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Title:
REGULATION OF PRODUCTION PATHWAYS IN HOST CELLS
Document Type and Number:
WIPO Patent Application WO/2015/002919
Kind Code:
A2
Abstract:
The invention relates to recombinant host cells that comprise controlled biocatalyst polypeptides and methods for producing fermentation products employing the same. In some embodiments, various controlled polypeptides and control cues are employed during the propagation and production phases of a fermentation process.

Inventors:
DAUNER MICHAEL (US)
JAHIC MEHMEDALIJA (US)
O'KEEFE DANIEL P (US)
PRASAD JAHNAVI CHANDRA (US)
Application Number:
PCT/US2014/044996
Publication Date:
January 08, 2015
Filing Date:
July 01, 2014
Export Citation:
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Assignee:
BUTAMAX ADVANCED BIOFUELS LLC (US)
International Classes:
C12P7/16
Attorney, Agent or Firm:
EDWARDS, Mark, A. (Legal Patent Records CenterChestnut Run Plaza 721/2340,974 Centre Roa, PO Box 2915 Wilmington DE, US)
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Claims:
WHAT IS CLAIMED IS:

1. A method of producing butanol or 2-butanone comprising:

a) contacting a recombinant host cell comprising a controlled biocatalyst polypeptide and a butanol or 2-butanone biosynthetic pathway with a carbon substrate for a first phase; and

b) providing a control cue such that the activity of the controlled biocatalyst polypeptide is altered for a second phase

wherein butanol or 2-butanone is produced during the first phase, the second phase, or both; and

wherein the production of butanol or 2-butanone is different between the first phase and the second phase.

2. The method of Claim 1, wherein providing a control cue comprises at least one of changing the temperature, increasing the concentration of the controlled biocatalyst polypeptide substrate, increasing the concentration of the controlled biocatalyst polypeptide cofactor, or reducing the concentration of controlled biocatalyst polypeptide inhibitor.

3. The method of Claim 1 or Claim 2, wherein the controlled biocatalyst polypeptide is a propagation polypeptide.

4. The method of Claim 1 or Claim 2, wherein the controlled biocatalyst polypeptide is a biosynthetic pathway polypeptide.

5. The method of any one of the previous claims wherein the butanol or 2-butanone biosynthetic pathway comprises the substrate to product conversion pyruvate to acetolactate.

6. The method of any one of the previous claims wherein the butanol or 2-butanone biosynthetic pathway is an isobutanol biosynthetic pathway.

7. The method of claim 5 wherein the substrate to product conversion pyruvate to acetolactate is catalyzed by an acetolactate synthase.

8. The method of claim 5 or claim 7 wherein the acetolactate synthase comprises at least about 90% identity to SEQ ID NO: 1.

9. The method of any one of claims 5-8 wherein the control cue comprises changing the temperature.

10. The method of any one of claims 5-8 wherein the control cue comprises increasing the concentration of the controlled biocatalyst polypeptide substrate and wherein said substrate is pyruvate.

11. The method of claim 10 wherein the biocatalyst polypeptide comprises acetolactate synthase activity and a KM for pyruvate of at least that of B. subtilis.

12. The method of claim 11 wherein the acetolactate synthase comprises at least about 90% identity to SEQ ID NO: 267, 268, 269, 270, 271, 271, or an active fragment thereof.

13. The method of any one of claims 5-8 wherein the control cue comprises increasing the concentration of the controlled biocatalyst polypeptide cofactor and wherein said cofactor is TPP.

14. The method of claim 13 wherein the biocatalyst polypeptide comprises acetolactate synthase activity and a KD for TPP greater than that of SEQ ID NO: 1.

15. The method of claim 13 or 14 wherein the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises a substitution at least one position corresponding to position 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 of SEQ ID NO: 1.

16. The method of claim 13 or 14 wherein the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises a substitution at least one position corresponding to positions T94, G399, H401, G450, G453, V545 or Y547 of SEQ ID NO: 1.

17. The method of any one of claims 13 to 16 wherein the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises at least about 90% identity to SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266.

18. The method of any one of claims 5-8 wherein the control cue comprises reducing the concentration of controlled biocatalyst polypeptide inhibitor.

19. The method of claim 18 wherein the controlled biocatalyst polypeptide inhibitor reduces or eliminates acetolactate synthase activity.

20. The method of claim 18 or 19 wherein the controlled biocatalyst polypeptide inhibitor is a herbicide.

21. The method of any one of claims 18-20 wherein the controlled biocatalyst polypeptide inhibitor comprises a sulfonylurea, an imidazolinone, a triazolopyrimidine, a pyrimidyloxybenzoate, or a pyrimidylthiobenzoate.

22. A synthetic polypeptide comprising acetolactate synthase activity and a substitution at a position corresponding to position 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 of SEQ ID NO: 1.

23. The synthetic polypeptide of Claim 22 wherein said polypeptide comprises at least about 90% identity to SEQ ID NO: 1.

24. The synthetic polypeptide of Claim 22 or 23 wherein said polypeptide comprises a KD for TPP greater than that of SEQ ID NO: 1.

25. The synthetic polypeptide of any one of Claims 22-24 wherein said polypeptide comprises at least about 99% identity to SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, or 363 or an active fragment thereof.

26. A polynucleotide encoding the polypeptide of any one of claims 22-25.

27. A recombinant microbial host cell comprising a biosynthetic pathway, the pathway comprising the polypeptide of any one of claims 22-25.

28. The recombinant microbial host cell of Claim 27 wherein the biosynthetic pathway is an isobutanol biosynthetic pathway.

29. The recombinant microbial host cell of Claim 28 wherein the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

(a) pyruvate to acetolactate;

(b) acetolactate to 2,3-dihydroxyisovalerate;

(c) 2,3-dihydroxyisovalerate to a-ketoisovalerate;

(d) a-ketoisovalerate to isobutyraldehyde; and

(e) isobutyraldehyde to isobutanol.

30. The recombinant microbial host cell of Claim 28 or 29 wherein the host cell is a yeast host cell.

31. The recominbinant microbial host cell of Claim 30 wherein the yeast cell is S. cerevisiae.

32. The recombinant microbial host cell of Claim 31 comprising a deletion, mutation, and/or substitution in an endogenous gene encoding FRA2.

33. The recombinant microbial host cell of any one of Claims 30-32 comprising reduced or eliminated pyruvate decarboxylase activity.

34. The recombinant microbial host cell of any one of Claim 30-33 comprising a modification to reduce glycerol-3 -phosphate dehydrogenase activity.

35. A recombinant microbial host cell comprising a polypeptide of any one of claims 22-25.

36. A method of converting pyruvate to acetolactate comprising contacting a polypeptide of any one of claims 22-25 with acetolactate in the presence of an effective amount of TPP.

37. A method of converting pyruvate to acetolactate comprising contacting a recombinant host cell of any one of claims 27-35 with acetolactate in the presence of an effective amount of thiamine.

Description:
REGULATION OF PRODUCTION PATHWAYS IN HOST CELLS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of priority from United States Provisional Application

No. 61/842,881, filed July 3, 2013, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to the fields of industrial microbiology and alcohol production.

Embodiments of the invention relate to the differential flux through the target product biosynthetic pathway during various phases of the manufacturing process.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

[0003] The content of the electronically submitted Sequence Listing, (Name:

20140701_CL5504WOPCT_SequenceListing.txt; Size: 1,622,909 bytes; Date of Creation: June 23, 2014) filed herewith, is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

[0005] Methods for the chemical synthesis of the butanol isomer isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, 5:716-719 (6th ed. 2003)) and Guerbet condensation of methanol with n-propanol (Carlini et al, J. Molec. Catal. A:Chem.

220:215-220 (2004)). These processes use starting materials derived from petrochemicals and are generally expensive. The production of isobutanol from plant-derived raw materials represents an advance in the art. BRIEF SUMMARY OF THE INVENTION

[0006] Provided herein are methods of producing butanol or 2-butanone comprising: A) contacting a recombinant host cell comprising a controlled biocatalyst polypeptide and a butanol or 2-butanone biosynthetic pathway with a carbon substrate for a first phase; and B) providing a control cue such that the activity of the controlled biocatalyst polypeptide is altered for a second phase. In embodiments, butanol or 2-butanone is produced during the first phase, the second phase, or both. In embodiments, the production of butanol or 2-butanone is different between the first phase and the second phase. In embodiments, the rate of production of butanol or 2-butanone is greater in the second phase than in the first phase.

[0007] In embodiments, providing a control cue comprises at least one of changing the temperature, increasing the concentration of the controlled biocatalyst polypeptide substrate, increasing the concentration of the controlled biocatalyst polypeptide cofactor, or reducing the concentration of controlled biocatalyst polypeptide inhibitor.

[0008] In embodiments, the controlled biocatalyst polypeptide is a propagation polypeptide.

In embodiments, the controlled biocatalyst polypeptide is a biosynthetic pathway polypeptide.

[0009] In embodiments, a butanol or 2-butanone biosynthetic pathway comprises the substrate to product conversion pyruvate to acetolactate. In embodiments, a butanol or 2-butanone biosynthetic pathway is an isobutanol biosynthetic pathway. In embodiments, the substrate to product conversion pyruvate to acetolactate is catalyzed by an acetolactate synthase. In

embodiments, the acetolactate synthase comprises at least about 90% identity to SEQ ID NO: 1.

[0010] In embodiments, the control cue comprises increasing the concentration of the controlled biocatalyst polypeptide substrate and wherein said substrate is pyruvate. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and a K M for pyruvate of at least that of B. subtilis. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and a K M for pyruvate of greater than that of SEQ ID NO: 1. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and a K D for pyruvate of greater than twice that of SEQ ID NO: 1. In embodiments, the acetolactate synthase comprises at least about 90% identity to SEQ ID NO: 267, 268, 269, 270, 271, 271, or an active fragment thereof.

[0011] In embodiments, the control cue comprises increasing the concentration of the controlled biocatalyst polypeptide cofactor. In embodiments, the cofactor is TPP. In embodiments, concentration of TPP is increased by adding thiamine to the medium. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and a K D for TPP greater than that of SEQ ID NO: 1. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises a substitution at least one position corresponding to position 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 of SEQ ID NO: 1. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises a substitution at least one position corresponding to positions T94, G399, H401, G450, G453, V545 or Y547 of SEQ ID NO: 1. In embodiments, the biocatalyst polypeptide comprises acetolactate synthase activity and wherein said polypeptide comprises at least about 90% identity to SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266.

[0012] In embodiments, the control cue comprises reducing the concentration of a controlled biocatalyst polypeptide inhibitor. In embodiments, the controlled biocatalyst polypeptide inhibitor reduces or eliminates acetolactate synthase activity. In embodiments, the controlled biocatalyst polypeptide inhibitor is a herbicide. In embodiments, the controlled biocatalyst polypeptide inhibitor comprises a sulfonylurea, an imidazolinone, a triazolopyrimidine, a pyrimidyloxybenzoate, or a pyrimidylthiobenzoate.

[0013] Also provided herein are polypeptides comprising acetolactate synthase activity and a substitution at a position corresponding to position 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 62, 66, 94, 397, 398, 399, 400, 401, 402, 421, 423, 424, 425, 426, 427, 449, 450, 451, 452, 453, 454, 456, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 487, 498, 500, 545, 547, or 550 of SEQ ID NO: 1. In embodiments, the polypeptide comprises at least about 90%> identity to SEQ ID NO: 1. In embodiments, the polypeptide comprises a K D for TPP greater than that of SEQ ID NO: 1. In embodiments, the polypeptide comprises a K D for TPP greater than 2 times that of SEQ ID NO: 1. In embodiments, the polypeptide comprises at least about 99% identity to SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, or 363 or an active fragment thereof.

[0014] Also provided herein are polynucleotides encoding such polypeptides and

recombinant microbial host cells comprising biosynthetic pathways, the pathways comprising such polypeptides. In embodiments, the biosynthetic pathway is an isobutanol biosynthetic pathway. In embodiments, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: (a) pyruvate to acetolactate; (b) acetolactate to 2, 3 -dihydroxyiso valerate; (c) 2,3- dihydroxyisovalerate to a-ketoisovalerate; (d) a-ketoisovalerate to isobutyraldehyde; and (e) isobutyraldehyde to isobutanol.

[0015] In embodiments, each substrate to product conversion is catalyzed by polypeptide encoded by a polynucleotide heterologous to the host cell. In embodiments, the host cell is a yeast host cell. In embodiments, the yeast cell is S. cerevisiae. In embodiments, the host cell further comprises a deletion, mutation, and/or substitution in an endogenous gene encoding FRA2. In embodiments, the host cell further comprises reduced or eliminated pyruvate decarboxylase activity. In embodiments, the host cell further comprises a modification to reduce glycerol-3 -phosphate dehydrogenase activity.

[0016] Also provided herein are methods of converting pyruvate to acetolactate comprising contacting a polypeptide disclosed herein with acetolactate in the presence of an effective amount of TPP. Also provided herein are methods of converting pyruvate to acetolactate comprising contacting a recombinant host cell disclosed herein with acetolactate in the presence of an effective amount of thiamine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 depicts different isobutanol biosynthetic pathways. The steps labeled "a",

"b", "c", "d", "e", "f ', "g", "h", "i", "j", and "k" represent substrate to product conversions described below, "a" may be catalyzed, for example, by acetolactate synthase, "b" may be catalyzed, for example, by acetohydroxyacid reductoisomerase. "c" may be catalyzed, for example, by

acetohydroxy acid dehydratase, "d" may be catalyzed, for example, by branched-chain keto acid decarboxylase, "e" may be catalyzed, for example, by branched chain alcohol dehydrogenase, "f ' may be catalyzed, for example, by branched chain keto acid dehydrogenase, "g" may be catalyzed, for example, by acylating aldehyde dehydrogenase, "h" may be catalyzed, for example, by transaminase or valine dehydrogenase, "i" may be catalyzed, for example, by valine decarboxylase, "j" may be catalyzed, for example, by omega transaminase.

[0018] Figure 2 depicts consumed glucose ("D glucose", in mM) and produced isobutyric acid ("IBCOOH", in mM), isobutanol ("IBOH", in mM) and biomass measured as optical density "OD" for PNY 2092, D351 and E61A during aerobic growth in the time interval from elapsed process time EPT = 0 - lOh (Figure 2A), and during anaerobic production in the time interval from elapsed process time EPT = 10 - 24 h (Figure 2B) as described in Example 1. Concentrations in the "add" cultures were corrected to reflect the additional volume from the thiamine supplementation.

[0019] Figure 3 depicts the yield of major metabolites produced (positive values) or consumed (negative values) on glucose during the aerobic growth phase from EPT = 0 to 10 h as described in Example 1. Biomass represents an arbitrary value proportional to the difference in OD measured.

[0020] Figure 4 depicts yield of major metabolites produced (positive values) or consumed

(negative values) on glucose during the anaerobic production phase from EPT = 10 to 48 h as described in Example 1. Biomass represents an arbitrary value proportional to the difference in OD measured.

[0021] Figure 5 shows Specific isobutanol production rates (qp) during mainly aerobic growth (EPT = 0 - 10 h) and mainly anaerobic production phase (EPT = 10 - 24 h) in PNY 2092 and D35 IE with no thiamine and in medium without thiamine with addition of thiamine at EPT = 10 h ("add") as described in Example 1.

[0022] Figure 6 shows the model of B. subtilis ALS (SEQ ID NO: 1) described herein. The

TPP molecule is in stick-format and the Mg 2+ ion is being shown in solid fill.

[0023] Figure 7 shows sequence relationships of acetolactate synthase (als) coding regions that were retrieved by BLAST analysis using the sequence of B. subtilis AlsS, limiting to the 100 closest neighbors as described in U.S. Appn. Pub. No. 20090305363, incorporated herein by reference. The als encoding sequence is identified by its source organism.

[0024] Figure 8 shows a graph depicting the average growth rate of indicated strains from hour 3 to hour 9.25. [0025] Figure 9 shows a graph depicting the final OD 6 oo for the indicated strains at 27 hours.

DETAILED DESCRIPTION

[0026] The invention is directed to recombinant host cells that produce fermentation products and comprise biocatalyst polypeptides that provide differential activity in differing phases, eg. the propagation vs. production phases, of a process, as well as methods for using the same.

Promoters that can be used to provide differential expression of biocatalyst polypeptides in different phases of a process, and means of identifying such promoters, are described in Int'l Appl. No.

PCT/US2012/072186, which is incorporated herein by reference in its entirety. However, as described herein, biocatalyst polypeptides may be selected or engineered to act as control elements, providing controlled activity of the polypeptide and thus controlled flux through the biosynthetic pathway. Such controlled activity may provide flexibility in a manufacturing process such that production via a biosynthetic pathway may be turned up or down, depending on the desired result at a particular stage in the process.

[0027] In embodiments, recombinant host cells described herein produce butanol or 2- butanone from plant derived carbon sources. Accordingly, provided herein are methods for the production of butanol or 2-butanone using recombinant host cells comprising control elements that regulate the activity of particular polypeptides catalyzing substrate to product conversions during the propagation and production phases of a fermentation process. In one embodiment, a polypeptide catalyzing the first step in a butanol biosynthetic pathway can be preferentially active during the production phase. In one embodiment, a polypeptide catalyzing a substrate to product conversion in an isobutanol biosynthetic pathway can be preferentially active during the production phase. In one embodiment, acetolactate synthase may be preferentially active during the production phase. In one embodiment, ketol-acid reductoisomerase may be preferentially active during the production phase. In one embodiment, dihydroxyacid dehydratase may be preferentially active during the production phase.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

[0029] A used herein, the terms "comprises," "comprising," "includes," "including," "has,"

"having," "contains," or "containing," or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements not expressly listed or inherent to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to 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).

[0030] 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.

[0031] The term "invention" or "present invention" 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 application.

[0032] As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about," the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value. [0033] The term "environmental cue" or "process cue" refers to any change in cellular environmental conditions, including, but not limited to, changes in fermentable carbon substrates, oxygen levels, glucose levels, pH, temperature, and butanol levels.

[0034] The term "control cue" refers to a directed change in the cellular environment.

[0035] The term "controlled biocatalyst polypeptide" refers to a biocatalyst polypeptide which exhibits an alteration in activity in the presence of a control cue.

[0036] The term "growth phase" or "propagation phase" refers to the process steps during which yeast biomass is produced and inoculum build-up occurs.

[0037] The term "production phase" refers to the fermentation process steps during which a desired fermentation product, including, but not limited to butanol, isobutanol, 1-butanol, 2-butanol and/or 2-butanone production, occurs.

[0038] In some instances, "biomass" refers to the cell biomass of the fermentation product- producing microorganism, typically provided in units g/L dry cell weight (dew).

[0039] The term "fermentation product" includes any desired product of interest, including lower alkyl alcohols including, but not limited to butanol.

[0040] The term "lower alkyl alcohol" refers to any straight-chain or branched, saturated or unsaturated, alcohol molecule with 1-10 carbon atoms.

[0041] The term "butanol" refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.

Isobutanol is also known as 2-methyl-l-propanol.

[0042] The term "butanol biosynthetic pathway" as used herein refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol. For example, isobutanol biosynthetic pathways are disclosed in U.S. Patent No. 7,851,188, which is incorporated by reference herein. Components of the pathways consist of all substrates, cofactors, byproducts, intermediates, end-products, and enzymes in the pathways.

[0043] The term "2-butanone biosynthetic pathway" as used herein refers to an enzyme pathway to produce 2-butanone.

[0044] The term "propagation polypeptide" includes polypeptides associated with the production of biomass, and polypeptides associated with the performance of an enzyme that is associated with the production of biomass. [0045] The term "biocatalyst polypeptide" includes polypeptides associated with the substrate to product conversions of an indicated biosynthetic pathway, for example a butanol or 2- butanone biosynthetic pathway, and polypeptides associated with the propagation or performance of a biocatalyst that is associated with the indicated biosynthetic pathway, including, but not limited to, cell integrity polypeptides and propagation polypeptides. For example, a polypeptide that is a part of an NADPH generating pathway or a polypeptide that is part of a non-butanol NADH consuming product pathway may be biocatalyst polypeptides.

[0046] The term "biosynthetic pathway polypeptide" includes polypeptides that catalyze substrate to product conversions of a recited biosynthetic pathway.

[0047] The term "cell integrity polypeptide" includes polypeptides involved in cell integrity, including polypeptides required for constituting the cellular architecture.

[0048] A "recombinant microbial host cell" is defined as a host cell that has been genetically manipulated. In embodiments, recombinant microbial host cells have been genetically manipulated to express a biosynthetic production pathway, wherein the host cell either produces a biosynthetic product in greater quantities relative to an unmodified host cell or produces a biosynthetic product that is not ordinarily produced by an unmodified host cell.

[0049] The term "fermentable carbon substrate" refers to a carbon source capable of being metabolized by the microorganisms such as those disclosed herein. Suitable fermentable carbon substrates include, but are not limited to, monosaccharides, such as glucose or fructose;

disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch, cellulose, or lignocellulose, hemicellulose; one-carbon substrates, fatty acids; and a combination of these.

[0050] "Fermentation medium" means the mixture of water, sugars (fermentable carbon substrates), dissolved solids, microorganisms producing fermentation products, fermentation product and all other constituents of the material held in the fermentation vessel in which the fermentation product is being made by the reaction of fermentable carbon substrates to fermentation products, water and carbon dioxide (C0 2 ) by the microorganisms present. From time to time, as used herein the term "fermentation broth" and "fermentation mixture" can be used synonymously with

"fermentation medium."

[0051] The term "aerobic conditions" means conditions in the presence of oxygen. [0052] The term "microaerobic conditions" means conditions with low levels of dissolved oxygen. For example, the oxygen level may be less than about 1% of air-saturation.

[0053] The term "anaerobic conditions" means conditions in the absence of oxygen. It will be understood that in many fermentation processes, an initial amount of oxygen is present at the onset of the process, but such oxygen is depleted over the course of the fermentation such that the majority of the process takes place in the absence of detectable oxygen.

[0054] The term "yield" refers to the amount of product per amount of carbon source in g/g.

The yield may be exemplified for glucose as the carbon source. It is understood unless otherwise noted that yield is expressed as a percentage of the theoretical yield. In reference to a

microorganism or metabolic pathway, "theoretical yield" is defined as the maximum amount of product that can be generated per total amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isopropanol is 0.33 g/g. As such, a yield of isopropanol from glucose of 29.7 g/g would be expressed as 90% of theoretical or 90% theoretical yield. It is understood that while in the present disclosure the yield is exemplified for glucose as a carbon source, the invention can be applied to other carbon sources and the yield may vary depending on the carbon source used. One skilled in the art can calculate yields on various carbon sources.

[0055] The terms "acetohydroxyacid synthase," "acetolactate synthase" and "acetolactate synthetase" (abbreviated "ALS", "AlsS", "alsS" and/or "AHAS" herein) are used interchangeably to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and C0 2 . Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB07802.1 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), CAB15618 (SEQ ID NO: 214), Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:3), M73842 (SEQ ID NO:4)), and Lactococcus lactis (GenBank Nos: AAA25161 (SEQ ID NO:5), L16975 (SEQ ID NO:6)). Additional ALS enzymes are provided in U.S. Appn. Pub. No. 20090305363 and are incorporated herein by reference. Additional ALS enzymes are disclosed herein. [0056] The terms "ketol-acid reductoisomerase" ("KARI"), and "acetohydroxy acid isomeroreductase" will be used interchangeably and refer to enzymes capable of catalyzing the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP_418222 (SEQ ID NO: 7), NC_000913 (SEQ ID NO: 8)), Saccharomyces cerevisiae (GenBank Nos: NP 013459 (SEQ ID NO: 9), NC OOl 144 (SEQ ID NO: 10)),

Methanococcus maripaludis (GenBank Nos: CAF30210 (SEQ ID NO: 11), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (GenBank Nos: CAB 14789 (SEQ ID NO: 13), Z99118 (SEQ ID NO: 14)). KARIs include Anaerostipes caccae KARI variants "K9G9", "K9D3", and "K9JB4P" (SEQ ID NOs: 143, 142, and 215 respectively). In some embodiments, KARI utilizes NADH. In some embodiments, KARI utilizes NADPH.

[0057] The terms "acetohydroxy acid dehydratase" and "dihydroxyacid dehydratase"

("DHAD") refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to a- ketoiso valerate. Example acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. Such enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP 026248 (SEQ ID NO: 15), NC 000913 (SEQ ID NO: 16)), S. cerevisiae (GenBank Nos: NP_012550 (SEQ ID NO: 17), NC 001142 (SEQ ID NO: 18)), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO: 19), BX957219 (SEQ ID NO: 20)), B. subtilis (GenBank Nos: CAB14105 (SEQ ID NO: 21), Z99115 (SEQ ID NO: 22)), L. lactis, N. crassa, and S. mutans. DHADs include S. mutans variant "12 V5" (SEQ ID NO: 216)

[0058] The terms "branched-chain a-keto acid decarboxylase" or "a-ketoacid decarboxylase" or "a-ketoiso valerate decarboxylase" or "2-ketoiso valerate decarboxylase" ("KIVD") refer to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyraldehyde and C0 2 . Example branched-chain a-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 (SEQ ID NO: 23), AY548760 (SEQ ID NO: 24); CAG34226 (SEQ ID NO: 25), AJ746364 (SEQ ID NO: 26), Salmonella typhimurium (GenBank Nos: NP_461346 (SEQ ID NO: 27), NC_003197 (SEQ ID NO: 28)), Clostridium acetobutylicum (GenBank Nos: NPJ49189 (SEQ ID NO: 29), NC 001988 (SEQ ID NO: 30)), M. caseolyticus (SEQ ID NO: 141), and L. grayi (SEQ ID NO: 140).

[0059] The term "alcohol dehydrogenase" ("ADH") refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol, 2-butanone to 2-butanol, and/or butyraldehyde to 1- butanol. Alcohol dehydrogenases may be "branched chain alcohol dehydrogenases" or may be referred to as "butanol dehydrogenases." Example alcohol dehydrogenases suitable for

embodiments disclosed herein may be known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases, for example, according to published utilization of NADH (typically 1.1.1.1) or NADPH (typically 1.1.1.2) as co factors. Such enzymes are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP 010656 (SEQ ID NO: 31), NC OOl 136 (SEQ ID NO: 32); NP 014051 (SEQ ID NO: 33) NC_001145 (SEQ ID NO: 34)), E. coli (GenBank Nos: NP 417484 (SEQ ID NO: 35), NC 000913 (SEQ ID NO: 36)), C. acetobutylicum (GenBank Nos: NP_349892 (SEQ ID NO: 37), NC_003030 (SEQ ID NO: 38); NP 349891 (SEQ ID NO: 39), NC 003030 (SEQ ID NO: 40)), Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169), Acinetobacter sp. (GenBank Nos: AAG10026, AF282240), Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307), A. xylosoxidans (SEQ ID NO: 222), and B. indica (SEQ ID NO: 224).

[0060] The term "branched-chain keto acid dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), typically using NAD + (nicotinamide adenine dinucleotide) as an electron acceptor. Example branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. Such branched-chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B. subtilis (GenBank Nos: CAB14336 (SEQ ID NO: 41), Z99116 (SEQ ID NO: 42); CAB14335 (SEQ ID NO: 43), Z99116 (SEQ ID NO: 44); CAB14334 (SEQ ID NO: 45), Z99116 (SEQ ID NO: 46); and CAB14337 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48)) and Pseudomonas putida (GenBank Nos: AAA65614 (SEQ ID NO: 49), M57613 (SEQ ID NO: 50); AAA65615 (SEQ ID NO: 51), M57613 (SEQ ID NO: 52); AAA65617 (SEQ ID NO: 53), M57613 (SEQ ID NO: 54); and AAA65618 (SEQ ID NO: 55), M57613 (SEQ ID NO: 56)). [0061] The term "acylating aldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using either NADH or NADPH as an electron donor. Example acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO: 57), AF157306 (SEQ ID NO: 58)), C. acetobutylicum (GenBank Nos: NPJ49325 (SEQ ID NO: 59), NC_001988 (SEQ ID NO: 60); NP 149199 (SEQ ID NO: 61), NC 001988 (SEQ ID NO: 62)), P. putida (GenBank Nos:

AAA89106 (SEQ ID NO: 63), U13232 (SEQ ID NO: 64)), and Thermus thermophilus (GenBank Nos: YP 145486 (SEQ ID NO: 65), NC 006461 (SEQ ID NO: 66)).

[0062] The term "transaminase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, using either alanine or glutamate as an amine donor. Example transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026231 (SEQ ID NO: 67), NC 000913 (SEQ ID NO: 68)) and Bacillus licheniformis (GenBank Nos: YP 093743 (SEQ ID NO: 69), NC 006322 (SEQ ID NO: 70)). Examples of sources for glutamate-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026247 (SEQ ID NO: 71), NC 000913 (SEQ ID NO: 72)), S cerevisiae (GenBank Nos: NP_012682 (SEQ ID NO: 73), NC_001142 (SEQ ID NO: 74)) and

Methanobacterium thermoautotrophicum (GenBank Nos: NP_276546 (SEQ ID NO: 75),

NC 000916 (SEQ ID NO: 76)).

[0063] The term "valine dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor. Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP_628270 (SEQ ID NO: 77), NC_003888 (SEQ ID NO: 78)) and B. subtilis (GenBank Nos: CAB14339 (SEQ ID NO: 79), Z99116 (SEQ ID NO: 80)).

[0064] The term "valine decarboxylase" refers to an enzyme that catalyzes the conversion of

L-valine to isobutylamine and C0 2 . Example valine decarboxylases are known by the EC number 4.1.1.14. Such enzymes are found in Streptomyces, such as for example, Streptomyces viridifaciens (GenBank Nos: AAN10242 (SEQ ID NO: 81), AY116644 (SEQ ID NO: 82)). [0065] The term "omega transaminase" refers to an enzyme that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor. Example omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 83), AY330220 (SEQ ID NO: 84)), Ralstonia eutropha (GenBank Nos: YP_294474 (SEQ ID NO: 85), NC_007347 (SEQ ID NO: 86)), Shewanella oneidensis (GenBank Nos: NP_719046 (SEQ ID NO: 87),

NC_004347 (SEQ ID NO: 88)), and P. putida (GenBank Nos: AAN66223 (SEQ ID NO: 89), AE016776 (SEQ ID NO: 90)).

[0066] 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 (Co A). Example 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 (SEQ ID NO: 91), NC 000913 (SEQ ID NO: 92); NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP 349476.1 (SEQ ID NO: 93), NC 003030 (SEQ ID NO: 94); NP 149242 (SEQ ID NO: 95), NC 001988 (SEQ ID NO: 96), Bacillus subtilis (GenBank Nos: NP 390297 (SEQ ID NO: 97), NC_000964 (SEQ ID NO: 98)), and Saccharomyces cerevisiae (GenBank Nos: NP_015297 (SEQ ID NO: 99), NC OOl 148 (SEQ ID NO: 100)).

[0067] The term "3-hydroxybutyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3 -Example 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. Examples may be 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: 101), NC 003030 (SEQ ID NO: 102)), B. subtilis (GenBank NOs: AAB09614 (SEQ ID NO: 103), U29084 (SEQ ID NO: 104)), Ralstonia eutropha (GenBank NOs: YP 294481 (SEQ ID NO: 105), NC_007347 (SEQ ID NO: 106)), and Alcaligenes eutrophus (GenBank NOs:

AAA21973 (SEQ ID NO: 107), J04987 (SEQ ID NO: 108)).

[0068] The term "crotonase" refers to an enzyme that catalyzes the conversion of 3- hydroxybutyryl-CoA to crotonyl-CoA and H 2 0. Example crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and may be 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: 109), NC 000913 (SEQ ID NO: 110)), C. acetobutylicum (GenBank NOs: NP 349318 (SEQ ID NO: 111), NC 003030 (SEQ ID NO: 112)), B. subtilis (GenBank NOs: CAB13705 (SEQ ID NO: 113), Z99113 (SEQ ID NO: 114)), and Aeromonas caviae (GenBank NOs: BAA21816 (SEQ ID NO: 115), D88825 (SEQ ID NO: 116)).

[0069] The term "butyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Example butyryl-CoA dehydrogenases may be NADH- dependent, NADPH-dependent, or flavin-dependent and may be classified as E.C. 1.3.1.44, E.C. 1.3.1.38, and E.C. 1.3.99.2, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP_347102 (SEQ ID NO: 117), NC_ 003030 (SEQ ID NO: 118))), Euglena gracilis (GenBank NOs: Q5EU90 SEQ ID NO: 119), AY741582 SEQ ID NO: 120)), Streptomyces collinus (GenBank NOs: AAA92890 (SEQ ID NO: 121), U37135 (SEQ ID NO: 122)), and Streptomyces coelicolor (GenBank NOs: CAA22721 (SEQ ID NO: 123), AL939127 (SEQ ID NO: 124)).

[0070] The term "isobutyryl-CoA mutase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme Bi 2 as cofactor. Example isobutyryl-CoA mutases are known by the EC number 5.4.99.13. These enzymes are found in a number of Streptomyces, including, but not limited to, Streptomyces cinnamonensis (GenBank Nos: AAC08713 (SEQ ID NO: 125), U67612 (SEQ ID NO: 126); CAB59633 (SEQ ID NO: 127), AJ246005 (SEQ ID NO: 128)), S coelicolor (GenBank Nos: CAB70645 (SEQ ID NO: 129), AL939123 (SEQ ID NO: 130); CAB92663 (SEQ ID NO: 131), AL939121 (SEQ ID NO: 132)), and Streptomyces avermitilis (GenBank Nos: NP_824008 (SEQ ID NO: 133), NC_003155 (SEQ ID NO: 134); NP 824637 (SEQ ID NO: 135), NC 003155 (SEQ ID NO: 136)). [0071] The term "butyraldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as co factor. Example butyraldehyde dehydrogenases are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841, AF 157306) and C. acetobutylicum (GenBank NOs: NPJ49325, NC_001988).

[0072] The term "acetolactate decarboxylase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Example 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 (GenBank Nos: AAU43774, AY722056).

[0073] The term "acetoin aminase" or "acetoin transaminase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 3-amino-2- butanol. Acetoin aminase may utilize the cofactor pyridoxal 5'-phosphate or NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate). The resulting product may have (R) or (S) stereochemistry at the 3 -position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor. The NADH- and NADPH-utilizing enzymes may use ammonia as a second substrate. A suitable example of an NADH utilizing acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688). An example of a pyridoxal- dependent acetoin aminase is the amine :pyruvate aminotransferase (also called amine :pyruvate transaminase) described by Shin and Kim, J. Org. Chem. 67:2848-2853 (2002)).

[0074] The term "acetoin kinase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymes that catalyze the analogous reaction on the similar substrate dihydroxyacetone, for example, include enzymes known as EC 2.7.1.29 (Garcia-Alles et al, Biochemistry 45:13037-13046 (2004)).

[0075] The term "acetoin phosphate aminase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of phosphoacetoin to 3-amino-2- butanol O- phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal 5'-phosphate, NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate. The NADH and NADPH-utilizing enzymes may use ammonia as a second substrate. There is a pyridoxal phosphate- dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta et al, Appl. Environ. Microbial. (57:4999-5009 (2001)).

[0076] The term "aminobutanol phosphate phospho lyase", also called "amino alcohol O- phosphate lyase", refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Amino butanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5'-phosphate. There are reports of enzymes that catalyze the analogous reaction on the similar substrate l-amino-2-propanol phosphate (Jones et al., Biochem J. 754:167-182 (1973)). U.S. Appl. Pub. No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.

[0077] The term "aminobutanol kinase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3 -amino-2 -butanol to 3-amino-2butanol O- phosphate. Amino butanol kinase may utilize ATP as the phosphate donor. Although there are no reports of enzymes catalyzing this reaction on 3-amino-2-butanol, there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and 1 -amino-2 -propanol (Jones et al, supra). U.S. Appl. Pub. No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.

[0078] The terms "butanediol dehydrogenase" and "acetoin reductase" refer to a polypeptide

(or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3- butanediol. Butanedial dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)- stereo chemistry 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, 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).

[0079] The terms "butanediol dehydratase," "dial dehydratase" or "propanediol dehydratase" refer to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B12; although vitamin B 12 may refer also to other forms of cobalamin that are not coenzyme B12). Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (Note all three subunits are required for activity)], and Klebsiella pneumonia (GenBank Nos:

AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064). Other suitable dial dehydratases include, but are not limited to, B12-dependent dial dehydratases available from Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza et al., J. Agric. Food Chem. 45:3476-3480 (1997)), and nucleotide sequences that encode the corresponding enzymes. Methods of dial dehydratase gene isolation are well known in the art (e.g., U.S. Pat. No. 5,686,276).

[0080] The term "pyruvate decarboxylase" refers to an enzyme that catalyzes the

decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenases are known by the EC number 4.1.1.1. These enzymes are found in a number of yeast, including

Saccharomyces cerevisiae (GenBank Nos: CAA97575 (SEQ ID NO: 137), CAA97705 (SEQ ID NO: 138), CAA97091 (SEQ ID NO: 139)).

[0081] The term "phosphoketolase" refers to an enzyme that catalyzes the conversion of xyulose 5 -phosphate to glyceraldehyde 3 -phosphate and acetyl phosphate. Example

phosphoketolases are known by the EC number 4.1.2.9. In some embodiments, the phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 146; amino acid SEQ ID NO: 147).

[0082] The term "phosphotransacetylase" refers to an enzyme that catalyzes the conversion of acetyl-CoA and phosphate to Co A and acetyl phosphate. Example phosphotransacetylases are known by the EC number 2.3.1.8. In some embodiments, the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 144; amino acid SEQ ID NO: 145). [0083] The term "thiamine" or "thiamin" as used herein refers to the compound added to a medium which is then converted by a host cell to the cofactor for acetolactate synthase known as "thiamine pyrophosphate" ("TPP") or "thiamine diphosphate" ("ThDP") (used interchangeably herein). Suitable forms of thiamine are readily available to those of skill in the art.

[0084] The term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. In embodiments, the polypeptides provided herein, including, but not limited to biosynthetic pathway polypeptides, cell integrity polypeptides, propagation

polypeptides, and other enzymes comprise full-length polypeptides and active fragments thereof.

[0085] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment.

Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0086] A polypeptide of the invention may be of a size of about 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. [0087] Also included as polypeptides of the present invention are derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "active variant," "active fragment," "active derivative," and "analog" refer to polypeptides of the present invention and include any polypeptides that are capable of catalyzing the indicated substrate to product conversion. Variants of polypeptides of the present invention include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non- conservative amino acid substitutions, deletions and/or additions. Derivatives of polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein a "derivative" of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4- hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0088] A "fragment" is a unique portion of sequence used in the invention which is identical in sequence to but shorter in length than the parent full-length sequence. A fragment may comprise up to the entire length of the defined sequence, minus one amino acid or nucleotide residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues. A fragment may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 100 or 200 amino acids of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present

embodiments. Similarly, "active fragment", when used in reference to a polypeptide, is a portion of a polypeptide which retains the functionality of the subject polypeptide, but comprises less than the entire sequence of the polypeptide.

[0089] Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a host cell system.

[0090] Amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they can be result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements.

"Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, "non-conservative" amino acid substitutions can be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. "Insertions" or "deletions" are preferably in the range of about 1 to about 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

[0091] Polypeptides suitable for use in the present invention and fragments thereof are encoded by polynucleotides. The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mR A), virally-derived R A, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond {e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. Polynucleotides according to the present invention further include such molecules produced synthetically. Polynucleotides of the invention may be native to the host cell or heterologous. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0092] A polynucleotide or polypeptide sequence can be referred to as "isolated," in which it has been placed in an environment other than its native environment or is produced synthetically or is a non-naturally occurring, or engineered, sequence. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having enzymatic activity (e.g., the ability to convert a substrate to xylulose) contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant

polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide fragment in the form of a polymer of DNA can be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0093] 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.

[0094] A "coding region" or "ORF" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' non-translated regions, and the like, are not part of a coding region. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem- loop structures.

[0095] 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.

[0096] In certain embodiments, a polynucleotide or nucleic acid is DNA. In the case of

DNA, a polynucleotide comprising a nucleic acid, which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" or "coupled" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide. Suitable promoters and other transcription control regions are disclosed herein. An "expression construct", as used herein, comprises a promoter nucleic acid sequence operably linked to a coding region for a polypeptide and, optionally, a terminator nucleic acid sequence.

[0097] A variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES). In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded. [0098] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.

[0099] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "recombinant" or "transformed" organisms.

[00100] The terms "expression," and "expressed" refer to the transcription and stable accumulation of sense (mR A) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Differentially expressed" refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene depending on the environment of the host cell. A differentially expressed gene may be overexpressed or underexpressed as compared to the expression level under other conditions. In one aspect, it refers to a differential that is 1, 2, 3, 4, 5, 10, or 20 times higher or lower than the expression level detected in a reference environment. The term "differentially expressed" also refers to nucleotide sequences in a cell which are expressed where silent or not expressed in a control environment or not expressed where expressed in a control cell.

[00101] 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 cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific construct containing a gene and having elements in addition to the gene that allow for expression of that gene.

[00102] "Native" refers to the form of a polynucleotide, gene, or polypeptide as found in nature with its own regulatory sequences, if present. [0100] "Endogenous" refers to the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism. "Endogenous polynucleotide" includes a native polynucleotide in its natural location in the genome of an organism. "Endogenous gene" includes a native gene in its natural location in the genome of an organism. "Endogenous polypeptide" includes a native polypeptide in its natural location in the organism.

[0101] "Heterologous" refers to a polynucleotide, gene, or polypeptide not normally found in the host organism but that is introduced into the host organism. "Heterologous polynucleotide" may include a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native polynucleotide. "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 can 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. "Heterologous polypeptide" includes a native polypeptide that is reintroduced into the source organism in a form that is different from the corresponding native polypeptide.

[0102] By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

[0103] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. Appl. Biosci. (5:237-245 (1990). In a sequence alignment the query and subject sequences are both DNA sequences. An R A sequence can be compared by converting U's to T's. The result of the global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are:

Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty-30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequences, whichever is shorter.

[0104] If the subject sequence is shorter than the query sequence because of 5 ' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

[0105] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not

matched/total number of bases in the query sequence) so 10%> is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%>. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.

[0106] Polypeptides used in the invention are encoded by nucleic acid sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences described elsewhere in the specification, including active variants, fragments or derivatives thereof.

[0107] The terms "active variant," "active fragment," "active derivative," and "analog" refer to polynucleotides of the present invention and include any polynucleotides that encode biocatalyst polypeptides used in the invention that retain their respective substrate-to-product conversion activity, although aspects of their activity (kinetics, cofactor affinity, etc.) may be altered as compared to the reference enzyme. Variants of polynucleotides of the present invention include polynucleotides with altered nucleotide sequences due to base pair substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring (i.e. "engineered"). Non- naturally occurring variants may be produced using art-known mutagenesis techniques. Derivatives of polynucleotides of the present invention, are polynucleotides which have been altered so that the polypeptides they encode exhibit additional features not found on the native polypeptide. Examples include polynucleotides that encode fusion proteins. Variant polynucleotides may also be referred to herein as "polynucleotide analogs." As used herein a "derivative" of a polynucleotide refers to a subject polynucleotide having one or more nucleotides chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those polynucleotides which contain one or more naturally occurring nucleotide derivatives. For example, 3-methylcytidine may be substituted for cytosine; ribothymidine may be substituted for thymidine; and N4-acetylcytidine may be substituted for cytosine.

[0108] The term "K D " is known to those skilled in the art as the dissociation constant for a given enzyme. Accordingly, the lower the K D value, the higher the enzyme's affinity for a substrate or cofactor. For example, with regard to acetolactate synthase and the cofactor TPP, a higher KD value reflects a lower affinity of the enzyme for the cofactor. The K D of a particular enzyme for a cofactor can be readily determined using methods well-known to those of skill in the art, for example, by varying the concentration of cofactor in the presence of saturating substrates and measuring the rate of product formation.

[0109] The term "K M " is known to those skilled in the art and is described in Enzyme

Structure and Mechanism, 2nd ed. (Ferst; W.H. Freeman Press, NY, 1985; pp 98-120). K M , the Michaelis constant, is used herein to refer to the concentration of substrate that leads to half-maximal velocity and as a means of characterising an enzyme's affinity for a substrate. Accordingly, the lower the K M value, the higher the enzyme's affinity for a substrate or cofactor. The K M of a particular enzyme for a particular substrate can be readily determined using methods well-known to those of skill in the art.

[0110] The term "specific activity" as used herein is defined as the units of activity in a given amount of protein. Thus, the specific activity is not directly measured but is calculated by dividing 1) the activity in units/ml of the enzyme sample by 2) the concentration of protein in that sample, so the specific activity is expressed as units/mg, where an enzyme unit is defined as moles of product formed/minute. The specific activity of a sample of pure, fully active enzyme is a characteristic of that enzyme. The specific activity of a sample of a mixture of proteins is a measure of the relative fraction of protein in that sample that is composed of the active enzyme of interest.

[0111] A "fragment" when used in reference to a promoter sequence is a unique portion of the promoter nucleic acid sequence or the nucleic acid sequence encoding the biocatalyst

polypeptide used in the invention which is identical in sequence to but shorter in length than the parent nucleic acid sequence. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides. A fragment used as a probe, primer, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide or amino acid.

Fragments may be preferentially selected from certain regions of a molecule. For example, a polynucleotide fragment may comprise a certain length of contiguous nucleotides selected from the first 100 or 200 nucleotides of a polynucleotide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

[0112] The term "codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting 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.

[0113] The term "codon optimized coding region" means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

[0114] Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is reproduced herein as Table A. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

Table A: The Standard Genetic Code

[0115] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0116] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage.

Codon usage tables are readily available, for example, at the "Codon Usage Database" available at http://www.kazusa.or.jp/codon/ (visited March 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 February 2002], are reproduced below as Table B. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The Table has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

Table B: Codon Usage Table for Saccharomyces cerevisiae Genes

[0117] By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon- optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. [0118] Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTl Suite, available from InforMax, Inc., Bethesda, MD, and the "backtranslate" function in the GCG— Wisconsin Package, available from Accelrys, Inc., San Diego, CA. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the "backtranslation" function at http://www.entelechon.com/bioinformatics/ backtranslation.php?lang=eng (visited April 15, 2008) and the "backtranseq" function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited July 9, 2002). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.

[0119] Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as "synthetic gene designer"

(userpages.umbc.edu/~vyugl/codon/sgd/, visited March 19, 2012).

Control elements

[0120] Industrial fermentation processes may employ a stage of biomass production in order to provide sufficient biocatalyst for the fermentation stage to have desired yield and production rate. Ethanologen S. cerevisiae for example is typically propagated using fed-batch technology, in which low sugar concentrations and non-limiting aeration favor respiratory metabolism with high biomass yields, e.g., Y xs ~ 0.5 g biomass/g glucose. The maintenance of low sugar concentrations in a fed- batch regime may be important for a Crabtree-positive yeast like S. cerevisiae, in which the fraction of respiratory metabolism on overall metabolism is negatively correlated with increasing

extracellular glucose concentrations. Due to the low sugar concentrations, specific glucose uptake rate is limited and respiratory capacity is sufficient to completely metabolize pyruvic acid formed in catabolism of the carbohydrate substrates to C0 2 . Under fermentative conditions with no oxygen or at higher glucose concentrations under aerobic conditions with the Crabtree effect in action, ethanologen yeasts like, e.g., S. cerevisiae produce ethanol and only low biomass yields are achieved, e.g., Y xs ~ 0.15g biomass/g glucose.

[0121] In addition, considerations for the propagation of biocatalysts that produce lower alkyl alcohols, such as butanologen biocatalysts include (i) the negative effect of toxic products, such as butanol or 2-butanone, (ii) the accumulation of inhibitory pathway byproducts or

intermediates, and (iii) the loss of substrate to the formation of fermentation byproducts resulting in lower yields of biocatalyst and fermentation product formation. For example, when a butanol production pathway functions constitutively in yeast, then the butanol produced may inhibit growth during the propagation phase of a production process and may add cost and inefficiency to either or both the infrastructure and the operation of the biocatalyst production phase. Control, particularly reduction or elimination, of butanol production during the biomass-forming phase would represent an advance in the art.

[0122] In some embodiments, the polypeptide activity is sensitive to one or more directed differences between propagation and production stages of fermentation. In embodiments, the polypeptide is selected or engineered such that its activity is sensitive to the concentration of a cofactor. In embodiments, the polypeptide is selected or engineered such that its activity is sensitive to temperature. In some embodiments, the polypeptide's kinetic characteristics are exploited such that the activity varies between different stages of a fermentation-based manufacturing process due to different levels of substrate during the different stages. Such control elements allow for controlled activity of a target polypeptide during propagation and production stages of fermentation.

[0123] One desirable feature of the polypeptides, polynucleotides, recombinant host cells, and methods disclosed herein is that accumulation of inhibitory by-products due to flux via enzymes of the butanol production pathway can be avoided during the growth phase. A non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is isobutyric acid. Another non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is isobutyraldehyde. Yet another non- limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is acetic acid. Some acetolactate synthase enzymes demonstrate a significant oxygenase side reaction in which molecular oxygen electrophilically attacks a highly reactive carbanion/enamine to form a peroxy-adduct that decomposes to ThDP and peracetic acid. See Tse, M.T. and Schloss, J.V., Biochemistry 52: 10398-10403 (1993). The peracetic acid can further react with pyruvate to form two moles of acetate. In addition to the growth inhibitory effects and the loss of metabolic energy for fighting off the stress generated by the presence of by-products, carbon lost to the by-products adds to a lower yield of biocatalyst on the employed carbon substrate.

[0124] Another desirable feature of some embodiments is that with oxygen supply, reduction equivalents produced in metabolic pathways leading to pyruvate can be oxidized to a higher fraction by the respiratory chain rather than by a biosynthetic pathway such as a butanol biosynthetic pathway. A higher fraction of pyruvate can transition the mitochondrial membrane and be further metabolized by pyruvate dehydrogenase and the tricarboxylic acid cycle. Another desirable feature of some embodiments is that without oxygen supply, reduction equivalents produced in metabolic pathways leading to pyruvate may be oxidized by the butanol production pathway and a lower fraction by the glycerol production pathway. This way a lower yield of glycerol and a higher yield of butanol may be achieved.

[0125] Yet another desirable feature of some embodiments is that in case of excess pyruvic acid production, pyruvic acid can be excreted into the medium. See van Maris, A.J., et ah, Appl. Environ. Micriobiol. 70: 159-166 (2004). However, pyruvic acid even at very high concentrations is not growth inhibiting.

[0126] Such desirable features may be achieved using polypeptides, polynucleotides, recombinant host cells and methods provided herein. For example, as shown in the Examples, compositions and methods herein may be used to provide preferential activity of the acetolactate synthase of an isobutanol production pathway during the production phase of an isobutanol fermentation-based production process.

Biosynthetic pathways

[0127] Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase; - b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase;

- d) α-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for example, by a branched- chain keto acid decarboxylase; and,

- e) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0128] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;

- d) α-ketoisovalerate to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase;

- e) valine to isobutylamine, which may be catalyzed, for example, by valine decarboxylase;

- f) isobutylamine to isobutyraldehyde, which may be catalyzed by, for example, omega transaminase; and,

- g) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0129] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase; - d) α-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for example, by branched- chain keto acid dehydrogenase;

-e) isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for example, by aclylating aldehyde dehydrogenase; and,

- f) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0130] In another embodiment, the isobutanol biosynthetic pathway comprises the substrate to product conversions shown as steps k, g, and e in Figure 1.

[0131] Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Appl. Pub. No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl-CoA acetyl transferase;

- b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be catalyzed, for example, by 3- hydroxybutyryl-CoA dehydrogenase;

- c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed, for example, by crotonase;

- d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase;

- e) butyryl-CoA to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase; and,

- f) butyraldehyde to 1-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[0132] Biosynthetic pathways for the production of 2-butanol that may be used include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase; - b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 3 -amino-2 -butanol, which may be catalyzed, for example, acetonin aminase;

- d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase;

- e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase; and,

- f) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol dehydrogenase.

[0133] In another embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol

dehydrogenase;

- d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by dial dehydratase; and,

- e) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol dehydrogenase.

[0134] Biosynthetic pathways for the production of 2-butanone that may be used include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 3 -amino-2 -butanol, which may be catalyzed, for example, acetonin aminase;

- d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase; and, - e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase.

[0135] In another embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol

dehydrogenase;

- d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by diol dehydratase.

[0136] In another embodiment, the biocatalyst polypeptide comprises or is selected from the group of enzymes having the following Enzyme Commission Numbers: EC 2.2.1.6, EC 1.1.1.86, EC 4.2.1.9, EC 4.1.1.72, EC 1.1.1.1, EC 1.1.1.265, EC 1.1.1.2, EC 1.2.4.4, EC 1.3.99.2, EC 1.2.1.57, EC 1.2.1.10, EC 2.6.1.66, EC 2.6.1.42, EC 1.4.1.9, EC 1.4.1.8, EC 4.1.1.14, EC 2.6.1.18, EC 2.3.1.9, EC 2.3.1.16, EC 1.1.130, EC 1.1.1.35, EC 1.1.1.157, EC 1.1.136, EC 4.2.1.17, EC 4.2.1.55, EC 1.3.1.44, EC 1.3.1.38, EC 1.3.1.44, EC 1.3.1.38, EC 5.4.99.13, , EC 4.1.1.5, EC 1.1.1.1, 2.7.1.29, 1.1.1.76, and 4.2.1.28, or the enzymes acetonin aminase, acetoin phosphate aminase, aminobutanol phosphate phospholyase, and aminobutanol kinase.

[0137] In some embodiments, the biocatalyst polypeptide which catalyzes the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol utilize NADH as a cofactor.

[0138] In some embodiments, enzymes from the biosynthetic pathway are localized to the cytosol. In some embodiments, enzymes from the biosynthetic pathway that are usually localized to the mitochondria are localized to the cytosol. In some embodiments, an enzyme from the

biosynthetic pathway is localized to the cytosol by removing the mitochondrial targeting sequence. In some embodiments, mitochondrial targeting is eliminated by generating new start codons as described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference in its entirety.

[0139] Genes and polypeptides that may be used for the substrate to product conversions described above as well as those for additional isobutanol pathways, are described herein and in the art, for example, in U.S. Patent Appl. Pub. No. 20070092957, PCT Pub. No. WO 2011/019894, and in PCT App. No. WO2012/129555, all incorporated by reference herein. US Appl. Pub. Nos. 2011/019894, 20070092957, 20100081154, describe dihydroxyacid dehydratases including those from Lactococcus lactis (SEQ ID NO: 218) and Streptococcus mutans (SEQ ID NO: 217) and variants thereof, eg. S. mutans 12 V5 (SEQ ID NO: 216). Ketoiso valerate decarboxylases include those derived from Lactococcus lactis (SEQ ID NO: 219), Macrococcus caseolyticus (SEQ ID NO: 797) and Listeria grayi (SEQ ID NO: 220). U.S. Patent Appl. Publ. No. 2009/0269823 and U.S. Appl. Publ. No. 2011/0269199, incorporated by reference, describe alcohol dehydrogenases.

Alcohol dehydrogenases include SadB from Achromobacter xylosoxidans (SEQ ID NO: 222) disclosed in U.S. Patent 8,188,250, incorporated herein by reference. Additional alcohol dehydrogenases include horse liver ADH (SEQ ID NO: 223) and Beijerinkia indica ADH (SEQ ID NO: 224), and those that utilize NADH as a cofactor. KARI enzymes are described for example, in U.S. Patent Nos. 7,910,342 and 8,129,162; U.S. Publication No. 2008/0261230, 2009/0163376, 2010/0197519, 2013/0071898, 2014/0051133, and 2014/0093930; International Publication No. WO 2012/129555, all of which are incorporated by reference. KARIs include Pseudomonas fluorescens KARI (SEQ ID NO: 225) and variants thereof and Anaerostipes caccae KARI (SEQ ID NO: 226) and variants thereof (eg. "K9G9", "K9D3", and "K9JB4P"; SEQ ID NOs: 143, 142, and 215 respectively). In one embodiment an isobutanol biosynthetic pathway comprises a) a ketol-acid reductoisomerase that has a KM for NADH less than about 300 μΜ, less than about 100 μΜ, less than about 50 μΜ, less than about 20 μΜ or less than about 10 μΜ; b) an alcohol dehydrogenase that utilizes NADH as a cofactor; or c) both a) and b).

Cell Integrity Polypeptides

[0140] Another embodiment of the invention is directed a biocatalyst polypeptide for cell integrity. In one embodiment, the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance. In one embodiment, the biocatalyst polypeptide is YER150W (SPI1) (nucleic acid SEQ ID NO: 150; amino acid SEQ ID NO: 153), or a homolog thereof. In another embodiment, the biocatalyst polypeptide is encoded by a cell wall integrity gene activated by Rlml such as a polypeptide listed in Table 5 or a homolog thereof. Table 5 Biocatalyst polypeptides for cell integrity

[0141] In embodiments, cell integrity polypeptides are preferentially expressed during the production phase. While not wishing to be bound by theory, it is believed that expression of such polypeptides may contribute to improved tolerance of a host cell to a fermentation product (e.g., butanol), thus improving production.

Propagation Polypeptides

[0142] Another embodiment of the invention is directed to a biocatalyst polypeptide for cell propagation. In some embodiments, the propagation polypeptide comprises phosphoketolase activity. In some embodiments, the propagation polypeptide comprises phosphotransacetylase activity. Example phosphoketolases and phosphotransacetylases are described in PCT Publication No. WO/2011/159853 and U.S. App. Pub. No. 20120156735A1, incorporated by reference herein. In some embodiments, the phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 146; amino acid SEQ ID NO: 147). In some embodiments, the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 144; amino acid SEQ ID NO: 145).

[0143] In embodiments, host cells comprising such biocatalyst polypeptides for cell propagation have reduced or eliminated pyruvate decarboxylase activity.

Biosynthetic pathway by-product producing polypeptides

[0144] DHMB can be produced as a result of a side-reaction that occurs when host cells are genetically manipulated to include a biosynthetic pathway, e.g. , a biosynthetic pathway that involves the production of acetolactate. The presence of DHMB indicates that not all of the pathway substrates are being converted to the desired product. Thus, yield may be lowered. In addition, DHMB present in the fermentation media may have inhibitory effects on product production. For example, DHMB can decrease the activity of enzymes in the biosynthetic pathway or have other inhibitory effects on cell growth and/or productivity during fermentation. Thus, described herein are isolated polynucleotides resulting in lower expression of DHMB during the production phase of fermentation than in the propagation phase. The ability of a host cell to convert acetolactate to DHMB can be reduced or eliminated, for example, by reducing the expression of a polypeptide having acetolactate reductase activity. In some embodiments, the polypeptide having acetolactate reductase activity is YMR226C (nucleic acid SEQ ID NO: 227, amino acid SEQ ID NO: 228) or a homolog thereof.

[0145] The last step in the biosynthesis of isobutanol via a pyruvate-utilizing biosynthetic pathway is the conversion of isobutyraldehyde to isobutanol (Figure 1). A side reaction in this pathway is the conversion of isobutyraldehyde to isobutyric acid which results in reduced amounts of isobutyraldehyde available to convert into isobutanol and reduced isobutanol yield. Reducing or eliminating the conversion of isobutyraldehyde to isobutyric acid may result in increased amounts of isobutyraldehyde available for conversion to isobutanol. The conversion of isobutyraldehyde to isobutyric acid can be reduced or eliminated, for example, by reducing the expression of an aldehyde dehydrogenase. Thus, provided herein are isolated polynucleotides resulting in lower expression of an aldehyde dehydrogenase during the production phase of fermentation than in the propagation phase. In embodiments, a recombinant host cell of the invention can be S. cerevisiae, and a polypeptide having aldehyde dehydrogenase activity can be ALD2 (nucleic acid SEQ ID NO: 192; amino acid SEQ ID NO: 193), ALD3 (nucleic acid SEQ ID NO: 194; amino acid SEQ ID NO: 195), ALD4 (nucleic acid SEQ ID NO: 196; amino acid SEQ ID NO: 197), ALD5 (nucleic acid SEQ ID NO: 198; amino acid SEQ ID NO: 199), ALD6 (nucleic acid SEQ ID NO: 148; amino acid SEQ ID NO: 149), or combinations thereof. In other embodiments, a recombinant host cell can be

Kluyveromyces lactis, and a polypeptide having aldehyde dehydrogenase activity can be

KLLA0F00440, KLLA0E23057, KLLAOD 10021, KLLA0D09999G, or combinations thereof. In other embodiments, a recombinant host cell can be Pichia stipitis, and a polypeptide having aldehyde dehydrogenase activity can be ALD2, ALD3, ALD4, ALD5, ALD7, or combinations thereof. In other embodiments, a recombinant host cell can be Lactobacillus plantarum, and a polypeptide having aldehyde dehydrogenase activity can be AldH. In other embodiments, a recombinant host cell can be E. coli, and a polypeptide having aldehyde dehydrogenase activity can be aldA, aldB, or combinations thereof.

Glycerol biosynthesis pathway polypeptides

[0146] Endogenous NAD-dependent glycerol-3 -phosphate dehydrogenase is a key enzyme in glycerol synthesis, converting dihydroxyacetone phosphate (DHAP) to glycerol-3 -phosphate and playing a role in cellular oxidation of NADH. Yeast strains may have one or more genes encoding NAD-dependent glycerol-3 -phosphate dehydrogenase (GPD). In some yeasts, such as S. cerevisiae, S. pombe, and . stipitis, GPDl and GPD2 are functional homo logs for NAD-dependent glycerol-3 - phosphate dehydrogenase. Provided herein are isolated polynucleotides that resulting in lower expression of glycerol-3 -phosphate dehydrogenase activity during the production phase of fermentation than in the propagation phase. In one embodiment, the biocatalyst polypeptide is GPDl (nucleic acid SEQ ID NO: 200; amino acid SEQ ID NO: 201) or GPD2 (nucleic acid SEQ ID NO: 202; amino acid SEQ ID NO: 203), or a homolog thereof.

Acetolactate synthase

[0147] In one non-limiting embodiment, the biocatalyst polypeptide is biosynthetic pathway polypeptide acetolactate synthase. As described above, ALS catalyzes the first substrate to production conversion in isobutanol, 2-butanol, and 2-butanone biosynthetic pathways. Accordingly, controlled activity of acetolactate synthase may be employed to control flux through the pathway.

[0148] Two groups of enzymes are classified as AHAS (acetohydroxyacid synthase, EC

2.2.1.6): FAD-dependent biosynthetic enzymes that readily catalyze the formation of

acetohydroxybutyrate from pyruvate and 2-oxobutyrate, as well as of acetolactate from two molecules of pyruvate. These reactions represent the first committed steps in the biosynthesis of branched-chain amino acids, such as valine, leucine and isoleucine. The enzymes are generally susceptible to inhibition by one or more of the branched-chain amino acids. The second group of acetolactate synthases are apparently devoted to post-exponential growth phase production of acetoin in bacteria. These enzymes are not FAD-dependent and they are insensitive to inhibition by the branched-chain amino acids. To the extent that their specificity has been examined, they have a low capability for acetohydroxybutyrate synthesis and tend to show relatively low pH optima (Chipman et al, 1998, Biochim Biophy Acta, 1385(2): 401-419).

[0149] In embodiments, AHAS may be from the second group of acetolactate synthases, like e.g. the AlsS gene from Bacillus subtilis (Renna et al, 1993, J. Bacteriol 175(12): 3863-3875.) and may avoid feedback inhibition of the enzyme. Notwithstanding the different cofactor requirements, subunit structure, sensitivity to inhibition by branched-chain amino acids and substrate specificity, the two groups of acetolactate synthases are related and show substantial sequence similarities. Comparison between the FAD-dependent AHAS of S. cerevisiae and the FAD-independent AHAS of K. pneumonia revealed similar secondary and tertiary structures as well. Also, in both enzymes the active site is located at a dimer interface, situated at the bottom of a funnel about 15-20 A from the surface of the protein (Pang et al, 2004, J Biol Chem 279(3): 2242-2253).

[0150] Screening of chemical compound libraries to identify compounds or scaffolds for their inhibitory effects on AHAS within whole cells in vivo which are dependent on the biosynthesis of branched chain amino acids (BCAA), e.g. (Cho et al, 2013, Biochimie 95(7): 1411-1421), or alternatively based on isolated enzyme (in vitro), as for example described by Choi et al. (Choi et al., 2005, FEBS Lett 579(21): 4903-4910), is known to those of skill in the art. Also design of AHAS inhibitors based on structural knowledge and in silico screening (e.g. applying molecular docking approaches) are well established techniques in the field (Wang et al, 2007, Bioorganic and

Medicinal Chemistry 15(1): 374-380). [0151] Many known inhibitors of AHAS, e.g. most of the herbicides, bind within the tunnel leading to the active site, thereby blocking substrate access (McCourt et al, 2006, PNAS USA, 103(3): 569-573; Wang et al, 2009, FEBS J 276(5) 1282-1290). At least 17 amino acid residues have been identified in bacteria, fungi, or plants where mutation results in AHAS herbicide resistance (Table 6 (Duggleby et al., 2008, Plant Physiol and Biochem: PPB/Societa Francaise De Physiologie Vegetale 46(3): 309-324)). Herbicide-resistant AHAS variants that have been catalogued include both natural isolates from resistant organisms and those that have been engineered in the laboratory. In both whole plants, and at the molecular level, mutations can lead to cross-tolerance among AHAS inhibitors. Cross-tolerance generally exists between sulfonylureas and triazolopyrimidines, or between imidazolinones and pyrimidyl(oxy/thio)benzoates; however some mutations result in broad cross-resistance to all four classes of herbicide (Tranel and Wright, 2002, Weed Science, 50(6): 700-712). When bound to A. thaliana AHAS, the sulfonylurea chlorimuron ethyl makes contact with 16 amino acids. Of these, 14 are completely conserved in yeast AHAS and there is only one further difference in E. coli AHASII. The residues comprising the herbicide- binding site have been maintained through the two billion years since eukaryotes and bacteria shared a common ancestor (Duggleby et al., 2008). Construction of branch chain amino acid feedback resistant AHAS, for example by changing amino acid sequence of the regulatory domain and subsequently selecting feedback resistant AHAS enzymes, is described in literature and known to those of skill in the art (Elisakova et al., 2005, Appl Environ Microbiol 71(1): 207-213). Leader sequences/peptides which direct proteins expressed in the cytoplasm to the mitochondria are known to those of skill in the art (Neupert and Herrmann, 2007, Annual Review of Biochemistry, 76:723- 749; Mossmann et al, 2012, Biochim Biophys Acta 1819(9-10): 1098-1106). Accordingly, one of skill in the art may readily select, for example, an inhibitor-resistant and branched-chain amino acid feedback-inhibited AHAS to be localized in mitochondria or an inhibitor-sensitive AHAS, preferably de-sensitized for feedback-inhibition by branched-chain amino acids to be localized in the cytoplasm of a butanologen yeast. Table 6. Herbicide -resistant mutations of AHAS. Data from Duggleby et al. (Duggleby et al., 2008).

Yeast F590[CGLNR] SU

Yeast F590L SU/IM

Tobacco F577[DE] SU/IM/TP

A. thaliana S653N IM

A. thaliana S653T SUS/IMR

A. thaliana S653F SU/IM

A. thaliana S653N SUS/IMR

Tobacco S652T SUS/IMR/TPS

[0152] In Duggleby, herbicide "resistance" is only reported for variant enzymes with > 10- fold increases in the inhibition constant (K lapp , IC 50 ) relative to the wild-type enzyme. ResistantR and sensitiveS types are indicated in cases where one or more class of herbicide was tested but resistance was only evident for one class. Herbicide classes are abbreviated: SU, sulfonylurea; IM,

imidazolinone; TP, triazolopyrimidine; POB, pyrimidyloxybenzoate; PSF, pyrimidylthiobenzoate (Duggleby et al, 2008).

[0153] In some embodiments, an inhibitor is present with a recombinant host cell comprising a biosynthetic pathway comprising acetolactate synthase during a first phase of a fermentation process, such as a propagation phase. Decreasing the concentration of the inhibitor, for example, by increasing the volume of the fermentation mixture, may reduce the inhbitition of acetolactate synthase, increasing activity and increasing flux through the biosynthetic pathway.

Acetolactate Synthase Structure

[0154] A BLAST search using the B. subtilis ALS (GI number 1929340; CAB07802.1) sequence as query to the Research CoUaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) yielded 35 hits at an E-value of le-10. Structure 10ZG of the ALS from Klebsiella pneumoniae, was the top hit and was chosen as the template for the protein chain. The target (1929340) and the template lOZG A (chain A) were aligned using profile data with SwissProt hits. 1929340 and lOZG A share 51% identity.

[0155] A model of the B. subtilis ALS was built using 10ZG as template. Thiamine pyrophosphate (TPP) ligand and Mg2+ cofactor ion were included from 10ZG in the model. ALS structures are similar to the acetohydroxyacid synthase (AHAS) structures but do not have the groove that binds to FAD.

[0156] Just as in the structure 10ZG, the model is in the 'resting phase' of the enzyme, with three distinct but connected α, β and γ domains (see figure 6). In Figure 6, the TPP molecule is in stick-format and the Mg 2+ ion is being shown in solid fill. For B. subtilis ALS, a-domain is from Ml - P191, β-domain is from K192 - E344, and γ-domain is from A368 - L571. These domains are not fully shown in the figure because the first eight residues and last thirteen residues were not modeled. Residues F345 - P367 were not included in any of the three domains because they are part of a linker helix between the β and γ domains.

[0157] Residues at a distance of at most 6 A from the TPP molecule are considered binding site residues (a total of 38 residues). They fall into three categories based on neighborhood of the TPP molecule according to visual inspection - diphosphate end that also contains the cofactor ion (Mg 2+ in this case), methyl end at the other end, and catalytic site at the center of the TPP molecule.

[0158] The 38 positions identified as TPP binding sites are as follows: Q62, F66, T94, D397,

1398, G399, S400, H401, A402, N421, M423, Q424, T425, L426, G427, S449, G450, D451, G452, G453, F454, F456, W476, N477, D478, S479, T480, Y481, D482, M483, V484, A485, Q487, V498, F500, V545, Y547, N550. They were further classified broadly based on function and location as follows (with 6A distance cutoff and visual inspection): (A)S400, H401, S449, G450, D451, G452, G453, W476, D478, T480, Y481, D482, V545, Y547, N550 - Diphosphate-end of TPP (Of these, D451, D478, T480 are further classified as Mg2+ anchoring); (B)Q62, F66, T94, F454, F456 - Methyl end of TPP; (C) 1398, G399, M423, Q424, T425, L426, M483, V484, Q487 - Catalytic part of TPP; and (D) D397, A402, N421, G427, N477, S479, A485, V498, F500 - Other parts of TPP.

Cofactor affinity

[0159] TPP is a cofactor for ALS. Control of thiamine levels in fermentation processes may be exploited as a control cue to regulate the activity of ALS. In one embodiment, a recombinant host cell comprising a polypeptide having ALS activity is contacted with with a carbon substrate in the presence of an amount of thiamine for a first phase; subsequently, the thiamine concentration is increased such that the ALS activity is increased for a second phase. In embodiments, butanol or 2- butanone is produced during the first phase, the second phase, or both. In embodiments, the production of butanol or 2-butanone is increased in the second phase. In some embodiments the first phase is a propagation phase and the second phase is a production phase. In some embodiments, the production increase is an increase in butanol or 2-butanone production rate. [0160] It will be appreciated that the amount of thiamine to be used as a control cue will depend on the affinity of a particular ALS for the cofactor. One of skill in the art will also appreciate that other enzymes in a biosynthetic pathway or otherwise important for cellular function may likewise be affected by the same cofactor. Accordingly, it may be desireable to select or engineer an ALS with a low affinity for TPP, such that thiamine can be limiting for ALS activity without substantially altering the health and maintenance of a cell.

[0161] For E. coli AHAS isozyme II it was described (Bar-Ilan, Balan et al. 2001,

Biochemistry 40(39): 11946-54) that mutagenesis of E47 decreased the rate of proton exchange at C2 of bound ThDP by nearly 2 orders of magnitude and decreased the turnover rate for the mutants by about 10-fold. Variant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of E428, carried out on the basis of the model to be the crucial carboxylate ligand to Mg 2+ in the "ThDP motif, led to a decrease in the affinity of AHAS II for Mg 2+ . On saturation with Mg 2+ , D428E had a decreased affinity for ThDP as compared to the native enzyme. The mutation also led to dependence of the enzyme on K + .

[0162] Provided herein are acetolactate synthase substitutions and the corresponding variants. While not wishing to be bound by theory, it is believed that variants provided herein may be employed in biosynthetic pathways comprising the substrate to product conversion pyruvate to acetolactate. In embodiments, variants provided herein are employed as controlled biocatalyst polypeptides in methods to produce biosynthetic pathway products.

[0163] Based on the model of B. subtilis ALS described herein above, it is proposed that the polar diphosphate end and the non-polar methyl end of the TPP are not being chemically transformed and thus the protein residues interacting exclusively with those regions are not 'catalytic'. Accordingly, substitutions at those residues are candidates to increase the K D without affecting catalysis. Substitutions that may increase the K D of TPP were identified by calculating binding energies as well as proposing a more select group of changes to decrease the favorable interactions with TPP. The residues near the central 5-membered ring with the sulfur were deemed catalytic, and altering them was not preferred, though not ruled out. Each of the candidate 38 positions was mutated to all other 19 residues and ligand-affinity (where ligand in this case is TPP) was calculated using the following energy function.

[0164] AG = Δ Ghbond+ Δ Gi on i c + Δ Gmii g + Δ Gh y drophobic+ Δ Gh p + Δ G aa [0165] where the individual terms are: hbond = Interactions between hydrogen bond donor- acceptor pairs; ionic = ionic interactions; mlig = Metal ligation. Interactions between Nitrogens/ Sulfurs and transition metals are assumed to be metal ligation interactions; hydrophobic =

Hydrophobic interactions, e.g. between alkyl carbons; hp = Interactions between hydrophobic and polar atoms; aa = An interaction between any two atoms.

[0166] Each residue change was followed by a side-chain conformation search and repacking including neighboring side chains within 4.5A from the mutating one. The software MOE (by Chemical Computing Group Inc) was used to do these calculations.

[0167] From the above analysis, variants of acetolactate synthase provided herein comprise substitutions as given in Table 8. While the substitutions are indicated by the positions in SEQ ID NO: 1, it is envisioned that such substitutions can be made in any acetolactate synthase. Example suitable acetolactate synthase enzymes are disclosed in U.S. Appn. Pub. No. 20090305363 and are incorporated herein by reference. Example acetolactate synthase sequences and/or source organisms are provided in Table 7 and Figure 7.

Table 7. Acetolactate synthase sequences and source organisms

Table 8. Substitutions in acetolactate synthase activity

[0168] Accordingly, provided herein are polypeptides comprising acetolactate synthase activity. In embodiments, such polypeptides comprise at least one substitution at a position corresponding to T94, G399, H401, G450, G453, V545 or Y547 of SEQ ID NO: 1. Also provided herein are polypeptides comprising acetolactate synthase activity and at least one, at least two, at least three, or at least 4 substitutions at positions corresponding to positions indicated in Table 8 for SEQ ID NO: 1. In embodiments, the substitution at a position corresponding to a position indicated in Table 8 for SEQ ID NO: 1 is at least one of the energy-based substitutions indicated in the Table. In embodiments, the substitution at a position corresponding to a position indicated in Table 8 for SEQ ID NO: 1 is at least one of the selected substitutions indicated in the Table. In embodiments, polypeptides comprising acetolactate synthase activity are variants of SEQ ID NO: 1. In

embodiments, the polypeptides are acetolactate synthase enzymes comprising at least about 90%, at least about 95%>, or at least about 98%> identity to SEQ ID NO: 1. Also provided herein are polypeptides comprising acetolactate synthase activity and wherein said polypeptide comprises at least about 90%, at least about 95%, or at least about 98% identity to SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266 or an active fragment thereof. In embodiments, polypeptides comprising acetolactate synthase activity comprises the sequence of SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, or 266 or an active fragment thereof. [0169] In embodiments, polypeptides comprising acetolactate synthase activity are variants of SEQ ID NO: 1 and comprise at least one substitution at position 94, 399, 401, 450, 453, 547 or 545. In embodiments, the substitution at position 547 is F, M, L, A, or V. In embodiments, the substitution at position 545 is M, W, or F. In embodiments, polypeptides comprising acetolactate synthase activity comprise at least about 90%, at least about 95%, or at least about 98% identity to SEQ ID NO: 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, or 363 or an active variant thereof. In embodiments, polypeptides comprising acetolactate synthase activity comprise the sequence of SEQ ID NO: 335, 336, 337, 338, 339, 340, 341, or 342 or an active variant thereof. In embodiments, polypeptides comprising acetolactate synthase activity comprise the sequence of SEQ ID NO: 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, or 363 or an active fragment thereof.

[0170] In embodiments, such polypeptides are employed as biocatalyst polypeptides in host cells and methods provided herein. Accordingly, also provided herein are polynucleotides encoding such polypeptides. In embodiments, polynucleotides are codon-optimized for a recombinant host cell provided herein.

[0171] It will be appreciated that suitable substitutions may be determined by those of skill in the art, for example, by preparing and inspecting multiple-sequence alignments wherein homologs, particularly homologs known to have a desired feature such as decreased affinity for TPP, are compared to a desired sequence. In such embodiments, substitutions in the desired sequence may be made based on the amino acids found in regions or residues of interest in the homologs.

[0172] In embodiments, multiple sequence alignments are employed to select homologs which have a desired feature such as decreased affinity for TPP, based on sequence diversity in regions or residues believed to be involved in cofactor binding. Such homologs may thus be employed in host cells and methods provided herein.

[0173] In embodiments, variants with lower affinity for TPP without substantial alteration of specific activity as compared to the native sequence are employed.

[0174] In embodiments, variants provided herein comprise lower affinity for TPP than the native sequence. In embodiments, variants provided herein comprise K D greater than that of SEQ ID NO: 1. [0175] One of skill in the art, equipped with this disclosure, will readily be able to alter the affinity of acetolactate synthase, and determine an appropriate amount of thiamine as a control cue for a recombinant host cell comprising such an acetolactate synthase.

Substrate concentration

[0176] In embodiments, the activity of a biocatalyst polypeptide is controlled by controlling the amount of substrate available. One of skill in the art will be readily able to determine a suitable amount of substrate for a given enzyme using standard methods and/or kinetic characteristics of the enzyme.

[0177] In embodiments, the controlled biocatalyst polypeptide is an acetolactate synthase.

Accordingly, control of the amount of pyruvate provides a control mechanism, with pyruvate concentration as a control cue. The amount of pyruvate produced by a recombinant host cell contacted by a fermentable carbon substrate such as glucose, and thus available as a substrate for acetolactate synthase, may be controlled by limiting the flux through the glycolytic pathway which converts glucose to pyruvate. Such control can be achieved by employing glucose-limited fed batch. When increased activity of acetolactate synthase is desired (eg. in the production phase of a fermentation), glucose supply is increased, thus increasing the supply of pyruvate available as a substrate for the biosynthetic pathway.

[0178] An acetolactate synthase with high K M for pyruvate may be selected or engineered to ensure a dynamic range. In embodiments, K M for pyruvate is greater than about 15 mM, about 20 mM, about 25 mM or about 50 mM. Candidate acetolactate synthase sequences are available to those of skill in the art (http://www.brenda-enzymes.org, last visited June 27, 2013) and include, but are not limited to those in Table 9.

Table 9. Candidate acetolactate synthase enzymes

Temperature

[0179] One process that is temperature sensitive is folding of the primary protein sequence into its tertiary structure. Within the aqueous environment of the cell polypeptide chains do not fold through two-state transitions but pass through well-defined intermediate states. These folding intermediates have to overcome a variety of problems: avoiding aggregate states; avoiding proteolysis; reaching particular cellular compartments; transiently binding chaperonins and other auxiliary proteins; and finally, reaching the native state (Mitraki and King, 1992, FEBS Lett 307(1): 20-25). If folding is responsible for the temperature sensitive (Ts) phenotype of a mutant at the permissive temperature a large fraction of the newly synthesized folding intermediates reach the native state. However, at non-permissive temperatures, a fraction ends up as inactive aggregates.

[0180] A number of in vitro mutagenesis approaches have been developed to generate Ts alleles for a particular gene of interest. One approach is to clone the gene of interest and randomly mutagenize it via low- fidelity PCR. The mutagenized DNA is then introduced into a host organism and screened in the context of a null background (Tan et al., 2009, Genetics 183(1): 13-22). For example a plasmid shuffle technique can be applied that relies upon the use of a counter-selection method for a functional marker 1 gene, such as for example 5-FOA to select against yeast cells possessing a functional URA3 (Boeke et al, 1987, Methods Enzymol 154: 164-175). An AHAS library is created on a plasmid carrying a second marker (marker 2, e.g. LEU2) and subsequently introduced into a haploid strain with the marker 1 -based plasmid and the non-temperature sensitive AHAS enzyme. Conditions that will not allow for growth of the strain without functional AHAS (e.g. under anaerobic conditions in a PDC deletion strain and with expressing complete isobutanol pathway apart from AHAS) are applied. Subsequently the transformed cells are screened for mutants unable to grow on the counter agent of marker 1 at restrictive temperature (Harris et al, 1992, J Mol Biol 225(1): 53-65). Other techniques to obtain Ts mutants of a protein comprise the replacement of charged amino acid clusters with alanines (Wertman et al, 1992, Genetics 132(2): 337-350), or the modification of buried hydrophobic cores within a protein (Chakshusmathi et al., 2004, PNAS USA 101(21):7925-7930). A more general method for creating TS alleles for genome-wide application in S. cerevisiae uses a degron (Dohmen et al, 1994, Science 263(5151): 1273-1276). At 37°C, the degron and its fusion protein are targeted for degradation via an ubiquitin mediated "N-end rule" pathway. At permissive temperatures, the degron does not activate the N-end rule pathway.

[0181] Alternatively a more general method that utilizes conditionally splicing inteins to generate TS mutations is known to those skilled in the art (Zeidler et al., 2004, Nat Biotechnol 22(7): 871-876). An intein is a self-excising stretch of amino acids, which is removed during protein maturation. An intein switch splices itself only at permissive temperatures. At non-permissive temperatures, it fails to splice and remains within the host protein. Intein splicing, also known as protein splicing, is a post-translational process in which the intein is precisely excised from a nascent protein precursor, and the two flanking sequences (N and C exteins) are ligated together through a normal peptide bond. Thus, no intein footprint is left behind after protein splicing. Furthermore, protein splicing is a self-catalytic reaction that does not require any exogenous proteins, cofactors, or energy sources. A broad set of inteins with different permissive temperatures are for example described by Tan et al. (Tan et al., 2009).

[0182] It is known to those skilled in the art that the methods for identifying temperature- sensitive mutants described previously are suited to find Ts enzymes that (i) are expressed and/or active up to a certain temperature (permissive temperature range) and are not expressed and/or inactive at higher temperatures (non-permissive temperature range), as well as Ts enzymes that (ii) are not expressed and/or inactive up to a certain temperature (non-permissive temperature range) and that are expressed and/or active at higher temperature (permissive temperature range).

Recombinant Microbial Host Cells

[0183] Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A

Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,

Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al, Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods are in Methods in

Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, CA). Molecular tools and techniques are known in the art and include splicing by overlapping extension polymerase chain reaction (PCR) (Yu, et al., Fungal Genet. Biol. 41 :973-981 (2004)), positive selection for mutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J.D. et al., Mol. Gen. Genet. 197, 345-346 (1984); M A Romanos, et al, Nucleic Acids Res. 19(1): 187 (1991)), the cre-lox site- specific recombination system as well as mutant lox sites and FLP substrate mutations (Sauer, B.m Mol Cell Biol 7: 2087-2096 (1987); Senecoff, et al, Journal of Molecular Biology 201(2):405-421 (1988); Albert, et al, The Plant Journal 7(4,): 649-659 (1995)), "seamless" gene deletion (Akada, et al, (2006) Yeast 23(5):399-405 (2006)), and gap repair methodology (Ma, et al, Genetics 58:201- 216 (1981)).

[0184] The genetic manipulations of a recombinant host cell disclosed herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202).

[0185] Non-limiting examples of host cells for use in the invention include bacteria, cyanobacteria, filamentous fungi and yeasts. [0186] In one embodiment, the recombinant host cell of the invention is a bacterial or a cyanobacterial cell. In another embodiment, the recombinant host cell comprises or is selected from the group consisting of: Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium,

Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus, Streptomyces,

Zymomonas, Escherichia, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Serratia, Shigella, Alcaligenes, Erwinia, Paenibacillus, and Xanthomonas . In some embodiments, the recombinant host cell is E. coli, S. cerevisiae, or L. plantarum.

[0187] In another embodiment, the recombinant host cell of the invention is a filamentous fungi or yeast cell. In another embodiment, the recombinant host cell comprises or is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces, Galactomyces, Schizosaccharomyces, Torulaspora, Debayomyces, Williopsis, Dekkera, Kloeckera, Metschnikowia, and Candida. In another embodiment, the host cell does not express an enzyme or has reduced expression of an enzyme having the following Enzyme Commission Number: EC 4.1.1.1.

[0188] In some embodiments, the yeast is crabtree-positive. Crabtree-positive yeast cells demonstrate the crabtree effect, which is a phenomenon whereby cellular respiration is inhibited when a high concentration of glucose is present in aerobic culture medium. Suitable crabtree- positive yeast are viable in culture and include, but are not limited to, Saccharomyces,

Schizosaccharomyces, and Issatchenkia. Suitable species include, but are not limited to,

Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces thermotolerans, Candida glabrata, Issatchenkia orientalis.

[0189] Crabtree-positive yeast cells may be grown with high aeration and in low glucose concentration to maximize respiration and cell mass production, as known in the art, rather than butanol production. Typically the glucose concentration is kept to less than about 0.2 g/L. The aerated culture can grow to a high cell density and then be used as the present production culture. Alternatively, yeast cells that are capable of producing butanol may be grown and concentrated to produce a high cell density culture.

[0190] In some embodiments, the yeast is crabtree-negative. Crabtree-negative yeast cells do not demonstrate the crabtree effect when a high concentration of glucose is added to aerobic culture medium, and therefore, in crabtree-negative yeast cells, alcoholic fermentation is absent after an excess of glucose is added. Suitable Crabtree-negative yeast genera are viable in culture and include, but are not limited to, Hansenula, Debaryomyces, Yarrowia, Rhodotorula, and Pichia. Suitable species include, but are not limited to, Candida utilis, Hansenula nonfermentans,

Kluyveromyces marxianus, Kluyveromyces lactis, Pichia stipitis, and Pichia pastoris.

[0191] Suitable microbial hosts include, but are not limited to, members of the genera

Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter,

Corynebacterium, Brevibacterium, Pichia, Candida, Issatchenkia, Hansenula, Kluyveromyces, and Saccharomyces. Suitable hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida,

Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae. In some embodiments, the host cell is

Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 1 13-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio- Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

[0192] Recombinant microorganisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to a desired product (eg. butanol) can be constructed using techniques well known in the art. For example, genes encoding the enzymes of one of the isobutanol biosynthetic pathways of the invention, for example, acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain a-keto acid decarboxylase, and branched-chain alcohol dehydrogenase, can be obtained from various sources, as described above.

[0193] Methods of obtaining desired genes from a genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries can be created by restriction endonuclease digestion and can be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available (described elsewhere herein).

[0194] Once the relevant pathway genes are identified and isolated they can be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE® (Madison, WI), Invitrogen Corp. (Carlsbad, CA), Stratagene (La Jolla, CA), and New England Biolabs, Inc. (Beverly, MA). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.

[0195] Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements, including those used in the Examples, is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL 10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, 1PL, 1PR, T7, tac, and trc (useful for expression in Escherichia coli, Alcaligenes, and Pseudomonas) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans. For yeast recombinant host cells, a number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1; and the following inducible promoters suitable for use in yeast: GAL1, GAL 10, OLE1, and CUP1. Other yeast promoters include hybrid promoters UAS(PGKl)-FBAlp, UAS(PGKl)-EN02p, UAS(FBAl)-PDClp, UAS(PGKl)-PDClp, and UAS(PGK)-OLElp. [0196] Promoters, transcriptional terminators, and coding regions can be cloned into a yeast

2 micron plasmid and transformed into yeast cells (Ludwig, et al. Gene, 132: 33-40, 1993; US Appl. Pub. No. 20080261861A1).

[0197] Adjusting the amount of gene expression in a given host may be achieved by varying the level of transcription, such as through selection of native or artificial promoters. In addition, techniques such as the use of promoter libraries to achieve desired levels of gene transcription are well known in the art. Such libraries can be generated using techniques known in the art, for example, by cloning of random cDNA fragments in front of gene cassettes (Goh et al. (2002) AEM 99, 17025), by modulating regulatory sequences present within promoters (Ligr et al. (2006) Genetics 172, 2113),or by mutagenesis of known promoter sequences (Alper et al. (2005) PNAS, 12678; Nevoigt et al. (2006) AEM 72, 5266).

[0198] Termination control regions can also be derived from various genes native to the hosts. Optionally, a termination site can be unnecessaryor can be included.

[0199] Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors-pR 437, pR 442, and pR 442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid, 50: 74-79, 2003). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.

[0200] Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWVlOl has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin, et al, J. BacterioL, 174: 5633-5638 (1992)). Additionally, in vitro transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®.

[0201] The expression of a biosynthetic pathway in various microbial hosts is described in more detail in the Examples herein and in the art.U.S. Patent 7,851,188 and PCT App. No.

WO2012/129555, both incorporated by reference, which disclose the engineering of recombinant microorganisms for production of isobutanol. U.S. Appl. Pub. No. 2008/0182308A1, incorporated by reference, discloses the engineering of recombinant microorganisms for production of 1-butanol. U.S. Appl. Pub. Nos. 2007/0259410A1 and 2007/0292927A1, both incorporated by reference, disclose the engineering of recombinant microorganisms for production of 2-butanol. Multiple pathways are described for biosynthesis of isobutanol and 2-butanol. The methods disclosed in these publications can be used to engineer the recombinant host cells of the present invention. The information presented in these publications is hereby incorporated by reference in its entirety.

Modifications

[0202] In some embodiments, the host cells comprising a biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Appl. Pub. No.

2009/0305363, incorporated herein by reference, discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. Modifications to reduce glycerol-3- phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Appl. Pub. No. 2009/0305363, incorporated herein by reference, modifications to a host cell that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in U.S. Appl. Pub. No. 2010/0120105, incorporated herein by reference. Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate -utilizing biosynthetic pathway. Other modifications are described in PCT. Pub. No. WO2012/129555, incorporated herein by reference. Modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In embodiments, the polypeptide having acetolactate reductase activity is YMR226C of Saccharomyces cerevisae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from

Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Appl. Pub. No. 2011/0124060, incorporated herein by reference. In some embodiments, the pyruvate decarboxylase that is deleted or downregulated is selected from the group consisting of: PDC1, PDC5, PDC6, or combinations thereof. In some embodiments, host cells contain a deletion or downregulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3 -phosphate to glycerate 1,3- bisphosphate. In some embodiments, the enzyme that catalyzes this reaction is glyceraldehyde-3 -phosphate dehydrogenase.

[0203] Recombinant host cells may further comprise (a) at least one heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity; and (b)(i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe-S cluster biosynthesis; and/or (ii) at least one heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster biosynthesis, described in PCT Publication No. WO2011/103300, incorporated herein by reference. In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is encoded by AFTl, AFT2, FRA2, GRX3, or CCCl . In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFTl L99A, AFTl L102A, AFTl C291F, or AFTl C293F.

Differential Activity

[0204] As demonstrated in the Examples, a recombinant host cell comprising an enzyme with controlled activity may be subjected to directed conditions wherein such conditions result in controlled activity between the phases. Such controlled activity may be confirmed using methods known in the art and/or provided herein. Controlled of a biocatalyst polypeptide can be confirmed by comparing production rate, titer, or yield of the biosynthetic pathway target in the presence or absence of a control cue. For example, where ALS is the biocatalyst polypeptide, the activity of ALS present in host cells subjected to conditions with and without a control cue may be determined (using, for example, methods described in Westerfeld, W.W., J. Biol. Chem. 7(57:495-502 (1945). A difference in ALS activity can be used to confirm controlled activity of the ALS. Controlled activity of a biocatalyst polypeptide can be confirmed indirectly by measurement of downstream products or byproducts. For example, a decrease in production of isobutyraldehyde between phases may be indicative of controlled ALS activity. [0205] It will be appreciated that other useful methods to confirm controlled activity include measurement of biomass and/or measurement of biosynthetic pathway products under different conditions. For example, spectrophotometric measurement of optical density (O.D.) can be used as an indicator of biomass. Measurement of pathway products or by-products, including, but not limited to butanol or isobutyric acid can be carried out using methods known in the art and/or provided herein such as high pressure liquid chromatography (HPLC; for example, see PCT. Pub. No. WO2012/129555, incorporated herein by reference) Likewise, the rate of biomass increase, the rate of glucose consumption, or the rate of butanol production can be determined, for example by using the indicated methods. Biomass yield and product (eg. butanol) yield can likewise be determined using methods disclosed in the art and/or herein.

Methods for Producing Fermentation Products

[0206] Another embodiment of the present invention is directed to methods for producing various fermentation products including, but not limited to, lower alkyl alcohols. These methods employ the recombinant host cells and controlled polypeptides of the invention. In one embodiment, the method of the present invention comprises providing a recombinant host cell as discussed above, contacting the recombinant host cell with a fermentable carbon substrate in a fermentation medium under conditions whereby the fermentation product is produced and, optionally, recovering the fermentation product.

[0207] It will be appreciated that a manufacturing process for producing fermentation products may comprise multiple phases. For example, a process may comprise a first biomass production phase, a second biomass production phase, a fermentation production phase, and a product recovery phase. In embodiments, processes provided herein comprise more than one, more than two, or more than three phases. It will be appreciated that process conditions and media composition may vary from phase to phase. For example, one phase of a process may be

substantially aerobic, while the next phase may be substantially anaerobic. Other differences between phases may include, but are not limited to, source of carbon substrate (e.g., feedstock from which the fermentable carbon is derived), carbon substrate (e.g.,. glucose) concentration, media component concentration, dissolved oxygen, pH, temperature, or concentration of fermentation product (e.g., butanol). In embodiments, a biocatalyst polypeptide has activity preferentially in at least one phase.

[0208] The propagation phase generally comprises at least one process by which biomass is increased. In embodiments, the temperature of the propagation phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C. In embodiments, the pH in the propagation phase may be at least about 4, at least about 5, at least about 5.5, at least about 6, or at least about 6.5. In embodiments, the propagation phase continues until the biomass concentration reaches at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 50, at least about 70, or at least about 100 g/L. In embodiments, the average glucose or sugar concentration is about or less than about 2 g/L, about or less than about 1 g/L, about or less than about 0.5 g/L or about or less than about 0.1 g/L. In embodiments, the dissolved oxygen

concetration may average as undetectable, or as at least about 10%, at least about 20%, at least about 30%, or at least about 40%.

[0209] In one non-limiting example, a stage of the propagation phase comprises contacting a recombinant yeast host cell with at least one carbon substrate at a temperature of about 30 to about 35°C and a pH of about 4 to about 5.5, until the biomass concentration is in the range of about 20 to about 100 g/L. The dissolved oxygen level over the course of the contact may average from about 20 to 40%) (0.8 - 3.2 ppm). The source of the carbon substrate may be molasses or corn mash, or pure glucose or other sugar, such that the glucose or sugar concentration is from about 0 to about 1 g/L over the course of the contacting or from about 0 to about 0.1 g/L. In a subsequence or alternate stage of the propagation phase, a recombinant yeast host cell may be subjected to a further process whereby recombinant yeast at a concentration of about 0.1 g/L to about 1 g/L is contacted with at least one carbon substrate at a temperature of about 25 to about 35°C and a pH of about about 4 to about 5.5 until the biomass concentration is in the range of about 5 to about 15 g/L. The dissolved oxygen level over the course of the contact may average from undetectable to about 30% (0-2.4 ppm). The source of the carbon substrate may be corn mash such that the glucose concentration averages about 2 to about 30 g/L over the course of contacting.

[0210] It will be understood that the propagation phase may comprise one, two, three, four, or more stages, and that the above non-limiting example stages may be practiced in any order or combination. [0211] The production phase typically comprises at least one process by which a product is produced. In embodiments, the average glucose concentration during the production phase is at least about 0.1, at least about 1, at least about 5, at least about 10 g/L, at least about 30 g/L, at least about 50 g/L, or at least about 100 g/L. In embodiments, the temperature of the production phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C. In embodiments, the pH in the production phase may be at least about 4, at least about 5, or at least about 5.5. In

embodiments, the production phase continues until the product titer reaches at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 30 g/L, at least about 35 g/L or at least about 40 g/L. In embodiments, the dissolved oxygen concetration may average as less than about 5%, less than about 1%, or as negligible such that the conditions are substantially anaerobic.

[0212] In one non-limiting example production phase, recombinant yeast cells at a concentration of about 0.1 to about 6 g/L are contacted with at least one carbon substrate at a concentration of about 5 to about 100 g/L, temperature of about 25 to about 30°C, pH of about 4 to about 5.5. The dissolved oxygen level over the course of the contact may be negligible on average, such that the contact occurs under substantially anaerobic conditions. The source of the carbon substrate may mash such as corn mash, such that the glucose concentration averages about 10 to about 100 g/L over the course of the contacting, until it is substantially completely consumed.

[0213] In embodiments, the glucose concentration is about 100-fold to about 1000-fold higher in the production phase than in the propagation phase. In embodiments, the glucose concentration in production is at least about 5X, at least about 10X, at least about 5 OX, at least about 100X, or at least about 500X higher than that in propagation. In embodiments, the temperature in the propagation phase is about 5 to about 10 degrees lower in the production phase than in the propagation phase. In embodiments, the average dissolved oxygen concentration is anaerobic in the production phase and microaerobic to aerobic in the propagation phase.

[0214] One of skill in the art will appreciate that the conditions for propagating a host cell and/or producing a fermentation product utilizing a host cell may vary according to the host cell being used. In one embodiment, the method for producing a fermentation product is performed under anaerobic conditions. In one embodiment, the method for producing a fermentation product is performed under microaerobic conditions. [0215] Further, it is envisioned that once a recombinant host cell comprising a suitable control element has been identified, the process may be further refined to take advantage of the regulated activity afforded thereby. For example, if the controlled biocatalyst polypeptide provides preferential activity in high glucose conditions, one of skill in the art will be able to readily determine the glucose levels necessary to maintain suitable activity. As such, the glucose concentration in the phase of the process under which minimal expression is desired can be controlled so as to maintain desired activity. In one non-limiting example, polymer-based slow- release feed beads (available, for example, from Kuhner Shaker, Basel, Switzerland) may be used to maintain a low glucose condition. A similar strategy can be employed to refine the propagation or production phase conditions relevant to the host cells and methods provided herein.

[0216] Carbon substrates may include, but are not limited to, monosaccharides (such as fructose, glucose, mannose, rhamnose, xylose or galactose), oligosaccharides (such as lactose, maltose, or sucrose), polysaccharides such as starch, maltodextrin, or cellulose, fatty acids, or mixtures thereof and unpurified mixtures from renewable feedstocks such as corn mash, cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate, or glycerol.

[0217] Additionally, the carbon substrate may also be a one carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion, et al., Microb. Growth CI 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 (Suiter, 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.

[0218] Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof may be suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Appl. Pub. No. 2007/0031918 Al, which is herein incorporated by reference. Biomass in reference to a carbon source refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.

[0219] The carbon substrates may be provided in any media that is suitable for host cell growth and reproduction. Non-limiting examples of media that can be used include M122C, MOPS, SOB, TSY, YMG, YPD, 2XYT, LB, Ml 7, or M9 minimal media. Other examples of media that can be used include solutions containing potassium phosphate and/or sodium phosphate. Suitable media can be supplemented with NADH or NADPH.

[0220] In one embodiment, the method for producing a fermentation product results in a titer of at least about 20 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 30 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L or 40 g/L of fermentation product. [0221] In embodiments, the rate of production of a fermentation product is increased. In embodiments, the rate of biomass production is increased. In embodiments, the yield of

fermentation product is increased. In embodiments, the yield of biomass is increased. Such improvements may be observed by comparison to that obtained using the control recombinant host cell without a genetic switch.

[0222] Non- limiting examples of lower alkyl alcohols which may be produced by the methods of the invention include butanol (for example, isobutanol), propanol, isopropanol, and ethanol. In one embodiment, isobutanol is produced.

[0223] In one embodiment, the recombinant host cell of the invention produces a

fermentation product at a yield of greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of theoretical. In one embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 25% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 40% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 50% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 75% of theoretical.

[0224] Non- limiting examples of lower alkyl alcohols produced by the recombinant host cells of the invention include butanol, isobutanol, propanol, isopropanol, and ethanol. In one embodiment, the recombinant host cells of the invention produce isobutanol. In another

embodiment, the recombinant host cells of the invention do not produce ethanol.

Methods for Isobutanol Isolation from the Fermentation Medium

[0225] Bioproduced butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot, et al, Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation. [0226] Because butanol forms a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques.

Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

[0227] 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.

[0228] The butanol can 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.

[0229] Distillation in combination with adsorption can 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).

[0230] 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)). [0231] In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields. One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction. In general, with regard to butanol fermentation, for example, the fermentation medium, which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the fermentation medium form a biphasic mixture. The butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.

[0232] Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 2009/0305370, the disclosure of which is hereby

incorporated in its entirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono- unsaturated, poly-unsaturated (and mixtures thereof) C 12 to C 22 fatty alcohols, C 12 to C 22 fatty acids, esters of C 12 to C 22 fatty acids, C 12 to C 22 fatty aldehydes, and mixtures thereof. The extractant(s) for ISPR can be non-alcohol extractants. The ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.

[0233] In some embodiments, the alcohol can be formed by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst capable of esterfiying the alcohol with the organic acid. In such embodiments, the organic acid can serve as an ISPR extractant into which the alcohol esters partition. The organic acid can be supplied to the

fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol. The catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock. When the catalyst is supplied to the fermentation vessel, alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel. Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant. The extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration. In addition, unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.

[0234] In situ product removal can be carried out in a batch mode or a continuous mode. In a continuous mode of in situ product removal, product is continually removed from the reactor. In a batchwise mode of in situ product removal, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. For in situ product removal, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some

embodiments of the present invention, the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel. The ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved. In some embodiments, the ester- containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.

[0235] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

Examples

[0236] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating 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

[0237] Standard recombinant DNA, molecular cloning techniques and transformation protocols used in the Examples are well known in the art and are described by Sambrook et al.

(Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, here in referred to as Maniatis), by Ausubel et al. (Ausubel et al, Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience, 1987) and by Amberg et al (Amberg, D. C, Burke, D. J. and

Strathern, J. N. (Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Press, 2005). Materials and methods suitable for the maintenance and growth of bacterial 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 et al., eds., American Society for Microbiology, Washington, DC, 1994) or by Thomas D. Brock in (Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Sigma-Aldrich Chemicals (St. Louis, MO), BD Diagnostic Systems (Sparks, MD), Invitrogen (Carlsbad, CA), HiMedia (Mumbai, India), SD Fine chemicals (India), or Takara Bio Inc. (Shiga,, Japan), unless otherwise specified.

[0238] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "nm" means nanometers, "uL" means microliter(s), "mL" means milliliter(s), "mg/mL" means milligram per milliliter, "L" means liter(s), "nm" means nanometers, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "μιηοΐε" means micromole(s), "kg" means kilogram, "g" means gram(s), '^g" 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" can also mean the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "kb" means kilobase, "%" means percent, "% w/v" means weight/volume percent, "% v/v" means volume/volume percent, "HPLC" means high performance liquid chromatography, "g/L" means gram per liter, '^g/L" means microgram per liter, "ng^L" means nanogram per microliter,

"ριηοΐ/μυ' means picomol per microliter, "RPM" means rotation per minute, '^mol/min/mg" means micromole per minute per milligram, "w/v" means weight per volume, "v/v" means volume per volume.

Construction of strains used in Examples

Construction of PNY2068

[0239] Saccharomyces cerevisiae strain PNY0827 is used as the host cell for further genetic manipulation. PNY0827 refers to a strain derived from Saccharomyces cerevisiae which has been deposited at the ATCC under the Budapest Treaty on September 22, 2011 at the American Type Culture Collection, Patent Depository 10801 University Boulevard, Manassas, VA 20110-2209 and has the patent deposit designation PTA-12105.

Deletion of URA3 and sporulation into haploids

[0240] In order to delete the endogenous URA3 coding region, a deletion cassette was PCR- amplified from pLA54 (SEQ ID NO: 272) which contains a V TE Fi-kanMX4-TEFlt cassette flanked by loxP sites to allow homologous recombination in vivo and subsequent removal of the KANMX4 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and primers BK505 (SEQ ID NO: 273) and BK506 (SEQ ID NO: 274). The URA3 portion of each primer was derived from the 5' region 180bp upstream of the URA3 ATG and 3' region 78bp downstream of the coding region such that integration of the kanMX4 cassette results in replacement of the URA3 coding region. The PCR product was transformed into PNY0827 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202) and transformants were selected on YEP medium supplemented 2% glucose and 100 μg/ml Geneticin at 30°C. Transformants were screened by colony PCR with primers LA468 (SEQ ID NO: 275) and LA492 (SEQ ID NO: 276) to verify presence of the integration cassette. A heterozygous diploid was obtained: NYLA98, which has the genotype MAT a/a XJRA3/ura3::loxP-kanMX4-loxF. To obtain haploids, NYLA98 was sporulated using standard methods (Codon AC, Gasent-Ramirez JM, Benitez T. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker's yeast. Appl Environ Microbiol. 1995 PMID: 7574601). Tetrads were dissected using a micromanipulator and grown on rich YPE medium supplemented with 2% glucose. Tetrads containing four viable spores were patched onto synthetic complete medium lacking uracil supplemented with 2% glucose, and the mating type was verified by multiplex colony PCR using primers AK109-1 (SEQ ID NO: 277), AK109-2 (SEQ ID NO: 278), and AK109-3 (SEQ ID NO: 279). The resulting indentified haploid strain called NYLA 103, which has the genotype: MATa ura3A::loxP-kanMX4-loxP, and NYLA106, which has the genotype: MATa ura3 A::loxV-kanMX4-loxV .

Deletion of His3

[0241] To delete the endogenous HIS3 coding region, a scarless deletion cassette was used.

The four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, CA). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 280) and primer oBP453 (SEQ ID NO: 281), containing a 5' tail with homology to the 5' end of HIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO: 282), containing a 5' tail with homology to the 3' end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 283) containing a 5' tail with homology to the 5' end of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 284), containing a 5' tail with homology to the 3' end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 285), containing a 5' tail with homology to the 5' end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 286), containing a 5' tail with homology to the 3' end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 287). PCR products were purified with a PCR Purification kit (Qiagen). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 280) and oBP455 (SEQ ID NO: 283). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO: 284) and oBP459 (SEQ ID NO: 287). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 280) and oBP459 (SEQ ID NO: 287). The PCR product was purified with a PCR Purification kit (Qiagen). Competent cells of NYLA106 were transformed with the HIS3 ABUC PCR cassette and were plated on synthetic complete medium lacking uracil supplemented with 2% glucose at 30 °C. Transformants were screened to verify correct integration by replica plating onto synthetic complete medium lacking histidine and supplemented with 2% glucose at 30°C. Genomic DNA preps were made to verify the integration by PCR using primers oBP460 (SEQ ID NO: 288) and LA135 (SEQ ID NO: 289) for the 5 * end and primers oBP461 (SEQ ID NO: 290) and LA92 (SEQ ID NO: 291) for the 3' end. The URA3 marker was recycled by plating on synthetic complete medium supplemented with 2% glucose and 5-FOA at 30°C following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD -URA medium to verify the absence of growth. The resulting identified strain, called PNY2003 has the genotype: MATa ura3A::loxP-kanMX4-loxP his3A.

Deletion of PDCl

[0242] To delete the endogenous PDC1 coding region, a deletion cassette was PCR- amplified from pLA59 (SEQ ID NO: 292), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and primers LA678 (SEQ ID NO: 293) and LA679 (SEQ ID NO: 294). The PDCl portion of each primer was derived from the 5' region 50bp downstream of the PDCl start codon and 3' region 50bp upstream of the stop codon such that integration of the URA3 cassette results in replacement of the PDCl coding region but leaves the first 50bp and the last 50bp of the coding region. The PCR product was transformed into PNY2003 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 2% glucose at 30°C. Transformants were screened to verify correct integration by colony PCR using primers LA337 (SEQ ID NO: 295), external to the 5' coding region and LA135 (SEQ ID NO: 289), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA692 (SEQ ID NO: 296) and LA693 (SEQ ID NO: 297), internal to the PDCl coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 298) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 2% glucose at 30°C. Transformants were plated on rich medium

supplemented with 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 2% glucose to verify absence of growth. The resulting identified strain, called PNY2008 has the genotype: MATa ura3A::loxP-kanMX4-loxP his3A /¾ ciA::loxP71/66.

Deletion of PDC5

[0243] To delete the endogenous PDC5 coding region, a deletion cassette was PCR- amp lifted from pLA59 (SEQ ID NO: 292), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and primers LA722 (SEQ ID NO: 299) and LA733 (SEQ ID NO: 300). The PDC5 portion of each primer was derived from the 5' region 50bp upstream of the PDC5 start codon and 3' region 50bp downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire PDC5 coding region. The PCR product was transformed into PNY2008 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers LA453 (SEQ ID NO: 301), external to the 5' coding region and LAI 35 (SEQ ID NO: 289), an internal primer to URA3. Positive transformants were then screened by colony PCR using primers LA694 (SEQ ID NO: 302) and LA695 (SEQ ID NO: 303), internal to the PDC5 coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 298) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30°C. Transformants were plated on rich YEP medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, called PNY2009 has the genotype: MATa ura3A::loxP-kanMX4-loxP his3A /¾ ciA::loxP71/66 /¾/c5A::loxP71/66.

Deletion of FRA2

[0244] The FRA2 deletion was designed to delete 250 nucleotides from the 3' end of the coding sequence, leaving the first 113 nucleotides of the FRA2 coding sequence intact. An in- frame stop codon was present 7 nucleotides downstream of the deletion. The four fragments for the PCR cassette for the scarless FRA2 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, CA). FRA2 Fragment A was amplified with primer oBP594 (SEQ ID NO: 304) and primer oBP595 (SEQ ID NO: 305), containing a 5' tail with homology to the 5' end of FRA2 Fragment B. FRA2 Fragment B was amplified with primer oBP596 (SEQ ID NO: 306), containing a 5" tail with homology to the 3' end ofFRA2 Fragment A, and primer oBP597 (SEQ ID NO: 307), containing a 5' tail with homology to the 5' end of FRA2 Fragment U. FRA2 Fragment U was amplified with primer oBP598 (SEQ ID NO: 308), containing a 5' tail with homology to the 3' end of FRA2 Fragment B, and primer oBP599 (SEQ ID NO: 309), containing a 5' tail with homology to the 5' end of FRA2 Fragment C. FRA2 Fragment C was amplified with primer 0BP6OO (SEQ ID NO: 310), containing a 5' tail with homology to the 3' end of FRA2 Fragment U, and primer 0BP6OI (SEQ ID NO: 311). PCR products were purified with a PCR Purification kit (Qiagen). FRA2 Fragment AB was created by overlapping PCR by mixing FRA2 Fragment A and FRA2 Fragment B and amplifying with primers oBP594 (SEQ ID NO: 304) and oBP597 (SEQ ID NO: 307). FRA2 Fragment UC was created by overlapping PCR by mixing FRA2 Fragment U and FRA2 Fragment C and amplifying with primers oBP598 (SEQ ID NO: 308) and 0BP6OI (SEQ ID NO: 31 1). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The FRA2 ABUC cassette was created by overlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment UC and amplifying with primers oBP594 (SEQ ID NO: 304) and 0BP6OI (SEQ ID NO: 31 1). The PCR product was purified with a PCR Purification kit (Qiagen).

[0245] To delete the endogenous FRA2 coding region, the scarless deletion cassette obtained above was transformed into PNY2009 using standard techniques and plated on synthetic complete medium lacking uracil and supplemented with 1% ethanol. Genomic DNA preps were made to verify the integration by PCR using primers oBP602 (SEQ ID NO: 312) and LA135 (SEQ ID NO: 289) for the 5* end, and primers oBP602 (SEQ ID NO: 312) and oBP603 (SEQ ID NO: 313) to amplify the whole locus. The URA3 marker was recycled by plating on synthetic complete medium supplemented with 1% ethanol and 5-FOA (5-Fluoroorotic Acid) at 30°C following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify the absence of growth. The resulting identified strain, PNY2037, has the genotype: MATa wra3A: :loxP- kanMX4-loxP his3A pdcl A: ΛοχΡΊ 1/66 pdc5A: :\oxPl\l66fra2A.

Addition of 2 micron plasmid

[0246] The loxP71 -URA3-loxP66 marker was PCR-amplified using Phusion DNA polymerase (New England BioLabs; Ipswich, MA) from pLA59 (SEQ ID NO: 29), and transformed along with the LA81 1x817 (SEQ ID NOs: 314, 315) and LA812x818 (SEQ ID NOs: 316, 317) 2- micron plasmid fragments into strain PNY2037 on SE -URA plates at 30°C. The resulting strain PNY2037 2μ: :1οχΡ71-υΡχΑ3-1οχΡ66 was transformed with pLA34 (pRS423 : xre) (also called, pLA34) (SEQ ID NO: 298) and selected on SE -HIS -URA plates at 30°C. Transformants were patched onto YP-1% galactose plates and allowed to grow for 48 hrs at 30°C to induce Cre recombinase expression. Individual colonies were then patched onto SE -URA, SE -HIS, and YPE plates to confirm URA3 marker removal. The resulting identified strain, PNY2050, has the genotype: MATa ura3A: :loxP-kanMX4-loxP, his3A pdcl A: ΛοχΡΊ 1/66 pdc 5 A: ΛοχΡΊ 1/66 fralA 2- micron.

Deletion of GPD2

[0247] To delete the endogenous GPD2 coding region, a deletion cassette was PCR- amp lifted from pLA59 (SEQ ID NO: 292), which contains a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using Phusion High Fidelity PCR Master Mix (New England BioLabs; Ipswich, MA) and primers LA512 (SEQ ID NO: 318) and LA513 (SEQ ID NO: 319). The GPD2 portion of each primer was derived from the 5' region 50bp upstream of the GPD2 start codon and 3' region 50bp downstream of the stop codon such that integration of the URA3 cassette results in replacement of the entire GPD2 coding region. The PCR product was transformed into PNY2050 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers LA516 (SEQ ID NO: 320), external to the 5' coding region and LA135 (SEQ ID NO: 289), internal to URA3. Positive transformants were then screened by colony PCR using primers LA514 (SEQ ID NO: 321) and LA515 (SEQ ID NO: 322), internal to the GPD2 coding region. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 298) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30°C. Transformants were plated on rich medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain,

PNY2056, has the genotype: MATa ura3A: :loxP-kanMX4-loxP his 3 A /¾ ciA::loxP71/66

/¾ c5A::loxP71/66 fra2A 2-micron gpd2A.

Deletion of YMR226 and integration of AlsS

[0248] To delete the endogenous YMR226C coding region, an integration cassette was PCR- amp lifted from pLA71 (SEQ ID NO: 323), which contains the gene acetolactate synthase from the species Bacillus subtilis with a FBA1 promoter and a CYC1 terminator, and a URA3 marker flanked by degenerate loxP sites to allow homologous recombination in vivo and subsequent removal of the URA3 marker. PCR was done by using KAPA HiFi from Kapa Biosystems, Woburn, MA and primers LA829 (SEQ ID NO: 324) and LA834 (SEQ ID NO: 325). The YMR226C portion of each primer was derived from the first 60bp of the coding sequence and 65bp that are 409bp upstream of the stop codon. The PCR product was transformed into PNY2056 using standard genetic techniques and transformants were selected on synthetic complete medium lacking uracil and supplemented with 1% ethanol at 30°C. Transformants were screened to verify correct integration by colony PCR using primers N1257 (SEQ ID NO: 326), external to the 5' coding region and LA740 (SEQ ID NO: 328), internal to the FBA1 promoter. Positive transformants were then screened by colony PCR using primers N1257 (SEQ ID NO: 326) and LA830 (SEQ ID NO: 327), internal to the YMR226C coding region, and primers LA830 (SEQ ID NO: 327), external to the 3' coding region, and LA92 (SEQ ID NO: 291), internal to the URA3 marker. The URA3 marker was recycled by transforming with pLA34 (SEQ ID NO: 298) containing the CRE recombinase under the GAL1 promoter and plated on synthetic complete medium lacking histidine and supplemented with 1% ethanol at 30°C. Transformants were plated on rich medium supplemented with 1% ethanol and 0.5% galactose to induce the recombinase. Marker removal was confirmed by patching colonies to synthetic complete medium lacking uracil and supplemented with 1% ethanol to verify absence of growth. The resulting identified strain, PNY2061, has the genotype: MATa ura3A: :loxP-kanMX4-loxP his 3 /¾ ciA::loxP71/66 /¾/c5A::loxP71/66 fra2A 2-micro gpd2Aymr226cA::P F BAi-alsS_Bs-CYCR- loxP71/66.

[0249] Strain PNY2092 was constructed by plasmid transformation of the base strain

PNY2061 (described above) which has genotype: MATa ura3A: :loxP-kanMX4-loxP his3A

/¾ ciA::loxP71/66 /¾ c5A::loxP71/66 fra2A 2-micro gpd2Aymr226cA::P FBA1 -alsS_Bs-CYCR- loxP71/66 with plasmids: pHR81-ILV5p-R8B2y2 (SEQ ID NO: 333) and pLA84 (SEQ ID NO: 334).

[0250] pHR81 -ILV5p-R8B2y2 (SEQ ID NO : 82) contains P. fluorescens R8B2 KARI

(codon-optimized for yeast) driven by ILV5 promoter and ILV5 terminator in pHR81 plasmid backbone. pLA84 (SEQ ID NO: 83) contains IlvD from S. mutans driven by FBA1 promoter and FBA1 terminator, ADH from B. indica driven by GPM1 promoter and ADH1 terminator and KivD from L. grayi driven by TDH3 promoter and TDH3 terminator in pRS423 plasmid backbone. Construction of PNY2286 and PNY2167

[0251] QuickChange primers were designed to substitute residues E61 and D351 into Ala and Glu, respectively in B. subtilis ALS (0E6IAI and oE61Alr, SEQ ID NOs: 329 and 330, respectively and oD351E and oD351Er, SEQ ID NOs: 330 and 331, respectively). Purified primers (polyacrylamide gel electrophoresis) were used with an Agilent QuickChange Lightening kit with alsS integration construct pLA71 (SEQ ID NO: 323) as the template.

[0252] Five colonies from each transformation were sequenced (TempliPhi). Plasmids from clones 2-5 (E61A) and 8-10 (D351E) were prepared and submitted for additional sequencing.

Clones 2 and 8 were used as template DNA in a PCR reaction (KAPA HIFI polymerase) using primers LA829 (SEQ ID NO: 324) and LA834 (SEQ ID NO: 325) to add flanking sequences for integration. The expected product sizes (4352 bp) were confirmed by agarose gel and cleaned using phenol-chloroform extraction. About 2.7-3.0 mg of each DNA were transformed into PNY2056.

[0253] Transformants were screened by PCR. Two colonies with expected PCR product were transformed with pLA34 (SEQ ID NO: 298) for URA3 marker recycling. Transformants were obtained on SE minus His plates. Four clones of each were streaked on YPE Gal for isolated colonies. Colonies were patched and screened by PCR. After proper marker removal, the expected PCR product size was 706 bp. Clones of each alsS variant containing strain were transformed with pLA84 (SEQ ID NO: 333) and pHR81 ::R8B2y2 (SEQ ID NO: 334). Transformant colonies (3 of each variant-containing strain) were inoculated directly into 0.3% glucose/2 mM acetate medium (5mL). Two clones of the strain containing the D351E variant were transferred to fresh medium and stocked. One clone was designated PNY2286. Transformants for E61A clones did not grow as well in the 0.3% glucose/2 mM acetate medium. Additional clones from the transformation plate were patched onto SE-ura-his plates. One clone was designated PNY2167.

Construction of Strain PNY1628

[0254] Strain PNY1628 was constructed from strain PNY2145 (Described in U.S. Patent

Application No. 14/208,474, which is incorporated herein by reference). The chimeric gene on chromosome XII in PNY2145 consisting of the PDC1 promoter, alsS coding region, CYC I terminator, and loxP71/66 site was deleted from 750 bp upstream of the alsS coding region through 1244 bp of the 1716 bp alsS coding region. The region was deleted using CRE-lox mediated marker removal (methodology described above). A loxP71/66 site flanked by two priming sites remained upstream of the deletion after CRE -mediated marker removal. The sequence of the resulting locus, which had the promoter and the majority of alsS deleted, was confirmed by sequencing and/or PCR. The sequence of the modified locus is provided in SEQ ID NO:378 (native upstream region = nt 1- 100; priming site-loxP71/66-priming site = nt 109-203; remaining alsS coding region = nt 210-681; CYC1 terminator = nt 689-936; priming site-loxP71/66-priming site = nt 942-1036; native downstream region = nt 1037-1136). PNY1628 has the genotype MATa ura3A::loxP his3A pdc5A::P[FBA(L8)]-XPK|xpkl_Lp-CYCt-loxP66/71 fra2A 2-micron plasmid (CEN.PK2) pdclA::loxP71/66-AP[PDCl]AalsS pdc6A::(UAS)PGKl-P[FBAl]-KIVD|Lg(y)-TDH3t-loxP71/66 adhlA::P[ADHl]-ADH|Bi(y)-ADHt-loxP71/66 fra2A::P[ILV5]-ADH|Bi(y)-ADHt-loxP71/66 gpd2A::loxP71/66 amnlA::AMNl .

Construction of ALS expression plasmids

[0255] Variants of ALS were cloned into expression plasmid pBP2986 (SEQ ID NO:379). pBP2986 is based on pRS413 (ATCC# 87518), a centromeric shuttle vector, and contains the FBA1 promoter (nt 2119-2708) and ADH1 terminator (nt 2725-3040) from Saccharomyces cerevisiae.

[0256] pBP4790 (SEQ ID NO:380) was constructed to contain the wild type coding region of alsS from Bacillus subtilis (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119- 2708) and followed by the ADH1 terminator (nt 4444-4759). pBP4791 (SEQ ID NO:381) was constructed to contain the coding region of the G453A alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADH1 terminator (nt 4444-4759). pBP4792 (SEQ ID NO:382) was constructed to contain the coding region of the V545L alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADH1 terminator (nt 4444-4759). pBP4793 (SEQ ID NO:383) was constructed to contain the coding region of the V545W alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADH1 terminator (nt 4444-4759). pBP4794 (SEQ ID NO:384) was constructed to contain the coding region of the Y547A alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADH1 terminator (nt 4444-4759). pBP4795 (SEQ ID NO:385) was constructed to contain the coding region of the Y547L alsS mutant (nt 2717- 4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADHl terminator (nt 4444-4759).

[0257] pBP5256 (SEQ ID NO:386) was constructed to contain the coding region of the

H401A/V545M alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADHl terminator (nt 4444-4759). pBP5258 (SEQ ID NO:387) was constructed to contain the coding region of the H401A/Y547L alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADHl terminator (nt 4444-4759).

pBP5260 (SEQ ID NO:388) was constructed to contain the coding region of the G453A/Y547F alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 21 19-2708) and followed by the ADHl terminator (nt 4444-4759). pBP5262 (SEQ ID NO:389) was constructed to contain the coding region of the V545M/Y 547L alsS mutant (nt 2717-4435) expressed from the yeast FBA1 promoter (nt 2119-2708) and followed by the ADHl terminator (nt 4444-4759).

Construction of yeast ALS expression strains

[0258] pLMHl 1-JM44 (SEQ ID NO: 390) was constructed to contain a chimeric gene having the coding region of the ilvD gene from Streptococcus mutans containing the L2V4 mutation (nt position 5362-3629) expressed from the yeast TEF1 mutant 7 promoter (nt 5772-5372; Nevoigt et al. 2006. Applied and Environmental Microbiology, v72 p5266) and followed by the FBA1 terminator (nt 3617-3305) for expression of DHAD, and a chimeric gene having the coding region of the K9JM44 mutant ilvC gene from Anaeropstipes cacae (nt 1628-2644) expressed from the yeast ILV5 promoter (nt 427-1620) and followed by the ILV5 terminator (nt 2670-3292) for expression of KARL

[0259] Yeast ALS expression strains were constructed by transforming the alsS deletion strain PNY1628 with pLMH-JM44 and an ALS expression plasmid. pBP2986 was used to construct a no-ALS control strain. PNY1628 was transformed using a Yeast Transformation Kit (Sigma- Aldrich, St. Louis, MO). Trans formants were selected for growth on synthetic complete media lacking uracil and histidine and supplemented with 1% ethanol at 30°C. Three independent transformants were selected for each transformation. Example 1 : Cofactor affinities

[0260] The purpose of this is example is to demonstrate the use of acetolactate synthase harboring strains that exhibit low acetolactate synthase flux with low extracellular thiamine concentrations and increased acetolactate synthase flux at increased extracellular thiamine concentration in the medium. Furthermore, the example demonstrates the effect of modifications of the thiamine binding affinity of acetolactate synthase that result in more preferable properties of the enzyme for use as a "switch", i.e. a higher ratio of specific acetolactate synthase flux at high extracellular thiamine concentrations as compared to low extracellular thiamine concentrations. For this purpose, previously described strains PNY2092, also referred to as "wt", PNY2167, also referred to as "E61A", as well as PNY2286, also referred to as "D351E" were inoculated from frozen glycerol stock cultures each into 125 ml aerobic shake flasks prefilled with 10 ml SEED medium. SEED medium was composed of 50% (v/v) Yeast Synthetic Medium (2x), 10%> (v/v) Double Drop-Out Supplements Complete Supplement Mixture (CSM) without histidine and uracil (Formedium, DSCK162, Hunstanton, UK) and 40.00% (v/v) of water. Yeast Synthetic Medium (2x) in turn was composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3), 40 mg/1 thiamine, 40 mg/1 niacin, 7 ml/1 ethanol, 36 ml/L 50% glucose solution, 2 ml/1 of Tween &

Ergosterol solution and 200 ml/1 of a 1 M MES buffer, pH = 5.5 in distilled water. 1 1 of Tween & Ergosterol solution contained 10 g of ergosterol dissolved in 500 ml ethanol and 500 ml Tween 80.

[0261] After 24 h, 5 ml of the PNY2092 seed culture were inoculated into 95 ml of STAGE 1 medium in a 500 ml aerobic shake flask. Also 2 ml of the "E61A" and "D351E" seed culture were inoculated each into a 38 ml of STAGE 1 medium in a 250 ml aerobic shake flask. STAGE 1 medium was composed of 6.7 g/L, Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3); 3.7 g/L Double Drop-Out Supplements Complete Supplement Mixture (CSM) without histidine and uracil

(Formedium, DSCK162, Hunstanton, UK), 20 mg/L thiamine, 20 mg/L niacin, 3.5 mL/L of ethanol, 5.5 mL/L of a 50%> glucose solution and 0.6 mL/L of acetic acid. The stage 1 cultures were incubated at 30°C and 250 rpm in an Innova Laboratory Shaker (New Brunswick Scientific, Edison, NJ). After 24 h, the measured ODs at λ = 600 nm were 1.600, 1.330 and 1.430 for PNY2092, "E61A" and "D351E", respectively.

[0262] Subsequently 7.50 ml, 9.02 ml and 8.40 ml of the PNY2092, "E61A" and "D351E" seed cultures were transferred into 3 x 15 ml centrifuge tubes, and 15.00 ml, 18.04 ml and 16.80 ml into 3 x 50 ml centrifuge tubes, respectively. The cell suspensions were spun down at 9500 rpm for 20 minutes and the supernatant discarded. 12 ml of STAGE 2 medium with amino acids and with 100.4 mg/L thiamine were added to the 15 ml centrifuge tubes and 24 ml of STAGE 2 medium with amino acids and w/o thiamine to the 50 ml centrifuge tubes. STAGE 2 medium with amino acids and with 100.4 mg/L thiamine was composed of 50.00% (v/v) Yeast synthetic medium w/o amino acids, glucose and thiamine (2x), 10.00%) (v/v) of a 37 g/L Double Drop-Out Supplements Complete Supplement Mixture (CSM) without histidine and uracil (Formedium, DSCK162, Hunstanton, UK), 16.00%) (v/v) of a 250 g/1 glucose solution, 2.86%> (v/v) of a 3.5 g/L thiamine stock solution and 21.14% (v/v) of distilled water. STAGE 2 medium with amino acids and w/o thiamine was composed of 50.00%) (v/v) Yeast synthetic medium w/o amino acids, glucose and thiamine (2x), 10.00%) (v/v) of a 37 g/L Double Drop-Out Supplements Complete Supplement Mixture (CSM) without histidine and uracil (Formedium, DSCK162, Hunstanton, UK), 16.00%> (v/v) of a 250 g/1 glucose solution and 24.00%) (v/v) of distilled water. Yeast synthetic medium w/o amino acids, glucose and thiamine (2x) was composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3), 40 mg/L niacin, 2 mL/L of Tween & Ergosterol solution and 200 ml/1 1 M MES buffer, pH = 5.5. 1 1 of Tween & Ergosterol solution contains 10 g of ergosterol dissolved in 500 ml ethanol and 500 ml Tween 80.

[0263] Next the cell pellets were re-suspended by vortexing. The medium "without" thiamine still contained approximately 0.4 mg/1 thiamine from the Yeast Nitrogen Base addition. The cell suspensions were distributed in 11 ml portions into nine 25mL Balch tubes in the Biohood, and 1 ml was saved for analysis of OD and organic metabolites at EPT = 0 h. Each Balch tube was fitted with a butyl rubber septum and cramped to the tube with a sheet metal with circular opening to allow samples withdrawal by syringes. This experiment was a shift experiment, which means it started with air in the head space. For addition of thiamine during the stage 2 experiments, 200 μΐ of a sterile 3.5 g/1 thiamine stock solution were injected in each tube of the second set of 0.4 mg/L thiamine cultures (set "2") at EPT = 10 h to increase the thiamine concentration in the Balch tube cultures by about 100 mg/L.

[0264] Biomass concentrations of the cultures were monitored with help of OD measured with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at λ = 600 nm. Cell dry weight concentration was deferred from the OD readings assuming an OD-DW-correlation of 0.33 g(DW)/OD. Extracellular compound analysis in supernatant was accomplished by HPLC. A BIO-RAD Aminex HPX-87H column was used in an isocratic method with 0.01 N sulfuric acid as eluent on a Waters Alliance 2695 Separations Module (Milford, MA). Flow rate was 0.60 ml/min, column temperature 40°C, injection volume 10 μΐ and run time 58 min. Detection was carried out with a refractive index detector (Waters 2414 RI) operated at 40°C and an UV detector (Waters 2996 PDA) at 210nm. Mumax values were determined by applying the exponential regression function of Microsoft Excel (Microsoft Office Excel 2003, SP 3). Outliers were discarded until good fit of the regression curve with measurements was confirmed by visual inspection.

[0265] Maximum specific growth rates of the cultures found for the first 6 h of the cultivations (EPT = 0 - 6 h) are represented in table 14. It was found that (i) E61A under all conditions exhibited a considerably higher specific maximum growth rate than the other two cultures, and (ii) that in PNY 2092 and D35 IE the cultures with thiamine addition at the beginning showed lower specific maximum growth rates than the cultures without thiamine or thiamine addition at a later time point.

[0266] Glucose consumed, isobutyric acid, isobutanol and biomass produced was determined for the mostly aerobic growth phase and the mostly anaerobic production ranging from ETP = 0 h - 10 h and 10 h - 24 h, respectively. Results are depicted in Figure 2. It was found that for all three strains the consumption and production pattern of the "no thiamine" cultures ("no thiamine" and "add") were similar and distinct from the cultures with thiamine ("thiamine"). In turn after addition of thiamine to the cultures without initial thiamine addition ("add") the consumption and production pattern of the now thiamine containing cultures ("thiamine" and "add") were found to be similar and distinct from the cultures still without thiamine ("no thiamine"Error! Reference source not found.) (figure 2). In general consumption of glucose and increase of extracellular isobutyric acid, isobutanol and biomass concentrations as measured in ODs during the aerobic growth phase were higher in the cultures without thiamine than in the thiamine cultures. On the other hand during the anaerobic production phase from EPT = 10 - 24 h glucose consumption as well as production of isobutyric acid, isobutanol and biomass was higher in case of cultures with thiamine ("thiamine" and "add") as compared to the cultures without thiamine ("no thiamine"Error! Reference source not found.). The only exceptions from this pattern during anaerobic production were the E61A cultures. The E61A cultures produced only low amounts of isobutyric acid and isobutanol, indicative for comparatively low flux through the isobutanol pathway via acetolactate synthase (Figure 2). [0267] In examining the yield of produced/consumed metabolites and biomass on glucose during the aerobic growth phase, it was observed that all strains and their cultures either with or without thiamine produced pyruvic acid, glycerol, acetic acid, isobutanol and biomass (Tables 10- 12). Formation of ketoisovaleric and isobutyric acid was detected only for PNY 2092 and D351E cultures, but not E61A cultures. The same applies to acetoin, with the exception of the 2092 "add" culture. The production of significant amounts of DHIV/DHMB, meso-BDO, d/l-BDO or formic acid was not discovered. Interestingly, E61A produced significant amounts of ethanol, lactic acid and succinic acid, as opposite to wt and D351E (figure 3).

[0268] During the anaerobic production phase from EPT = 10 h to 24 h, it was observed that all strains and their cultures either with or without thiamine produced ethanol, pyruvic acid, glycerol, isobutanol, acetoine and succinic acid (figure 4). Formation of ketoisovaleric acid, DHIV(/DHMB), isobutyric acid, meso- and d/l-BDO was detected only for PNY2092 and D351E, but not E61A. A small amount of acetic acid was consumed by PNY2092 and D35 IE, and produced by E61 A. Also PNY2092 and D351E continued to grow under anaerobic conditions, while E61A not only stopped to grow, but even showed a decrease in biomass concentration as derived from OD measurements in some of the experiments. In general, two different fermentation regimes were detected in PNY 2092 and D351E as compared to E61A. PNY2092 and D351E produced isobutanol and glycerol as major fermentation products, while E61A mainly produced ethanol, glycerol and lactic acid.

[0269] In the experiment it was found that the average specific isobutanol production during anaerobic production phase is higher than during aerobic growth phase in the measured time intervals (figure 5). However, if specific isobutanol production (q p ) is compared between PNY2092 and D35 IE, it can be seen that without thiamine in the medium D35 IE exhibited a considerably lower qp than PNY2092 during aerobic growth (0.068 g/g/h vs. 0.106 g/g/h) as well as during anaerobic production (0.170 g/g/h vs. 0.208 g/g/h). In both, the PNY 2092 as well as the D351E culture aerobic average specific isobutanol production was comparable in the two runs with initially no thiamine in the medium. However, while with addition of thiamine at EPT = 10 h the average anaerobic qp of PNY2092 was only slightly increased (0.227 g/g/h as compared to 0.208 g/g/h w/o thiamine), the average anaerobic qp in D351E increased significantly more, to 0.207 g/g/h as compared to 0.170 g/g/h, which was almost at the level of PNY2092 with addition of thiamine (0.227 g/g/h). [0270] Comparing the thiamine vs. non-thiamine cultures at EPT = 48 h the differences become less pronounced, in some instances the cultures even show opposite trends than at EPT = 24 h. It may be that, in this experiment, higher butanol concentrations mask effects of cultures containing high vs. low thiamine cultures, and possibly turn initial performance benefits into detrimental effects. However, other phenomena may be of importance as well, for example an increased catabolism and/or sequestration of thiamine in combination with repressed thiamine biosynthesis in the supplemented cultures.

[0271] The example illustrates the use of acetolactate synthase harboring strains that exhibit low acetolactate synthase flux with low extracellular thiamine concentrations and increased acetolactate synthase flux at increased extracellular thiamine concentration in the medium.

Furthermore, experiments with D351E demonstrate that modifications of the protein sequence comprising for example the thiamine binding affinity result can result in advantageous properties of the enzyme for use as a controlled biocatalyst polypeptide, i.e. a higher ratio of specific acetolactate synthase flux at high extracellular thiamine concentrations as compared to low extracellular thiamine concentrations.

Table 10. Substrate, product and byproduct measurements by HPLC for PNY2092 in cultures with initial thiamine supplementation ("+ thiamine '), no thiamine supplementation ("no thiamine ") and thiamine addition at EPT = 10 h ("add'). The symbol « designates that no signal was found in respective sample. Concentrations of the culture with thiamine addition at EPT = 10 h ("add') were corrected for volume change caused by addition.

Sample EPT Glue. etoh pyruKIV DHX glyc. aceIBA IBOH mBDO d/1- Acetoirj LacsuccHMB PET( vate tate BDO tate inate

[h] [mM] [mM] [mM] [mM] [mM] [mM [mM; [mM] [mM] [mM] [mM] [mM] [mM; [mM; [mM] [mM

2092 + thiamine 0 222.02 6.47 « « « 0.12 « « « « « « « « « «

2092 + thiamine 3 219.99 6.13 0.11 0.03 « 0.34 0.33 « « « « « « « « «

2092 + thiamine 6 217.48 6.42 0.31 0.21 « 0.52 0.32 1.23 1.96 « « « « « « «

2092 + thiamine 10 207.09 5.95 0.76 0.78 « 0.84 0.40 2.52 6.55 « « 0.49 « 0.16 « «

2092 + thiamine 24 153.94 7.31 1.87 2.21 0.66 3.98 « 5.02 40.48 1.22 0.57 1.73 0.17 0.33 « «

2092 + thiamine 48 90.25 9.48 2.56 2.87 1.22 13. U « 5.29 85.50 3.29 1.09 2.10 0.63 0.45 0.47 0.06

2092 - no thiamine 0 222.26 6.43 « « « 0.11 « « « « « « « « « «

2092 - no thiamine 3 220.27 6.10 0.14 0.04 « 0.36 « 0.48 « « « « « « « «

2092 - no thiamine 6 215.70 6.22 0.36 0.25 « 0.53 « 1.43 2.27 « « « « « « «

2092 - no thiamine 10 203.67 6.52 0.82 0.90 « 0.90 0.47 2.86 8.04 « « 0.70 « « « «

2092 - no thiamine 24 159.29 7.49 1.48 1.76 0.55 5.16 « 4.53 38.80 0.92 0.41 1.37 « 0.26 « «

2092 - no thiamine 48 90.19 9.43 2.30 2.64 1.25 17.6* « 4.60 85.65 3.44 1.11 2.76 0.66 0.43 0.48 0.04

2092 - no thiamine 0 222.26 6.43 « « « 0.11 « « « « « « « « « «

2092 + add 3 220.13 6.13 0.14 0.04 « 0.35 « 0.49 « « « « « « « «

2092 + add 6 216.08 6.19 0.37 0.26 « 0.54 « 1.37 2.36 « « « « « « «

2092 + add 10 203.35 6.14 0.88 0.90 « 0.90 0.36 2.87 7.98 « « « « « « «

2092 + add 24 151.81 7.83 2.06 2.12 0.78 3.96 « 5.36 42.43 1.42 0.50 1.89 « 0.39 « «

2092 + add 48 94.54 9.80 2.66 2.60 1.44 12.81 « 5.40 81.67 3.31 1.24 1.79 0.59 0.48 0.30 0.06

Table 11. Substrate, product and byproduct measurements by HPLC for D351E in cultures with initial thiamine supplementation ("+ thiamine '), no thiamine supplementation ("no thiamine ') and thiamine addition at EPT = 10 h ("add'). The symbol « designates that no signal was found in respective sample. Concentrations of the culture with thiamine addition at EPT = 10 h ("add') were corrected for volume change caused by addition. sample EPT Glue. etoh pyruv KIV DHX glyc. aceIBA IBOH mBDC d/1- acetoii lacsuccHMB PET( tate BDO tate inate

[h] [mM] [mM] [mM] [mM] [mM] [mM] [mM; [mM] [mM] [mM] [mM] [mM] [mM [mM] [mM] [mM

D351E + thiamine 0 221.80 6.51 « « « 0.13 « « « « « « « « « «

D351E + thiamine 3 220.64 6.16 0.15 0.04 « 0.39 0.32 « « « « « « « « «

D351E + thiamine 6 218.14 6.26 0.40 0.18 « 0.58 0.45 0.81 1.19 « « « « « « «

D351E + thiamine 10 211.67 5.96 1.05 0.60 « 0.88 0.62 1.62 3.88 « « 0.51 « « « «

D351E + thiamine 24 165.44 8.29 4.44 2.36 0.61 3.40 « 3.29 30.46 1.20 0.42 2.74 0.14 0.36 « «

D351E + thiamine 48 104.99 11.62 5.87 2.98 1.32 12.56 « 3.46 71.65 2.72 1.28 1.84 0.71 0.51 0.46 0.06

D351E - no thiamine 0 221.70 6.42 0.01 « « 0.14 « « « « « « « « « «

D351E - no thiamine 3 220.00 6.14 0.19 0.06 « 0.41 0.38 « « « « « « « « «

D351E - no thiamine 6 218.01 6.46 0.55 0.24 « 0.62 « 1.08 1.55 « « « « « « «

D351E - no thiamine 10 209.28 7.50 1.54 0.76 « 0.95 0.53 1.83 4.96 « « 0.58 « 0.19 « «

D351E - no thiamine 24 170.01 10.01 4.60 1.87 0.47 4.49 « 2.52 27.81 1.01 0.34 1.85 0.26 0.34 0.37 «

D351E - no thiamine 48 101.14 13.07 6.56 2.87 1.41 17.48 « 2.59 72.52 2.96 1.17 2.79 0.87 0.50 0.49 0.09

D351E - no thiamine 0 221.70 6.42 0.01 « « 0.14 « « « « « « « « « «

D351E + add 3 219.98 6.14 0.19 0.06 « 0.42 0.40 « « « « « « « « «

D351E + add 6 216.87 6.08 0.55 0.24 « 0.62 « 0.92 1.66 « « « « « « «

D351E + add 10 208.81 5.79 1.52 0.77 « 0.94 0.59 1.65 4.83 « « 0.59 « « « «

D351E + add 24 159.90 8.84 5.14 2.40 0.76 3.82 « 3.35 33.98 1.52 0.62 2.87 0.19 0.37 « «

D351E + add 48 98.69 12.10 6.35 2.93 1.62 13.60 « 3.44 75.42 2.82 1.59 1.99 0.75 0.55 0.23 0.08

Table 12. Substrate, product and byproduct measurements by HPLC for E61A in cultures with initial thiamine supplementation ("+ thiamine '), no thiamine supplementation ("no thiamine ') and thiamine addition at EPT = 10 h ("add'). The symbol « designates that no signal was found in respective sample. Concentrations of the culture with thiamine addition at EPT = 10 h ("add') were corrected for volume change caused by addition. sample EPT Glue. etoh pyruKIV DHX glyc. aceIBA IBOH mBDC d/1- ace- lacsuccHMB PET( vate tate BDO toin tate inate

[h] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM] [mM

E61A + thiamine 0 221.94 6.73 0.14 « « 0.16 « « « « « « « « « «

E61A + thiamine 3 216.63 6.94 3.00 « « 0.55 0.78 « « « « « « « « «

E61A + thiamine 6 211.11 9.73 7.49 « « 1.08 1.06 « « « « « « 0.23 « «

E61A + thiamine 10 207.63 13.31 8.48 « « 2.75 1.27 « 0.37 « « « 0.36 0.35 « «

E61A + thiamine 24 196.15 23.34 9.48 « « 5.69 1.27 « 0.58 « « 0.75 1.17 0.68 « 0.01

E61A + thiamine 48 189.93 30.56 10.35 « « 7.37 1.34 « 0.69 0.40 0.32 « 1.95 0.96 « 0.02

E61A - no thiamine 0 221.51 6.74 0.17 « « 0.19 « « « « « « « « « «

E61A - no thiamine 3 215.79 7.04 2.99 « « 0.57 0.79 « « « « « « « « «

E61A - no thiamine 6 212.07 9.86 6.77 « « 1.33 1.01 « 0.28 « « « 0.17 0.20 « «

E61A - no thiamine 10 208.42 13.02 7.43 « « 2.80 1.07 « 0.34 « « « 0.34 0.33 « «

E61A - no thiamine 24 198.15 22.22 8.42 « « 5.60 1.23 « 0.53 « « 0.73 1.04 0.51 « 0.00

E61A - no thiamine 48 192.30 28.83 9.26 « « 7.15 1.22 « 0.67 0.35 0.22 « 1.67 0.92 « 0.03

E61A - no thiamine 0 221.51 6.74 0.17 « « 0.19 « « « « « « « « « «

E61A + add 3 216.05 6.98 2.99 « « 0.57 0.76 « « « « « « « « «

E61A + add 6 210.29 9.62 7.42 « « 1.15 1.11 « 0.27 « « « 0.17 0.25 « «

E61A + add 10 207.52 13.25 8.36 « « 2.75 1.23 « 0.35 « « « 0.36 0.35 « «

E61A + add 24 196.70 23.55 9.52 « « 5.76 1.24 « 0.56 « « 0.83 1.16 0.70 « 0.01

E61A + add 48 189.75 30.17 10.44 « « 7.43 1.41 « 0.69 0.42 0.34 « 1.89 0.99 « 0.04

Table 13. OD measurements for PNY2092 (2092), D351E and E61A in cultures with initial thiamine supplementation ("+ thiamine '), no thiamine supplementation ("no thiamine ') and thiamine addition at EPT = 10 h ("add'). OD measurements of the culture with thiamine addition at EPT = 10 h ("add') were corrected for volume change caused by addition. sample\EPT [h] 0.00 1.50 3.00 4.50 6.00 10.00 24.00 48.00

OD 600 [ ]

2092 + thiamine 1.096 1.216 1.341 1.431 1.551 1.991 2.781 2.921

2092 - no thiamine 1.071 1.246 1.361 1.481 1.581 2.051 2.681 2.831

2092 + add 1.071 1.251 1.461 1.521 1.701 2.071 2.799 2.902

D351 E + thiamine 1.111 1.236 1.341 1.401 1.491 1.721 2.551 2.611

D351E - no thiamine 1.061 1.261 1.341 1.501 1.591 1.861 2.441 2.691

D351E + add 1.061 1.256 1.271 1.401 1.581 1.871 2.644 2.788

E61A + thiamine 1.026 1.336 1.541 1.791 1.911 1.951 1.711 1.771

E61A - no thiamine 1.021 1.311 1.511 1.791 1.841 1.861 1.801 1.671

E61A + add 1.021 1.286 1.541 1.751 1.901 1.931 1.914 1.791

Table 14. Mumax values determined from the OD measurements taken in the first 6 h of the experiment culture mumax [1/h]

2092 + thiamine 0.057

2092 - no thiamine 0.063

2092 + add 0.075

D351E + thiamine 0.048

D351E - no thiamine 0.066

D351E + add 0.060

E61A + thiamine 0.121

E61A - no thiamine 0.122

E61A + add 0.120

Example 2: AHAS inhibitor (Prophetic)

[0272] The example uses a recombinant host cell comprising a butanol biosynthetic pathway and further comprising (i) an inhibitor-resistant and branched-chain amino acid feedback-inhibited AHAS localized in the mitochondria, as well as (ii) an inhibitor-sensitive AHAS, preferably de-sensitized for feedback-inhibition by branched- chain amino acids in the cytoplasm. This strain is referred to as NORTH 1002. In the example improved butanologen biomass generation in batch and fed-batch cultivations by NORTH 1002 is demonstrated in cultures supplemented with an AHAS inhibitor as compared to the cultivation of the same strain without AHAS inhibitor supplementation.

[0273] A frozen vial of NORTH 1002 is inoculated into a sterilized pre-pure culture tank and grown in an aerobic batch regime with and without supplementation of AHAS inhibitor. Following growth, the content of the vessel is transferred to a larger pure culture fermentor where propagation is carried out with some aeration, again under sterile conditions in a batch regime with and without AHAS inhibitor. In the cultivations, NORTH 1002 is brought into contact with a media, comprising a carbon source (e.g. glucose, fructose, molasses, corn mash, ...), certain minerals, vitamins, and salts, as well as a nitrogen source (e.g. ammonium, urea, ...). During the scale up of NORTH 1002 in the pre-pure culture tank and pure culture fermentor under the same culture conditions, the NORTH 1002 cultures with AHAS inhibitor exhibit a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyraldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002 cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the main carbon substrate.

[0274] From the pure culture vessel with or without inhibitor, NORTH 1002 is transferred to a series of progressively larger seed and semi-seed fermentors. During the fed-batch fermentations in these larger seed and semi-seed fermenters under aerobic conditions with or without AHAS inhibitor, a carbon source (e.g. molasses, glucose, fructose, corn mesh, ...), phosphoric acid, a nitrogen source (e.g. ammonium, urea, ...), optionally AHAS inhibitor and minerals are fed at a controlled rate. At the end of the semi-seed fermentations with or without supplementation of an AHAS inhibitor, the contents of the vessels are each pumped to a series of separators that separate the biomass from the spent media. Each of the cultures are then washed with cold water and pumped to semi-seed yeast storage tanks where the yeast creams of NORTH 1002 are held at temperatures of 5°C or below. During the scale up of NORTH 1002 in the seed and semi-seed fermenters with and without supplementation of AHAS inhibitor, the NORTH 1002 with supplementation of AHAS inhibitor exhibit a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002 cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the substrate.

[0275] The commercial fermentations in fermentation tanks with a working volume up to 50,000 gallons are started by pumping water, referred to as set water, into the fermentors. Next, in a process referred to as pitching, semi-seed yeasts of NORTH

1002 from the storage tank are transferred into fermentor with and without

supplementation of AHAS inhibitor. After the addition, aeration, cooling and nutrient additions, optionally addition of AHAS inhibitor, are started. At the start of the fermentation, the liquid seed yeast and additional water may occupy only about one-third to one -half of the fermentor volumes. Constant additions of nutrients and optionally AHAS inhibitor during the course of fermentation (fed-batch) bring the fermentors to their final volume. The rate of nutrient addition and optionally AHAS inhibitor increases throughout the fermentation because more nutrients have to be supplied to support growth of the increasing cell population. Air is provided to the fermentors at or below about one volume of air per fermentor volume per minute. Cooling is accomplished by internal cooling coils or by pumping the fermentation liquid, also known as broth, through an external heat exchanger. The addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. Throughout the fermentation, the pH is kept in the range of 4.0-7.0.

[0276] At the end of the fermentations, the fermentor broths of NORTH 1002 with and without supplementation of AHAS inhibitor are each separated, e.g. by centrifuges, washed with water and re-centrifuged to yield yeast creams. The yeast creams are each cooled to 10°C or below and stored in separate, refrigerated stainless steel cream tanks. Alternatively, the yeast creams of NORTH 1002 with or without AHAS inhibitor are pumped to a plate and frame filter press and dewatered to a cake-like consistency. This press cake yeasts are crumbled into pieces and packed into 50-pound bags that are stacked on a pallet and cooled in a refrigerator for a period of time with adequate ventilation. During the commercial fermentations of NORTH 1002 with and without AHAS inhibitor, the NORTH 1002 culture with AHAS inhibitor exhibits a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002 cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the substrate. Final biomass concentration of NORTH 1002 with supplementation of AHAS inhibitor at the end of the commercial fermentation is higher than of NORTH 1002 without addition of AHAS inhibitor.

[0277] Same amounts of produced cake and yeast creams of NORTH 1002 cultivated with or without addition of AHAS inhibitor are re-suspended in 12 ml of production medium and transferred into 25 ml Balch tubes, respectively. Production medium is composed of 50% Yeast Synthetic Medium (2x), 10%> Complete Supplement Mixture (CSM) with adenine (Formedium, DCS0031, Hunstanton, UK), 16% of a 250 g/1 glucose solution, and 24% of water. Yeast Synthetic Medium (2x) in turn is composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3), 40 mg/1 thiamine, 40 mg/1 niacin, 7 ml/1 ethanol and 200 ml/1 of a 1 M MES buffer, pH = 5.5. Each Balch tube is fitted with a butyl rubber septum and cramped to the tube with a sheet metal with circular opening to allow samples withdrawal by syringes. For sample withdrawal, 1 ml syringes (25G 5/8 (0.5 mm x 16 mm) Safety-Lok, Becton Dickinson, Franklin Lakes, NJ) are employed. Growth of the cultures is monitored with help of OD measured with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at λ = 600 nm. Extracellular compound analysis in supernatant is accomplished by HPLC. A BIO-RAD Aminex HPX-87H column is used in an isocratic method with 0.01 N sulfuric acid as eluent on a Waters Alliance 2695 Separations Module (Milford, MA). Flow rate is 0.60 ml/min, column temperature 40°C, injection volume 10 μΐ and run time 58 min. Detection is carried out with a refractive index detector (Waters 2414 RI) operated at 40°C and an UV detector (Waters 2996 PDA) at 210nm. Isobutanol formation is observed in all the NORTH 1002 cultures, respectively. At least one of the final isobutanol rate, titer, or specific productivity of a NORTH 1002 cultures reconstituted from a NORTH 1002 culture previously cultivated with AHAS inhibitor is not substantially different than that parameter of a cultures reconstituted from a NORTH 1002 culture that was not contacted with AHAS inhibitor during scale up.

Example 3 : Temperature stability (Prophetic)

[0278] Strain NORTH 1000 comprises a chromosomally integrated B. subtilis

AHAS for cytoplasmatic expression, and strain NORTH 1001 comprises a

chromosomally integrated B. subtilis temperature sensitive (Ts) cytoplasmatic AHAS mutant with a permissive temperature of 30°C and a non-permissive temperature of 37°C. Both strains comprise a native, biosynthetic and end-product inhibited AHAS in the mitochondrium.

[0279] A frozen vial of NORTH 1000 and NORTH 1001 are inoculated each into a sterilized pre-pure culture tank and grown in a batch regime at 37°C. Following growth, the contents of these vessels are each transferred to a larger pure culture fermentor where propagation is carried out at 37°C with some aeration, again under sterile conditions and a batch regime. In the cultivations, NORTH 1000 and NORTH 1001 cultures are each provided with the same media, comprising a carbon source (e.g. glucose, fructose, molasses, corn mash, ...), certain minerals, vitamins, and salts, as well as a nitrogen source (e.g. ammonium, urea, ...). During the scale up of NORTH 1001 and NORTH 1000 in the pre-pure culture tank and pure culture fermentor under the same culture conditions, the NORTH 1001 culture exhibits a significantly lower isobutanol as well as total isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate.

[0280] From the pure culture vessel, the grown cells of NORTH 1000 and

NORTH 1001 are each transferred to a series of progressively larger seed and semi-seed fermentors. During the fed-batch fermentations in these larger seed and semi-seed fermenters under aerobic conditions at 37°C, a carbon source (e.g. molasses, glucose, fructose, corn mash, ...), phosphoric acid, a nitrogen source (e.g. ammonium, urea, ...) and minerals are fed to each NORTH 1000 and NORTH 1001 at a controlled rate. At the end of the semi-seed fermentation, the contents of the vessels are each pumped to a series of separators that separate the biomass of NORTH 1000 and NORTH 1001 from the spent medium. Each of the cultures are then washed with cold water and pumped to semi-seed yeast storage tanks where the yeast creams of NORTH 1000 and NORTH 1001 are held at temperatures of 5°C or below. During the scale up of NORTH 1001 and NORTH 1000 in the seed and semi-seed fermenters, the NORTH 1001 culture exhibits a significantly lower isobutanol as well as a total isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate. Final biomass concentration of NORTH 1001 at the end of the seed and semi-seed

fermentations is higher than of NORTH 1000.

[0281] The commercial fermentations in fermentation tanks with a working volume up to 50,000 gallons are started by pumping water, referred to as set water, into the fermentors. Next, in a process referred to as pitching, semi-seed yeasts of NORTH

1000 and NORTH 1001 from the storage tank are each transferred into the respective fermentor. After the addition of NORTH 1000 and NORTH 1001, aeration, cooling and nutrient additions are started. At the start of the fermentation, the liquid seed yeast and additional water may occupy only about one-third to one-half of the fermentor volumes.

Constant additions of nutrients during the course of fermentation (fed-batch) bring the fermentor to its final volume. The rate of nutrient addition increases throughout the fermentation because more nutrients have to be supplied to support growth of the increasing cell population. Air is provided to the fermentors at or below about one volume of air per fermentor volume per minute. Cooling is accomplished by internal cooling coils or by pumping the fermentation liquid, also known as broth, through an external heat exchanger. The temperature of each of the cultivations of NORTH 1000 and NORTH 1001 is controlled at 37°C. The addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. Throughout the fermentation, the pH is kept in the range of 4.0-7.0.

[0282] At the end of the fermentations, the fermentor broths of NORTH 1000 and NORTH 1001 are each separated, e.g. by centrifuges, washed with water and re- centrifuged to yield yeast creams. The yeast creams are each cooled to 10°C or below and stored in separate, refrigerated stainless steel cream tanks. Alternatively, the yeast creams of NORTH 1000 or NORTH 1001 are pumped to a plate and frame filter press and dewatered to a cake-like consistency. This press cake yeasts are crumbled into pieces and packed into 50-pound bags that are stacked on a pallet and cooled in a refrigerator for a period of time with adequate ventilation.

[0283] During the commercial fermentations of NORTH 1001 and NORTH

1000, the NORTH 1001 culture exhibits a significantly lower isobutanol as well as total isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyraldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate. Final biomass concentration of NORTH 1001 at the end of the commercial fermentation is higher than of NORTH 1000.

[0284] The equivalent of 250 mg dry weight of produced cake and/or yeast creams of NORTH 1000 and NORTH 1001 per liter are each re-suspended in 12 ml of

PRODUCTION medium and transferred into 25 ml Balch tubes, respectively.

PRODUCTION medium is composed of 50% Yeast Synthetic Medium (2x), 10%

Complete Supplement Mixture (CSM) with adenine (Formedium, DCS0031,

Hunstanton, UK), 16%> of a 250 g/1 glucose solution, and 24%> of water. Yeast Synthetic

Medium (2x) in turn is composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids

(Difco 0919-15-3), 40 mg/1 thiamine, 40 mg/1 niacin, 7 ml/1 ethanol and 200 ml/1 of a 1

M MES buffer, pH = 5.5. Each Balch tube is fitted with a butyl rubber septum and cramped to the tube with a sheet metal with circular opening to allow samples withdrawal by syringes. For sample withdrawal, 1 ml syringes (25G 5/8 (0.5 mm x 16 mm) Safety-Lok, Becton Dickinson, Franklin Lakes, NJ) are employed. Cultures are incubated at 30°C. Growth of the cultures is monitored with help of OD measured with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at λ = 600 nm. Extracellular compound analysis in supernatant is accomplished by HPLC. A BIO- RAD Aminex HPX-87H column is used in an isocratic method with 0.01 N sulfuric acid as eluent on a Waters Alliance 2695 Separations Module (Milford, MA). Flow rate is 0.60 ml/min, column temperature 40°C, injection volume 10 μΐ and run time 58 min. Detection is carried out with a refractive index detector (Waters 2414 RI) operated at 40°C and an UV detector (Waters 2996 PDA) at 210nm. Isobutanol formation is observed in all the NORTH 1001 and NORTH 1000 cultures, respectively. At least one of final isobutanol titers, rates, or specific productivities of the NORTH 1001 cultures is not substantially different as compared to the same final parameter of the respective NORTH 1000 cultures.

Example 4: Temperature stability (Prophetic)

[0285] Strain NORTH 1000a comprises a chromosomally integrated B. subtilis

AHAS for cytoplasmatic expression, and strain NORTH 1001a comprises a

chromosomally integrated B. subtilis temperature sensitive (Ts) cytoplasmatic AHAS mutant with a permissive temperature of 30°C and a non-permissive temperature of 37°C. Both strains have also the native, biosynthetic and end-product inhibited AHAS in the mitochondrium (ILV2) deleted.

[0286] A frozen vial of NORTH 1000a and NORTH 1001 a are inoculated each into a sterilized pre-pure culture tank and grown in a batch regime at 37°C. Following growth, the contents of these vessels are each transferred to a larger pure culture fermentor where propagation is carried out at 37°C with some aeration, again under sterile conditions and a batch regime. In the cultivations, NORTH 1000a and NORTH 1001a cultures are each provided with the same media, comprising a carbon source (e.g. glucose, fructose, molasses, corn mash, ...), certain minerals, vitamins, amino acids and salts, as well as a nitrogen source other than amino acids (e.g. ammonium, urea, ...). During the scale up of NORTH 1001a and NORTH 1000a in the pre-pure culture tank and pure culture fermentor under the same culture conditions, the NORTH 1001a culture exhibits a significantly lower isobutanol as well as total isobutanol pathway

intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate.

[0287] From the pure culture vessel, the grown cells of NORTH 1000a and

NORTH 1001a are each transferred to a series of progressively larger seed and semi-seed fermentors. During the fed-batch fermentations in these larger seed and semi-seed fermenters under aerobic conditions at 37°C, a carbon source (e.g. molasses, glucose, fructose, corn mash, ...), phosphoric acid, a nitrogen source (e.g. ammonium, urea, ...), sufficient branched-chain amino acids (BCA) and BCA-derived vitamins, and minerals are fed to each NORTH 1000a and NORTH 1001a at a controlled rate. At the end of the semi-seed fermentation, the contents of the vessels are each pumped to a series of separators that separate the biomass of NORTH 1000a and NORTH 1001a from the spent medium. Each of the cultures are then washed with cold water and pumped to semi-seed yeast storage tanks where the yeast creams of NORTH 1000a and NORTH

1001a are held at temperatures of 5°C or below. During the scale up of NORTH 1001a and NORTH 1000a in the seed and semi-seed fermenters, the NORTH 1001a culture exhibits a significantly lower isobutanol as well as a total isobutanol pathway

intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid,

isobutyraldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate. Final biomass concentration of NORTH 1001a at the end of the seed and semi-seed fermentations is higher than of NORTH 1000a.

[0288] The commercial fermentations in fermentation tanks with a working volume up to 50,000 gallons are started by pumping water, referred to as set water, into the fermentors. Next, in a process referred to as pitching, semi-seed yeasts of NORTH

1000a and NORTH 1001a from the storage tank are each transferred into the respective fermentor. After the addition of NORTH 1000a and NORTH 1001a, aeration, cooling and nutrient additions are started. At the start of the fermentation, the liquid seed yeast and additional water may occupy only about one-third to one-half of the fermentor volumes. Constant additions of nutrients during the course of fermentation (fed-batch) bring the fermentor to its final volume. The rate of nutrient addition increases throughout the fermentation because more nutrients have to be supplied to support growth of the increasing cell population. Air is provided to the fermentors at or below about one volume of air per fermentor volume per minute. Cooling is accomplished by internal cooling coils or by pumping the fermentation liquid, also known as broth, through an external heat exchanger. The temperature of each of the cultivations of NORTH 1000a and NORTH 1001a is controlled at 37°C. The addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. Throughout the fermentation, the pH is kept in the range of 4.0-7.0.

[0289] At the end of the fermentations, the fermentor broths of NORTH 1000a and NORTH 1001a are each separated, e.g. by centrifuges, washed with water and re- centrifuged to yield yeast creams. The yeast creams are each cooled to 10°C or below and stored in separate, refrigerated stainless steel cream tanks. Alternatively, the yeast creams of NORTH 1000a or NORTH 1001a are pumped to a plate and frame filter press and dewatered to a cake-like consistency. This press cake yeasts are crumbled into pieces and packed into 50-pound bags that are stacked on a pallet and cooled in a refrigerator for a period of time with adequate ventilation.

[0290] During the commercial fermentations of NORTH 1001 a and NORTH

1000a, the NORTH 1001a culture exhibits a significantly lower isobutanol as well as total isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyraldehyde, acetolactate, butadiol, acetoin, diacetyl) yield but a higher biomass yield on the substrate. Final biomass concentration of NORTH 1001a at the end of the commercial fermentation is higher than of NORTH 1000a.

[0291] The equivalent of 250 mg dry weight of produced cake and/or yeast creams of NORTH 1000a and NORTH 1001a per liter are each re-suspended in 12 ml of

PRODUCTION medium and transferred into 25 ml Balch tubes, respectively.

PRODUCTION medium is composed of 50% Yeast Synthetic Medium (2x), 10%

Complete Supplement Mixture (CSM) with adenine (Formedium, DCS0031,

Hunstanton, UK), 16%> of a 250 g/1 glucose solution, and 24%> of water. Yeast Synthetic

Medium (2x) in turn is composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids

(Difco 0919-15-3), 40 mg/1 thiamine, 40 mg/1 niacin, 7 ml/1 ethanol and 200 ml/1 of a 1

M MES buffer, pH = 5.5. Each Balch tube is fitted with a butyl rubber septum and cramped to the tube with a sheet metal with circular opening to allow samples withdrawal by syringes. For sample withdrawal, 1 ml syringes (25G 5/8 (0.5 mm x 16 mm) Safety-Lok, Becton Dickinson, Franklin Lakes, NJ) are employed. Cultures are incubated at 30°C. Growth of the cultures is monitored with help of OD measured with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at λ = 600 nm. Extracellular compound analysis in supernatant is accomplished by HPLC. A BIO-

RAD Aminex HPX-87H column is used in an isocratic method with 0.01 N sulfuric acid as eluent on a Waters Alliance 2695 Separations Module (Milford, MA). Flow rate is 0.60 ml/min, column temperature 40°C, injection volume 10 μΐ and run time 58 min. Detection is carried out with a refractive index detector (Waters 2414 RI) operated at 40°C and an UV detector (Waters 2996 PDA) at 210nm. Isobutanol formation is observed in all the NORTH 1001a and NORTH 1000a cultures, respectively. At least one of final isobutanol rates, titers, or specific productivities of the NORTH 1001a cultures are not substantially different than the same final parameter of the respective NORTH 1000a cultures.

Example 5: Glucose limited fed-batch technology for control of acetolactate synthase substrate concentration (Prophetic)

[0292] The recombinant microbial host cell comprising an isobutanol

biosynthetic pathway comprising an acetolactate synthase with high K M for pyruvate is provided as an inoculum for fed-batch fermentation and is pregrown on glucose in shake- flask cultures at pH 5.0 and 30°C. The medium containing per liter: KH 2 PO 4 , 10.0 g; MgS0 4 , 2.5 g; trace element solution, 10 mL. After autoclaving (121°C, 20min), filter- sterilized urea and vitamin solution are added to a final concentration of 3.0 g/L and 15 ml/L, respectively. Glucose is added separately to a final concentration of 10 g/L after sterilization at 110°C for 20 min. One-liter shake flask containing 200 mL of culture volume is incubated at 260 rpm for 24 h.

Vitamin and trace element solutions for fed batch cultivation

[0293] A concentrated trace element solution is made to contain per liter: EDTA,

15 g; ZnS0 4 , 5.75 g; MnCl 2 , 0.32 g; CuS0 4 , 0.50 g; CoCl 2 , 0.47 g; Na 2 Mo0 4 , 0.48 g; CaCl 2 , 2.9 g; FeS0 4 , 2.8 g. The trace element solution is sterilized at 121°C for 20 min. The vitamin solution contains per liter: biotin, 0.05 g; calcium pantothenate, 1.0 g;

nicotinic acid, 1.0 g; myoinositol, 25.0 g; thiamine hydrochloride, 1 g; pyridoxol hydrochloride, 1 g; p-aminobenzoic acid, 0.2 g. The vitamin solution is filter-sterilized before use.

Fermentor scale cultivation

[0294] Fed-batch cultivation is carried out in a fermentor (Sartorius Biostat B-

DCU Twin 2L, NY, USA) with an initial working volume of 1 L, at 30°C and at pH 5.0. Ammonium hydroxide (14.7 mM) is used as the titrant. The dissolved-oxygen concentration is continuously measured with a polarographic oxygen electrode

(Hamilton Oxyferm FDA 225, NV, USA) and kept above 20% of air saturation at a constant impeller speed of 1500 rpm. The air flow is maintained at 0.5 L/h-1 using internal Sartorius mass flow meter (Sartorius Biostat B-DCU, NY, USA). The amount of medium added in the fed-batch phase is recorded by continuous monitoring of the mass of the reservoir vessels by electronic balances.

Batch Phase

[0295] The medium for the batch phase contains per liter: (NH 4 ) 2 S0 4 , 15 g;

KH 2 P0 4 , 8.0 g; MgS0 4 , 3.0 g; trace element solution, 10 mL; anti-foaming agent Struktol J673, 0.3 mL; ZnS0 4 , 0.4 g. The medium was sterilized in autoclave at 121°C for 45 min. After sterilization, 12 mL/L of vitamin solution, 200 mL L of inoculum and sterile glucose are added to give a final concentration of 10 g/L glucose in a total culture volume of 1 L. After complete exhaustion of carbon sources, which is indicated by a rise in the dissolved oxygen concentration and a decrease in C0 2 production and 0 2 consumption, the fed-batch is started immediately.

Fed-Batch Phase

[0296] The medium for the fed-batch phase contains per liter: KH 2 P0 4 , 9.0 g;

MgS0 4 , 2.5 g; K 2 S0 4 , 3.5 g; Na 2 S0 4 , 0.28 g; glucose, 500 g; and trace-element solution, 10 mL. After sterilization of the medium at 110°C for 20 min, 12 mL/L of vitamin solution is added. The medium is pumped into the reactor using a controllable peristaltic pump (SciLog, Tandem model 1081, WI, USA). The specific growth rate is kept at 0.14 /h. The exponential feed was calculated by

f = μ-Χο-Vo Γ μ·£

St-Yx/s

[0297] F is the flow rate of the medium feed (L/h),), Y s is the biomass yield on substrate (g cells/g substrate), Xo is the initial biomass concentration (g/L), Vo is the initial culture volume (L), Si is the substrate concentration in the feed (g / L), and t is the time (h) after starting the feed. When the exponential feed can no longer be maintained as a result of a low-dissolved-oxygen concentration, a constant feed is employed. Gas Analysis

[0298] The exhaust gas is cooled in a condenser (12°C). 0 2 and C0 2

concentrations are both continuously determined with mass spectrometer (Thermo Electron VG Prima δ B Process MS, Cheshire, UK).

Determination of Culture Dry Weight

[0299] Culture samples (5 mL) are centrifuged in pre-weighted 15 mL round bottom centrifuge tubes (Kimble HS 45500-15, Thermo Fisher Scientific, NH, US) at 5000 rpm for 10 min using high speed centrifuge (Eppendorf 5804R, NY, USA ). The supernatant is decanted and pellet washed with 5 mL of distilled water. After repeated centrifugation and decanting the pellet is kept on 80 C in oven until constant weight.

Results

[0300] Starting biomass concentration in the batch fermentor stage is 0.4 g/L.

Glucose is completely exhausted after 8 hours and results in about 2 g/L biomass for the start of fed-batch stage. During this stage isobutanol concentration in the culture supernatant is not detectable and intracellular concentration of pyruvate is such that flux through the biosynthetic pathway is low. After 17 hours of glucose limited fed-batch stage cell concentration is about 20 g/L and part of the culture is transformed to a fresh batch medium containing 30 g/L of glucose. In the new batch fermentation intracellular pyruvate concentration increases such that flux through the biosynthetic pathway is higher and isobutanol starts to accumulate in the culture supernatant.

Example 6: AHAS inhibitor (Prophetic)

[0301] The example uses a butanologen strain comprising (i) a non-expressed

(deleted) and/or non-functional native yeast AHAS (Ilv2p), as well as (ii) an inhibitor- sensitive AHAS, preferably de-sensitized for feedback-inhibition by branched-chain amino acids localized in the cytoplasm. This strain is referred to as NORTH 1002a. In the example improved butanologen biomass generation in batch and fed-batch cultivations by NORTH 1002aa is demonstrated in cultures supplemented with an AHAS inhibitor as compared to the cultivation of the same strain without AHAS inhibitor supplementation. [0302] A frozen vial of NORTH 1002a is inoculated into a sterilized pre-pure culture tank and grown in an aerobic batch regime with and without supplementation of AHAS inhibitor. Following growth, the content of the vessel is transferred to a larger pure culture fermentor where propagation is carried out with some aeration, again under sterile conditions in a batch regime with and without AHAS inhibitor. In the cultivations, NORTH 1002a is brought into contact with a media, comprising a carbon source (e.g. glucose, fructose, molasses, corn mash, ...), certain minerals, vitamins, amino acids and salts, as well as a nitrogen source (e.g. ammonium, urea, ...). During the scale up of NORTH 1002a in the pre-pure culture tank and pure culture fermentor under the same culture conditions, the NORTH 1002a cultures with AHAS inhibitor exhibit a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002a cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the main carbon substrate.

[0303] From the pure culture vessel with or without inhibitor, NORTH 1002a is transferred to a series of progressively larger seed and semi-seed fermentors. During the fed-batch fermentations in these larger seed and semi-seed fermenters under aerobic conditions with or without AHAS inhibitor, a carbon source (e.g. molasses, glucose, fructose, corn mash, ...), certain amino acids and vitamins, phosphoric acid, a nitrogen source (e.g. ammonium, urea, ...), optionally AHAS inhibitor and minerals are fed at a controlled rate. At the end of the semi-seed fermentations with or without

supplementation of an AHAS inhibitor, the contents of the vessels are each pumped to a series of separators that separate the biomass from the spent media. Each of the cultures are then washed with cold water and pumped to semi-seed yeast storage tanks where the yeast creams of NORTH 1002a are held at temperatures of 5°C or below. During the scale up of NORTH 1002a in the seed and semi-seed fermenters with and without supplementation of AHAS inhibitor, the NORTH 1002a with supplementation of AHAS inhibitor exhibit a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002a cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the substrate. [0304] The commercial fermentations in fermentation tanks with a working volume up to 50,000 gallons are started by pumping water, referred to as set water, into the fermentors. Next, in a process referred to as pitching, semi-seed yeasts of NORTH 1002a from the storage tank are transferred into fermentor with and without

supplementation of AHAS inhibitor. After the addition, aeration, cooling and nutrient additions, optionally addition of AHAS inhibitor, are started. At the start of the fermentation, the liquid seed yeast and additional water may occupy only about one-third to one -half of the fermentor volumes. Constant additions of nutrients and optionally AHAS inhibitor during the course of fermentation (fed-batch) bring the fermentors to their final volume. The rate of nutrient addition and optionally AHAS inhibitor increases throughout the fermentation because more nutrients have to be supplied to support growth of the increasing cell population. Air is provided to the fermentors at or below about one volume of air per fermentor volume per minute. Cooling is accomplished by internal cooling coils or by pumping the fermentation liquid, also known as broth, through an external heat exchanger. The addition of nutrients and regulation of pH, temperature and airflow are carefully monitored and controlled by computer systems during the entire production process. Throughout the fermentation, the pH is kept in the range of 4.0-7.0.

[0305] At the end of the fermentations, the fermentor broths of NORTH 1002a with and without supplementation of AHAS inhibitor are each separated, e.g. by centrifuges, washed with water and re-centrifuged to yield yeast creams. The yeast creams are each cooled to 10°C or below and stored in separate, refrigerated stainless steel cream tanks. Alternatively, the yeast creams of NORTH 1002a with or without AHAS inhibitor are pumped to a plate and frame filter press and dewatered to a cake-like consistency. This press cake yeasts are crumbled into pieces and packed into 50-pound bags that are stacked on a pallet and cooled in a refrigerator for a period of time with adequate ventilation. During the commercial fermentations of NORTH 1002a with and without AHAS inhibitor, the NORTH 1002a culture with AHAS inhibitor exhibits a significantly lower isobutanol yield as well as total yield of isobutanol pathway intermediates and derivatives (e.g. DHIV, DHMB, KIV, isobutyric acid, isobutyr aldehyde, acetolactate, butadiol, acetoin, diacetyl) than the NORTH 1002a cultures without addition of an AHAS inhibitor, as well as a higher biomass yield on the substrate. Final biomass concentration of NORTH 1002a with supplementation of AHAS inhibitor at the end of the commercial fermentation is higher than of NORTH 1002a without addition of AHAS inhibitor.

[0306] Same amounts of produced cake and yeast creams of NORTH 1002a cultivated with or without addition of AHAS inhibitor are re-suspended in 12 ml of PRODUCTION medium and transferred into 25 ml Balch tubes, respectively.

PRODUCTION medium is composed of 50% Yeast Synthetic Medium (2x), 10% Complete Supplement Mixture (CSM) with adenine (Formedium, DCS0031,

Hunstanton, UK), 16%> of a 250 g/1 glucose solution, and 24%> of water. Yeast Synthetic Medium (2x) in turn is composed of 13.4 g/1 Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3), 40 mg/1 thiamine, 40 mg/1 niacin and 200 ml/1 of a 1 M MES buffer, pH = 5.5. Each Balch tube is fitted with a butyl rubber septum and cramped to the tube with a sheet metal with circular opening to allow samples withdrawal by syringes. For sample withdrawal, 1 ml syringes (25G 5/8 (0.5 mm x 16 mm) Safety-Lok, Becton Dickinson, Franklin Lakes, NJ) are employed. Growth of the cultures is monitored with help of OD measured with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, NJ) at λ = 600 nm. Extracellular compound analysis in supernatant is accomplished by HPLC. A BIO-RAD Aminex HPX-87H column is used in an isocratic method with 0.01 N sulfuric acid as eluent on a Waters Alliance 2695 Separations Module (Milford, MA). Flow rate is 0.60 ml/min, column temperature 40°C, injection volume 10 μΐ and run time 58 min. Detection is carried out with a refractive index detector (Waters 2414 RI) operated at 40°C and an UV detector (Waters 2996 PDA) at 210nm. Isobutanol formation is observed in all the NORTH 1002a cultures, respectively. At least one of the final isobutanol rate, titer, or specific productivity of a NORTH 1002a culture reconstituted from a NORTH 1002a culture previously cultivated with AHAS inhibitor is not substantially different than that parameter of a culture reconstituted from a NORTH 1002a culture that was not contacted with AHAS inhibitor during scale up.

Example 7: Bacillus subtilis ALS variants with decreased affinity for TPP [0307] In this example, variants of Bacillus subtilis ALS were created. Bacillus subtilis ALS variants demonstrated increased apparent dissociation constants for TPP. These variants, with decreased affinity for TPP, can be used as a switchable step in yeast by requiring supplementation with thiamine to activate ALS. Strain and media

[0308] Escherichia coli TOP10 was obtained from Life Technologies Corp. (Cat.

# C404003, Grand Island, NY). Expression plasmid pBAD was previously described (U.S. Patent No. 7,910,342, which is incorporated herein by reference in its entirety). Bacillus subtilis ALS gene was previously described (U.S. Patent Publication No.

2009/0305363, which is incorporated herein by reference in its entirety). Cells were grown at 37°C in Miller's LB broth (Cat. # 46-050-CM, Mediatech, Inc., Herndon, VA) with 0.02% L-(+)-arabinose (Cat. # A3256, Sigma-Aldrich, Inc., St. Louis, MO) and 100μg/mL ampicillin (Cat. # A1066, Sigma-Aldrich, Inc., St. Louis, MO). Cells were plated on LB agar plates with 100μg/mL ampicillin (Cat. # LI 004, Teknova, Inc., Hollister, CA).

Construction of ALS variants

[0309] The amino acid substitutions at 7 different positions as indicated in Table

8 were introduced into B. subtilis ALS using the primers as indicated in the Table 15. Mutagenesis was performed using a QuikChange Lightning Kit (Cat. #210519, Agilent Technologies, La Jolla, CA), according to manufacturer's directions. Mutagenesis primers were obtained from Sigma-Aldrich Co. LLC, St. Louis MO. Reactions were thermocycled in a Gene Amp 9700 (Perkin Elmer Applied Biosystems, Norwalk, CT). Escherichia coli TOP 10 cells were transformed with 1 μΐ of QuikChange reaction product according to manufacturer's directions and transformants were selected on LB agar plates with 100μg/mL ampicillin. DNA sequences were obtained for multiple isolates from each transformation in order to identify those with the desired mutations.

Table 15 : Primers used to create Bacillus subtilis ALS variants.

G399S gtaacttgcgatatctcttcgcacgccatttgg (409) ccaaatggcgtgcgaagagatatcgcaagttac (410)

HIS401 gcgatatcggttcgcacgccatttggatgtcac (411)

H401A gcgatatcggttcggcagccatttggatgtcac (412) gtgacatccaaatggctgccgaaccgatatcgc (413)

H401G gcgatatcggttcgggcgccatttggatgtcac (414) gtgacatccaaatggcgcccgaaccgatatcgc (415)

GLY450 gtggtttctgtctctggtgacggcggtttcttattc (416)

G450A gtggtttctgtctctgcagacggcggtttcttattc (417) gaataagaaaccgccgtctgcagagacagaaaccac (418)

G450D gtggtttctgtctctgatgacggcggtttcttattc (419) gaataagaaaccgccgtcatcagagacagaaaccac (420)

G450V gtggtttctgtctctgttgacggcggtttcttattc (421) gaataagaaaccgccgtcaacagagacagaaaccac (422)

G450S gtggtttctgtctcttctgacggcggtttcttattc (423) gaataagaaaccgccgtcagaagagacagaaaccac (424)

GLY453 gtctctggtgacggcggtttcttattctcagcaatg (425)

G453A gtctctggtgacggcgcattcttattctcagcaatg (426) cattgctgagaataagaatgcgccgtcaccagagac (427)

G453M gtctctggtgacggcatgttcttattctcagcaatg (428) cattgctgagaataagaacatgccgtcaccagagac (429)

G453L gtctctggtgacggcttattcttattctcagcaatg (430) cattgctgagaataagaataagccgtcaccagagac (431)

G453I gtctctggtgacggcatcttcttattctcagcaatg (432) cattgctgagaataagaagatgccgtcaccagagac (433)

G453V gtctctggtgacggcgttttcttattctcagcaatg (434) cattgctgagaataagaaaacgccgtcaccagagac(435)

VAL545 gtcatcatcgatgtcccggttgactacagtgataac (436)

V545L gtcatcatcgatgtcccgttagactacagtgataac (437) gttatcactgtagtctaacgggacatcgatgatgac (438)

V545M gtcatcatcgatgtcccgatggactacagtgataac (439) gttatcactgtagtccatcgggacatcgatgatgac (440)

V545F gtcatcatcgatgtcccgttcgactacagtgataac (441 ) gttatcactgtagtcgaacgggacatcgatgatgac (442)

V545W gtcatcatcgatgtcccgtgggactacagtgataac (443) gttatcactgtagtcccacgggacatcgatgatgac (444)

TYR547 gatgtcccggttgactacagtgataacattaatttagc (445)

Y547F gatgtcccggttgacttcagtgataacattaatttagc (446) gctaaattaatgttatcactgaagtcaaccgggacatc (447)

Y547M gatgtcccggttgacatgagtgataacattaatttagc (448) gctaaattaatgttatcactcatgtcaaccgggacatc (449)

Y547L gatgtcccggttgacttaagtgataacattaatttagc (450) gctaaattaatgttatcacttaagtcaaccgggacatc (451)

Y547V gatgtcccggttgacgttagtgataacattaatttagc (452) gctaaattaatgttatcactaacgtcaaccgggacatc (453)

Y547A gatgtcccggttgacgcaagtgataacattaatttagc (454) gctaaattaatgttatc acttgcgtcaac cgggac ate (455 )

ALS assay

[0310] Soluble fraction cell extracts were prepared from 5ml of culture by bead beating 2 x 10 seconds in lOOmM MOPS pH6.8, lOmM MgC12, ImM EDTA in a Mini- Bead-beater (Cat. #3110BX, Biospec Products, Bartlesville, OK). Cell extract protein concentration was determined by Pierce BCA assay (Cat. #23224 and 23228, Thermo Fisher Scientific, Inc., Rockford, IL).

[0311] Assays were performed in a lOOul volume containing lOOmM MOPS pH6.8, ImM EDTA, varying concentrations of pyruvate (Cat#P2256, Sigma-Aldrich, Inc., St. Louis, MO) , varying concentrations of TPP (Cat#C8754, Sigma-Aldrich, Inc., St. Louis, MO) and varying concentrations of cell extract. Reactions were terminated by 4-fold dilution into 60mM EDTA (pH 4.5) in water (Omnisolv, #WX0001-1, EMD Chemicals, Gibbstown, NJ). Acetolactate production was measured by LC/MS. LC/MS method

[0312] 2μΙ, of each sample were injected on a Waters Acuity UPLC/SQD

System, using a HSS T3 column (2.1 x 100 mm, 1.8 μπι, #186003539, Waters, Milford, MA) at a temperature of 30°C. UPLC mobile phases consisted of 0.1% formic acid in water (Mobile A) and 0.1% formic acid in acetonitrile (Omnisolv, #AX0156-1, EMD Chemicals, Gibbstown, NJ) (Mobile B) with a constant flow rate of 0.5mL/min. The gradient consisted of an initial 1 minute period at 99% A, followed by a 0.5 minute linear gradient ending at 75% B and then a 0.5 minute linear gradient back down to 99% A before injecting the next sample. MS analysis was performed by electrospray negative ionization at a cone voltage of 20V and m/z =130.9. Retention time and peak intensities were determined using MassLynx4.1 software (Waters, Milford, MA). External standard, (i?,5)-acetolactate, (synthesized according to Aulabaugh & Schloss,

Biochemistry 29: 2824-2830, 1990) analyzed in the same manner was used for quantitation.

TPP binding measurements

[0313] Standard measurement of TPP binding consisted of measuring the ALS activity in crude cell extracts as described above, using 1 mM pyruvate and with varying concentrations of TPP. Although not consumed in the reaction, the rate at each concentration of TPP was used in the Michaelis-Menton equation to solve for K M , which was interpreted for these experiments as the apparent dissociation constant, K D . Of the 36 single amino acid substitutions, there was no detectable ALS activity at 50 μΜ TPP, with the following substitutions: T94D, T94E, T94R, T94K, T94S, G399A, G399D, G399V, G399S, G450A, G450D, G450V, G450S, G453L, G453I, G453V. These were not characterized any further. The remaining variants were tested at varying TPP concentrations and the apparent dissociation constants are shown in the Table 16.

Table 16: Apparent dissociation constants for Bacillus subtilis ALS variants.

- I l l - V545W 3546

Y547F 206

Y547L 204

Y547M 157

Y547V 4

Y547A 45

T94S 7

H401A 57

H401 G 55

G453A 344

V545L 13

[0314] Based on these results, variants with two amino acid changes were produced from H401G, H401A, G453A, V545M, V545W, Y547F, Y547M, and Y547L. The 23 variants containing all possible combinations of the changes in 2 positions included: H401A/G453A; H401A/V545M; H401A/V545W; H401A/Y547F;

H401A/Y547L; H401A/Y547M; H401G/G453A; H401G/V545M; H401G/V545W; H401G/Y547F; H401G/Y547L; ; H401G/Y547M; G453A/V545M; G453A/V545W; G453A/Y547F; G453A/Y547L; G453A/Y547M; V545M/Y547F; V545M/Y547L; V545M/Y547M; V545W/Y547F; V545W/Y547L; and V545W/Y547M.

[0315] All of the variants were prepared and tested for activity by a preliminary measurement with 1 mM pyruvate and varying concentrations of TPP. One variant, H401G/V545W, had no detectable activity. Of the remaining variants, 10 had activity and K D values near to or greater than the single variant V545W. The apparent K D values for these variants are shown in the Table 17.

Table 17: Apparent dissociation constants for Bacillus subtilis ALS variants

Example 7: Determination of growth rate of strains comprising mutant ALS constructs

[0316] The growth rate of strains containing the wild-type ALS plasmid

(pBP4790), the V545W mutant ALS plasmid (pBP4793), the H401A/Y547L mutant ALS plasmid (pBP5258), the H401A/Y547L mutant ALS plasmid (pBP5262), and the no-ALS control plasmid (pBP2986) was measured under low glucose conditions. Two transformants for each construct were tested. Polymer-based slow-release feed beads (Kuhner Shaker, Basel, Switzerland) were used for the low glucose condition.

[0317] The strains were grown overnight in 70 ml of synthetic medium (See table

18) supplemented with 30 g/L thiamine hydrochloride and two 12 mm Kuhner Shaker FeedBead Glucose discs in 500 mL vented Erlenmeyer flasks at 30°C, 250 RPM in a New Brunswick Scientific 124 shaker. The overnight cultures were centrifuged at 4,000 x g for 5 minutes at 22°C and resuspended in the same synthetic medium, supplemented with 30 μg/L thiamine hydrochloride, to a final OD 6 oo of 0.1. 75 ml of the culture was added to 500 mL vented Erlenmeyer flasks and one 12 mm Kuhner Shaker FeedBead Glucose disc was added to each flask. The cultures were grown at 30°C, 250 RPM in a New Brunswick Scientific 124 shaker and the OD 6 oo was monitored for 27 hours. The growth rate of the cultures was calculated during the period of growth between 3 and 9.25 hours. The average growth rate and standard deviation for each strain type is shown in Figure 8. The final OD 60 o at 27 hours and standard deviation for each strain type is shown in Figure 9.

Table 18: Growth Curve Medium

Table 19: Trace Element Solution

Table 20: Vitamin Solution