# Patent

## Title
Yeast with increased butanol tolerance involving cell wall integrity pathway

## Abstract

Increasing tolerance to butanol in yeast has been accomplished by increasing activity of the cell wall integrity pathway. Yeast with increased expression of SLT2p, a mitogen activated protein kinase of the MAPK module of the cell wall integrity pathway had increased tolerance to isobutanol. These yeast may be used for improved butanol production.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 61/141,009, filed Dec. 29, 2008, the entirety of which is herein incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the field of microbiology and genetic engineering. More specifically, yeast genes that are involved in the cell response to butanol were identified. These genes may be engineered to improve growth yield in the presence of butanol.
BACKGROUND OF THE INVENTION
Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Butanol may be made through chemical synthesis or by fermentation. Isobutanol is produced biologically 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 this group of fungi. Isobutanol is specifically produced from catabolism of L-valine and the yield is typically very low. Additionally, recombinant microbial production hosts, expressing a 1-butanol biosynthetic pathway (Donaldson et al., U.S. Patent Application Publication No. US20080182308A1), a 2-butanol biosynthetic pathway (Donaldson et al., U.S. Patent Publication Nos. US 20070259410A1 and US 20070292927), and an isobutanol biosynthetic pathway (Maggio-Hall et al., U.S. Patent Publication No. US 20070092957) have been described.
Biological production of butanols is believed to be limited by butanol toxicity to the host microorganism used in fermentation for butanol production. Yeast are typically sensitive to butanol in the medium. Using a screen for 1-butanol insensitive Saccharomyces cerevisiae mutants, Lorenz et al. (Molec. Biol. of the Cell (2000) 11:183-199) identified proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG. 1), mitochondrial function (MSM1, MRP21, and HM11), and a transcriptional regulator (CHD1). They also found that 1-butanol stimulates filamentous growth in haploid cells and induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology. Ashe et al. (The EMBO Journal (2001) 20:6464-6474) found that butanol brings about a rapid inhibition of translation at the initiation step in Saccharomyces cerevisiae. The GCD1-P180 allele has a single amino acid change in Gcd1p, which is part of the eIF2B guanine nucleotide complex that is responsible for recycling eIF2-GDP to eIF2-GTP, that allows translational regulation upon butanol addition. Smirnova et al. (Molecular and Cellular Bioloty (2005) 25:9340-9340) found by using microarray analysis that with addition of fusel alcohol, there is widespread translational reprogramming in yeast. These studies all indicate the complexity of butanol sensitivity in yeast.
Van Voorst et al. (Yeast 23(5)351-359 (2006)) found that the MAP kinase of the cell wall integrity pathway, Slt2p, was phosphorylated when cells were treated with 6% ethanol. Isoamyl alcohol induces a phenotype that resembles pseudohyphae in S. cerevisiae, that requires the activity of SWE1 and SLT2 (Martinez-Anaya et al. (2003) Journal of Cell Science 116, 3423-3431, (2003); Levin, D. E. (2005) Microbiol. Mol. Biol. Rev.: 262-291). A complete set of S. cerevisiae homozygous deletions was screened for tolerance to the aliphatic alcohols ethanol, propanol and pentanol (Fuijta et al. (2006) FEMS Yeast Res 6:744-750). Mutant SLT2 deletion strains were hypersensitive to ethanol and propanol but not to pentanol,
There remains a need for yeast cells with increased tolerance to butanol, as well as methods of producing butanols using yeast host strains that are more tolerant to these chemicals. To this end applicants have Identified genes in yeast that are involved in butanol tolerance, that can be engineered to increase the level of butanol tolerance in yeast cells used for butanol production.
SUMMARY OF THE INVENTION
Provided herein are recombinant yeast cells comprising: a) a butanol biosynthetic pathway; and b) at least one genetic modification which increases activity of the cell wall integrity pathway; wherein the butanol biosynthetic pathway comprises at least one gene that is heterologous to the yeast cell and wherein the yeast cell has an increase in tolerance to butanol as compared with a yeast cell that lacks the at least one genetic modification of (b).
In some embodiments, the cell has at least about a 25% improvement in growth yield in 1% (w/v) isobutanol as compared to a parental cell having no increase in activity of the cell wall integrity pathway. In some embodiments, the yeast is selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia. 
In some embodiments, the genetic modification increases activity of the mitogen-activated protein kinase module of the cell wall integrity pathway. In some embodiments, the genetic modification increases SLT2p serine/threonine MAP kinase activity. In some embodiments, the modification increasing SLT2 serine/threonine MAP kinase activity is overexpression of an SLT2 protein encoding gene.
In some embodiments, the SLT2 protein encoding gene encodes a protein having an amino acid sequence with at least about 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs:50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74 based on Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. In some embodiments, the genetic modification increasing the activity of the mitogen-activated protein kinase module is the overexpression of a gene selected from the group consisting of WSC1, WSC2, WSC3, MID2, MTL1, HOR2, YPT7, ROM2, BEM2, ABC1, RHO1, EPT1, STB5, YDJ1, SSD1, CNA1, KRE11, EXG1, EXG2, DGK1, HCR1, LOC1, PDR2, PDR8 and RLM1.
Also provided herein is a recombinant yeast cell comprising a butanol biosynthetic pathway and at least one heterologous SLT2 protein encoding gene. In some embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the yeast cell. In some embodiments, the yeast cell has an increased tolerance to butanol as compared to a yeast cell that does not comprise at least one heterologous SLT2 protein encoding gene.
In some embodiments, the butanol biosynthetic pathway is selected from the group consisting of: a) a 1-butanol biosynthetic pathway; b) a 2-butanol biosynthetic pathway; and c) an isobutanol biosynthetic pathway. In some embodiments, the 1-butanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions: a) acetyl-CoA to acetoacetyl-CoA, as catalyzed by acetyl-CoA acetyltransferase; b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed by 3-hydroxybutyryl-CoA dehydrogenase; c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed by crotonase; d) crotonyl-CoA to butyryl-CoA, as catalyzed by butyryl-CoA dehydrogenase; e) butyryl-CoA to butyraldehyde, as catalyzed by butyraldehyde dehydrogenase; and f) butyraldehyde to 1-butanol, as catalyzed by 1-butanol dehydrogenase. In some embodiments, the 2-butanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions: a) pyruvate to alpha-acetolactate, as catalyzed by acetolactate synthase; b) alpha-acetolactate to acetoin, as catalyzed by acetolactate decarboxylase; c) acetoin to 2,3-butanediol, as catalyzed by butanediol dehydrogenase; d) 2,3-butanediol to 2-butanone, as catalyzed by butanediol dehydratase; and e) 2-butanone to 2-butanol, as catalyzed by 2-butanol dehydrogenase. In some embodiments, the isobutanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions: a) pyruvate to acetolactate, as catalyzed by acetolactate synthase; b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by acetohydroxy acid isomeroreductase; c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed by acetohydroxy acid dehydratase or dihydroxyacid dehydratase; d) α-ketoisovalerate to isobutyraldehyde, as catalyzed by a branched-chain keto acid decarboxylase; and e) isobutyraldehyde to isobutanol, as catalyzed by a branched-chain alcohol dehydrogenase.
Also provided herein are methods for the production of 1-butanol comprising growing a recombinant yeast cell under conditions where 1-butanol is produced and optionally recovering the 1-butanol. Also provided are methods for the production of 2-butanol comprising growing a recombinant yeast cell under conditions where 2-butanol is produced and optionally recovering the 2-butanol. Also provided are methods for the production of isobutanol comprising growing a recombinant yeast cell of under conditions where isobutanol is produced and optionally recovering the isobutanol.
Also provided are methods for producing a recombinant yeast cell having increased tolerance to butanol comprising: a) providing a recombinant yeast cell comprising a butanol biosynthetic pathway selected from the group consisting of : i) a 1-butanol biosynthetic pathway; ii) a 2-butanol biosynthetic pathway; and iii) an isobutanol biosynthetic pathway; and b) engineering the yeast cell of (a) to comprise at least one genetic modification which increases activity of the cell wall integrity pathway wherein the genetic modification increases SLT2p serine/threonine MAP kinase activity.
Also provided herein is a method for improving fermentative production of butanol comprising:
a) providing a recombinant yeast cell comprising a butanol biosynthetic pathway selected from the group consisting of:
i) a 1-butanol biosynthetic pathway
ii) a 2-butanol biosynthetic pathway; and
iii) an isobutanol biosynthetic pathway;
wherein said yeast cell also comprises at least one genetic modification that increases activity of the cell wall integrity pathway; and
b) contacting said yeast cell with fermentable sugar whereby said yeast cell produces butanol and wherein said yeast cell has improved tolerance to said butanol as compared to a yeast cell without at least one genetic modification that increases activity of the cell wall integrity pathway. Said method also provides for improved production of butanol as compared to a yeast cell without at least one genetic modification that increases activity of the cell wall integrity pathway.



BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
The various embodiments of the invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this application.
FIG. 1 depicts isobutanol biosynthetic pathways.
FIG. 2 depicts 1-butanol biosynthetic pathways.
FIG. 3 depicts 2-butanol biosynthetic pathways.
FIG. 4 is a cell wall integrity (CWI) pathway schematic.



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 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 37 C.F.R. §1.822.







TABLE 1







Summary of Coding Region and Protein SEQ ID Numbers for 1-


Butanol Biosynthetic Pathway










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












Acetyl-CoA acetyltransferase thlA from
1
2


Clostridium acetobutylicum ATCC 824


Acetyl-CoA acetyltransferase thlB from
3
4


Clostridium acetobutylicum ATCC 824


Acetyl-CoA acetyltransferase from
39
40


Saccharomyces cerevisiae


3-Hydroxybutyryl-CoA dehydrogenase
5
6


from Clostridium acetobutylicum ATCC


824


Crotonase from Clostridium
7
8


acetobutylicum ATCC 824


Putative trans-enoyl CoA reductase from
9
10


Clostridium acetobutylicum ATCC 824


Butyraldehyde dehydrogenase from
11
12


Clostridium beijerinckii NRRL B594


1-Butanol dehydrogenase bdhB from
13
14


Clostridium acetobutylicum ATCC 824


1-Butanol dehydrogenase
15
16


bdhA from Clostridium acetobutylicum


ATCC 824
















TABLE 2







Summary of Coding Region and Protein SEQ ID Numbers for 2-


Butanol Biosynthetic Pathway










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












budA, acetolactate decarboxylase from
17
18


Klebsiella pneumoniae ATCC 25955


budB, acetolactate synthase from
19
20


Klebsiella pneumoniae ATCC 25955


budC, butanediol dehydrogenase from
21
22


Klebsiella pneumoniae IAM1063


pddA, butanediol dehydratase alpha
23
24


subunit from Klebsiella oxytoca ATCC


8724


pddB, butanediol dehydratase beta
25
26


subunit from Klebsiella oxytoca ATCC


8724


pddC, butanediol dehydratase gamma
27
28


subunit from Klebsiella oxytoca ATCC


8724


sadH, 2-butanol dehydrogenase from
29
30


Rhodococcus ruber 219
















TABLE 3







Summary of Coding Region and Protein SEQ ID Numbers for


Isobutanol Biosynthetic Pathway










SEQ ID NO:
SEQ ID NO:


Description
Nucleic acid
Peptide












Klebsiella pneumoniae budB
19
20


(acetolactate synthase)


Bacillus subtilis alsS
41
42


(acetolactate synthase)


E. coli ilvC (acetohydroxy acid
31
32


reductoisomerase)


S. cerevisiae ILV5
43
44


(acetohydroxy acid reductoisomerase)


B. subtilis ilvC (acetohydroxy acid
45
46


reductoisomerase)


E. coli ilvD (acetohydroxy acid
33
34


dehydratase)


S. cerevisiae ILV3
47
48


(Dihydroxyacid dehydratase)


Lactococcus lactis kivD (branched-chain
35
36


α-keto acid decarboxylase), codon


optimized


E. coli yqhD (branched-chain alcohol
37
38


dehydrogenase)
















TABLE 4







Summary of SLT2 Coding Region and Protein SEQ ID Numbers












SEQ ID NO:
SEQ ID NO:



Organism
Nucleic acid
Peptide















Saccharomyces cerevisiae
49
50



Candida glabrata
51
52



Candida albicans
53
54



Yarrowia lipolytica
55
56



Pichia stipitis
57
58



Debaryomyces hansenii
59
60



Kluyveromyces lactis
61
62



Ashbya gossypii
63
64



Apergillus fumigatus
65
66



Apergillus terreus
67
68



Coprinopsis cinerea
69
70



Schizosaccharomyces pombe
71
72



Ustilago maydis
73
74










SEQ ID NOs:75 and 76 are primers used for sequencing the ends of the S. cerevisiae genomic DNA insert of the plasmid in the yBUT11 clone.
SEQ ID NOs:77 and 78 are primers for PCR of iYDR006C.
SEQ ID NOs:79 and 80 are primers for PCR of iYDR008C.
SEQ ID NOs:81 and 82 are primers for PCR of upTRP1 DR UR.
SEQ ID NOs:83 and 84 are primers for PCR of RA3* DR downTRP1.
SEQ ID NO:85 is the nucleotide sequence of the CUP1 promoter.
SEQ ID NO:86 is the nucleotide sequence of the CYC1 terminator.
SEQ ID NO:87 is the nucleotide sequence of the FBA promoter.
SEQ ID NO:88 is the nucleotide sequence of the ADH1 terminator.
SEQ ID NO:89 is the nucleotide sequence of the GPM promoter.
SEQ ID NOs:90 and 91 are primers for PCR of the SLT2 gene and flanking DNA.
DETAILED DESCRIPTION
The present invention relates to recombinant yeast cells that are engineered for production of butanol and that additionally are engineered to have increased activity of the cell wall integrity (CWI) pathway. The present yeast cells may have increased expression or activity of at least one protein involved in promoting activity of the CWI pathway including proteins that are CWI pathway receptors of external stimuli, proteins that are in the mitogen-activated protein kinase (MAPK) module of the CWI pathway, and proteins that are downstream targets of the MAPK module of the CWI pathway. These yeast cells have increased tolerance to butanol and may be used for production of butanol which is valuable as a fuel or fuel additive to reduce demand for fossil fuels.
The following abbreviations and definitions will be used for the interpretation of the specification and the claims.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value
The term “butanol” as used herein, refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.
The terms “butanol tolerant yeast cell” and “tolerant” when used to describe a modified yeast cell of the invention, refers to a modified yeast that shows better growth in the presence of butanol than the parent strain from which it is derived.
The term “butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.
The term “1-butanol biosynthetic pathway” refers to an enzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).
The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.
The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.
The term “mitogen activated protein (MAP) kinase” refers to proteins with EC number EC 2.7.11.24, which are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis.
The term “SLT2” refers to a gene encoding a type of MAP kinase that is a part of the mitogen-activated protein kinase module of the cell wall integrity pathway. SLT2p is the protein encoded by SLT2, which is an example of proteins classified as EC 2.7.11.24. Proteins with the same function as SLT2p encoded by SLT2 may be referred to in the art as being encoded by genes with other names including BYC2, MPK1, SLK2, MPKA, SPM1, MKC1, PMK1, and PIM1. The term SLT2p refers herein to any of these encoded proteins that function similarly to SLT2p in the mitogen-activated protein kinase module of the cell wall integrity pathway and that have sequence identity to an SLT2p amino acid sequence that is at least about 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.
The term “acetyl-CoA acetyltransferase” refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Preferred acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP—416728, NC—000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP—349476.1 (SEQ ID NO:2), NC—003030; NP—149242 (SEQ ID NO:4), NC—001988), Bacillus subtilis (GenBank Nos: NP—390297, NC—000964), and Saccharomyces cerevisiae (GenBank Nos: NP—015297, NC—001148 (SEQ ID NO:39)).
The term “3-hydroxybutyryl-CoA dehydrogenase” refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3-Hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide (NADH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP—349314 (SEQ ID NO:6), NC—003030), B. subtilis (GenBank NOs: AAB09614, U29084), Ralstonia eutropha (GenBank NOs: ZP—0017144, NZ_AADY01000001, Alcaligenes eutrophus (GenBank NOs: YP—294481, NC—007347), and A. eutrophus (GenBank NOs: P14697, J04987).
The term “crotonase” refers to an enzyme that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H2O. Crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, E. coli (GenBank NOs: NP—415911 (SEQ ID NO:8), NC—000913), C. acetobutylicum (GenBank NOs: NP—349318, NC—003030), B. subtilis (GenBank NOs: CAB13705, Z99113), and Aeromonas caviae (GenBank NOs: BAA21816, D88825).
The term “butyryl-CoA dehydrogenase”, also called trans-enoyl CoA reductase, refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Butyryl-CoA dehydrogenases may be NADH-dependent or NADPH-dependent and are classified as E.C. 1.3.1.44 and E.C. 1.3.1.38, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP—347102 (SEQ ID NO:10), NC—003030), Euglena gracilis (GenBank NOs: Q5EU90, AY741582), Streptomyces collinus (Gen Bank NOs: AAA92890, U37135), and Streptomyces coelicolor (GenBank NOs: CAA22721, AL939127).
The term “butyraldehyde dehydrogenase” refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841 (SEQ ID NO:12), AF157306) and C. acetobutylicum (GenBank NOs: NP—149325, NC—001988).
The term “1-butanol dehydrogenase” refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol. 1-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 1-butanol dehydrogenase may be NADH- or NADPH-dependent. 1-butanol dehydrogenases are available from, for example, C. acetobutylicum (GenBank NOs: NP—149325, NC—001988; NP—349891 (SEQ ID NO:14), NC—003030; and NP—349892 (SEQ ID NO:16), NC—003030) and E. coli (GenBank NOs: NP—417484, NC—000913).
The term “acetolactate synthase”, also known as “acetohydroxy acid synthase”, refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of two molecules of pyruvic acid to one molecule of alpha-acetolactate. Acetolactate synthase, known as EC 2.2.1.6 [formerly 4.1.3.18] (Enzyme Nomenclature 1992, Academic Press, San Diego) may be dependent on the cofactor thiamin pyrophosphate for its activity. Suitable acetolactate synthase enzymes are available from a number of sources, for example, Bacillus subtilis (GenBank Nos: AAA22222 NCBI (National Center for Biotechnology Information) amino acid sequence (SEQ ID NO:42), L04470 NCBI nucleotide sequence (SEQ ID NO:41)), Klebsiella terrigena (GenBank Nos: AAA25055, L04507), and Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:20), M73842 (SEQ ID NO:19).
The term “acetolactate decarboxylase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Acetolactate decarboxylases are known as
EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (SEQ ID NO:18 (amino acid) SEQ ID NO:17 (nucleotide)).
The term “butanediol dehydrogenase” also known as “acetoin reductase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of R- or S-stereochemistry in the alcohol product. S-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085 (SEQ ID NO:22), D86412. R-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP—830481, NC—004722; AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323).
The term “butanediol dehydratase”, also known as “diol dehydratase” or “propanediol dehydratase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone, also known as methyl ethyl ketone (MEK). Butanediol dehydratase may utilize the cofactor adenosyl cobalamin. Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: BAA08099 (alpha subunit) (SEQ ID NO:24), BAA08100 (beta subunit) (SEQ ID NO:26), and BBA08101 (gamma subunit) (SEQ ID NO:28), (Note all three subunits are required for activity), D45071).
The term “2-butanol dehydrogenase” refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2-butanone to 2-butanol. 2-butanol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. 2-butanol dehydrogenase may be NADH- or NADPH-dependent. The NADH-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475 (SEQ ID NO:30), AJ491307 (SEQ ID NO:29)). The NADPH-dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169).
The term “acetohydroxy acid isomeroreductase” or “acetohydroxy acid reductoisomerase” refers to an enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADPH (reduced nicotinamide adenine dinucleotide phosphate) as an electron donor. Preferred acetohydroxy acid isomeroreductases are known by the EC number 1.1.1.86 and sequences are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP—418222 (SEQ ID NO:32), NC—000913 (SEQ ID NO:31)), Saccharomyces cerevisiae (GenBank Nos: NP—013459 (SEQ ID NO:44), NC—001144 (SEQ ID NO:43)), Methanococcus maripaludis (GenBank Nos: CAF30210, BX957220), and Bacillus subtilis (GenBank Nos: CAB14789 (SEQ ID NO:46), Z99118 (SEQ ID NO:45)).
The term “acetohydroxy acid dehydratase” or “dihydroxy 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 (SEQ ID NO:34), NC—000913 (SEQ ID NO:33)), S. cerevisiae (GenBank Nos: NP—012550 (SEQ ID NO:48), NC—001142 (SEQ ID NO:47)), 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 lactis (GenBank Nos: AAS49166, AY548760; CAG34226 (SEQ ID NO:36), 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 (SEQ ID NO:38), NC—000913 (SEQ ID NO:37)), and C. acetobutylicum (GenBank Nos: NP—349892, NC—003030).
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 an organism. A “heterologous” or “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign or heterologous genes can comprise native genes inserted into a non-native organism, or chimeric genes. “Heterologous gene” includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. 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 codes 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 a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” 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 vector” 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.
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 organism without altering the polypeptide encoded by the DNA.
A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to identify putatively a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
As used herein, “substantially similar” enzymes will refer to enzymes belonging to a family of proteins in the art known to share similar structures and function. It is well within the skill of one in the art to identify substantially similar proteins given a known structure. Typical methods to identify substantially similar structures will rely upon known sequence information (nucleotide sequence and/or amino acid sequences) and may include PCR amplification, nucleic acid hybridization, and/or sequence identity/similarity analysis (e.g., sequence alignments between partial and/or complete sequences and/or known functional motifs associated with the desired activity).
A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Given the nucleic acid sequences described herein, one of skill in the art can identify substantially similar nucleic acid fragments that may encode proteins having similar activity. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (2001), particularly Chapter 11 and Table 11.1 therein. The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS at 65° C. followed by 0.1×SSC, 0.1% SDS at 65° C., for example.
In one aspect, suitable nucleic acid fragments encode polypeptides that are at least about 70% identical to the amino acid sequences reported herein. In another aspect, the nucleic acid fragments encode amino acid sequences that are about 85-90% identical to the amino acid sequences reported herein. In a further aspect, the nucleic acid fragments encode amino acid sequences that are at least about 90-100% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least about 50 amino acids, preferably at least about 100 amino acids, more preferably at least about 150 amino acids, still more preferably at least about 200 amino acids, and most preferably at least about 250 amino acids.
The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the Clustal method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10), unless otherwise specified. Default parameters for pairwise alignments using the Clustal method are: KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 75% identical, and more preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “homology” refers to the relationship among sequences whereby there is some extent of likeness, typically due to descent from a common ancestral sequence. Homologous sequences can share homology based on genic, structural, functional and/or behavioral properties. The term “ortholog” or “orthologous sequences” refers herein to a relationship where sequence divergence follows speciation (i.e., homologous sequences in different species arose from a common ancestral gene during speciation). In contrast, the term “paralogous” refers to homologous sequences within a single species that arose by gene duplication. One skilled in the art will be familiar with techniques required to identify homologous, orthologous and paralogous sequences.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein, “default values” will mean any set of values or parameters (as set by the software manufacturer) which originally load with the software when first initialized
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987.
“Fermentable sugars” refers to a sugar content primarily comprising monosaccharides and some disaccharides that can be used as the carbon source by microorganisms in a fermentation process to produce a target product. Sugars may from from any source, including cellulosic, hemicellulosic or ligcellulosic biomass.
Screening for Butanol Tolerance: Involvement of CWI Pathway
The invention relates to the discovery that increasing activity of the CWI pathway has the effect of increasing tolerance of yeast cells to butanol. The discovery came from screening studies to identify yeast cells containing random fragments of yeast genomic DNA that had increased tolerance to butanol. In these studies, yeast containing a library of random genomic DNA fragments were grown in the presence of isobutanol to identify clones with improved growth relative to controls. In one clone with increased tolerance to butanol the random genomic DNA fragment was found to include the STL2 gene. In further experiments herein, deletion of the SLT2 gene was found to increase sensitivity of yeast to butanol while overexpression of the SLT2 coding region was found to increase tolerance of yeast to butanol, A yeast strain which overexpressed the SLT2p product of the SLT2 coding region had a 29% improvement in growth yield over the parental strain in 1% (w/v) isobutanol (Example 3 herein). In some embodiments, yeast cells provided herein have at least about a 20% or at least about a 25% improvement in growth yield.
The SLT2p product of the SLT2 gene plays an important role in the CWI pathway. SLT2 encodes a protein kinase that phosphorylates serine and threonine residues of its target proteins. SLT2 is a mitogen activated protein kinase (MAPK) that responds to environmental signals and is involved in regulating the maintenance of cell wall integrity. Specifically, SLT2p is the MAPK of the MAPK module of the CWI pathway. Thus increase in SLT2p expression increases activity of the CWI pathway.
Increase in CWI Pathway Activity by Directly Engineering SLT2p Expression
In the present engineered yeast cell any SLT2p may be expressed in increased amount above the amount found in the cell without SLT2p engineering to provide increased butanol tolerance. In the present yeast cell the endogenous SLT2p of the target yeast cell may be overexpressed, or a heterologous SLT2p may be expressed in the cell to provide increased activity. Examples of SLT2p that may be expressed include those from Saccharomyces cerevisiae (coding region SEQ ID NO:49; protein SEQ ID NO:50), Candida glabrata (coding region SEQ ID NO:51; protein SEQ ID NO:52), Candida albicans (coding region SEQ ID NO:53; protein SEQ ID NO:54), Yarrowia lipolytica (coding region SEQ ID NO:55; protein SEQ ID NO:56), Pichia stipitis (coding region SEQ ID NO:57; protein SEQ ID NO:58), Debaryomyces hansenii (coding region SEQ ID NO:59; protein SEQ ID NO:60), Kluyveromyces lactis (coding region SEQ ID NO:61; protein SEQ ID NO:62), Ashbya gossypii (coding region SEQ ID NO:63; protein SEQ ID NO:64), Aspegillus fumigatus (coding region SEQ ID NO:65; protein SEQ ID NO:66), Aspergillus terreus (coding region SEQ ID NO:67; protein SEQ ID NO:68), Coprinopsis cinerea (coding region SEQ ID NO:69; protein SEQ ID NO:70), Schizosaccharomyces pombe (coding region SEQ ID NO:71; protein SEQ ID NO:72), and Ustilago maydis (coding region SEQ ID NO:73; protein SEQ ID NO:74).
Some proteins that can be recognized as performing the same function as SLT2p, that have high sequence identity to SLT2p, may have a different name in the art. Other names for the SLT2 gene include BYC2, MPK1, SLK2, MPKA, SPM1, MKC1, PMK1, and PIM1. Although there is this variation in naming, a protein may be readily recognized as an SLT2p homolog by its sequence and by its activity as a MAP kinase in the MAP kinase module of the cell wall integrity pathway.
Because the sequences of SLT2 coding regions and the encoded proteins are known, as exemplified in the SEQ ID NOs listed above and given in Table 4, suitable SLT2ps may be readily identified by one skilled in the art on the basis of sequence similarity using bioinformatics approaches. Typically BLAST (described above) searching of publicly available databases with known SLT2p amino acid sequences, such as those provided herein, is used to identify SLT2ps, and their encoding sequences, that may be used in the present strains. These proteins may have at least about 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 95%-99% sequence identity to any of the SLT2ps of SEQ ID NOS:50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, or 74 while having SLT2p activity. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
In addition to using protein or coding region sequence and bioinformatics methods to identify additional SLT2ps, the sequences described herein or those recited in the art may be used to experimentally identify other homologs in nature. For example each of the of SLT2 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.
For example, genes encoding similar proteins or polypeptides to the SLT2p encoding genes described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.
Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, “The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders”, in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).
Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding microbial genes.
Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL, Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).
Alternatively, the described SLT2p encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are “hybridizable” to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.
Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).
Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).
Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.
Expression of SLT2p is achieved by transforming with a gene comprising a sequence encoding an SLT2p. When using a heterologous coding region, the sequence may be codon-optimized for maximal expression in the target yeast host cell, as well known to one skilled in the art. Methods for gene expression in yeasts are known in the art (see for example Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding an SLT2p, including, but not limited to constitutive promoters FBA, GPD, ADH1, TEF, 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.
Suitable promoters, transcriptional terminators, and SLT2 coding regions may be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells. These vectors allow propagation in both E. coli and yeast strains. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2μ origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Construction of expression vectors with a chimeric gene encoding an SLT2p may be performed by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast.
The gap repair cloning approach takes advantage of the highly efficient homologous recombination in yeast. Typically, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain a ≧21 by sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X”, a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 by overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g. TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.
Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. Typically, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding regionX-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 by of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA. In addition the endogenous promoter of a gene may be replaced with a stronger promoter to increase expression by homologous recombination.
Additional Engineering to Increase CWI Pathway Activity
Increased expression of other genes of the CWI pathway may be engineered to provide yeast cells of the present invention that have increased tolerance to butanol. Target genes and their encoded proteins for increased expression in the present yeast cells include any gene whose increased expression causes increased activity of the CWI pathway. Target genes may include those that increase activity of SLT2p as well as any gene whose activity is increased by increased SLT2p activity. Genes of the CWI pathway are known in the art. For example, genes of the CWI pathway are disclosed in Kim et al. (Mol. Cell. Biol. (2008) 281:2579-89), Kim et al. (Yeast (2007) 241: 335-42), Jimenez-Sanchez and Molina (J. Biol. Chem. (2007) 282: 31174-85), Levin, D. E. (Microbiol. Mol. Biol. Rev. (2005): 262-291), Chen and Thorner (Biochimica et Biophysica Acta (2007) 1773: 1311-1340), all herein incorporated by reference. For example, genes that may be targets are in the MAPK module of the CWI pathway which includes SLT2. These genes include the MAPK kinases (MAPKK) MKK1 and MKK2, and the MAPKK kinase (MAPKKK) BCK1. Another target gene is PKC1, whose product PKC1p activates the MAPK module of the CWI pathway by phosphorylating BCK1p. Other target proteins activate PKC1p and are the products of the genes WSC1, WSC2, WSC3, MID2, MTL1, HOR2, YPT7, ROM2, BEM2, ABC1 and RHO1. In addition proteins in TOR2 and PKH1/PKH2 branches of the CWI pathway network activate PKC1p and may be targets in the present cells. Target genes for increased CWI pathway activity that are downstream of the MAPK module include EPT1, STB5, YDJ1, SSD1, CNA1, KRE11, EXG1, EXG2, DGK1, HCR1, LOC1, PDR2, PDR8, SWI4, SWI6, and RLM1. The SKN7 product is downstream of the RHO1 product and is also a target gene for increased CWI pathway activity.
The activity of any of the proteins encoded by these target genes may be increased by overexpressing the endogenous encoding sequence in a yeast cell or by expressing a heterologous sequence encoding the protein. Expression of any of these proteins may be accomplished as described above for SLT2p. The coding sequences and encoded proteins that may be used in the present cells may be readily identified in publicly available databases by one skilled in the art using the gene names and functions listed above. Any coding region to be expressed may be codon optimized for the host cell to be engineered, as well known to one skilled in the art.
Host Yeast Cells
The target genes and proteins that are engineered to provide an increase in CWI pathway activity to confer butanol tolerance may be engineered in any yeast cell that is additionally engineered for production of butanol. Suitable yeasts include, but are not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, and Pichia. Suitable strains include, but are not limited to, Saccharomyces cerevisiae Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata, Candida albicans, Pichia stipitis and Yarrowia lipolytica. 
Butanol Biosynthetic Pathway
In the present invention, a genetic modification conferring increased butanol tolerance, as described above, is engineered in a yeast cell that is engineered to express a butanol biosynthetic pathway. Either genetic modification may take place prior to the other. The butanol biosynthetic pathway may be a 1-butanol, 2-butanol, or isobutanol biosynthetic pathway.
Suitable biosynthetic pathways are known in the art, and certain suitable pathways are described herein. In some embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the yeast cell. In some embodiments, genes encoding proteins which catalyze each substrate to product conversion of the butanol biosynthetic pathway are heterologous to the yeast cell. In some embodiments, the butanol biosynthetic pathway comprises more than one gene that is heterologous to the yeast cell. In some embodiments, genes encoding proteins which catalyze each substrate to product conversion of the butanol biosynthetic pathway are heterologous to the yeast cell. In some embodiments, the yeast cell comprises heterologous genes encoding the proteins for each substrate to product conversion of a butanol biosynthetic pathway.
Likewise, certain suitable proteins having the ability to catalyze the indicated substrate to product conversions are described herein and other suitable proteins are described in the art. For example, US Published Patent Application Nos. US20080261230 and US20090163376, incorporated herein by reference, describe acetohydroxy acid isomeroreductases; US Patent Application No. 12/569,636, incorporated by reference, describes suitable dihydroxyacid dehydratases; a suitable alcohol dehydrogenase is described in US Published Patent Application US20090269823, incorporated herein by reference.
1-Butanol Biosynthetic Pathway
A suitable biosynthetic pathway for the production of 1-butanol that may be used is described by Donaldson et al. in U.S. Patent Application Publication No. US20080182308A1, incorporated herein by reference. This biosynthetic pathway comprises the following substrate to product conversions:

    a) acetyl-CoA to acetoacetyl-CoA, as catalyzed for example by acetyl-CoA acetyltransferase with protein sequence such as SEQ ID NO:2, 4 or 40 (which may be encoded, for example, by the genes given as SEQ ID NO:1, 3 or 39);
    b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, as catalyzed for example by 3-hydroxybutyryl-CoA dehydrogenase with protein sequence such as SEQ ID NO:6 (which may be encoded, for example, by the gene given as SEQ ID NO:5);
    c) 3-hydroxybutyryl-CoA to crotonyl-CoA, as catalyzed for example by crotonase with protein sequence such as SEQ ID NO:8 (which may be encoded, for example, by the gene given as SEQ ID NO:7);
    d) crotonyl-CoA to butyryl-CoA, as catalyzed for example by butyryl-CoA dehydrogenase with protein sequence such as SEQ ID NO:10 (which may be encoded, for example, by the gene given as SEQ ID NO:9);
    e) butyryl-CoA to butyraldehyde, as catalyzed for example by butyraldehyde dehydrogenase with protein sequence such as SEQ ID NO:12 (which may be encoded, for example, by the gene given as SEQ ID NO:11); and
    f) butyraldehyde to 1-butanol, as catalyzed for example by 1-butanol dehydrogenase with protein sequence such as SEQ ID NO:14 or 16 (which may be encoded, for example, by the genes given as SEQ ID NO:13 or 15).


The pathway requires no ATP and generates NAD+ and/or NADP+, thus, it balances with the central, metabolic routes that generate acetyl-CoA.
Other suitable biosynthetic pathways for the production of 1-butanol will be apparent to those of skill in the art. It will be appreciated that yeast cells may be engineered to express proteins that retain the ability to catalyze the indicated substrate to product conversion but have less than 100% sequence identity to the protein sequences provided herein. In one embodiment, yeast cells may be engineered to express a 1-butanol biosynthetic pathway comprising a sequence that has at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 95%-99% sequence identity to a 1-butanol pathway protein provided herein.
2-Butanol Biosynthetic Pathway
Suitable biosynthetic pathways for the production of 2-butanol that may be used are described by Donaldson et al. in U.S. Patent Application Publication Nos. US20070259410A1 and US 20070292927A1, each incorporated herein by reference. One 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

    a) pyruvate to alpha-acetolactate, as catalyzed for example by acetolactate synthase with protein sequence such as SEQ ID NO:20 (which may be encoded, for example, by the gene given as SEQ ID NO:19);
    b) alpha-acetolactate to acetoin, as catalyzed for example by acetolactate decarboxylase with protein sequence such as SEQ ID NO:18 (which may be encoded, for example, by the gene given as SEQ ID NO:17);
    c) acetoin to 2,3-butanediol, as catalyzed for example by butanediol dehydrogenase with protein sequence such as SEQ ID NO:22 (which may be encoded, for example, by the gene given as SEQ ID NO:21);
    d) 2,3-butanediol to 2-butanone, catalyzed for example by butanediol dehydratase with protein sequence such as SEQ ID NO:24, 26, or 28 (which may be encoded, for example, by genes given as SEQ ID NO:23, 25, or 27); and
    e) 2-butanone to 2-butanol, as catalyzed for example by 2-butanol dehydrogenase with protein sequence such as SEQ ID NO:30 (which may be encoded, for example, by the gene given as SEQ ID NO:29).


Other suitable biosynthetic pathways for the production of 2-butanol will be apparent to those of skill in the art. It will be appreciated that yeast cells may be engineered to express proteins that retain the ability to catalyze the indicated substrate to product conversion but have less than 100% sequence identity to the protein sequences provided herein. In one embodiment, yeast cells may be engineered to express a 2-butanol biosynthetic pathway comprising a sequence that has at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 95%-99% sequence identity to a 2-butanol pathway protein provided herein.
Isobutanol Biosynthetic Pathway
Suitable biosynthetic pathways for the production of isobutanol that may be used are described by Maggio-Hall et al. in U.S. Patent Application Publication No. US20070092957 A1, incorporated herein by reference. One isobutanol biosynthetic pathway comprises the following substrate to product conversions:

    a) pyruvate to acetolactate, as catalyzed for example by acetolactate synthase with protein sequence such as SEQ ID NO:20 or 42 (which may be encoded, for example, by genes given as SEQ ID NO:19 or 41);
    b) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed for example by acetohydroxy acid isomeroreductase with protein sequence such as SEQ ID NO:32, 44 or 46 (which may be encoded, for example, by genes given as SEQ ID NO:31, 43 or 45);
    c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed for example by acetohydroxy acid dehydratase with protein sequence such as SEQ ID NO:34 (which may be encoded, for example, by the gene given as SEQ ID NO:33); or dihydroxyacid dehydratase with protein sequence such as SEQ ID NO:48 (which may be encoded, for example, by the gene given as SEQ ID NO:47);
    d) α-ketoisovalerate to isobutyraldehyde, as catalyzed for example by a branched-chain keto acid decarboxylase with protein sequence such as SEQ ID NO:36 (which may be encoded, for example, by the gene given as SEQ ID NO:35); and
    e) isobutyraldehyde to isobutanol, as catalyzed for example by a branched-chain alcohol dehydrogenase with protein sequence such as SEQ ID NO:38 (which may be encoded, for example, by the gene given as SEQ ID NO:37).


Other suitable biosynthetic pathways for the production of isobutanol will be apparent to those of skill in the art. It will be appreciated that yeast cells may be engineered to express proteins that retain the ability to catalyze the indicated substrate to product conversion but have less than 100% sequence identity to the protein sequences provided herein. In one embodiment, yeast cells may be engineered to express an isobutanol biosynthetic pathway comprising a sequence that has at least about 70%-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or 95%-99% sequence identity to an isobutanol pathway protein provided herein.
Construction of Yeast Strains for Butanol Production
Any yeast strain that is genetically modified for butanol tolerance as described herein is additionally genetically modified (before or after modification to tolerance) to incorporate a butanol biosynthetic pathway by methods well known to one skilled in the art. Genes encoding the enzyme activities described above, or homologs that may be identified and obtained by commonly used methods, such as those described above, that are well known to one skilled in the art, are introduced into a yeast host. Representative coding and amino acid sequences for pathway enzymes that may be used are given in Tables 1, 2, and 3, with SEQ ID NOs:1-48. Methods for gene expression in yeasts that may be used for butanol pathway genes are described above for expression of SLT2.
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, cornsteep 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 organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic 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. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, 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 the growth of the cultures and promotion of the enzymatic pathway necessary for butanol production.
Culture Conditions
Typically cells are grown at a temperature in the range of about 20° C. to about 37° C. in an appropriate medium. Suitable growth media in the present invention are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media 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.
Suitable pH ranges for the fermentation are between pH 3.0 to pH 7.5, where pH 4.5.0 to pH 6.5 is preferred as the initial condition.
Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.
The amount of butanol produced in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).
Industrial Batch and Continuous Fermentations
The present process may employ 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 organism or organisms, 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 media. 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 V., 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 media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. 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 can be altered continuously while the cell concentration, measured by media 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 1-butanol production.
Methods for Butanol Isolation from the Fermentation Medium
The bioproduced butanol may be isolated from the fermentation medium using methods known in the art. For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium, which has been treated to remove solids as described above, using methods such as distillation, liquid-liquid extraction, or membrane-based separation. Because butanol forms a low boiling point, azeotropic mixture with water, distillation can only be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see for example Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol. In this method, the butanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
The butanol may also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent. Distillation in combination with adsorption may also be used to isolate butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al. Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).
Additionally, distillation in combination with pervaporation may be used to isolate and purify the butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).
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) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, 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, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).
Materials and methods suitable for the maintenance and growth of bacterial and yeast cultures are well known in the art. Techniques suitable for use in the following Examples may be found as set out 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) or in Yeast Protocols, Second Edition (Wei Xiao, ed; Humana Press, Totowa, N.J. (2006))). 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.
Methods for Determining Isobutanol Concentration in Culture Media
The concentration of isobutanol in the culture media can be determined by a number of methods known in the art. For example, a specific high performance liquid chromatography (HPLC) method utilizes a Shodex SH-1011 column with a Shodex SH-G guard column, both purchased from Waters Corporation (Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol has a retention time of 46.6 min under the conditions described. Alternatively, gas chromatography (GC) methods are available. For example, a specific GC method utilizes an HP-INNOWax column (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies, Wilmington, Del.), with a flame ionization detector (FID). The carrier gas is helium at a flow rate of 4.5 mL/min, measured at 150° C. with constant head pressure; injector split is 1:25 at 200° C.; oven temperature ias 45° C. for 1 min, 45 to 220° C. at 10° C/min, and 22° C. for 5 min; and FID detection is employed at 240° C. with 26 mL/min helium makeup gas. The retention time of isobutanol is 4.5 min.
The meaning of abbreviations is as follows: “s” means second(s), “min” means minute(s), “h” means hour(s), “psi” means pounds per square inch, “nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm” means nanometers, “mM” means millimolar, “μM” means micromolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s)“, “g” means gram(s), “μg” means microgram(s) and “ng” means nanogram(s), “PCR” means polymerase chain reaction, “OD” means optical density, “OD600” means the optical density measured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” means the gravitation constant, “bp” means base pair(s), “kbp” means kilobase pair(s), “% w/v” means weight/volume percent, % v/v” means volume/volume percent, “HPLC” means high performance liquid chromatography, and “GC” means gas chromatography.
CM refers to synthetic complete medium which is described in Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Example 1
Identification of SLT2 from an Isobutanol Tolerance Screen
A yeast genomic library in the E. coli/yeast shuttle vector YEp13 was obtained from the American Type Culture Collection (ATCC 37323; Reed et al. (1989) J. Cell Sci. Suppl. 12:29-37). The library was provided in an E. coli host and supplies complete coverage of the Saccharomyces cerevisiae genome with 10,000 clones containing random and/or overlapping fragments of genomic DNA. The YEp13 vector includes a leu marker. The library was amplified by growth of the E. coli mixed culture in LB with ampicillin (50 μg/ml) for 16 hours at 37° C. with shaking, isolated from E. coli using a Qiaprep Spin Miniprep Kit (Cat. No. 27104) and transformed into S. cerevisiae BY4741 cells (ATCC 201388) using a lithium acetate transformation procedure (Gietz et al. (1995) Yeast 11:355-360). Transformants were washed from selection plates (SD Agar minus leucine, called CSM-Leu; Cat #YPL-1540 of KD Medical; Columbia, Md.) and combined to form a pool of at least 10,000 S. cerevisiae BY4741 transformants. The pool was mixed with 25% glycerol and stored at −70° C.
Isolation of Isobutanol Tolerance Clones by Screening
The pool was plated on selective media (CM-Leu) and colonies grown at 30° C. were picked to liquid selective medium in microtiter wells and then grown for 2 days at 30° C. with shaking. Glycerol was added to a final concentration of 12% (v/v) and mixed by repeated pipetting prior to freezing at −80° C. Microtiter contents were thawed prior to printing to control and isobutanol containing agar plates (CM-Leu). Leu+ transformants of BY4741 (haploid) were obtained and picked to microtiter wells. Growth of more than 10,000 clones was compared on control plates (complete synthetic medium lacking leucine) and on the same plates containing 1.8% (w/v) isobutanol after 2 days at 30° C. This primary screen identified 638 putative isobutanol tolerant colonies. Positive-scoring colonies were consolidated by inoculating new microtiter plates containing CM-Leu liquid media that were incubated at 30° C. overnight. A secondary screen for isobutanol tolerant clones was accomplished by printing from the consolidated microtiter plates on 1.6%, 1.8% and 2% (w/v) isobutanol containing agar plates and observing growth. This secondary screen identified 14 tolerant clones with improved growth relative to controls, termed BUT (isoBUtanol Tolerant) clones.
Molecular Identity of the BUT Clones
The fragment of genomic DNA present in one of the 14 isobutanol tolerant strains named yBUT11 was characterized as follows. DNA was prepared from 2 μl of well mixed frozen cell glycerol stock (20% final) that was mixed with 9 μl of Genomiphi Sample Buffer prior to the addition of 1 μl zymolase [5 units] (ZymoResearch, Cat#E1004). Samples were incubated for 15 minutes at 37° C. The reaction was terminated by heating for 3 minutes at 95° C. (no longer). The heated sample was cooled at 4° C. for 5 minutes.
To amplify DNA we used the GE/Amersham Biosciences IllustraTM GenomiPhi V2 DNA Amplification Kit (Product number: 25-6600-31) which was stored at −70° C. The kit was not allowed to warm above 4° C. prior to initiation of DNA amplification. Next amplification reactions were set up. A reaction mix was prepared by combining 19.5 μl Reaction Buffer+0.5 μl Enzyme Mix (Genomiphi Version 2). 10 μl of the Reaction Mix was added to the heated and cooled template sample described in the prior paragraph. Amplification reactions were incubated at 30° C. overnight. Amplification reactions were terminated by heating at 65° C. for 10 minutes prior to cooling on ice. This amplified, derivative genomic DNA sample, including the YEp13 clone, was suitable for sequencing without any further manipulation.
DNA sequencing was performed as follows. For each 20 μl Genomiphi amplified sample, 8 μl was removed and added to 8 μl of Big Dye v3.1 Sequencing reagent (PN #4337457 Applied Biosystems, Foster City, Calif., 94404 USA), 4 μl of 10 μM primer (ype13-fwd: 5′-CTATGCGCACCCGTTCTCGGAGC SEQ ID NO:75, or ype13-rev: 5′-CGCTCATGAGCCCGAAGTGGCG SEQ ID NO:76; Sigma Genosys, Woodlands, Tex.), 4 μl 5× Sequencing buffer (PN #4336699 Applied Biosystems, Foster City, Calif.) and 16 μl Molecular Biology Grade water (Mediatech, Inc., Herndon, Va.). The sequencing reactions were then thermal cycled as follows; 3 minutes at 96° C. followed by 200 cycles of (95° C. 30 sec+55° C. 20 sec+60° C. 2 min) then stored at 4° C. The unincorporated ddNTPs were removed prior to sequencing using Edge Biosystems (Gaithersburg, Md.) clean-up plates. For each sequencing reaction the total 40 μl was pipetted into one well of a pre-spun 96 well clean up plate. The plate was then spun for 5 min at 5,000×g in a Sorvall RT-7 refrigerated centrifuge. The cleaned up reactions were then placed directly onto an Applied Biosystems 3730 DNA sequencer and sequenced with automatic base-calling.
The sequences of the two DNA vector-insert junction fragments from each clone were aligned to the yeast genome (Saccharomyces Genome Database) via the BLAST method (Altschul et al. (1997) Nucleic Acids Research 25:3389-3402). Both insert ends were homologous to segments of chromosome VIII. One sequence read matched 813 of 954 nucleotides between nucleotide positions 165109 and 165960 on chromosome VIII while the other junction matched 471 of 477 nucleotides between nucleotide positions 171100 and 170625. These data demonstrated that a 5991 by fragment resides on the plasmid of yBUT11 that contains nucleotides 165109 to 171100 of chromosome VIII of the S. cerevisiae genome. This region contains the following ORFs and genetic elements:

    DAP2′ YHR028W-A YHI9 ARS809 SLT2 ‘RRM3
    where DAP2′ indicates a 3′ truncation and ‘RRM3_indicates a 5′truncation.


ARS809 is a DNA replication element, YHR028W-A is annotated as a dubious open reading frame, and YHI9 encodes a protein of unknown function. The other ORF was SLT2 which encodes a cell wall integrity specific MAP Kinase. This MAP Kinase is part of a regulatory cascade that controls cell wall integrity in yeast (Chen and Thorner (2007) Biochimica et Biophysica Acta 1773: 1311-1340).
Example 2
Isobutanol Sensitivity of SLT2 Deletion Strain
A strain of diploid BY4743 having homozygous deletion of the SLT2 gene from the collection described by Brachmann et al. (Yeast (1998) 14:115-132) was assayed for sensitivity to isobutanol. The Δslt2 strain was plated on YPD medium and incubated for 3 days at 30° C. Colonies were picked and used to inoculate complete synthetic medium with glucose (2%) as the carbon source. They were diluted in the same medium to an OD600 of 0.01. 2 ml aliquots of this suspension were appended with pure isobutanol to the concentrations indicated in Table 5. Cultures were agitated on a roller drum at 30° C. for 2 days. Cultures were tested for growth yield after 1 and 2 days, assayed in triplicate. 1 day (20 hr) data is provided in Table 5.







TABLE 5







Growth of Δslt2 strain in isobutanol.









[ISO]
OD (A600)










(wt %)
BY4743
Δslt2/Δslt2












0.00
>1
>1


0.25
>1
0.07


0.50
>1
0.03


0.75
0.07
0.03


0.87
0.07
0.01


1.00
0.04
0.01


1.25
0.02
0.02


1.50
0.04
0.02





Both data sets indicated that the homozygous SLT2 deletion strain was isobutanol-hypersensitive at 0.25 and 0.5 wt % isobutanol






Example 3
Overexpression of SLT2 Coding Region
The effect of overexpression of the SLT2 coding region was assessed as follows. A Yeast ORF collection (Gelperin, White et al. (2005) Genes Dev. 19(23):2816-2826)_is available from Open Biosystems (Huntsville, Ala.). In this collection yeast ORFs are expressed from the GAL1 promoter in the vector BG1805. This vector can be introduced into ura3 yeast mutants and transformants selected for uracil prototrophy. In the presence of galactose and the absence of glucose, expression from the GAL1 promoter is elevated about 1000 fold relative to the barely detectable level observed with glucose grown cells (Johnston and Davis (1984) Mol. Cell. Biol. 4(8):1440-1448). Note that each ORF encodes a fusion protein of the form:
N terminal yeast ORF-C terminal epitope tag
A plasmid containing the SLT2 ORF was obtained from this collection (YSC3867-9522814) and transformed into yeast strain BY4741 selecting for uracil prototrophy. To measure the effects of isobutanol we compare the growth observed in an isobutanol-treated culture in galactose medium to an untreated culture grown in the same medium lacking isobutanol. Growth yield was measured by the following method. A fresh colony of a transformant was used to inoculate a tube in which CM galactose minus uracil medium had been placed. This medium provides selection for plasmid maintenance. The resultant culture was incubated at 30° C. on a roller drum to provide aeration. The parent BY4741 was cultured in this medium modified by the inclusion of uracil to satisfy its uracil requirement. The culture was diluted into fresh medium (10 ml) to OD600 of approximately 0.4. Absorbance was recorded. Cultures were grown for 2-3 hours to allow one doubling. Absorbance was recorded at this time and was called T=0. The culture was divided into two equal parts; each was diluted with an equal volume of pre-warmed fresh media. In one sample the fresh media was supplemented with 1 wt % isobutanol; in the other (control) the fresh media was unadulterated. OD600 was measured after incubation on a roller drum for 1 day. Absorbencies presented were averages of triplicate control and triplicate treated cultures. Percent growth was calculated as 100×[average A600 of the triplicate isobutanol challenged cultures/average A600 of the triplicate control cultures.
Results for a 22 hr experiment indicated that the parental strain (BY4741) grown in galactose displayed a 27% growth yield in the presence of 1 wt % isobutanol relative to the uninhibited culture (measures are averages of triplicate cultures with standard deviations of less than 6% under each of the conditions tested for both strains). In contrast, the SLT2p over-producing strain grown in galactose displayed a growth yield of 56% yield in the presence of 1 wt % isobutanol, a 29% improvement.
Example 4 (Prophetic)
Production of Isobutanol in Recombinant S. cerevisiae with Engineered Isobutanol Pathway and SLT2 Overexpression
The purpose of this prophetic example is to describe how to enhance isobutanol production in a yeast strain by combining an isobutanol biosynthetic pathway with SLT2 overexpression. To this end we need to disrupt TRP1, the gene encoding phosphoribosylanthranilate isomerase that catalyzes the third step in tryptophan biosynthesis, to provide a third selectable marker. BY4741 is the starting strain. A cassette containing DNA sequences that are located upstream and downstream just outside of TRP1 (up TRP1 and downTRP1) is created containing the following elements: upTRP1 DR URA3* DR downTRP1, where DR are direct repeat sequences and URA3* is a heterologous URA3 gene. The upTRP1 DR URA3* DR downTRP1 fragment is constructed by the method of Reid et al. ((2002) Yeast 19(4):319-328). Following this method the 5′ and 3′ flanking regions of the TRP1 gene, which contain the up TRP1 and downTRP1 sequences, are prepared. These are called intergenic DNAs iYHR029C and iYHR030C, respectively.
Intergenic DNA iYDR006C is amplified from S. cerevisiae genomic DNA using PCR with the following primers where the small letters are the adaptamers described in Reid et al. (ibid) and the capital letters are yeast genomic DNA:









Forward:







(SEQ ID NO: 77)









ccgctgctaggcgcgccgtgTCTGAAAACGGAAGAGGAGTAGG



 



Reverse:







(SEQ ID NO: 78)









gcagggatgcggccgctgacATAACAGACATACTCCAAGCTGCC






Intergenic DNA iYDR008C is amplified from S. cerevisiae genomic DNA using PCR with the following primers where the small letters are the adaptamers described in Reid et al. (ibid.) and the capital letters are yeast genomic DNA:







Forward:







(SEQ ID NO: 79)







ccgctgctaggcgcgccgtgCATTTGGCTTTTTGATTGATTGTAC


 


Reverse:







(SEQ ID NO: 80)







gcagggatgcggccgctgacACTTTTATTTTCTCTTTTTGCACTCCT






The two intergenic DNA PCR fragments are each used together with the plasmid pWJ1077, containing DR URA3*DR (Reid et al. ibid.), as template for PCR to produce DNA fragments containing each intergenic DNA sequence and a portion of the URA3* sequence, with overlap of the URA3* sequence between the two resulting fragments: upTRP1 DR UR and RA3* DR downTRP1. This is because there is complementarity between the right end of iYDR006C and a sequence on the plasmid pWJ1077 left of the DR upstream relative to URA3. Similarly, there is complementarity between the left end of iYDR006C and a sequence on the plasmid pWJ1077 right of the DR downstream relative to URA3. Primers for iYDR006C and pWJ1077 templates are C and kli3′ (SEQ ID NOS:81 and 82). Primers for iYDR008C and pWJ1077 templates are D and kli5′′ (SEQ ID NOs:83 and 84).

Co-transformation of these two fragments into yeast allows recombination between the two fragments to create a cassette containing an intact URA3* gene flanked by upTRP1 and downTRP1 sequences. Recombination of this cassette into the yeast chromosome results in the replacement of TRP1 by DR URA3* DR. Transformants with this recombination event are selected by demanding growth in the absence of pyrimidines but in the presence of tryptophan. The recombinant requires tryptophan to grow. Excision of URA3* is accomplished by homologous recombination between the DR's and its loss is selected for with 5-FOA to create BY4741ΔTRP1.
Construction of vectors pRS423::CUP1p-alsS+FBAp-IL V3 and pHR81::FBAp-ILV5-GPMp-kivD is described in US Patent Publication # US20070092957 A1, Example 17, which is herein incorporated by reference. pRS423::CUP1p-alsS+FBAp-ILV3 has a chimeric gene containing the CUP1 promoter (SEQ ID NO:85), the alsS coding region from Bacillus subtilis (SEQ ID NO:41), and CYC1 terminator (SEQ ID NO:86) as well as a chimeric gene containing the FBA promoter (SEQ ID NO:87), the coding region of the ILV3 gene of S. cerevisiae (SEQ ID NO:47), and the ADH1 terminator (SEQ ID NO:88). pHR81::FBAp-ILV5+GPMp-kivD is the pHR81 vector (ATCC #87541) with a chimeric gene containing the FBA promoter, the coding region of the ILV5 gene of S. cerevisiae (SEQ ID NO:43), and the CYC1 terminator as well as a chimeric gene containing the GPM promoter (SEQ ID NO:89), the coding region from kivD gene of Lactococcus lactis (SEQ ID NO:35), and the ADH1 terminator. pHR81 has URA3 and leu2-d selection markers.
Plasmid vectors pRS423::CUP1p-alsS+FBAp-ILV3 and pHR81::FBAp-ILV5+GPMp-kivD are transformed into BY4741ΔTRP1 using standard genetic techniques to yield the doubly transformed strain BY4741ΔTRP1-iso (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). BY4741ΔTRP1-iso is maintained on synthetic complete media lacking histidine and uracil.
Next the YRp7 plasmid (Botstein et al. (1979) Gene 8(1): 17-240), which is available from ATCC (catalog number 37060), and has unique restriction sites within the Tet gene (BamHI, SalI, EagI, NruI), is digested with BamHI to yield a linear fragment which is then dephosphorylated. To it is ligated the SLT2 gene fragment that is amplified, including 1 kbp of flanking sequence on both ends, from yeast chromosomal DNA using the primers:








ccatggtggatccGTGGTGAAAATGAAGGAAAT
(SEQ ID NO: 90)


and


 


ccatggggatccATCTTCATAAACGTTTATCA.
(SEQ ID NO: 91)






Prior to ligation the PCR product is digested with BamHI (restriction sites underlined and italicized) to yield compatible sticky ends. The resultant plasmid (YRp7-SLT2) is selected based upon ampicillin resistance in E. coli and is confirmed molecularly (liberation of an approximately 3.5 kbp fragment upon BamHI digestion) and phenotypically by being tetracycline sensitive. The plasmid is then isolated from E. coli and transformed into yeast strains, selecting for the ability to grow in the absence of tryptophan (TRP1 function).

BY4741ΔTRP1-iso is next transformed with yRP7-SLT2, selecting for the ability to grow without tryptophan supplementation, yielding BY4741ΔTRP1-iso-SLT2. BY4741ΔTRP1-iso is also transformed with yRP7 selecting for the ability to grow without tryptophan supplementation yielding BY4741ΔTRP1-iso-c. Aerobic cultures are grown in 250 ml flasks containing 50 ml synthetic complete media lacking histidine, tryptophan and uracil, and supplemented with 2% glucose in an Innova4000 incubator (New Brunswick Scientific, Edison, N.J.) at 30° C. and 225 rpm. Low oxygen cultures are prepared by adding 45 mL of medium to 60 mL serum vials that are sealed with crimped caps after inoculation and kept at 30° C. Approximately 24 h and 48 h after induction with 0.03 mM CuSO4 (final concentration), an aliquot of the broth is analyzed by HPLC (Shodex Sugar SH1011 column (Showa Denko America, Inc. NY) with refractive index (RI) detection and GC (HP-Innowax, 0.32 mm×0.25 μm×30 m (Agilent Technologies, Inc., Santa Clara, Calif.) with flame ionization detection (FID) for isobutanol content. Isobutanol is detected. More isobutanol is produced by BY4741ΔTRP1-iso-SLT than by BY4741ΔTRP1-iso-c.

## Claims

1. A recombinant yeast cell comprising:
a) an isobutanol biosynthetic pathway, wherein the isobutanol biosynthetic pathway comprises at least one gene encoding a polypeptide that performs at least one of the following substrate to product conversions:
i) pyruvate to acetolactate, as catalyzed by acetolactate synthase;
ii) acetolactate to 2,3-dihydroxyisovalerate, as catalyzed by acetohydroxy acid isomeroreductase;
iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, as catalyzed by dihydroxyacid dehydratase;
iv) α-ketoisovalerate to isobutyraldehyde, as catalyzed by branched-chain keto acid decarboxylase; and
v) isobutyraldehyde to isobutanol, as catalyzed by a branched-chain alcohol dehydrogenase; and

b) at least one genetic modification which increases cell wall integrity pathway activity, wherein the genetic modification increases activity of a mitogen-activated protein kinase module of the cell wall integrity pathway, and wherein the genetic modification is overexpression of a STL2 protein encoding gene;

wherein the isobutanol biosynthetic pathway comprises at least one gene that is heterologous to the yeast cell and wherein the yeast cell has an increase in tolerance to isobutanol and at least a 20% improvement in growth yield as compared with a yeast cell that lacks the at least one genetic modification of (b).

2. The recombinant yeast cell of claim 1 wherein the cell has at least a 29% improvement in growth yield in 1% (weight/volume) isobutanol as compared to a parental cell having no increase in activity of the cell wall integrity pathway.

3. The recombinant yeast cell of claim 1 selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.

4. The yeast cell of claim 1 wherein the genetic modification increases STL2p serine/threonine MAP kinase activity.

5. The yeast cell of claim 1 wherein the STL2 protein encoding gene is heterologous to the yeast cell.

6. The yeast cell of claim 1 wherein the at least one gene encoding the acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, dihydroxyacid dehydratase, branched-chain keto acid decarboxylase, or branched-chain alcohol dehydrogenase is heterologous to the yeast cell.

7. method for the production of isobutanol comprising growing the recombinant yeast cell of claim 1 under conditions where isobutanol is produced and optionally recovering the isobutanol.

8. A method for improving fermentative production of isobutanol comprising:
a) providing the recombinant yeast cell of claim 1; and
b) contacting said yeast cell with fermentable sugar whereby said yeast cell produces isobutanol and wherein said yeast cell has improved tolerance to said isobutanol and improved growth yield as compared to a yeast cell without the at least one genetic modification that increases activity of the mitogen-activated protein kinase module of the cell wall integrity pathway.