
−1349
−5421
−4248
−822
−4928
948
4920
−5248
−4838
−4039
−4539
−4860
−3558
−1557
−3044
−3030
40400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














288(K)
−1646
−2891
−1591
287
−3346
−526
−912
−2971
2831
−2832
−1997
−1146
−2554
−472
1762
−1527
−1524
−2597
−2885
1245
40500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














289(E)
−172
−2394
367
2205
−2713
−487
−545
−2465
−134
−2409
−1485
1305
−1957
663
831
−795
72
−2015
2577
−1891
40600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














290(C)
1574
3024
−4584
−4122
−2155
−3932
−3330
1746
−3870
−1406
−1109
−3691
−3957
−3613
−3805
−3144
−2046
2342
−3118
−2720
40700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














291(L)
−187
−2175
−4307
−3889
−898
−3779
−2344
−944
−3485
2855
−478
−3390
−3782
−3025
−3298
−2972
−2345
−1269
−1846
1565
40800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














292(K)
862
−2347
143
1211
−2665
−1855
873
−2414
1692
−2392
917
−492
−1949
889
−603
−763
783
−1968
−2532
−1851
40900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














293(D)
−2148
−3878
2790
1765
−4119
−2356
−1511
−3962
−1467
−3852
−3075
24
−2782
−1139
2142
−189
−2163
−3473
−4024
−3155
41000%



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




−8
−8139
−9181
−894
−1115
−701
−1378
*
*














294(I)
−2630
−2131
−5302
−4991
−2737
−5092
−5106
3464
−4941
−1495
−1447
−4789
−4862
−4869
−5086
−4473
−2627
2071
−4467
−3968
41100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














295(Q)
346
−3134
−1818
−1401
−3862
−2760
−1314
−3433
1329
−3271
−2513
−1545
−2936
3817
−430
−2018
−2031
−3060
−3278
−2908
41200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














296(S)
−1354
−2895
1712
354
−3192
−2068
−914
−2967
−621
−2903
−2012
−1817
−2288
724
−1177
1978
−96
−2508
−3076
−2340
41300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














297(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
41400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














298(E)
−437
−2374
−769
2013
−2697
−1895
−552
−2438
623
−2389
−1472
−536
−1991
−97
777
829
1488
−1999
−2559
−1890
41500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














299(F)
−4347
−3577
−4619
543
3858
−4820
−1320
−3438
−4609
−2900
−2894
−3532
−4750
−3628
−4209
−4078
−4237
−3546
−601
2917
41600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














300(A)
2827
−1603
−4068
−3628
−2047
−3165
−2823
1205
−3349
−1486
−1089
−3103
−3432
−3073
−3298
−2387
197
1100
−2812
−2447
41700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














301(K)
−2364
−3363
−2464
163
−4038
−3041
−1188
−3501
2928
−3234
−2787
−1693
−3047
−758
2488
−458
−2152
−3194
−3159
−2895
41800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














302(M)
−893
−2361
740
1780
−2680
−537
−524
−2429
930
−2376
1895
−498
−1958
−66
722
775
−833
−1982
−2545
−1864
41900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














303(W)
−2965
2533
−4795
−4482
3045
−4315
−1779
−1426
−4093
92
−987
−3564
−4179
−3330
−3743
−3475
−2878
664
4754
−84
42000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














304(I)
−2621
−2136
−5246
−4859
−2364
−4987
−4577
3052
−4747
−250
1043
−4641
−4713
−4488
−4769
−4301
−2597
2384
−3934
−3596
41200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














305(L)
684
−1319
−1741
761
−1375
−2223
−1037
−927
−1042
1693
−533
−1287
−2325
−915
−1349
767
−152
84
−1756
−1329
42200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














306(E)
364
−4165
621
3314
−4393
−2398
−1686
−4292
−1836
−4169
−3446
728
−2893
−1339
−2626
−2045
−2402
−3767
−4362
−3401
42300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














307(N)
−939
979
−1235
−681
−1738
1352
935
−1357
−549
−1572
−816
2186
−2155
−482
165
−1022
−880
30
−1990
1446
42400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














308(Q)
667
−2393
−773
511
−2721
−316
−544
−2465
1211
−2405
−1484
584
−1989
2135
1528
−812
−868
−2019
−2566
−1895
42500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














309(A)
2050
−1857
−1081
−526
−2013
−2012
188
−1645
−385
−1213
−1011
486
−2103
−346
1657
−953
306
−261
−2189
−1624
42600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














310(G)
−1848
−2469
−2089
−2292
1262
2944
−2358
−3563
−2904
−3628
−3017
2347
−3167
−2550
−3185
−1986
−2162
−3006
−2814
−1874
42700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














311(Y)
475
1019
−1606
−1042
225
−2192
−935
−946
−891
−1226
1222
357
−2267
1002
−1577
−1172
−888
−800
−1730
2446
42800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














312(P)
−87
−2372
−1362
756
−3738
−2205
−2007
−3445
−1924
−3575
−2761
−1555
3598
−1697
−2362
−1566
286
−2803
−3832
−3277
42900%


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



—
−2336
−8139
−325
−894
−1115
−701
−1378
*
*














313(K)
−804
−1483
−564
−230
−1920
−1335
−101
−1605
2889
−1630
−1021
−349
−1569
232
698
−786
−759
−1358
−1637
−1317
43000%


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



—
−38
−5840
−6882
−894
−1115
−3098
−179
*
*














314(E)
−766
−1695
521
2831
−2050
−1029
−293
−1804
−118
−1919
−1331
69
−1441
−4
−527
−653
−814
−1512
−1988
−1505
43100%


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



—
−38
−5840
−6882
−894
−1115
−109
−3375
*
*














315(T)
−942
−2382
−739
1086
−2714
151
−581
−2459
−171
−2415
−1499
414
−2004
−128
839
1365
1730
−2017
−2592
−1915
43200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














316(M)
−2196
−1920
−4499
−3891
1726
−3822
−2504
−645
−3523
1973
3030
−3442
−3673
−2938
−3257
−2944
−2114
326
−2014
1662
43300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














317(H)
−883
−2314
−747
517
−2618
−1863
1714
−647
1272
−2322
−1408
1011
472
−69
433
−772
1411
−299
−2507
−1836
43400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














318(A)
2474
−2397
−816
−367
−2797
−273
−722
−2529
555
−2507
−1610
592
−2110
837
−805
−138
−1006
−2092
−2699
−2039
43500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














319(M)
−154
−986
−2485
−337
1024
−375
−1232
325
−444
867
1235
−1752
−2474
−1419
1020
−1455
670
−411
831
535
43600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














320(R)
−1311
−2432
−1349
−724
−2724
−2272
−799
−2361
613
−644
1079
976
597
−382
2908
−1246
−1219
−2044
−2579
−2061
43700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














321(R)
897
−2364
−833
905
−2678
−1930
−568
−2405
1293
−2366
1485
−575
−2020
−117
2045
95
−893
−1984
−2543
−1892
43800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














322(N)
505
−2300
−750
523
−2596
121
−525
−594
485
95
−1395
1720
−1957
348
224
551
−821
−1910
−2497
−1828
43900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














323(E)
444
−2266
−766
1488
−2551
−1871
−533
−2275
889
−2266
1474
1478
−1963
682
−629
−781
12
−279
−2472
476
44000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














324(N)
1511
−1170
−728
−204
−2244
−1781
−426
282
601
−2121
−1133
1769
−1827
−1398
447
−748
−766
−1713
−1779
−1595
44100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














325(N)
1053
−3109
1756
1735
−3404
−2143
−1074
−3191
−846
−3124
−2254
2158
−2417
−657
−1430
−1361
197
−2727
−3303
−2540
44200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














326(H)
−2064
−3071
−1245
−1267
−3262
−2570
4711
−3611
−1060
−3525
−2812
−1479
−2961
1288
−1287
670
−2133
−3161
−3310
−2601
44300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














327(Q)
−891
−2294
332
948
−2585
−739
−537
−2316
−131
−138
−1391
−518
1404
2021
−634
−785
619
−561
−2495
−1830
44400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














328(I)
−1632
−1661
−2846
−86
−1626
−2983
−1663
3240
−1327
−191
−722
−2160
−3016
−1615
822
−2097
−1556
−560
−2157
−1805
44500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














329(E)
−2734
−3605
−1382
3593
−3624
−2986
−2317
−3317
−2175
−3167
1983
−1898
−3440
−2054
−2556
−2649
−2820
−3234
−3920
−3370
44600%


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



—
−2336
−8139
−325
−894
−1115
−701
−1378
*
*














330(W)
−1530
−1265
−2068
−1964
479
−1810
−483
−1181
−1470
−968
−802
−1648
−2104
−1454
−1405
−1757
−1583
−1218
5462
838
44700%


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



—
−38
−5840
−6882
−894
−1115
−109
−3775
*
*














331(K)
8
−2067
−905
437
−2275
−1941
−611
−307
2031
−2031
−1189
−636
−2030
1425
−709
−864
−350
1337
−2323
−1722
44800%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














332(V)
−2445
−2012
−5067
−4708
−2628
−4682
−4459
1586
−4580
−1533
−1402
−4414
−4575
−4456
−4653
−3990
1117
3227
−4065
−3597
44900%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














333(G)
−4435
−4203
−5092
−5462
−5893
3834
−5028
−6627
−5765
−6297
−5970
−5141
−4804
−5546
−5385
−4727
−4815
−5862
−4924
−5849
45000%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














334(E)
1477
−2744
−762
2410
−3114
−2113
−884
−2850
−445
−2792
−1912
619
−2297
−451
1346
−1191
−1280
−2412
−2952
−2288
45100%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














335(K)
−1204
−2643
366
−1309
−3998
−2086
−724
−2722
2626
−2637
−1741
−718
−2200
1198
862
−1073
−1133
−2287
−2770
−2133
45200%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














336(L)
−3571
−3023
−5954
−5375
−1321
−5646
−4390
−632
−5138
2962
1671
−5358
−4852
−4044
−4689
−4963
−3436
742
−3082
−3239
45300%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














337(R)
−4845
−4446
−5107
−4682
−5507
−4412
−3791
−5676
−2789
−5502
−5118
−4521
−4754
−3672
4219
−4989
−4832
−5644
−4538
−4993
45400%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














338(E)
943
−2422
1002
1200
−2741
377
−572
−2493
982
−2439
−1517
−522
−1998
−117
−681
1129
−534
−2044
−2609
−1921
45500%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














339(M)
−3391
−2886
−5774
−5202
−1338
−5407
−4210
−576
−4943
1721
4369
−5109
−4742
−3639
−4548
−4689
−3273
771
−3038
−3137
45600%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














340(M)
−1812
−1601
−3997
−3375
696
−3401
−2222
−516
−2983
441
4360
−2991
−3334
−2559
641
−2503
−1740
404
−1934
−1661
45700%


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



—
−8
−8139
−9181
−894
−1115
−701
−1378
*
*














341(P)
102
−1789
−1729
−1313
428
−2112
−1410
−2021
−1236
−2235
−1470
−1394
3205
695
−1607
703
−1207
−1700
−2613
−2121
45800%


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



—
−259
−8139
−2637
−894
−1115
−701
−1378
*
*














342(W)
−3486
−3022
−4312
−4121
1891
−4228
−1173
−2749
310
−2389
−2250
−3147
−4206
−2971
−3005
−3484
−3396
−2818
5644
611
45900%


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



—
−239
−7889
−2749
−894
−1115
−1590
−583
*
*














343(I)
−2220
−1737
−4860
−4531
−2271
−4617
−4497
3348
−4448
−1059
−1008
−4311
−4707
−4340
−4559
−3974
−2216
2150
−3915
−3445
46000%


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



—
−114
−7660
−3820
−894
−1115
−1149
−865
*
*














344(A)
1699
−2218
532
−33
−2555
−553
−413
−2304
1212
−2260
−1348
582
−1822
37
−536
966
−724
−1859
−2439
1755
46100%


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



—
−10
−7753
−8795
−894
−1115
−897
−1111
*
*














345(A)
1523
−2068
−769
−231
−2383
1040
−522
−2084
928
−2120
−1245
−517
−1922
1335
−611
251
−768
−123
−2361
−1732
46200%


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



—
−9
−7949
−8991
−894
−1115
−1432
−668
*
*














346(N)
−1650
−3264
1760
−348
−3555
−230
−1131
−3362
324
−3285
−2448
2847
1624
−733
−1607
−1438
−1645
−2887
−3466
−2662
46300%


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



—
−193
−7842
−3049
−894
−1115
−1432
−668
*
*














347(K)
150
−2932
−2433
−1483
−3710
−2747
−907
−3141
3369
−2897
−2178
−1487
−2774
−488
1175
−1994
−1888
−2848
−2822
−2610
46400%


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



—
−11
−7660
−8702
−894
−1115
−1824
−479
*
*














348(L)
−740
−922
−1768
154
−921
−2070
−829
−1384
247
1472
−100
−1202
−2134
−805
485
−1085
−677
471
−1340
−944
46500%


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



—
−11
−7660
−8702
−894
−1115
−943
−1059
*
*














349(V)
138
−1046
−3186
−2599
−1089
−2803
−1711
589
645
945
−236
−2305
−2803
−2012
−2236
−1892
−1154
−253
−1701
−1344
46600%


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



—
−9
−7842
−8885
−894
−1115
−380
−2109
*
*














350(D)
−2086
−3722
2888
1158
−4001
1601
−1510
−3811
−1573
−3773
−3018
−904
−2733
−1154
−2294
−1833
−2125
575
−3993
−3117
46700%


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



—
−8
−8046
−9088
−894
−1115
−701
−1378
*
*














351(K)
−78
−2308
398
−137
−2626
−483
542
−2374
2441
−2323
−1400
−447
531
720
−562
−724
290
−1929
−2493
−1812
46800%


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



—
−8
−8046
−9088
−894
−1115
−701
−1378
*
*














352(D)
898
−1911
1604
−335
−2089
−1892
−573
−1746
489
−144
811
−609
34
−182
−708
541
−74
−281
−2188
−1605
46900%


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



—
−10
−7745
−8787
−894
−1115
−701
−1378
*
*














353(K)
−1676
−2544
−1956
−1135
−2823
−2531
−800
−2469
2955
−2426
−1683
−1204
−2548
−407
1469
−1628
−1517
−119
−2497
1619
47000%


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



—
−11
−7649
−8691
−894
−1115
−701
−1378
*
*














354(N)
−1074
−1925
−1092
−1190
−3523
−1781
−1760
−3345
−1701
−3432
−2589
3429
1327
−1460
−2119
1182
−1362
−2558
−3601
3046
47100%


—
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*



—
*
*
*
*
*
*
*
*
0

## Claims

1. A recombinant host cell comprising a mutant ketol-acid reductoisomerase enzyme, wherein said enzyme has a ketol-acid reductoisomerase HMM search profile E value of <10−3 using the hmmsearch program and comprises at least one mutation within the phosphate binding loop of said enzyme wherein said at least one mutation comprises a substitution at the residue corresponding to T52 of SEQ ID NO:17 wherein the substitution at residue T52 is a substitution to a polar, negatively charged amino acid.

2. The recombinant host cell of claim 1, wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.

3. The recombinant host cell of claim 2, wherein the host cell is a Saccharomyces.

4. The recombinant host cell of claim 1, wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.

5. The recombinant host cell of claim 1, wherein the host cell further comprises an acetolactate synthase enzyme.

6. The recombinant host cell of claim 5, wherein the acetolactate synthase enzyme is a Bacillus subtilis acetolactate synthase enzyme.

7. A method of converting acetolactate to 2,3-dihydroxyisovalerate, the method comprising:
(a) providing a recombinant host cell comprising a mutant ketol-acid reductoisomerase enzyme, wherein said enzyme has a ketol-acid reductoisomerase HMM search profile E value of <10−3 using the hmmsearch program and comprises at least one mutation within the phosphate binding loop of said enzyme wherein said at least one mutation comprises a substitution at the residue corresponding to T52 of SEQ ID NO:17 wherein the substitution at residue T52 is a substitution to a polar, negatively charged amino acid; and
(b) contacting the recombinant host cell with acetolactate, wherein the recombinant host cell converts acetolactate to 2,3-dihydroxyisovalerate.

8. The method of claim 7, wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.

9. The method of claim 8, wherein the recombinant host cell is a Saccharomyces.

10. The method of claim 7, wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.

11. A method of producing isobutanol, the method comprising:
(a) providing a recombinant host cell comprising an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises a mutant ketol-acid reductoisomerase enzyme, wherein the enzyme has a ketol-acid reductoisomerase HMM search profile E value of <10−3 using the hmmsearch program and comprises at least one mutation within the phosphate binding loop of said enzyme wherein said at least one mutation comprises a substitution at the residue corresponding to T52 of SEQ ID NO:17 wherein the substitution at residue T52 is a substitution to a polar, negatively charged amino acid; and
(b) contacting the recombinant host cell with a fermentable carbon substrate, wherein the recombinant host cell produces isobutanol.

12. The method of claim 11, wherein the recombinant host cell is a member of a genera selected from Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.

13. The method of claim 12, wherein the recombinant host cell is a Saccharomyces.

14. The method of claim 11, wherein the mutant ketol-acid reductoisomerase enzyme comprises a GxGxx(G/A) cofactor binding motif.

15. The method of claim 11, wherein the recombinant host cell further comprises an acetolactate synthase enzyme.

16. The method of claim 15, wherein the acetolactate synthase enzyme is a Bacillus subtilus acetolactate synthase enzyme.