# Patent

## Title
Use of thiamine and nicotine adenine dinucleotide for butanol production

## Abstract

The invention relates generally to the field of industrial microbiology and alcohol production. More specifically, the invention relates to the use of thiamine, biosynthetic precursors of thiamine, nicotinic acid, nicotinamid, nicotinic acid riboside, nicotinamid riboside, or other biosynthetic precursors of nicotine adenine dinucleotide (NAD) to improve butanol production. Butanol production can be improved by providing sufficient amounts of thiamine, biosynthetic precursors of thiamine, nicotinic acid, nicotinamid, nicotinic acid riboside, nicotinamid riboside, or other biosynthetic precursors of nicotine adenine dinucleotide (NAD) in the production media.

## Background
FIELD OF THE INVENTION
The invention relates generally to the field of industrial microbiology and fermentative alcohol production. More specifically, the invention relates to the use of thiamine or biosynthetic precursors of thiamine, nicotinic acid, nicotinamid, nicotinic acid riboside, nicotinamid riboside, or other biosynthetic precursors of nicotine adenine dinucleotide (NAD) to improve the ability of a recombinant host cell to produce a product alcohol such as butanol. Recombinant host cells of the invention can produce butanol.
Additionally, the invention relates to methods of increasing butanol yield and methods for increasing specific butanol production rates.
BACKGROUND OF THE INVENTION
Global demand for liquid transportation fuel is projected to strain the ability to meet certain environmentally driven goals, for example, the conservation of oil reserves and limitation of green house gas emissions. Such demand has driven the development of technology which allows utilization of renewable resources to mitigate the depletion of oil reserves and to minimize green house gas emissions. This invention addresses the need for improved processes for the conversion of plant-derived raw materials to a product stream useful as a liquid transportation fuel. Such processes would satisfy both fuel demands and environmental concerns.
Butanol is an important industrial chemical and is 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.
Methods for the chemical synthesis of isobutanol are known such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) 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, are generally expensive, and are not environmentally friendly. The production of butanol from plant-derived raw materials would minimize green house gas emissions and would represent an advance in the art.
Some fungi desire supplementation of the vitamin biotin, like many wild isolates of the yeast Saccharomyces cerevisiae (Leonian, et al., Science 95:658, 1942; Stolz, et al., J. Biol. Chem. 274:18741-18746, 1999; Hall, et al. Genetics 177:2293-2307, 2007). For a biotin auxotrophic organisms to grow, sufficient amount of biotin is provided exogenously. Alternatively, endogenous biosynthesis of biotin can be accomplished, for example, by directed evolution (Leonian, et al., supra) or complementing the retained biosynthesis pathway of yeast with the missing enzyme activities (Hall, et al., supra).
Another vitamin that is often supplemented to yeast cultures is pantothenic acid (Leonian, et al., supra). However, although Saccharomyces cerevisiae is capable of de novo pantothenic acid biosynthesis, an increase of biosynthesis, especially the enzyme activity in the rate-limiting step of amine oxidase, may be achieved by directed evolution (Leonian, et al., supra) or recombinant DNA technology (White, et al., J. Biol. Chem. 276:10794-10800, 2001) which is typically required to provide sufficient activity to remedy growth, respectively. No further vitamin requirements for aerobic growth of Saccharomyces cerevisiae have been identified (Henry, Appl. Environ. Microbiol. 31:395-398, 1976).
A commercial defined medium without yeast extract and containing only the vitamins inositol, biotin, pantothenic acid, and pyridoxine was used to aerobically produce factor XIII with Saccharomyces cerevisiae (see, e.g., U.S. Pat. No. 6,750,045). A commercial medium for the aerobic production of protein in Saccharomyces cerevisiae supplemented with a vitamin mixture comprising biotin, pantothenic acid, myo-inositol, and pyridoxine is described in U.S. Pat. No. 5,795,771. U.S. Patent Application Publication No. 2005/0112737 reports a chemically defined medium supplemented with the vitamins biotin, inositol, and thiamine to grow a pyruvate-decarboxylase (PDC)-negative Saccharomyces cerevisiae with an exogenous lactate dehydrogenase activity. The non-Crabtree and PDC-KO yeast Kluyveromyces marxianus has been shown to produce lactic acid in shake flask cultivations with complex yeast extract peptone-dextrose medium (YPD) media comprising 10 g/L yeast extract, 20 g/L peptone, additionally glucose, and occasionally agar. No additional specific media requirements are described (see, e.g., U.S. Pat. No. 7,534,597). An example of a non-Crabtree yeast Kluyveromyces marxianus capable of generating biomass when cultured with corn fiber hydrolyzate supplemented with yeast minimal medium and a vitamin cocktail (Kiers, et al., Yeast 14:459-469, 1998) where 5 mg/L of nicotinic acid was added to the fiber hydrolysate is described in U.S. Pat. No. 7,700,332. However, no link of bioprocess performance to specific compounds of the vitamin mixture is made.
Very high gravity (VHG) fermentation with Saccharomyces cerevisiae and corn flour mash for the production of ethanol was optimized through supplementations of Mg2+, glycine, yeast extract, biotin, acetaldehyde, and peptone (Wang, et al., Biotechnol. Lett. 29:233-236, 2007). Ethanol production with brewing yeast was also improved by adding a nitrogen source, ergosterol, and oleic acid to high-gravity worts of 16 to 18% dissolved solids (Casey, et al., Appl. Environ. Microbiol. 48:639-646, 1984). It is also reported that in a high cell density fermentation, feeding biotin in combination with a vitamin mixture containing pantothenic acid, meso-inositol, nicotinic acid, thiamine, pyridoxine, and para-aminobenzoic acid during aerated fed-batch processes improves ethanol production and viability of Saccharomyces cerevisiae (Alfenore, et al., Appl. Microbiol. Biotechnol. 60:67-72, 2002). However, apart from biotin, the composition of the vitamin mixture was not further investigated to link observed performance increases to specific components.
A complex YPD medium consisting of 10 g/L yeast extract, 20 g/L peptone, and variable amounts of glucose was described in examples for culturing the yeasts Kluyveromyces marxianus and Saccharomyces cerevisiae to produce butanol (see, e.g., WO 2010/075504). No specific requirements or analyses of vitamin requirements are mentioned. Examples of butanol production in yeasts Kluyveromyces marxianus and Saccharomyces cerevisiae, some with reduced or completely deleted PDC-activities, were described using 6.7 g/L YNB medium without amino acids and 0.076 g/L histidine with nicotinic acid concentrations of about 0.4 mg/L (see, e.g., WO 2010/051527). An economic comparison of nutrient costs in example fermentations is disclosed in U.S. Patent Application Publication No. 2009/0215137. Synthetic fermentation medium (mineral medium) (SFM) is described for butanol production with a yeast cell using a vitamin mixture according to Verduyn, et al., (Yeast 8:501-517, 1992) with the medium containing 1 mg/L nicotinic acid (see, e.g., WO 2009/103533).
Genetic modification of microorganisms to produce new products frequently comes with changed and/or new nutritional requirements to ensure optimum performance of the biocatalyst. Identifying and optimizing nutritional requirements can be complex and feeding complex multi-vitamin mixtures to address nutritional needs can be costly. Consequently, technical inventions are necessary to address and solve these hurdles.
BRIEF SUMMARY OF THE INVENTION
Provided herein are methods for producing a fermentation product comprising: providing a production culture comprising recombinant cells capable of producing butanol and production media comprising 1) thiamine or a biosynthetic precursor thereof, 2) optionally nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD, and 3) a production feed derived from biomass comprising a fermentable carbon source wherein the production media contains less than 1 g/L of multi-component media additives. Methods further comprise contacting the production culture with the production media and the production feed in a fermentation vessel to form a production broth under conditions whereby a fermentation product is produced. In some embodiments, the production media is substantially free of multi-component media additives.
In some embodiments, the production broth contains at least about 5 mg/L thiamine or a biosynthetic precursor thereof. In other embodiments, the production broth comprises about 100 mg/L nicotinic acid and about 20 mg/L thiamine. In some embodiments, the production broth contains at least about 50 mg/L thiamine or a biosynthetic precursor thereof. In some embodiments, the production media contains at least about 20 mg/L thiamine or a biosynthetic precursor thereof. In other embodiments, the production broth contains at least about 0.1 mg/g dcw thiamine or a biosynthetic precursor thereof.
In some embodiments, the thiamine or a biosynthetic precursor thereof is present in an amount sufficient to provide at least about 30% of the rate, titer, or specific productivity observed in media that contains about 10 g/L yeast extract.
In some embodiments, the thiamine or a biosynthetic precursor thereof and/or nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD are components of the production feed.
Also, provided herein are methods for producing a fermentation product comprising: providing a production culture comprising recombinant cells capable of producing butanol and a production media comprising 1) nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD, 2) optionally thiamine or a biosynthetic precursor thereof, and 3) a production feed derived from biomass comprising a fermentable carbon source wherein the production media contains less than 1 g/L of multi-component media additives. Methods further comprise contacting the production culture with the production media and the production feed in a fermentation vessel to form a production broth under conditions whereby a fermentation product is produced. In some embodiments, the production media is substantially free of multi-component media additives.
In some embodiments, the production broth contains at least about 5 mg/L nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD. In other embodiments, the production broth contains at least about 10 mg/L nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD. In some embodiments, the production media contains at least about 20 mg/L nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD. In some embodiments, the production broth comprises about 100 mg/L nicotinic acid and about 20 mg/L thiamine. In some embodiments, the production broth contains at least about 0.1 mg/g dcw nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD.
In embodiments, nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD is present in an amount sufficient to provide at least about 30% of the rate, titer, or specific productivity observed in media that contains about 10 g/L yeast extract.
In some embodiments the nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD and/or thiamine or a biosynthetic precursor thereof are components of the production feed.
In some embodiments, the cell density in the fermentation vessel is at least about 10 g dcw/L. In other embodiments, the cell density in the fermentation vessel is less than about 10 g dcw/L.
In some embodiments, the recombinant cells are yeast. In some embodiments, the yeast is Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, Torulaspora, Hanseniaspora, Kluveromyces, Issatchenkia, Ashbya, or Candida. In some embodiments, the yeast is crabtree positive and in other embodiments, the yeast is crabtree negative.
In some embodiments, the recombinant cell comprises a modification of an endogenous gene encoding a thiamine-phyrophosphate-dependent (TPP) gene. In some embodiments, the TPP-dependent gene is a pyruvate decarboxylase gene. In some embodiments, the modification is a deletion. In some embodiments, the recombinant cell comprises a modification in a gene encoding PDC1, PDC5, PDC6, or combinations thereof. In embodiments, the modification is a deletion.
In some embodiments of the methods provided, the recombinant cell comprises a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be comprised of at least one heterologous polynucleotide encoding a polypeptide that catalyzes a substrate to product conversion of the pathway. The butanol biosynthetic pathway may be an isobutanol biosynthetic pathway. The isobutanol biosynthetic pathway may comprise the following substrate to product conversions:
a) pyruvate to acetolactate;
b) acetolactate to 2,3-dihydroxyisovalerate;
c) 2,3-dihydroxyisovalerate to α-ketoisovalerate;
d) α-ketoisovalerate to isobutyraldehyde; and
e) isobutyraldehyde to isobutanol.
In another embodiment, the recombinant host cell may comprise at least one DNA molecule encoding a polypeptide having aspartate oxidase or aspartate dehydrogenase activity. In one embodiment, the recombinant host cell may comprise at least one DNA molecule encoding a polypeptide having quinolinate synthase activity.
In some embodiments, butanol is produced at a specific butanol production rate of at least about 0.10 g/g/h
In some embodiments, the product is produced under aerobic conditions and in other embodiments, the product is produced under anaerobic conditions.
In some embodiments, the fermentation broth further comprises at least about 5 mg/L thiamine or other biosynthetic precursors of thiamine. In other embodiments, the media further comprises at least about 10 mg/L thiamine or other biosynthetic precursors of thiamine. In some embodiments, the product is produced at a titer of at least about 50% of the titer when the host cell is grown in media containing about 10 g/L yeast extract and no supplemental thiamine or nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD. In some embodiments, the product is produced at a specific production rate of at least about 50% of the rate when the host cell is grown in media containing about 10 g/L yeast extract and no supplemental thiamine or nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD.
Also provided are compositions comprising: production media consisting essentially of greater than about 10 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and, optionally, greater than about 10 mg/L thiamine or a biosynthetic precursor thereof; a production culture comprising a recombinant microorganism which comprises a butanol biosynthetic pathway; and liquefied biomass comprising a fermentable carbon substrate. In some embodiments, the production media consists essentially of greater than about 20 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and greater than about 20 mg/L thiamine or a biosynthetic precursor thereof. In other embodiments, the production broth comprises about 100 mg/L nicotinic acid and about 20 mg/L thiamine.
In addition, provided herein are compositions comprising: production media consisting essentially of greater than about 10 mg/L thiamine or a biosynthetic precursor thereof and, optionally, greater than about 10 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD; a production culture comprising a recombinant microorganism which comprises a butanol biosynthetic pathway; and liquefied biomass comprising a fermentable carbon substrate. In some embodiments, the production media consists essentially of greater than about 20 mg/L thiamine or a biosynthetic precursor thereof and greater than about 20 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD. In other embodiments, the production broth comprises about 100 mg/L thiamine and about 20 mg/L nicotinic acid.
Provided herein are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 1 mg/L thiamine or a thiamine biosynthetic precursor; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density less than about 10 g dcw/L.
Also provided are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 1 mg/L thiamine or a thiamine biosynthetic precursor; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density greater than about 10 g dcw/L
Also provided are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 0.1 mg/gdcw thiamine or a thiamine biosynthetic precursor; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density greater than about 10 g dcw/L
Provided herein are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 1 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density less than about 10 g dcw/L.
Also provided are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 1 mg/L nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density greater than about 10 g dcw/L
Also provided are methods for producing butanol comprising: (a) providing a production culture comprising recombinant cells comprising a butanol biosynthetic pathway; (b) providing production media containing greater than about 0.1 mg/gdcw nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD; and (c) contacting said production culture with said media under conditions whereby butanol is produced. In some embodiments, the production culture has a cell density greater than about 10 g dcw/L
In some embodiments, the amount of ketoisovaleric acid accumulated is less than that observed in media that contains about 10 g/L yeast extract. In some embodiments, the host cell further comprises ketoisovalerate decarboxylase.
In some embodiments, butanol is produced in a commercial scale fermentation and in some embodiments, the commercial scale is greater than about 1000 L.
In some embodiments, a production broth is provided with less than 1 g/L of multi-component media additives.
In some embodiments a butanol fermentation production media comprising;

    
    
        1. thiamine or a biosynthetic precursor thereof;
        2. optionally nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD; and
        3. a production feed derived from biomass comprising a fermentable carbon source; and

wherein the production media comprises less than 1 g/L of multi-component media additives is provided.

    
    


In some embodiments a butanol fermentation production media comprising;

    
    
        1. nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD;
        2. optionally thiamine or a biosynthetic precursor thereof; and
        3. a production feed derived from biomass comprising a fermentable carbon source; and

wherein the production media comprises less than 1 g/L of multi-component media additives is provided.

    
    





BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
The various embodiments of the invention can be more fully understood from the following detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.
FIG. 1 depicts the final biomass (X; g/L), isobutanol concentration (g/L), and specific isobutanol production rate (qP; g/g/h) in aerobic (left panels) and anaerobic (right panels) shake flask cultivations with the addition of either water (control), yeast extract (YE), or thiamine (TH).
FIG. 2 depicts the isobutanol concentrations in aerobic shake flask cultivations with the addition of 0 (control), 6, 60, or 100 mg/L of thiamine or 10 g/l yeast extract (YE).
FIG. 3 depicts the α-ketoisovalerate accumulated in aerobic shake flask fermentations with the addition of 0 (control), 6, 60, or 100 mg/L of thiamine (TH) or 10 g/l yeast extract (YE).
FIG. 4 shows the isobutanol titer during fermentations described in the Examples (F—140-100 (O), F—141-20 (□), F—142-10 (Δ), and F—143-0 (X)).
FIG. 5 shows the optical density measured during fermentations described in the Examples (F—140-100 (O), F—141-20 (□), F—142-10 (A), and F—143-0 (X)).
FIG. 6 shows the thiamine concentration in culture medium measured during fermentations described in the Examples (F—140-100 (O), F—141-20 (□), F—142-10 (Δ), and F—143-0 (X)).
FIG. 7 depicts the pathway of thiamin biosynthesis in yeast. The major intermediates and products are written in bold, and the genes involved are written in italics. The dashed arrows indicate processes whose steps have not yet been fully identified.
FIG. 8 depicts the biosynthesis pathway of NAD+ via the kynurenine pathway or by direct incorporation of nicotinic acid in Saccharomyces cerevisiae. 
FIG. 9 depicts the increase in isobutanol concentration (delta c(isobutanol); mM), decrease of glucose concentration (delta (glucose); mM), and increase in biomass concentration (delta (OD);) in aerobic (solid black bars) and anaerobic (striped gray bars) shake flask cultivations with the addition of either water (H2O, control), vitamin solution, yeast extract and peptone solution (YEP), or nicotinic acid (NA).
FIG. 10 depicts the increase in isobutanol concentration (delta c(isobutanol); mM), decrease of glucose concentration (delta (glucose); mM), and increase in biomass concentration (delta (OD);) in aerobic (A-C) and anaerobic (D-F) shake flask cultivations with the addition of either water (H2O, control) (Δ), vitamin solution (□), yeast extract and peptone solution (YEP) (Δ), or nicotinic acid (NA) (o).
FIG. 11 depicts the isobutanol concentrations (c(isobutanol); mM) and biomass concentration (OD600;) in aerobic shake flask cultivations with nicotinic acid (NA) added ad 0 mg/L (⋄; +H2O), ad 6 mg/L (Δ; +NA 6), ad 60 mg/L (o; +NA 60), or ad 100 mg/L (x; +NA 600) or yeast extract supplementation (YE) (□).
FIG. 12 depicts (A) the isobutanol concentration (c(isobutanol); g/L), (B) the biomass concentration (c(DE); g/L), and (C) the nicotinic acid (NA) concentration (c(NA); g/L) during fermentations described in the Example 9 with initial nicotinic acid supplementation of about 100 mg/L (⋄), about 20 mg/L (□), about 5 mg/L (Δ), and about 0 mg/L (●).



The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.
DETAILED DESCRIPTION OF THE INVENTION
Genetic modification of yeast to produce products other than biomass and ethanol, like e.g. butanol, requires genetic modification of the yeast biochemical network, resulting in changes of the genome, transcriptome, proteome and metabolome. Additionally, different products like butanol exert different physiological effects on cells due to their different physical properties.
Thiamine pyrophosphate (TPP) is an essential cofactor for a variety of metabolic enzymes. Such metabolic enzymes may be necessary for host organism production of fermentation products from a carbon substrate. Applicants have discovered that providing media with thiamine or biosynthetic precursors of thiamine leads to improved butanol production in a fermentation. Supplementation with thiamine has also been found to reduce or eliminate the need to supplement fermentation media with yeast extract. This is believed to be particularly advantageous for commercial scale fermentations. Furthermore, because the composition of yeast extract may vary from lot to lot, reducing or eliminating the need to provide yeast extract can advantageously reduce or eliminate variability of fermentation media composition and thus, variability in production of fermentation products.
Host cells such as yeast possess a biochemical pathway for the production of thiamine, but if thiamine or precursors are available in the medium, yeast typically use the externally provided compounds preferentially. Typically only minimal differences in aerobic growth are observed regardless of whether the thiamine is produced endogenously or provided in the medium. However, as shown herein, it has been surprisingly discovered that the addition of thiamine and/or its biosynthetic precursors improves butanol production in both aerobic and anaerobic conditions.
Applicants have also discovered that increased nicotine adenine dinucleotide (NAD) precursor availability improves alcohol production by recombinant microorganisms such as yeast under aerobic and/or anaerobic conditions. These findings come as a surprise, as it is known that yeast possesses the biochemical capability to endogenously produce NAD from carbon substrates. The major pathway for NAD biosynthesis in yeast is assumed to be the kynurenine pathway, starting from tryptophane. Accordingly, provided herein are methods of increasing the production of alcohols such as butanol using recombinant yeast organisms by increasing available nicotine adenine dinucleotide (NAD) via supplementation of NAD precursors to media and/or recombinant expression of genes involved in the synthesis of NAD precursors.
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. 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 as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference, unless only specific sections of patents or patent publications are indicated to be incorporated by reference.
Although methods and materials similar or equivalent to those disclosed herein can be used in practice or testing of the present invention, suitable methods and materials are disclosed below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
In order to further define this invention, the following terms, abbreviations and definitions are provided.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as disclosed in the application.
As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or 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, preferably within 5% of the reported numerical value.
The term “butanol” as used herein refers to 2-butanol, 1-butanol, isobutanol, or mixtures thereof.
The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.
The terms “PDC-,” “PDC knockout,” or “PDC-KO” as used herein, refer to a cell that has a genetic modification to inactivate or reduce expression of a gene encoding pyruvate decarboxylase (PDC) so that the cell substantially or completely lacks pyruvate decarboxylase enzyme activity. If the cell has more than one expressed (active) PDC gene, then each of the active PDC genes may be inactivated or have minimal expression thereby producing a PDC-cell. For example, PDC1, PDC5, and PDC6 may be knocked out in a PDC knockout.
“Multi-component media additives” as used herein, encompasses complex media additives such as yeast extract and concentrated plant hydrolysates. Also included are multi-vitamin cocktails which, when added to production media, provide 2 or more of the following vitamins at final concentrations of greater than about 0.1 mg/L: biotin, pantothenate, myoinositol, pyridoxine, riboflavin, p-aminobenzoic acid, and folic acid. Production feed is not a multi-component media additive.
“Production feed” as used herein, refers to the portion of the fermentation broth that is provided from the biomass and may be a component of the production media. The biomass is also the source of the carbon substrate. The term “carbon substrate” refers to a carbon source capable of being metabolized by the recombinant host cells disclosed herein. Non-limiting examples of carbon substrates are provided herein and include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, glucose, fructose, sucrose, xylose, arabinose, dextrose, or mixtures thereof.
“Production broth” as used herein, refers to the contents of the fermentation vessel and comprises the production media with production feed and the production culture.
The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, for example, messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5′ and 3′ sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or more typically, double-stranded or a mixture of single- and double-stranded regions. “Polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
A polynucleotide sequence can be referred to as “isolated,” in which it has been removed from its native environment. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having dihydroxy-acid dehydratase activity 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. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. 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.
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.
As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
As used herein, “pyruvate decarboxylase activity” refers to any polypeptide having a biological function of a pyruvate decarboxylase enzyme, including the examples provided herein. Such polypeptides include a polypeptide that catalyzes the conversion of pyruvate to acetaldehyde. Such polypeptides also include a polypeptide that corresponds to Enzyme Commission Number 4.1.1.1. Such polypeptides can be determined by methods well known in the art and disclosed herein.
As used herein, “reduced activity” can refer to any measurable decrease in a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the reduced activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. A reduced activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein. “Reduced activity” can also refer to any measurable decrease in a known biological activity in a cell when compared to the same biological activity in the cell prior to the change resulting in the decreased activity.
As used herein, “substantially eliminated activity” refers to measurable decrease in a known biological activity of a polypeptide that results in nearly complete abolishment of the activity when compared to the same biological activity of the polypeptide prior to the change resulting in the substantially eliminated activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. A substantially eliminated activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.
As used herein, “eliminated activity” refers to the complete abolishment of a known biological activity of a polypeptide when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity. Such a change can include a modification of a polypeptide or a polynucleotide encoding a polypeptide as described herein. An eliminated activity includes a biological activity of a polypeptide that is not measurable when compared to the same biological activity of the polypeptide prior to the change resulting in the eliminated activity. An eliminated activity of a polypeptide disclosed herein can be determined by methods well known in the art and disclosed herein.
As used herein, “native” refers to the form of a polynucleotide, gene, or polypeptide as found in nature with its own regulatory sequences, if present.
As used herein, “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.
As used herein, “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” 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 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.
As used herein, the term “modification” refers to a change in a polynucleotide disclosed herein that results in reduced, substantially eliminated or eliminated activity of a polypeptide encoded by the polynucleotide, as well as a change in a polypeptide disclosed herein that results in reduced, substantially eliminated or eliminated activity of the polypeptide. Such changes can be made by methods well known in the art including, but not limited to, deleting, mutating (e.g., spontaneous mutagenesis, random mutagenesis, mutagenesis caused by mutator genes, or transposon mutagenesis), substituting, inserting, down-regulating, altering the cellular location, altering the state of the polynucleotide or polypeptide (e.g., methylation, phosphorylation or ubiquitination), removing a cofactor, introduction of an antisense RNA/DNA, introduction of an interfering RNA/DNA, chemical modification, covalent modification, irradiation with UV or X-rays, homologous recombination, mitotic recombination, promoter replacement methods, and/or combinations thereof. Guidance in determining which nucleotides or amino acid residues can be modified, can be found by comparing the sequence of the particular polynucleotide or polypeptide with that of homologous polynucleotides or polypeptides, for example, yeast or bacterial, and maximizing the number of modifications made in regions of high homology (conserved regions) or consensus sequences.
As used herein, the term “variant” refers to a polypeptide differing from a specifically recited polypeptide of the invention by amino acid insertions, deletions, mutations, and substitutions, created using, for example, recombinant DNA techniques, such as mutagenesis. Guidance in determining which amino acid residues can be replaced, added, or deleted without abolishing activities of interest, can be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, for example, yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.
Alternatively, recombinant polynucleotide variants encoding these same or similar polypeptides can be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as silent changes which produce various restriction sites, can be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence can be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.
Amino acid “substitutions” can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they can be the result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. “Conservative” amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, “non-conservative” amino acid substitutions can be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. “Insertions” or “deletions” can be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed can be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or 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 can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.
The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression can also refer to translation of mRNA into a polypeptide.
The term “overexpression,” as used herein, refers to an increase in the level of nucleic acid or protein in a host cell. Thus, overexpression can result from increasing the level of transcription or translation of an endogenous sequence in a host cell or can result from the introduction of a heterologous sequence into a host cell. Overexpression can also result from increasing the stability of a nucleic acid or protein sequence.
As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
As used herein 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.
The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.







TABLE 1







The Standard Genetic Code












T
C
A
G















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



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



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



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


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



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



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



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


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



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



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



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


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



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



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



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









Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
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 Mar. 20, 2008), and these tables can be adapted in a number of ways (see, e.g., Nakamura, et al., Nucl. Acids Res. 28:292, 2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2. 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. Table 2 has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.







TABLE 2







Codon Usage Table for Saccharomyces cerevisiae Genes












Amino


Frequency per



Acid
Codon
Number
thousand
















Phe
UUU
170666
26.1



Phe
UUC
120510
18.4



Leu
UUA
170884
26.2



Leu
UUG
177573
27.2



Leu
CUU
80076
12.3



Leu
CUC
35545
5.4



Leu
CUA
87619
13.4



Leu
CUG
68494
10.5



Ile
AUU
196893
30.1



Ile
AUC
112176
17.2



Ile
AUA
116254
17.8



Met
AUG
136805
20.9



Val
GUU
144243
22.1



Val
GUC
76947
11.8



Val
GUA
76927
11.8



Val
GUG
70337
10.8



Ser
UCU
153557
23.5



Ser
UCC
92923
14.2



Ser
UCA
122028
18.7



Ser
UCG
55951
8.6



Ser
AGU
92466
14.2



Ser
AGC
63726
9.8



Pro
CCU
88263
13.5



Pro
CCC
44309
6.8



Pro
CCA
119641
18.3



Pro
CCG
34597
5.3



Thr
ACU
132522
20.3



Thr
ACC
83207
12.7



Thr
ACA
116084
17.8



Thr
ACG
52045
8.0



Ala
GCU
138358
21.2



Ala
GCC
82357
12.6



Ala
GCA
105910
16.2



Ala
GCG
40358
6.2



Tyr
UAU
122728
18.8



Tyr
UAC
96596
14.8



His
CAU
89007
13.6



His
CAC
50785
7.8



Gln
CAA
178251
27.3



Gln
CAG
79121
12.1



Asn
AAU
233124
35.7



Asn
AAC
162199
24.8



Lys
AAA
273618
41.9



Lys
AAG
201361
30.8



Asp
GAU
245641
37.6



Asp
GAC
132048
20.2



Glu
GAA
297944
45.6



Glu
GAG
125717
19.2



Cys
UGU
52903
8.1



Cys
UGC
31095
4.8



Trp
UGG
67789
10.4



Arg
CGU
41791
6.4



Arg
CGC
16993
2.6



Arg
CGA
19562
3.0



Arg
CGG
11351
1.7



Arg
AGA
139081
21.3



Arg
AGG
60289
9.2



Gly
GGU
156109
23.9



Gly
GGC
63903
9.8



Gly
GGA
71216
10.9



Gly
GGG
39359
6.0



Stop
UAA
6913
1.1



Stop
UAG
3312
0.5



Stop
UGA
4447
0.7










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.
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, Wis., the backtranslation function in the Vector NTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, for example, the “backtranslation” function at http://www.entelechon.com/bioinformatics/backtranslation.php?lang=eng (visited Apr. 15, 2008) and the “backtranseq” function available at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited Jul. 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.
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” (http://phenotype.biosci.umbc.edu/codon/sgd/index.php).
The term “percent identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods including, but not limited to, those disclosed in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).
Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (disclosed by Higgins and Sharp, CABIOS. 5:151-153, 1989; Higgins, et al., Comput. Appl. Biosci., 8:189-191, 1992) and found in the MegAlignTM program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, supra; Higgins, et al., supra) and found in the MegAlignTM v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.
It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.
The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol., 215:403-410, 1990); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.
Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional suitable 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, Calif.).
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, N.Y., pp. 201-202). In some embodiments, the recombinant host cell disclosed herein can be bacterial or fungal. In some embodiments, the recombinant host cell disclosed herein can be E. coli. In some embodiments, a recombinant host cell disclosed herein can be any yeast or fungi host useful for genetic modification and recombinant gene expression. In other embodiments, a recombinant host cell can be a member of the genera Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, Torulaspora, Hanseniaspora, Kluveromyces, Issatchenkia, and some species of Candida. In another embodiment, a recombinant host cell can be Saccharomyces cerevisiae. 
Exogenous Thiamine and/or Thiamine Biosynthetic Precursor Supplementation
The main biologically active thiamine derivative is thiaminediphosphate (TDP). Its primary function, recognized early after its discovery, is its contribution to the universal metabolic pathways including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, where TDP serves as a cofactor of enzymes such as pyruvate dehydrogenases, α-ketoglutarate dehydrogenases, branched-chain α-ketoacid dehydrogenases, transketolases, and pyruvate decarboxylases. The roles of thiamin and its phosphate derivatives are described in Kowalska and Kozik, (Cell Mol. Biol. Lett. 13:271-82, 2008).
A regulatory activity of TDP has been reported, but the mechanisms of this regulation at the nucleic acid and protein levels is still poorly understood, as elaborated by Hohmann and Meacock (Biochim Biophys Acta 1385:201-19, 1998). More recently, TDP was shown to bind to mRNA domains called “riboswitches” and to affect mRNA structure resulting in gene expression regulation, particularly regulation of the genes involved in thiamin biosynthesis (Lai, Curr. Biol. 13:R285-91, 2003).
A recent report on the accumulation of TTP in Escherichia coli in response to amino acid starvation raises a hypothesis that TTP may play a more universal role as a signal molecule in prokaryotic and eukaryotic cells (Lakaye, et al., J. Biol. Chem. 279:17142-7, 2004).
It has also recently been hypothesized that thiamin and its derivatives contribute to organism responses to various stress conditions, such as UV illumination when thiamin may be involved in the repair of DNA damage, or under conditions of oxidative stress and heat shock when thiamin may increase mitochondrial stability (Machado, et al., J. Mol. Biol. 273:114-21, 1997) and Medina-Silva, et al., Res. Microbiol. 157:275-81, 2006).
Knowledge on thiamin biosynthesis in yeast has been reviewed by Kowalska and Kozik (Cell Mol. Biol. Lett. 13: 271-82, 2008). According to Kowalska and Kozik, similar to other thiamin-synthesizing organisms, yeast first separately synthesize two precursors, 5-(2-hydroxyethyl)-4-methylthiazole phosphate (HET-P) and 4-amino-5-hydroxymethyl-2 methylpyrimidine diphosphate (HMP-PP), which are then condensed into TMP. In the absence of experimental evidence of the early steps of thiazole (HET-P) and pyrimidine (HMP-P) synthesis in yeast, intermediates have been predicted only by extrapolations from prokaryotic thiamin biosynthesis (Begley, et al., Arch. Microbiol. 171:293-300, 1999).
Kowalska and Kozik also indicate that substrates for yeast thiazole synthesis include cysteine as a sulfur donor, glycine, and D-pentulose-5-phosphate. The latter may be D-ribulose-5-phosphate or D-xylulose-5-phosphate, indicative of a link between thiamin biosynthesis and the pentose phosphate pathway (Hohmann and Meacock, supra). A mechanism of thiazole synthesis has been proposed in which NAD+ serves as the early source of a five-carbon carbohydrate and the advanced intermediate is an ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (Chatterjee, et al., J. Am. Chem. Soc. 129:2914-22, 2007). The final product of the thiazole synthesis pathway is HET-P.
Furthermore, according to Kowalska and Kozik, yeast also possess a salvage pathway through which external 5-(2-hydroxyethyl)-4-methylthiazole (HET) is taken up and then phosphorylated to HET-P (Nosaka, et al., J. Biol. Chem. 269:30510-6, 1994). HMP-P is synthesized in yeast cells from histidine and pyridoxal-5-phosphate, the latter linking the thiamin and vitamin B6 (pyridoxine) biosynthesis pathways (Zeidler, et al., J. Am. Chem. Soc. 125:13094-105, 2003). Yeast can also salvage HMP-P by uptake of 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) from the environment followed by its phosphorylation (Kawasak, et al., Curr. Genet. 47:156-62, 2005). The next phosphorylation step yields HMP-PP ready for condensation with HET-P to produce TMP.
Finally, Kowalska and Kozik state that unlike many bacteria, yeast cannot directly phosphorylate TMP to obtain the bioactive coenzyme, TDP. Therefore, TMP must first be dephosphorylated to free thiamin, which is then activated via one-step diphosphorylation (Nosaka, et al., J. Biol. Chem. 268: 17440-7, 1993). TDP may also be produced from free thiamin taken up by the yeast cells from the environment (Enjo, et al., J. Biol. Chem. 272: 19165-70, 1997). External thiamin phosphates, which cannot be transported across the cell membrane, are first dephosphorylated in the periplasm to be utilized by the yeast cells.
Provided herein are methods of increasing the production of butanol using recombinant host organisms by increasing available thiamine via addition of thiamine and/or its biosynthetic precursors to media. In some embodiments, the butanol is 1-butanol, 2-butanol, or isobutanol. In some embodiments, the host cell is E. coli. In other embodiments, the host cell is S. cerevisiae. At least two metabolic enzymes which can be expressed in host cells for butanol production, acetolactate synthase (AlsS) and ketoisovalerate decarboxylase, are TPP-dependent enzymes. Thus, the present invention provides methods for providing media containing sufficient thiamine for butanol production. The addition of thiamine or other biosynthetic precursors of thiamine to production media for recombinant butanol-producing yeast cells can increase butanol production. The thiamine or other biosynthetic precursor of thiamine can be added to the production media in the absence of other multi-component media additives such as yeast extract, corn steep liquor, and sugar cane concentrate. Thus, in some embodiments, the concentration of yeast extract in the production media is less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, or less than about 0.5 g/L. In some embodiments, the production media is substantially free of yeast extract. In some embodiments, the concentration of multi-component media additives in the production media is less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, or less than about 0.5 g/L. In some embodiments, the production media is substantially free of multi-component media additives. In some embodiments, the production broth contains less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, less than about 0.5 g/L, or less than about 0.2 g/L of multi-component media additives.
Thiamine or a biosynthetic precursor of thiamine can be added to the production media at a concentration of at least about 0.4 mg/L, at least about 1.2 mg/L, at least about 2 mg/L, at least about 5 mg/L, at least about 6 mg/L, at least about 10 mg/L, at least about 50 mg/L, at least about 60 mg/L, at least about 80 mg/L, at least about 100 mg/L, at least about 150 mg/L, at least about 250 mg/L, or at least about at least about 300 mg/L. In one aspect, thiamine or a biosynthetic precursor of thiamine is provided in the production media in an amount greater than about 1 mg/L. In another aspect, thiamine or a biosynthetic precursor of thiamine is provided in the production media based on the cell density. Thiamine or a biosynthetic precursor of thiamine may be provided in an amount greater than about 0.1 g/g of dry cell weight (dcw) in the production media. In some embodiments, the concentration of thiamine is determined in the production broth (which comprises the production media). Accordingly, the concentration of thiamine may be at least about 0.2 mg/L, at least about 1 mg/L, at least about 5 mg/L, at least about 10 mg/L, at least about 50 mg/L, or at least about 100 mg/L in the production broth.
In some embodiments, the amount of thiamine or biosynthetic precursor is greater than about 0.1 g/g of dry cell weight and the cell density is greater than about 7 gdcw/L, about 10 g dcw/L, or about 20 gdcw/L. In some embodiments, the amount of thiamine or biosynthetic precursor is greater than about 1 g/L of dry cell weight and the cell density is less than about 7 g dcw/L, less than about 10 g dcw/L, or less than about 20 g dcw/L.
Cell density can be determined using techniques known in the art. Dry weight of the cell suspension is determined, for example, by centrifugation of 5 mL cell broth in a pre-weighed centrifuge tube, followed by washing with distilled water and drying to constant weight at 80° C. in an oven
In some embodiments, the addition of thiamine and/or other biosynthetic precursor of thiamine improves fermentation product production parameters in aerobic conditions, anaerobic conditions, or both in aerobic and in anaerobic conditions. The addition of thiamine or other biosynthetic precursor of thiamine can increase butanol production as measured by butanol titer or specific butanol production rate. The addition of thiamine or other biosynthetic precursor of thiamine can increase butanol production as measured by butanol concentration or specific butanol production rate in aerobic conditions, anaerobic conditions, or both in aerobic and in anaerobic conditions. The addition of thiamine or other biosynthetic precursor of thiamine can increase butanol production as measured by less accumulation of the isobutanol pathway intermediates like α-ketoisovalerate.
In some embodiments, the host cell cultured in the media supplemented with thiamine or other biosynthetic precursor of thiamine can be a recombinant butanol producing host cell. In some embodiments, the recombinant butanol producing host cell can be an S. cerevisiae host cell. In other embodiments, the recombinant host cell can comprise a butanol biosynthetic pathway as described herein. In other embodiments, the butanol biosynthetic pathway can comprise polynucleotides encoding polypeptides having acetolactate synthase and ketoisovalerate decarboxylase and, optionally, alcohol dehydrogenase activity. In a particular embodiment, the butanol producing strain is S. cerevisiae. 
In some embodiments, thiamine is added to the media prior to or at the beginning of a fermentation. In other embodiments, thiamine is added over the course of a fermentation or during a fermentation. Concentrations of thiamine provided may be achieved, for example, via addition of a bolus of thiamine to production media or via aliquots to production broth.
In some embodiments, nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is added to the production media in addition to the thiamine or other biosynthetic precursor of thiamine. Nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD can be added to the production media at a concentration of at least about 5 mg/L, at least about 6 mg/L, at least about 7 mg/L, at least about 8 mg/L, at least about 9 mg/L, at least about 10 mg/L, at least about 20 mg/L, at least about 50 mg/L, at least about 80 mg/L, at least about 100 mg/L, at least about 150 mg/L, at least about 250 mg/L, at least about 250 mg/L, or at least about at least about 300 mg/L.
Because the methods provided herein are believed to be particularly advantageous for commercial scale fermentations, some embodiments include production media comprising added thiamine or biosynthetic precursors thereof and added nicotinic acid, nicotinamid, or biosynthetic precursor of NAD wherein the production media is substantially free of multi-component media additives. In some embodiments, the production media consists essentially of thiamine or biosynthetic precursors thereof and optionally nicotinic acid, nicotinamid, or biosynthetic precursor of NAD. In other embodiments, the production media consists essentially of thiamine or biosynthetic precursors thereof, and nicotinic acid, nicotinamid, or biosynthetic precursor of NAD. Some embodiments also include media for commercial-scale fermentations such as fermentations of volume greater than about 1000 L, greater than about 10,000 L, or greater than about 50,000 L.
Thi2, Thi3, and Pdc2 Activity
Pdc2 is a transcriptional regulator that coordinates the expression of THI (thiamine biosynthesis) regulon and the TPP-dependent enzymes (PDC1 and PDC5). Pdc2 interacts with Thi2 and Thi3 proteins in controlling the expression of many genes in the THI regulon. Thi2 is a transcription factor with a zinc finger DNA-binding motif. Overexpression of PDC2 protein has been shown to upregulate many genes in the THI regulon in a Thi2-independent manner, for example, THI4, THI5, THI7, THI13, PHO3, and PDC5. In addition, Thi2-dependent upregulation of THI6 and THI73 require Pdc2. Thi3 is an essential regulatory protein of the THI regulon. It binds to the TPP cofactor at high thiamine concentrations and leads to repression of THI genes. At low thiamine concentrations, it binds Thi2 and Pdc2 to activate THI gene transcription. Pdc2 mediates the upregulation of certain THI genes (e.g., THI20, PET18) together with Thi3.
Thus, in order to improve isobutanol production, a host cell such as yeast can be engineered to increase the activity of Thi2, Thi3, and/or Pdc2. The overall Thi2, Thi3, and/or Pdc2 activity in a yeast cell can be increased by increasing the levels of Thi2, Thi3, and/or Pdc2 or by increasing the activity of individual Thi2, Thi3, and/or Pdc2 molecules. Thus, for example, the levels of Thi2, Thi3, and/or Pdc2 can be increased by introducing sequences encoding Thi2, Thi3, and/or Pdc2 into a host cell, which may or may not express Thi2, Thi3, and/or Pdc2 endogenously. The levels of Thi2, Thi3, and/or Pdc2 can also be increased by increasing transcription of endogenous Thi2, Thi3, and/or Pdc2 sequences, for example, by creating mutations in the Thi2, Thi3, and/or Pdc2 promoter sequences that allow for increased transcription of Thi2, Thi3, and/or Pdc2. The activity of individual Thi2, Thi3, and/or Pdc2 molecules can be increased, for example, by introducing mutations into Thi2, Thi3, and/or Pdc2 coding sequences or proteins that result in increased transcriptional activity. For example, it has been demonstrated that a C-terminally truncated Pdc2 protein can transactivate THI11 gene transcription in a Thi3-independent manner (Nosaka, et al., FEBS Letters, 582:3991-3996, 2008). Thus, mutation of an endogenous Pdc2-coding sequence that results in the production of a C-terminally truncated Pdc2 protein increases Pdc2 activity. Similarly, introducing heterologous sequences encoding the truncated Pdc2 protein into a host cell can also increase Pdc2 activity. Therefore, the overall activity of Thi2, Thi3, and/or Pdc2 in a host cell can be increased by the introduction of heterologous nucleic acid and/or protein sequences or by mutation of endogenous nucleic acid and/or protein sequences. The introduction of Thi2, Thi3, and/or Pdc2 activity into a recombinant host cell can have the same, similar, or improved effects on butanol production as supplementation with thiamine or biosynthetic precursors thereof.
Heterologous Aspartate Oxidase/Dehydrogenase Activity
The introduction of heterologous aspartate oxidase and/or aspartate dehydrogenase activity into a recombinant host cell can increase isobutanol production. In some embodiments of the methods described herein, a heterologous polynucleotide encoding an aspartate oxidase and/or a heterologous polynucleotide encoding an aspartate dehydrogenase can be introduced into a cell using recombinant DNA technologies that are well known in the art. In some embodiments, the introduction of a heterologous polynucleotide encoding a polypeptide having aspartate oxidase or aspartate dehydrogenase activity results in an improved isobutanol concentrations and increased specific isobutanol production rates. In other embodiments, the NAD biosynthetic pathway can comprise a polynucleotide encoding a polypeptide that catalyzes the conversion of aspartic acid to iminoaspartic acid.
Examples of aspartate oxidase or aspartate dehydrogenase polynucleotides, genes and polypeptides that can be heterologously expressed in a host cell disclosed herein include, but are not limited to, those of the following Table 3.







TABLE 3





Aspartate Oxidase or Aspartate Dehydrogenase Sequences
















L-aspartate
Amino acid (SEQ ID NO: 97)


oxidase from E.
MNTLPEHSCDVLIIGSGAAGLSLALRLADQHQVIVLSKGPVTEGST


coli
FYAQGGIAAVFDETDSIDSHVEDTLIAGAGICDRHAVEFVASNAR



SCVQWLIDQGVLFDTHIQPNGEESYHLTREGGHSHRRILHAADAT



GREVETTLVSKALNHPNIRVLERSNAVDLIVSDKIGLPGTRRVVG



AWVWNRNKETVETCHAKAVVLATGGASKVYQYTTNPDISSGDG



IAMAWRAGCRVANLEFNQFHPTALYHPQARNFLLTEALRGEGAY



LKRPDGTRFMPDFDERGELAPRDIVARAIDHEMKRLGADCMFLDI



SHKPADFIRQHFPMIYEKLLGLGIDLTQEPVPIVPAAHYTCGGVM



VDDHGRTDVEGLYAIGEVSYTGLHGANRMASNSLLECLVYGWS



AAEDITRRMPYAHDISTLPPWDESRVENPDERVVIQHNWHELRLF



MWDYVGIVRTTKRLERALRRITMLQQEIDEYYAHFRVSNNLLEL



RNLVQVAELIVRCAMMRKESRGLHFTLDYPELLTHSGPSILSPGN



HYINR


 


L-aspartate
Amino acid (SEQ ID NO: 98):


oxidase from B.
MSKKTIAVIGSGAAALSLAAAFPPSYEVTVITKKSVKNSNSVYAQ


subtilis
GGIAAAYAKDDSIEAHLEDTLYAGCGHNNLAIVADVLHDGKMM



VQSLLERGFPFDRNERGGVCLGREGAHSYNRIFHAGGDATGRLLI



DYLLKRINSKIKLIENETAADLLIEDGRCIGVMTKDSKGRLKVRHA



DEVVLAAGGCGNLFLHHTNDLTVTGDGLSLAYRAGAELTDLEFT



QFHPTLLVKNGVSYGLVSEAVRGEGGCLVDENGRRIMAERHPLG



DLAPRDIVSRVIHEEMAKGNRVYIDFSAISDFETRFPTITAICEKAGI



DIHSGKIPVAPGMHFLMGGVSVNRWGETTVPGLYAIGETACSGL



HGANRLASNSLLEALVFGKRAAEHIIQKPVYNRQYQSGLETSVFY



EVPDIEGHELQSKMTSHMSILREQSSLIELSIWLHTLPFQEVNVKDI



TIRQMELSHLWQTAKLMTFSALLREESRGAHFRTDFPHAEVSWQ



GRQIVHTKKGTKIRKNEGIWNNESFTAEKITESLFS


 


Aspartate
Amino acid (SEQ ID NO: 99):


dehydrogenase
MTVLIIGMGNIGKKLVELGNFEKIYAYDRISKDIPGVVRLDEFQVP


from T. maritime
SDVSTVVECASPEAVKEYSLQILKNPVNYIIISTSAFADEVFRERFF



SELKNSPARVFFPSGAIGGLDVLSSIKDFVKNVRIETIKPPKSLGLD



LKGKTVVFEGSVEEASKLFPRNINVASTIGLIVGFEKVKVTIVADP



AMDHNIHIVRISSAIGNYEFKIENIPSPENPKTSMLTVYSILRTLRNL



ESKIIFG









Heterologous Quinolinate Synthase Activity

The introduction of heterologous quinolinate synthase activity into a recombinant host cell can increase alcohol production. In some embodiments of the methods described herein, a heterologous polynucleotide encoding a quinolinate synthase can be introduced into a cell using recombinant DNA technologies that are well known in the art. In some embodiments, the introduction of a heterologous polynucleotide encoding a polypeptide having quinolinate synthase activity results in an improved isobutanol concentrations and increased specific isobutanol production rates. In other embodiments, the NAD biosynthetic pathway can comprise a polynucleotide encoding a polypeptide that catalyzes the conversion of iminoaspartic acid to quinolate.
Examples of quinolinate synthase polynucleotides, genes and polypeptides that can be heterologously expressed in a host cell disclosed herein include, but are not limited to, those of the following Table 4.







TABLE 4





Quinolinate Synthase Sequences
















Quinolinate
Amino acid (SEQ ID NO: 100):


synthase from E.
MSVMFDPDTAIYPFPPKPTPLSIDEKAYYREKIKRLLKERNAVMV


coli
AHYYTDPEIQQLAEETGGCISDSLEMARFGAKHPASTLLVAGVRF



MGETAKILSPEKTILMPTLQAECSLDLGCPVEEFNAFCDAHPDRT



VVVYANTSAAVKARADWVVTSSIAVELIDHLDSLGEKIIWAPDK



HLGRYVQKQTGGDILCWQGACIVHDEFKTQALTRLQEEYPDAAI



LVHPESPQAIVDMADAVGSTSQLIAAAKTLPHQRLIVATDRGIFY



KMQQAVPDKELLEAPTAGEGATCRSCAHCPWMAMNGLQAIAEA



LEQEGSNHEVHVDERLRERALVPLNRMLDFAATLRG


 


Quinolinate
Amino acid (SEQ ID NO: 101):


synthase from B.
MSILDVIKQSNDMMPESYKELSRKDMETRVAAIKKKFGSRLFIPG


subtilis
HHYQKDEVIQFADQTGDSLQLAQVAEKNKEADYIVFCGVHFMA



ETADMLTSEQQTVVLPDMRAGCSMADMADMQQTNRAWKKLQ



HIFGDTIIPLTYVNSTAEIKAFVGKHGGATVTSSNAKKVLEWAFTQ



KKRILFLPDQHLGRNTAYDLGIALEDMAVWDPMKDELVAESGHT



NVKVILWKGHCSVHEKFTTKNIHDMRERDPDIQIIVHPECSHEVV



TLSDDNGSTKYIIDTINQAPAGSKWAIGTEMNLVQRIIHEHPDKQI



ESLNPDMCPCLTMNRIDLPHLLWSLEQIEKGEPSGVIKVPKAIQED



ALLALNRMLSIT


 


Quinolinate
Amino acid (SEQ ID NO: 102):


synthase from T.
MVDEILKLKKEKGYIILAHNYQIPELQDIADFVGDSLQLARKAME


maritime
LSEKKILFLGVDFMAELVKILNPDKKVIVPDRSATCPMANRLTPEII



REYREKFPDAPVVLYVNSTSECKTLADVICTSANAVEVVKKLDSS



VVIFGPDRNLGEYVAEKTGKKVITIPENGHCPVHQFNAESIDAVR



KKYPDAKVIVHPECPKPVRDKADYVGSTGQMEKIPEKDPSRIFVI



GTEIGMIHKLKKKFPDREFVPLEMAVCVNMKKNTLENTLHALQT



ESFEVILPKEVIEKAKKPILRMFELMG









Nicotinic Acid and/or Nicotinamid Supplementation

The addition of nicotinic acid, nicotinamid, or other biosynthetic precursors of nicotine adenine dinucleotide (NAD) to production media where recombinant alcohol-producing cells are cultured can increase alcohol production. In some embodiments, the alcohol is butanol and in other embodiments, the butanol is 1-butanol, 2-butanol, or isobutanol. In some embodiments, the host cell is yeast. In embodiments, the host cell is the yeast S. cerevisiae. The nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD including, but not limited to, nicotinic acid riboside or nicotinamid riboside can be added to the production media in the absence of other multi-component media additives such as yeast extract, corn steep liquor, and sugar cane concentrate. Thus, in some embodiments, the concentration of yeast extract in the production media is less than about 20 g/L, less than about 10 g/L, less than about 5 g/L, less than about 4 g/L, less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, or less than about 0.5 g/L. In some embodiments, the production media is substantially free of yeast extract. In some embodiments, the concentration of multi-component media additives in the production media is less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, or less than about 0.5 g/L. In some embodiments, the production media is substantially free of multi-component media additives. In some embodiments, the production broth contains less than about 3 g/L, less than about 2 g/L, less than about 1 g/L, less than about 0.5 g/L, or less than about 0.2 g/L of multi-component media additives.
In another aspect, nicotinic acid, nicotinamid, nicotinic acid riboside, nicotinamid riboside, or other biosynthetic precursor of NAD is provided in the production media based on the cell density. The nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD may be provided in an amount greater than about 0.5 mg/g of dry cell weight in the production media, greater than about 0.4 mg/g of dry cell weight in the production media, greater than about 0.3 mg/g of dry cell weight in the production media, greater than about 0.2 mg/g of dry cell weight in the production media, or greater than about 0.1 mg/g of dry cell weight in the production media.
In some embodiments, the amount of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is greater than about 0.1 g/g of dry cell weight and the cell density is greater than about 5 gdcw/L, about 7 g dcw/L, about 10 g dcw/L, or about 20 gdcw/L. In some embodiments, the amount of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is greater than about 5 mg/L of dry cell weight and the cell density is less than about 5 g dcw/L, less than about 7 g dcw/L, less than about 10 g dcw/L, or less than about 20 g dcw/L. Cell density may be determined as described above.
The nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD can be added to the production media at a total concentration of at least about 5 mg/L, at least about 6 mg/L, at least about 7 mg/L, at least about 8 mg/L, at least about 9 mg/L, at least about 10 mg/L, at least about 15 mg/L, at least about 20 mg/L, at least about 25 mg/L, at least about 30 mg/L, at least about 40 mg/L, at least about 50 mg/L, at least about 60 mg/L, at least about 80 mg/L, or at least about 100 mg/L, at least about 120 mg/L, at least about 150 mg/L, or at least about 300 mg/L. In some embodiments, the concentration of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is determined in the production broth (which comprises the production media). Accordingly, the concentration of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD may be at least about 0.2 mg/L, at least about 1 mg/L, at least about 5 mg/L, at least about 10 mg/L, at least about 50 mg/L, or at least about 70 mg/L in the production broth.
In some embodiments, the addition of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD improves one or more product production parameters like volumetric rate, specific rate, titer, or yield in aerobic conditions, anaerobic conditions, or both in aerobic and in anaerobic conditions. The addition of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD can increase alcohol production as measured by alcohol titer or specific alcohol production rate. In some embodiments, the addition of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD may increase cell mass. In some embodiments, the host cell cultured in the media supplemented with nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD can be a recombinant butanol producing host cell. In some embodiments, the recombinant butanol producing host cell can be an S. cerevisiae host cell. In other embodiments, the recombinant host cell can comprise a butanol biosynthetic pathway as described further herein. In other embodiments, the butanol biosynthetic pathway can comprise polynucleotides encoding polypeptides having acetolactate synthase and ketoisovalerate decarboxylase, and, optionally, alcohol dehydrogenase activity. In a particular embodiment, the butanol producing strain is S. cerevisiae. 
In some embodiments, the nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is added to the media prior to or at the beginning of a fermentation. In other embodiments, the nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is added over the course of a fermentation or during a fermentation. Concentrations of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD provided may be achieved, for example, via addition of a bolus of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD to media or via aliquots.
In some embodiments, thiamine or a biosynthetic precursor thereof is added to the production media in addition to the nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD. Thiamine or a biosynthetic precursor thereof can be added to the production media at a concentration of at least about 1 mg/L, at least about 2 mg/L, at least about 3 mg/L, at least about 4 mg/L, at least about 5 mg/L, at least about 6 mg/L, at least about 7 mg/L, at least about 8 mg/L, at least about 10 mg/L, at least about 15 mg/L, at least about 20 mg/L, at least about 30 mg/L, at least about 50 mg/L, at least about 70 mg/L, at least about 100 mg/L, or at least about at least about 200 mg/L.
In some embodiments, the production media contacts recombinant microorganisms and a fermentable carbon source in a fermentation vessel. Thus, provided herein is a composition comprising i) production media, the media comprising thiamine and nicotinic acid, ii) recombinant microorganisms comprising a biosynthetic pathway, and iii) liquefied biomass comprising a fermentable carbon source. In some embodiments, the production media consists essentially of thiamine and nicotinic acid. In some embodiments, the production media consists essentially of nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and optionally thiamine or a biosynthetic precursor thereof. In some embodiments, the production media consists essentially of thiamine or biosynthetic precursors thereof and nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD.
Modification of Pyruvate Decarboxylase
Functional deletion of the pyruvate decarboxylase gene has been used to increase the availability of pyruvate for utilization in biosynthetic product pathways. For example, U.S. Patent Application Publication No. 2007/0031950 A1 discloses a yeast strain with a disruption of one or more pyruvate decarboxylase genes and expression of a D-lactate dehydrogenase gene which is used for production of D-lactic acid. U.S. Patent Application Publication No. 2005/0059136 A1 discloses glucose tolerant two carbon source independent (GCSI) yeast strains with no pyruvate decarboxylase activity, which may have an exogenous lactate dehydrogenase gene. Nevoigt and Stahl (Yeast 12:1331-1337, 1996) describe the impact of reduced pyruvate decarboxylase and increased NAD-dependent glycerol-3-phosphate dehydrogenase in Saccharomyces cerevisiae on glycerol yield. U.S. patent application Ser. No. 12/477,942 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.
In some embodiments of the invention, a recombinant host cell disclosed herein can comprise a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase (PDC) activity or a modification in an endogenous polypeptide having PDC activity. In some embodiments, a recombinant host cell disclosed herein can have a modification or disruption of a polynucleotide, gene, and/or polypeptide encoding PDC. In some embodiments, a recombinant host cell comprises a deletion, mutation, and/or substitution in an endogenous polynucleotide or gene encoding a polypeptide having PDC activity, or in an endogenous polypeptides having PDC activity. Such modifications, disruptions, deletions, mutations, and/or substitutions can result in PDC activity that is reduced, substantially eliminated, or eliminated resulting, for example, in a PDC knock-out (PDC-KO) phenotype.
In some embodiments of the invention, an endogenous pyruvate decarboxylase activity of a recombinant host cell disclosed herein converts pyruvate to acetaldehyde which can then be converted to ethanol or to acetyl-CoA via acetate. In other embodiments, a recombinant host cell is Kluyveromyces lactis containing one gene encoding pyruvate decarboxylase, Candida glabrata containing one gene encoding pyruvate decarboxylase, or Schizosaccharomyces pombe containing one gene encoding pyruvate decarboxylase.
In other embodiments, a recombinant host cell is Saccharomyces cerevisiae containing three isozymes of pyruvate decarboxylase encoded by the PDC1, PDC5, and PDC6 genes. In a non-limiting example in S. cerevisiae, the PDC1 and PDC5 genes or the PDC1, PDC5, and PDC6 genes, are disrupted. In another non-limiting example in S. cerevisiae, polynucleotides or genes encoding pyruvate decarboxylase proteins such as those having about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to PDC1, PDC5, and/or PDC6 can be disrupted.
In some embodiments, a polypeptide having PDC activity or a polynucleotide or gene encoding a polypeptide having PDC activity corresponds to Enzyme Commission Number EC 4.1.1.1. In other embodiments, a PDC gene of a recombinant host cell disclosed herein is not active under the fermentation conditions used, and therefore such a gene would not need to be modified or inactivated.
Examples of recombinant host cells with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported, such as for Saccharomyces in Flikweert, et al. (Yeast 12:247-257, 1996), for Kluyveromyces in Bianchi, et al. (Mol. Microbiol. 19(1):27-36, 1996), and disruption of the regulatory gene in Hohmann (Mol. Gen. Genet. 241:657-666, 1993). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC with Accession No. 200027 and Accession No. 200028.
Examples of PDC polynucleotides, genes, and/or polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, those of the following Table 5.







TABLE 5







Pyruvate Decarboxylase Target Gene Coding Regions and Proteins










SEQ
SEQ



ID NO:
ID NO:



Nucleic
Amino


Description
acid
acid












PDC1 pyruvate decarboxylase from Saccharomyces
1
2


cerevisiae


PDC5 pyruvate decarboxylase from Saccharomyces
3
4


cerevisiae


PDC6 pyruvate decarboxylase from Saccharomyces
5
6


cerevisiae


pyruvate decarboxylase from Candida glabrata
7
8


PDC1 pyruvate decarboxylase from Pichia stipitis
9
10


PDC2 pyruvate decarboxylase from Pichia stipitis
11
12


pyruvate decarboxylase from Kluyveromyces lactis
13
14


pyruvate decarboxylase from Yarrowia lipolytica
15
16


pyruvate decarboxylase from Schizosaccharomyces
17
18


pombe


pyruvate decarboxylase from Zygosaccharomyces
18
20


rouxii









Other examples of PDC polynucleotides, genes, and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, PDC polynucleotides, genes and/or polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity (or identity) to any one of the sequences of Table 5, wherein such a polynucleotide or gene encodes, or such a polypeptide has, PDC activity. Still other examples of PDC polynucleotides, genes, and polypeptides that can be targeted for modification or inactivation in a recombinant host cell disclosed herein include, but are not limited to, an active variant, fragment, or derivative of any one of the sequences of Table 5, wherein such a polynucleotide or gene encodes, or such a polypeptide has, PDC activity.
In some embodiments, the sequences of other PDC polynucleotides, genes, and/or polypeptides can be identified in the literature and in bioinformatics databases well known to the skilled person using sequences disclosed herein and available in the art. For example, such sequences can be identified through BLAST (as described above) searching of publicly available databases with known PDC encoding polynucleotide or polypeptide sequences. In such a method, identities can be based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
Additionally, the PDC polynucleotide or polypeptide sequences described herein or known the art can be used to identify other PDC homologs in nature. For example, each of the PDC encoding nucleic acid fragments described herein can be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, (1) methods of nucleic acid hybridization; (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis, et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, et al., Proc. Natl. Acad. Sci. USA 82:1074, 1985; or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. USA 89:392, 1992]; and (3) methods of library construction and screening by complementation.
In some embodiments, pyruvate decarboxylase polynucleotides, genes, and/or polypeptides related to a recombinant host cell disclosed herein can be modified or disrupted. Many methods for genetic modification and disruption of target genes to reduce or eliminate expression are known to one of ordinary skill in the art and can be used to create a recombinant host cell disclosed herein. Modifications that can be used include, but are not limited to, deletion of the entire gene or a portion of the gene encoding a pyruvate decarboxylase protein, inserting a DNA fragment into the encoding gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less active protein is expressed. In other embodiments, expression of a target gene can be blocked by expression of an antisense RNA or an interfering RNA, and constructs can be introduced that result in cosuppression. In other embodiments, the synthesis or stability of the transcript can be lessened by mutation. In some embodiments, the efficiency by which a protein is translated from mRNA can be modulated by mutation. All of these methods can be readily practiced by one skilled in the art making use of the known or identified sequences encoding target proteins.
In other embodiments, DNA sequences surrounding a target pyruvate decarboxylase coding sequence are also useful in some modification procedures and are available, for example, for yeast such as Saccharomyces cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. An additional non-limiting example of yeast genomic sequences is that of Candida albicans, which is included in GPID #10771, #10701, and #16373. Other yeast genomic sequences can be readily found by one of skill in the art in publicly available databases.
In other embodiments, DNA sequences surrounding a target pyruvate decarboxylase coding sequence can be useful for modification methods using homologous recombination. In a non-limiting example of this method, pyruvate decarboxylase gene flanking sequences can be placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the pyruvate decarboxylase gene. In another non-limiting example, partial pyruvate decarboxylase gene sequences and pyruvate decarboxylase gene flanking sequences bounding a selectable marker gene can be used to mediate homologous recombination whereby the marker gene replaces a portion of the target pyruvate decarboxylase gene. In some embodiments, the selectable marker can be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the pyruvate decarboxylase gene without reactivating the latter. In some embodiments, the site-specific recombination leaves behind a recombination site which disrupts expression of the pyruvate decarboxylase protein. In other embodiments, the homologous recombination vector can be constructed to also leave a deletion in the pyruvate decarboxylase gene following excision of the selectable marker, as is well known to one skilled in the art.
In other embodiments, deletions can be made to a pyruvate decarboxylase target gene using mitotic recombination as described by Wach, et al. (Yeast 10:1793-1808, 1994). Such a method can involve preparing a DNA fragment that contains a selectable marker between genomic regions that can be as short as 20 bp, and which bound a target DNA sequence. In other embodiments, this DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. In some embodiments, the linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence (as disclosed, for example, in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.).
Moreover, promoter replacement methods can be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described by Mnaimneh, et al. (Cell 118(1):31-44, 2004).
In other embodiments, the pyruvate decarboxylase target gene encoded activity can be disrupted using random mutagenesis, which can then be followed by screening to identify strains with reduced or substantially eliminated activity. In this type of method, the DNA sequence of the target gene encoding region need not be known.
Methods for creating genetic mutations are common and well known in the art and can be applied to the exercise of creating mutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.
Chemical mutagenesis of host cells can involve, but is not limited to, treatment with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). Such methods of mutagenesis have been reviewed in Spencer, et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In some embodiments, chemical mutagenesis with EMS can be performed as disclosed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer, et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In some embodiments, the introduction of a mutator phenotype can also be used to generate random chromosomal mutations in host cells. In some embodiments, common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAGI, RAD18, or RAD51. In other embodiments, restoration of the non-mutator phenotype can be obtained by insertion of the wildtype allele. In other embodiments, collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced or eliminated pyruvate decarboxylase activity.
Genomes have been completely sequenced and annotated and are publicly available for the following yeast strains: Ashbya gossypii ATCC 10895, Candida glabrata CBS138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c, Schizosaccharomyces pombe 972h-, and Yarrowia lipolytica CLIB122. Typically BLAST (described above) searching of publicly available databases with known pyruvate decarboxylase polynucleotide or polypeptide sequences, such as those provided herein, is used to identify pyruvate decarboxylase-encoding sequences of other host cells, such as yeast cells.
Accordingly, it is within the scope of the invention to provide pyruvate decarboxylase polynucleotides, genes, and polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of the pyruvate decarboxylase polynucleotides or polypeptides disclosed herein (e.g., SEQ ID NOs: 1-20 of Table 5). Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.
The modification of PDC in the host cells disclosed herein to reduce or eliminate PDC activity can be confirmed using methods known in the art. For example, PCR methods well known in the art can be used to confirm deletion of PDC. Other suitable methods will be known to those of skill in the art and include, but are not limited to, lack of growth on yeast extract peptone-dextrose medium (YPD).
Isobutanol and Other Products
In some embodiments of the invention, methods for the production of a product of a biosynthetic pathway are provided which comprise (a) providing a recombinant host cell disclosed herein; and (b) providing media conditions whereby the product of the biosynthetic pathway is produced. In some embodiments, the biosynthetic pathway of the product is derived from pyruvic acid. In other embodiments, the product of the biosynthetic pathway is lactic acid, malic acid, succinic acid, fumaric acid or alanine. In other embodiments, the product is produced as a co-product along with ethanol. In still other embodiments, the product of the biosynthetic pathway is butanol. In still other embodiments, the product of the biosynthetic pathway is isobutanol. In other embodiments, the product is produced at a yield of at least about 50% of that observed when the same recombinant host cell is grown in the presence of yeast extract.
The product of the biosynthetic pathway can be produced at a greater yield or amount compared to the production of the same product in a recombinant host cell that is not provided with media supplemented with thiamine or biosynthetic precursors thereof and/or nicotinic acid, nicotinamid, or biosynthetic precursor of NAD. In some embodiments, this greater yield includes production at a yield of greater than about 10% of theoretical, at a yield of greater than about 20% of theoretical, at a yield of greater than about 25% of theoretical, at a yield of greater than about 30% of theoretical, at a yield of greater than about 40% of theoretical, at a yield of greater than about 50% of theoretical, at a yield of greater than about 60% of theoretical, at a yield of greater than about 70% of theoretical, at a yield of greater than about 75% of theoretical, at a yield of greater than about 80% of theoretical at a yield of greater than about 85% of theoretical, at a yield of greater than about 90% of theoretical, at a yield of greater than about 95% of theoretical, at a yield of greater than about 96% of theoretical, at a yield of greater than about 97% of theoretical, at a yield of greater than about 98% of theoretical, at a yield of greater than about 99% of theoretical, or at a yield of about 100% of theoretical. In other embodiments, the product is produced as a co-product along with ethanol. In still other embodiments, the product of the biosynthetic pathway is isobutanol.
The specific production rate can be at least about 0.10 g/g/h, at least about 0.11 g/g/h, at least about 0.12 g/g/h, at least about 0.13 g/g/h, at least about 0.14 g/g/h, at least about 0.15 g/g/h, at least about 0.16 g/g/h, at least about 0.17 g/g/h, at least about 0.18 g/g/h, at least about 0.19 g/g/h, or at least about 0.20 g/g/h. The specific production rate can also be about 0.05 g/g/h to about 0.50 g/g/h, about 0.05 g/g/h to about 0.40 g/g/h, about 0.05 g/g/h to about 0.30 g/g/h, or about 0.05 to about 0.20 g/g/h. The specific production rate can also be about 0.10 g/g/h to about 0.50 g/g/h, about 0.10 g/g/h to about 0.10 g/g/h, about 0.10 g/g/h to about 0.30 g/g/h, or about 0.10 to about 0.20 g/g/h. The specific production rate can also be about 0.15 g/g/h to about 0.15 g/g/h, about 0.15 g/g/h to about 0.40 g/g/h, about 0.15 g/g/h to about 0.30 g/g/h, or about 0.15 to about 0.20 g/g/h.
The titer (or butanol produced) can be at least about 8 g/L, at least about 10 g/L, at least about 15 g/L, at least about 30 g/L, at least about 35 g/L, at least about 50 g/L, at least about 60 g/L, at least about 70 g/L, at least about 80 g/L, at least about 90 g/L, or at least about 95 g/L. The titer can also be about 8 g/L to about 15 g/L, about 10 g/L to about 30 g/L, about 15 g/L to about 35 g/L, about 15 g/L to about 40 g/L, about 15 g/L to about 50 g/L, about 15 g/L to about 60 g/L, about 15 g/L to about 70 g/L, about 15 g/L to about 80 g/L, about 15 g/L to about 90 g/L, about 15 g/L to about 95 g/L, or about 8 g/L to about 95 g/L.
In some embodiments, the specific rate is at least about 50% of the rate observed when media containing about 20 g/L yeast extract and no additional thiamine or thiamine precursors is used. The rate can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the rate observed when media containing about 20 g/L yeast extract and no additional thiamine or thiamine precursors is used.
In some embodiments, the titer is at least about 50% of the titer observed when media containing about 20 g/L yeast extract and no additional thiamine or thiamine precursors is used. The titer can be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the titer observed when media containing about 20 g/L yeast extract and no additional thiamine or thiamine precursors is used.
In some embodiments, the volumetric rate is increased by at least about 50% as compared to the rate observed when production media containing no thiamine or thiamine precursors is used. The rate can be increased by at least about 20%, at least about 30%, at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least about 90% as compared to the rate observed when production media containing no thiamine or thiamine precursors and no multi-component media additives are employed. In some embodiments, the rate can be increased by at least about 200% or at least about 300% as compared to the rate observed when production media containing no thiamine or thiamine precursors and no multi-component media additives are employed.
In some embodiments, the titer is increased by at least about 10% as compared to the titer observed when media containing no thiamine or thiamine precursors and no multi-component media additives are employed. The titer can be increased by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least about 90% as compared to the titer observed when media containing no thiamine or thiamine precursors and no multi-component media additives are employed.
In some embodiments, the specific rate is at least about 50% of the rate observed when media containing about 20 g/L yeast extract and no additional nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is employed. The rate can be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the rate observed when media containing about 20 g/L yeast extract and no additional nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is employed.
In some embodiments, the titer is at least about 50% of the titer observed when media containing about 20 g/L yeast extract and no additional nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD. The titer can be at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the titer observed when media containing about 20 g/L yeast extract and no additional nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is employed.
In some embodiments, the volumetric rate is increased by at least about 50% as compared to the rate observed when media containing no nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD is used. The rate can be increased by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to the rate observed when media containing no nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and no yeast extract is used. In some embodiments, the rate can be increased by at least about 200% or at least about 300% as compared to the rate observed when media containing no nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and no yeast extract is used.
In some embodiments, the titer is increased by at least about 10% as compared to the titer observed when media containing no nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and no yeast extract is used. The titer can be increased by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least about 90% as compared to the titer observed when media containing no nicotinic acid, nicotinamid, or other biosynthetic precursor of NAD and no yeast extract is used.
Butanol Biosynthetic Pathways
Suitable biosynthetic pathways for production of butanol are known in the art, and certain suitable pathways are described herein. In some embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises more than one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises heterologous genes encoding polypeptides corresponding to every step of a biosynthetic pathway.
Likewise, certain suitable proteins having the ability to catalyze indicated substrate to product conversions are described herein and other suitable proteins are provided in the art. For example, U.S. Patent Application Publication No. 2008/0261230 and U.S. Patent Application Publication No. 2009/0163376, incorporated herein by reference, describe acetohydroxy acid isomeroreductases; U.S. patent application Ser. No. 12/569,636, incorporated by reference, describes dihydroxyacid dehydratases; and an alcohol dehydrogenase is described in U.S. Patent Application Publication No. 2009/0269823, incorporated herein by reference.
1-Butanol Biosynthetic Pathway
A biosynthetic pathway for the production of 1-butanol that may be used is described in U.S. Patent Application Publication No. 2008/0182308A1, incorporated herein by reference. This biosynthetic pathway comprises the following substrate to product conversions:

    
    
        a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl-CoA acetyltransferase;
        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 1-butanol dehydrogenase.
    
    


In some embodiments, the 1-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, at least four genes, or at least five genes that is/are heterologous to the yeast cell.
2-Butanol Biosynthetic Pathway
Biosynthetic pathways for the production of 2-butanol that may be used are described in U.S. Patent Application Publication No. 2007/0259410A1 and U.S. Patent Application Publication No. 2007/0292927A1, and in PCT Publication No. WO 2007/130521, all of which are incorporated herein by reference. One 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 butanediol dehydratase; and
        e) 2-butanone to 2-butanol, which may be catalyzed, for example, by 2-butanol dehydrogenase.
    
    


In some embodiments, the 2-butanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that is/are heterologous to the yeast cell.
Isobutanol Biosynthetic Pathway
Biosynthetic pathways for the production of isobutanol that may be used are described in U.S. Patent Application Publication No. 2007/0092957 A1 and PCT Publication No. WO 2007/050671, incorporated herein by reference. One 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;
        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.
    
    


In some embodiments, the isobutanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that is/are heterologous to the yeast cell.
Butanol Producing Strains
Microbial hosts for butanol production may be selected from bacteria, cyanobacteria, filamentous fungi, and yeast. The selection of a microbial host for butanol production is described in the art.
As mentioned above, microorganisms may be genetically modified to convert fermentable carbon sources into butanol, specifically 1-butanol, 2-butanol, or isobutanol, using methods known in the art. Suitable strains have been described in the art. Construction of example suitable yeast strains (“NYLA84” and “NYLA93”) is provided herein.
Construction of NYLA84 Strain
Construction of expression vectors for isobutanol pathway gene expression in S. cerevisiae 
Plasmid pLH532 Construction
The pLH532 plasmid (SEQ ID NO: 21) was constructed for expression of acetolactate synthase (ALS) and ketol-acid reductoisomerase (KARI) in yeast. pLH532 is a pHR81 vector (ATCC No. 87541) containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO: 24), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO: 95; protein SEQ ID NO: 96), and CYC1 terminator2 (SEQ ID NO: 25); 2) an ILV5 promoter (SEQ ID NO: 26), Pf5.IlvC coding region (SEQ ID NO: 23), and ILV5 terminator (SEQ ID NO: 27); and 3) the FBA1 promoter (SEQ ID NO: 28), S. cerevisiae KARI coding region (ILV5; SEQ ID NO: 22), and CYC1 terminator.
The Pf5.IlvC coding region is a sequence encoding KARI derived from Pseudomonas fluorescens that was described in U.S. Patent Application Publication No. 2009/0163376, which is herein incorporated by reference.
The Pf5.IlvC coding region was synthesized by DNA 2.0 (Palo Alto, Calif.) based on codons that were optimized for expression in Saccharomyces cerevisiae. 
Expression Vector pLH468
The pLH468 plasmid (SEQ ID NO: 33) was constructed for expression of dihydroxyacid dehydratase (DHAD), KivD, and HADH in yeast. Coding regions for Lactococcus lactis ketoisovalerate decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0 based on codons that were optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 30 and 31, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs: 29 and 32, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426::PTDH3-kivDy-TDH3t), vector pNY8 (SEQ ID NO: 36; also named pRS426.GPD-ald-GPDt, described in U.S. Patent Application Publication No. 2008/0182308, Example 17, which is herein incorporated by reference) was digested with AscI and SfiI enzymes, thus excising the GPD promoter and the ald coding region. A TDH3 promoter fragment (SEQ ID NO: 37) from pNY8 was PCR amplified to add an AscI site at the 5′ end and an SpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067 (SEQ ID NOs: 38 and 39). The AscI/SfiI digested pNY8 vector fragment was ligated with the TDH3 promoter PCR product digested with AscI and SpeI, and the SpeI-SfiI fragment containing the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. The triple ligation generated vector pLH467 (pRS426::PTDH3-kivDy-TDH3t). pLH467 was verified by restriction mapping and sequencing.
pLH435 (pRS425::PGPM1-Hadhy-ADH1t) was derived from vector pRS425::GPM-sadB (SEQ ID NO: 40) which is described in U.S. Provisional Application Ser. No. 61/058,970, Example 3, which is herein incorporated by reference. pRS425::GPM-sadB is the pRS425 vector (ATCC No. 77106) with a chimeric gene containing the GPM1 promoter (SEQ ID NO: 41), coding region from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB; DNA SEQ ID NO: 34; protein SEQ ID NO: 35; disclosed in U.S. Provisional Application Ser. No. 61/048,291), and ADH1 terminator (SEQ ID NO: 42). pRS425::GPMp-sadB contains BbvI and Pad sites at the 5′ and 3′ ends of the sadB coding region, respectively. A NheI site was added at the 5′ end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NO: 45 and 46) to generate vector pRS425-GPMp-sadB-NheI, which was verified by sequencing. pRS425::PGPM1-sadB-NheI was digested with NheI and Pad to drop out the sadB coding region, and ligated with the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadhy-DNA2.0 to create pLH435.
To combine KivD and HADH expression cassettes in a single vector, yeast vector pRS411 (ATCC No. 87474) was digested with Sad and NotI, and ligated with the Sad-SalI fragment from pLH467 that contains the PTDH3-kivDy-TDH3t cassette together with the SalI-NotI fragment from pLH435 that contains the PGPM1-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the vector pRS411::PTDH3-kivDy-PGPM1-Hadhy (pLH441), which was verified by restriction mapping.
In order to generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy and Hadhy, pRS423 FBA ilvD(Strep) (SEQ ID NO: 47) was used, which is described in commonly owned and co-pending U.S. Provisional Application Ser. No. 61/100,792, as the source of the IlvD gene. This shuttle vector contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector has an FBA1 promoter (nt 2111 to 3108; SEQ ID NO: 49) and FBA terminator (nt 4861 to 5860; SEQ ID NO: 48). In addition, it carries the His marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO: 43; protein SEQ ID NO: 44) from Streptococcus mutans UA159 (ATCC No. 700610) is between the FBA promoter and FBA terminator forming a chimeric gene for expression. In addition there is a lumio tag fused to the ilvD coding region (nt 4829-4849).
The first step was to linearize pRS423 FBA ilvD(Strep) (also called pRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio) with SacI and SacII (with SacII site blunt ended using T4 DNA polymerase), to give a vector with total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI site blunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment. This fragment was ligated with the 9,482 bp vector fragment from pRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio. This generated vector pLH468 (pRS423::PFBA1-ilvD(Strep)Lumio-FBA it-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.
Pyruvate Decarboxylase and Hexokinase Gene Inactivation
The following describes insertion-inactivation of endogenous PDC1, PDC5, and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase.
Construction of pdc6:: PGPM1-sadB Integration Cassette and PDC6 Deletion
A pdc6::PGPM1-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO: 50) from pRS425::GPM-sadB (described above) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO: 51) contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton, et al., Gene 77:61-68, 1989) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.; Catalog No. F-5405) and primers 114117-11A through 114117-11D (SEQ ID NOs: 52, 53, 54, and 55), and 114117-13A and 114117-13B (SEQ ID NOs: 56 and 57).
The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′ ̃50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC No. 200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 58 and 59) and 112590-34F and 112590-49E (SEQ ID NOs: 60 and 61) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-fluoro-orotic acid (5-FOA) at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::PGPM1-sadB-ADH1t.
Construction of pdc1:: PPDC1-ilvD Integration Cassette and PDC1 Deletion
A pdc1:: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO:62) from pLH468 (described above) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton, et al., supra) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.; Catalog No. F-5405) and primers 114117-27A through 114117-27D (SEQ ID NOs: 63, 64, 65, and 66).
The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′ ̃50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 67 and 68), and primers 112590-49E and 112590-30F (SEQ ID NOs: 61 and 69) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t.
HIS3 Deletion
To delete the endogenous HIS3 coding region, a his3:: URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 70). URA3r2 contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers 114117-45A and 114117-45B (SEQ ID NOs: 71 and 72) which generated a  ̃2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth. The resulting identified strain, called NYLA73, has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3.
Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion
A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs:73 and 74) which generated a  ̃2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 75 and 76). The identified correct transformants have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FRA1t Δhis3 pdc5::kanMX4. The strain was named NYLA74.
Deletion of HXK2 (hexokinase II)
A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (described above) using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers 384 and 385 (SEQ ID NOs: 77 and 78) which generated a  ̃2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5′ region upstream of the HXK2 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HXK2 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region using primers N869 and N871 (SEQ ID NOs: 79 and 80). The URA3r2 marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth, and by PCR to verify correct marker removal using primers N946 and N947 (SEQ ID NOs: 81 and 82). The resulting identified strain named NYLA83 has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.
Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion
A pdc5::kanMX4 cassette was PCR-amplified as described above. The PCR fragment was transformed into NYLA83, and transformants were selected and screened as described above. The identified correct transformants named NYLA84 have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4.
Plasmid vectors pLH468 and pLH532 were simultaneously transformed into strain NYLA84 (BY4700 pdc6::PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the resulting strain (“isobutanol producing NYLA 83 strain”) was maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol at 30° C.
Construction of NYLA93 (PNY2012)
Described below is insertion-inactivation of endogenous PDC1, PDC5, and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase. The resulting PDC inactivation strain was used as a host for expression vectors pYZ067 (SEQ ID NO: 86) and pYZ090 (SEQ ID NO: 85), the construction of which is described in U.S. Provisional Application No. 61/246,844, filed Sep. 29, 2009, herein incorporated by reference.
pYZ090 was constructed to contain a chimeric gene having the coding region of the alsS gene from Bacillus subtilis (nt position 457-2172) expressed from the yeast CUP1 promoter (nt 2-449) and followed by the CYC1 terminator (nt 2181-2430) for expression of ALS, and a chimeric gene having the coding region of the ilvC gene from Lactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4670-5292) for expression of KARI.
pYZ067 was constructed to contain the following chimeric genes: 1) the coding region of the ilvD gene from S. mutans UA159 (nt position 2260-3971) expressed from the yeast FBA1 promoter (nt 1161-2250) followed by the FBA terminator (nt 4005-4317) for expression of dihydroxy acid dehydratase (DHAD); 2) the coding region for horse liver ADH (nt 4680-5807) expressed from the yeast GPM promoter (nt 5819-6575) followed by the ADH1 terminator (nt 4356-4671) for expression of alcohol dehydrogenase; and 3) the coding region of the KivD gene from Lacrococcus lactis (nt 7175-8821) expressed from the yeast TDH3 promoter (nt 8830-9493) followed by the TDH3 terminator (nt 5682-7161) for expression of ketoisovalerate decarboxylase.
Construction of pdc6:: PGPM1-sadB Integration Cassette and PDC6 Deletion
A pdc6::PGPM1-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO: 50) from pRS425::GPM-sadB (SEQ ID NO: 40, described above) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO: 51) contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton, et al., supra) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass., Catalog No. F-5405) and primers 114117-11A through 114117-11D (SEQ ID NOs: 52-55), and 114117-13A and 114117-13B (SEQ ID NOs: 56 and 57).
The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′ ̃50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC No. 200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 58 and 59), and 112590-34F and 112590-49E (SEQ ID NOs: 60 and 61) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media 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-media to verify the absence of growth. The resulting identified strain has the genotype: BY4700pdc6::PGPM1-sadB-ADH1t.
Construction of pdc1:: PPDC1-ilvD Integration Cassette and PDC1 Deletion
A pdc1:: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO: 62) from pLH468 (described above) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton, et al., supra) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass., Catalog No. F-5405) and primers 114117-27A through 114117-27D (SEQ ID NOs: 63-66).
The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′ ̃50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 67 and 68), and primers 112590-49E and 112590-30F (SEQ ID NOs: 61 and 69) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t.
HIS3 Deletion
To delete the endogenous HIS3 coding region, a his3:: URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 70). URA3r2 contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers 114117-45A and 114117-45B (SEQ ID NOs: 71 and 72) which generated a  ̃2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth. The resulting identified strain, called NYLA73, has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3.
Deletion of Hexokinase 2:
A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (described above) using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers 384 and 385 (SEQ ID NOs: 77 and 78) which generated a  ̃2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5′ region upstream of the HXK2 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HXK2 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region using primers N869 and N871 (SEQ ID NO: 79 and 80). The URA3r2 marker was recycled by plating on synthetic complete media 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 media to verify the absence of growth, and by PCR to verify correct marker removal using primers N946 and N947 (SEQ ID NO: 81 and 82). The resulting identified strain named NYLA83 has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.
Deletion of NAD-Dependent Glycerol 3-Phosphate Dehydrogenase
A gpd2::loxP-URA3-loxP cassette was PCR-amplified from pUC19::loxP-URA3-loxP plasmid template using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers LA512 and LA513 (SEQ ID NOs: 87 and 88) which generated a  ̃1.6 kb PCR product. pUC19::loxP-URA3-loxP (SEQ ID NO: 83) contains the URA3 marker from (ATCC No. 77107) flanked by loxP recombinase sites. The GPD2 portion of each primer was derived from the 5′ region upstream of the GPD2 promoter and 3′ region downstream of the coding region such that integration of the loxP-URA3-loxP marker results in replacement of the GPD2 coding region. The PCR product was transformed into NYLA83 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the GPD2 locus with replacement of the HXK2 coding region using primers LA516 and N175 (SEQ ID NO: 89 and 76). The URA3 marker is recycled by transformation with pRS423::PGAL1-cre (SEQ ID NO: 84) and plating on synthetic complete media lacking histidine supplemented with 2% glucose at 30° C. Colonies are patched onto YP (1% galactose) plates at 30° C. to induce URA3 marker excision and are transferred onto YPD plates at 30° C. for recovery. Removal of the URA3 marker is confirmed by patching colonies from the YPD plates onto synthetic complete media lacking uracil to verify the absence of growth. The identified correct clones have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 Δgpd2::loxP. The strain was named NYLA92.
Construction of pdc5::loxP-kanMX-loxP Integration Cassette and PDC5 Deletion:
A pdc5::loxP-kanMX-loxP cassette was PCR-amplified from plasmid pUC19::loxP-kanMX-loxP (SEQ ID NO: 94) using Phusion® DNA polymerase (New England Biolabs Inc., Ipswich, Mass.) and primers LA249 and LA397 (SEQ ID NOs: 90 and 91) which generated a  ̃2.2 kb PCR product. pUC19::loxP-kanMX-loxP (SEQ ID NO: 94) contains the kanMX gene from pFA6 (Wach, et al., Yeast 10, 1793-1808, 1994) and K. lactis TEF1 promoter and terminator flanked by loxP recombinase sites. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the loxP-kanMX-loxP marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA92 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC5 locus with replacement of the PDC5 coding region using primers LA363 and LA364 (SEQ ID NOs: 92 and 93). The identified correct transformants have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 Δgpd2::loxP Δpdc5:loxP-kanMX-loxP. The strain was named NYLA93.
Plasmid vectors pYZ067 and pYZ090 were simultaneously transformed into strain NYLA93 (BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2 Δgpd2::loxP Δpdc5:loxP-kanMX-loxP) using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the resulting strain (“isobutanol producing NYLA93 strain”) was maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol at 30° C.
Construction of Saccharomyces cerevisiae Strain BP1083 (“NGCI-070”; PNY1504)
The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, PDC6, and GPD2. BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 85, described in U.S. Provisional Application Ser. No. 61/246,844) and pLH468 (SEQ ID NO: 33) to create strain NGCI-070 (BP1083, PNY1504).
Deletions, which completely removed the entire coding sequence, were created by homologous recombination with PCR fragments containing regions of homology upstream and downstream of the target gene and either a G418 resistance marker or URA3 gene for selection of transformants. The G418 resistance marker, flanked by loxP sites, was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion or if flanked by loxP sites, was removed using Cre recombinase.
The scarless deletion procedure was adapted from Akada, et al., (Yeast 23:399-405, 2006). In general, the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene). Fragments A and C, each 500 bp long, corresponded to the 500 bp immediately upstream of the target gene (Fragment A) and the 3′ 500 bp of the target gene (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target gene and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome. Using the PCR product ABUC cassette, the URA3 marker was first integrated into and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in the PCR cassette between fragments A and B.
URA3 Deletion
To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxP cassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 103). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP sites to allow recombination with Cre recombinase and removal of the marker. PCR was done using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers BK505 and BK506 (SEQ ID NOs: 104 and 105). The URA3 portion of each primer was derived from the 5′ region upstream of the URA3 promoter and 3′ region downstream of the coding region such that integration of the loxP-kanMX-loxP marker resulted in replacement of the URA3 coding region. The PCR product was transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YPD containing G418 (100 μg/mL) at 30° C. Transformants were screened to verify correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs: 106 and 107) and designated CEN.PK 113-7D Δura3::kanMX.
HIS3 Deletion
The four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact, kit (Qiagen, Valencia, Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 112) and primer oBP453 (SEQ ID NO: 113) 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: 114) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 115) 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: 116) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 117) 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: 118) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 119). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 112) and oBP455 (SEQ ID NO: 115). 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: 116) and oBP459 (SEQ ID NO: 119). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 112) and oBP459 (SEQ ID NO: 119). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a his3 knockout were screened for by PCR with primers oBP460 (SEQ ID NO: 120) and oBP461 (SEQ ID NO: 121) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correct transformant was selected as strain CEN.PK 113-7D Δura3::kanMX Δhis3::URA3.
KanMX Marker Removal from the Δura3 Site and URA3 Marker Removal from the Δhis3 Site
The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMX Δhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 164, described in U.S. Provisional Application No. 61/290,639) using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, Calif.) and plating on synthetic complete medium lacking histidine and uracil supplemented with 2% glucose at 30° C. Transformants were grown in YP supplemented with 1% galactose at 30° C. for  ̃6 hours to induce the Cre recombinase and KanMX marker excision and plated onto YPD (2% glucose) plates at 30° C. for recovery. An isolate was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (5-FOA, 0.1%) at 30° C. to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in and plated on YPD for removal of the pRS423::PGAL1-cre plasmid. Isolates were checked for loss of the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by assaying growth on YPD+G418 plates, synthetic complete medium lacking uracil plates, and synthetic complete medium lacking histidine plates. A correct isolate that was sensitive to G418 and auxotrophic for uracil and histidine was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletions and marker removal were confirmed by PCR and sequencing with primers oBP450 (SEQ ID NO: 122) and oBP451 (SEQ ID NO: 123) for Δura3 and primers oBP460 (SEQ ID NO: 120) and oBP461 (SEQ ID NO: 121) for Δhis3 using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.).
PDC6 Deletion
The four fragments for the PCR cassette for the scarless PDC6 deletion were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO: 124) and primer oBP441 (SEQ ID NO: 125) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B. PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 126), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment A, and primer oBP443 (SEQ ID NO: 127) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primer oBP444 (SEQ ID NO: 128) containing a 5′ tail with homology to the 3′ end of PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 129) containing a 5′ tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C was amplified with primer oBP446 (SEQ ID NO: 130) containing a 5′ tail with homology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO: 131). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). PDC6 Fragment AB was created by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment B and amplifying with primers oBP440 (SEQ ID NO: 124) and oBP443 (SEQ ID NO: 127). PDC6 Fragment UC was created by overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C and amplifying with primers oBP444 (SEQ ID NO: 128) and oBP447 (SEQ ID NO: 131). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC6 ABUC cassette was created by overlapping PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying with primers oBP440 (SEQ ID NO: 124) and oBP447 (SEQ ID NO: 131). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made and transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc6 knockout were screened for by PCR with primers oBP448 (SEQ ID NO: 132) and oBP449 (SEQ ID NO: 133) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 132) and oBP449 (SEQ ID NO: 133) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC6 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 134) and oBP555 (SEQ ID NO: 135). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designated as BP891.
PDC1 Deletion ilvDSm Integration
The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC No. 700610. The A fragment followed by the ilvD coding region from Streptococcus mutans for the PCR cassette for the PDC1 deletion-ilvDSm integration was amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and NYLA83 (described herein and in U.S. Provisional Application No. 61/246,709) genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment A-ilvDSm (SEQ ID NO: 167) was amplified with primer oBP513 (SEQ ID NO: 136) and primer oBP515 (SEQ ID NO: 137) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment B. The B, U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSm integration were amplified using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment B was amplified with primer oBP516 (SEQ ID NO: 138) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 139) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO: 140) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B, and primer oBP519 (SEQ ID NO: 141) containing a 5′ tail with homology to the 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primer oBP520 (SEQ ID NO: 142), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 143). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif. PDC1 Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513 (SEQ ID NO: 136) and oBP517 (SEQ ID NO: 139). PDC1 Fragment UC was created by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and amplifying with primers oBP518 (SEQ ID NO: 140) and oBP521 (SEQ ID NO: 143). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC1 A-ilvDSm-BUC cassette was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying with primers oBP513 (SEQ ID NO: 136) and oBP521 (SEQ ID NO: 143). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30° C. Transformants with a pdc1 knockout ilvDSm integration were screened for by PCR with primers oBP511 (SEQ ID NO: 144) and oBP512 (SEQ ID NO: 145) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC1 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 146) and oBP551 (SEQ ID NO: 147). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC1, integration of ilvDSm, and marker removal were confirmed by PCR and sequencing with primers oBP511 (SEQ ID NO: 144) and oBP512 (SEQ ID NO: 145) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm and designated as BP907.
PDC5 Deletion sadB Integration
The PDC5 gene was deleted and replaced with the sadB coding region from Achromobacter xylosoxidans. A segment of the PCR cassette for the PDC5 deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.
pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 gene from Saccaromyces cerevisiae situated within a multiple cloning site (MCS). pUC19 contains the pMB1 replicon and a gene coding for beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the sequences from upstream and downstream of this gene were included for expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.
The DNA encompassing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region from Saccaromyces cerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438 (SEQ ID NO: 110) containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 111) containing XbaI, Pad, and NotI restriction sites, using Phusion® High Fidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.). Genomic DNA was prepared using a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The PCR product and pUC19 were ligated with T4 DNA ligase after digestion with BamHI and XbaI to create vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO: 108) and oBP265 (SEQ ID NO: 109).
The coding sequence of sadB and PDC5 Fragment B were cloned into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. The coding sequence of sadB was amplified using pLH468-sadB (SEQ ID NO: 165) as template with primer oBP530 (SEQ ID NO: 148) containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 149) containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B. PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 150) containing a 5′ tail with homology to the 3′ end of sadB, and primer oBP533 (SEQ ID NO: 151) containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). sadB-PDC5 Fragment B was created by overlapping PCR by mixing the sadB and PDC5 Fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 148) and oBP533 (SEQ ID NO: 151). The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. The resulting plasmid was used as a template for amplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 152) and oBP546 (SEQ ID NO: 153) containing a 5′ tail with homology to the 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547 (SEQ ID NO: 154) containing a 5′ tail with homology to the 3′ end of PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 155). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). PDC5 sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C and amplifying with primers oBP536 (SEQ ID NO: 152) and oBP539 (SEQ ID NO: 155). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC5 A-sadB-BUC cassette was created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ ID NO: 156) containing a 5′ tail with homology to the 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 155). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).
Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR cassette using a Frozen-EZ Yeast Transformation IITM kit (Zymo Research Corporation, Irvine, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose) at 30° C. Transformants with a pdc5 knockout sadB integration were screened for by PCR with primers oBP540 (SEQ ID NO: 157) and oBP541 (SEQ ID NO: 158) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC5 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 159) and oBP553 (SEQ ID NO: 160). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3.
CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 was grown overnight in YPE (1% ethanol) and plated on synthetic complete medium supplemented with ethanol (no glucose) and containing 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lost the URA3 marker. The deletion of PDC5, integration of sadB, and marker removal were confirmed by PCR with primers oBP540 (SEQ ID NO: 157) and oBP541 (SEQ ID NO: 158) using genomic DNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB and designated as BP913.
GPD2 Deletion
To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxP cassette was PCR-amplified using loxP-URA3-loxP (SEQ ID NO: 166) as template DNA. loxP-URA3-loxP contains the URA3 marker from (ATCC No. 77107) flanked by loxP recombinase sites. PCR was done using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers LA512 and LA513 (SEQ ID NOs: 87 and 88). The GPD2 portion of each primer was derived from the 5′ region upstream of the GPD2 coding region and 3′ region downstream of the coding region such that integration of the loxP-URA3-loxP marker resulted in replacement of the GPD2 coding region. The PCR product was transformed into BP913 and transformants were selected on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose). Transformants were screened to verify correct integration by PCR using primers oBP582 and AA270 (SEQ ID NOs: 161 and 162).
The URA3 marker was recycled by transformation with pRS423::PGAL1-cre (SEQ ID NO: 164) and plating on synthetic complete media lacking histidine supplemented with 1% ethanol at 30° C. Transformants were streaked on synthetic complete medium supplemented with 1% ethanol and containing 5-fluoro-orotic acid (0.1%) and incubated at 30° C. to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid. The deletion and marker removal were confirmed by PCR with primers oBP582 (SEQ ID NO: 161) and oBP591 (SEQ ID NO: 163). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as PNY1503 (BP1064).
BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 85) and pLH468 (SEQ ID NO: 33) to create strain NGCI-070 (BP1083; PNY1504).
Growth for Production
Recombinant host cells disclosed herein are grown in production broth which contains suitable carbon substrates. Carbon substrates can include, but are not limited to, monosaccharides such as fructose; oligosaccharides such as lactose, maltose, galactose, or sucrose; polysaccharides such as starch or cellulose; or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates can include ethanol, lactate, succinate, or glycerol.
Additionally the carbon substrate can also be one-carbon substrates such as carbon dioxide or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine, and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion, et al., Microb. Growth C1-Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Sulter, et al., Arch. Microbiol. 153:485-489, 1990). Hence it is contemplated that the source of carbon utilized in the present invention can encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, in some embodiments, the carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose can be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose can be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. 2007/031918 A1, which is herein incorporated by reference.
Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass can also comprise additional components, such as protein and/or lipid. Biomass can be derived from a single source or biomass can comprise a mixture derived from more than one source; for example, biomass can comprise a mixture of corn cobs and corn stover or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, agave, and mixtures thereof.
Culture Conditions
Typically cells are grown at a temperature in the range of about 20° C. to about 40° C. in an appropriate medium. Suitable pH ranges for the fermentation are between about pH 5.0 to about pH 9.0. In one embodiment, the pH is about pH 6.0 to about pH 8.0. Suitable pH ranges for the fermentation of yeast are typically between about pH 3.0 to about pH 9.0. In one embodiment, the pH is about pH 5.0 to about pH 8.0. Suitable pH ranges for the fermentation of other microorganisms are between about pH 3.0 to about pH 7.5. In one embodiment, the pH is about pH 4.5 to about pH 6.5.
Fermentations can be performed under aerobic or anaerobic conditions. In one embodiment, anaerobic or microaerobic conditions are used for fermentations.
Industrial Batch and Continuous Fermentations
Butanol, or other products, can be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the production broth is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the production broth. Batch and fed-batch fermentations are common and well known in the art and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Appl. Biochem. Biotechnol. 36:227, 1992, herein incorporated by reference.
Butanol, or other products, can also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.
It is contemplated that the production of butanol, or other products, can be practiced using batch, fed-batch, or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for butanol production.
Methods for Isobutanol Isolation from the Fermentation Medium
Bioproduced butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Dune, 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.
Because butanol forms a low boiling point, azeotropic mixture with water, distillation may 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 can be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).
The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol. In this method, the butanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.
The butanol may also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent.
Distillation in combination with adsorption may also be used to isolate butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden, et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).
Additionally, distillation in combination with pervaporation may be used to isolate and purify the butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo, et al., J. Membr. Sci. 245:199-210, 2004).
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating 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.
All documents cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued or foreign patents, or any other documents, are each entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited documents.
General Methods
Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987), and by Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
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 can be found as set out in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass. (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of microbial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.) unless otherwise specified. Microbial strains were obtained from The American Type Culture Collection (ATCC), Manassas, Va., unless otherwise noted. Synthetic complete medium is described by Amberg, Burke and Strathern, 2005, Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Methods for Analyzing Cultivation Experiments
OD at λ=600 nm can be determined in a spectrophotometer by pipetting a well mixed broth sample into an appropriate cuvette. If biomass concentration of the sample exceeds the linear absorption range of the spectrophotometer (typically OD values from 0.000 to 0.600), sample should be diluted with 0.9% NaCl solution to yield values in the linear range. Dry weight of the cell suspension can be determined by centrifugating 5 mL cell broth in a pre-weighed centrifuge tube, followed by washing with distilled water, drying to constant weight at 80° C. in an oven and determining the weight difference.
Example 1
Thiamine Increases Isobutanol Production
Shake Flask Experimental Procedure
The medium contained the following ingredients per L of water: 6.7 g Yeast Nitrogen Base (YNB) w/o amino acids (Difco 0919-15-3); 2.8 g Yeast Synthetic Drop-out Medium Supplement Without histidine, leucine, tryptophan, and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 5 g ethanol; 3 g glucose; and 1.0 mL of the ergosterol and Tween 80. In order to obtain 10 mL ergosterol and Tween 80 solution, 100 mg of ergosterol were dissolved in 5 mL 100% EtOH and 5 mL Tween 80 and then heated until sterile and dissolved (10 min at 70° C.).
In the shake flask experiments described, the following were added to 125 mL shake flasks: 16 mL of the culture broth; 2 mL of the 1 M MES buffer; and 2 mL of the addition (H2O, YE, TH). The following additions were prepared: yeast extract (YE, final concentration 10 g/L); thiamine (TH, final concentration 6, 60, and 100 mg/L).
In order to provide the culture broth, seed cultures were started in 125 mL shake flasks with air permeable lids (10 mL medium inoculated with 1 mL inoculum from a frozen vial). After 24 hours OD (optical density) in the flask was about 0.7. The OD was measured at λ=600 nm (Spectrophotometer from Thermo Electron Corporation Heλios Alpha, USA). The correlation between OD reading and dry cell weight g/L concentration was experimentally determined as 2.6:1.0. Ergosterol and Tween 80 was added to the medium to a final concentration of 1 mL/L. Seed (10 mL) was inoculated into 2 L shake flask with 135 mL of the medium. Separately, 2 mL of the H2O, YE, TH additions were added to new 125 ml shake flasks. Into the 2 L shake flask, 16 mL of 1 M MES buffer was added and 5.3 mL of 50% (w/w) glucose solution. Eighteen (18) mL of this culture broth was distributed into the 125 mL shake flasks with prepared additions. The aerobic shake flasks with air permeable lid were incubated at 30° C. and 260 rpm. The anaerobic shake flasks were treated in anaerobic hood and incubated at 30° C. and 260 rpm. The anaerobic shake flasks were sampled in the anaerobic hood.
Effect of Thiamine on Isobutanol Production
The effect of thiamine on isobutanol production was analyzed using shake flask fermentation. Shake flask fermentations were performed under both aerobic and anaerobic conditions in the presence of water (negative control), yeast extract (YE, positive control, final concentration 10 g/L), or thiamine (TH, final concentration 100 mg/L) with isobutanol producing NYLA84 strain. The final biomass (X), isobutanol concentrations, and specific isobutanol production rates (qP) were measured and are shown in FIG. 1. The isobutanol pathway comprises a KARI enzyme that strongly prefers NADPH, and the resulting redox imbalance cannot be resolved under stringent anaerobic conditions. Therefore, only initial values for qP (during the first 6 fermentation hours) in anaerobic flasks are presented in FIG. 1F. Moreover, because of insufficient oxygen transfer rate at the higher cell densities in aerobic shake flasks, again only initial values for qP (during the first 24 fermentation hours) were considered (FIG. 1E). For this reason, aerobic and anaerobic shake flasks are not directly comparable.
The highest final biomass concentrations and isobutanol titers were achieved with YE addition in both aerobic and anaerobic conditions (FIGS. 1A-D). In aerobic shake flasks, a higher final biomass concentration was achieved with addition of thiamine compared to the control. In aerobic shake flasks, higher titers of isobutanol were achieved with the addition of thiamine compared to the control. In aerobic flasks with thiamine, the initial qP (0.14 g/g/h) was 75% higher than the control and about the same as achieved with yeast extract (FIG. 1E). Similar observations can be made about initial qP in anaerobic flasks (FIG. 1F).
In order to investigate influence of the addition of thiamine at different concentrations, aerobic shake flask experiments with final concentrations of 6, 60, and 100 mg/L thiamine were performed. The three concentrations of added vitamins resulted in similar isobutanol titers (FIG. 2). Thus, concentrations as low as 6 mg/L thiamine meet the requirements of the biomass concentrations used in these experiments. It was also observed that the accumulation of keto-isovaleric acid in control shake flasks was several times higher compared to the flasks with yeast extract or thiamine added (FIG. 3).
Examples 2-5
Experiments with Various Thiamine Concentrations in Production Medium
Fermentations with the isobutanol producing NYLA93 strain (construction described herein) were performed in defined medium with various thiamine concentrations in the range of 0 to 100 mg/L.
Example 2: F—140-100 defined medium supplemented with initial thiamine concentrations of 100 mg/L.
Example 3: F—141-20 defined medium supplemented with initial thiamine concentrations of 20 mg/L.
Example 4: F—142-10 defined medium supplemented with initial thiamine concentrations of 10 mg/L.
Example 5: F—143-0 defined medium supplemented with initial thiamine concentrations of 0 mg/L.
Methods:
Inoculum Preparation
1 L of inoculum medium contained: 6.7 g Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3); 2.8 g Yeast Synthetic Drop-out Medium Supplement Without histidine, leucine, tryptophan, and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 0.8 mL of ergosterol and Tween 80; 3 g of ethanol; 3 g of glucose
A 125 mL shake flask was inoculated directly from a frozen vial by pipetting the whole vial culture (approx. 1 ml) into 10 mL of the inoculum medium. The flask was incubated at 160 rpm and 30° C. The strain was grown overnight until OD was about 1.0. At this point, four 2 L shake flasks with 160 mL of the inoculum medium were inoculated from the overnight culture. For each fermentor, 160 mL of the inoculum was prepared in 2 L baffled shake flask at 260 rpm and 30° C. When OD at λ=600 nm in the shake flask reached about 1.0, 30 mL of 1M MES buffer and about 160 mL of oleyl alcohol were added to the shake flasks. Twenty-four (24) hours after this point, the oleyl alcohol was removed and fermentors inoculated.
Bioreactor Medium
One (1) L of bioreactor medium contained:

    
    
        salts: ammonium sulfate 10.0 g, potassium phosphate monobasic 5.33 g, magnesium sulfate heptahydrate 2.0 g, zinc sulfate heptahydrate, 0.47 g.
        vitamins: biotin (D−) 0.40 mg, Ca D(+) panthotenate 8.00 mg, myo-inositol 200.00 mg, pyridoxol hydrochloride 8.00 mg, p-aminobenzoic acid 1.60 mg, riboflavin 1.60 mg, folic acid 0.02 mg, niacin 100.0 mg.
        amino acids: yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan, and uracil (Sigma Y2001) 2.8 g, 1% (w/v) L-leucine 20 mL, 1% (w/v) L-tryptophan 4 mL.
        trace elements: EDTA (Titriplex 1117) 99.38 mg, zinc sulphate heptahydrate 29.81 mg, manganese chloride dehydrate 5.57 mg, cobalt(II)chloride hexahydrate 1.99 mg, copper(II)sulphate pentahydrate 1.99 mg, Di-sodium molybdenum dehydrate 2.65 mg, calcium chloride dehydrate 29.81 mg, iron sulphate heptahydrate 19.88 mg, boric acid, 6.63 mg, potassium iodide 0.66 mg.
    
    


Thiamine was added to fermentors according to Table 6.










TABLE 6







Fermentation
Thiamine start concentration mg/L



















F_140 - 11162009
100



F_141 - 11162009
20



F_142 - 11162009
10



F_143 - 11162009
0










Bioreactor Experimental Design

Experiments were executed in 2 L BIOSTAT B-DCU Tween2 L bioreactors from Sartorius (USA). The fermentors are connected to mass spectrometer from Thermo Electron Corporation (USA). Directly after inoculation with 80 mL of inoculum per bioreactor, the volume in bioreactors was about 800 mL, dissolved oxygen tension (DOT) was controlled at 15%, pH was controlled at 5.25, aeration was controlled at 0.5 L/min, 1.4 L of oleyl alcohol was added. Oleyl alcohol was used in order to extract isobutanol from culture broth. In this way, toxicity of isobutanol was lowered in the culture broth.
Analytics
Isobutanol was measured in oleyl alcohol, culture broth, and off-gas samples by GC method, HPLC method, and mass-spectrometry, respectively. The methods are described below.
GC Method
The GC method utilized an ZB-WAXplus column (30 m×0.25 mm ID, 0.25 μm film) from Phenomenex (Torrance, Calif.). The carrier gas was helium at a constant flow rate of 2.3 mL/min; injector split was 1:20 at 250° C.; oven temperature was 70° C. for 1 min, 70° C. to 160° C. at 10° C./min, and 160° C. to 240° C. at 30° C./min. FID detection was used at 260° C. with 40 mL/min helium makeup gas. Culture broth samples were filtered through 0.2 μm spin filters before injection. Depending on analytical sensitivity desired, either 0.1 μL or 0.5 μL injection volumes were used. Calibrated standard curves were generated for the following compounds: ethanol, isobutanol, acetoin, meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. (2S,3S)-2,3-butanediol retention time is 6.8 minutes. meso-2,3-butanediol retention time is 7.2 minutes. Analytical standards were also utilized to identify retention times for isobutryaldehyde, isobutyric acid, and isoamyl alcohol.
HPLC Method
Analysis for glucose and fermentation by-product composition is well known to those skilled in the art. For example, one high performance liquid chromatography (HPLC) method utilizes a Shodex® SH-1011 column with a Shodex® SH-G guard column (both available from Waters Corporation, Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol retention time is 47.6 minutes.
HPLC Method for Thiamine Analysis
Samples were prepared by centrifugation of the fermentation or shake flask samples and filtration using 0.2 micron filter.
The method was reversed phase chromatography using Zorbax SB-C8, 4.6×250 mm, 5 micron column. Mobile phase A was 0.05% TFA in H2O and B was methanol. Flow rate was 1.0 mL/min. Injection volume was 10 pt. Run time was 24 minutes. Column temperature and sample temperature were 30° C. and 10° C., respectively. Detector was PDA (205 nm to 400 nm). Wavelength was 210 nm and 260 nm. Gradient is shown in Table 7.












TABLE 7







Time (min)
Flow rate
% A
% B





















0
1.0
97
3



6.5
1.0
97
3



12
1.0
50
50



18
1.0
5
95



19
1.0
97
3



24
1.0
97
3










The final rate, titer, and yield values (RTY) are shown in Table 8. Isobutanol titers during the fermentation time are shown in FIG. 4 (or Table 9). Volumetric isobutanol production rates and titers were about 4 times higher in fermentations with added thiamine at 100 mg/L (F140) and 20 mg/L (F141) compared to the fermentation without thiamine (F143). Yeast cell concentration, measured as optical density at λ=600 nm in a HEλIOS a (Thermo Electron Corporation, USA) was increased as well (FIG. 5 or Table 10). Thiamine concentration in culture medium is shown in FIG. 6 (or Table 11).












TABLE 8







Examples
Rate g/L/h
Titer g/L
Yield g/g





















F_140-100
0.45
38.5
0.23



F_141-20
0.47
39.7
0.26



F_142-10
0.36
30.2
0.24



F_143-0
0.11
9.5
0.13

















TABLE 9







Isobutanol titers during the fermentation time











Time (h)
F_140
F_141
F_142
F_143














0.39
0.0
0.0
0.0
0.0


12.92
0.7
0.3
0.2
0.3


18.27
1.0
0.9
0.8
0.7


21.05
1.4
1.5
1.3
0.8


39.97
7.6
8.0
7.2
2.8


42.36
11.4
11.7
9.3


45.02
14.1
15.1
11.4
3.9


60.92
26.2
25.4
12.8
6.0


66.32
31.5
31.0
17.3
7.1


69.01
32.2
33.0
18.6
7.6


85.15
38.5
39.7
30.2
9.5
















TABLE 10







Yeast cell concentration, measured as optical density











Time (h)
F_140
F_141
F_142
F_143














0.39
2.42
1.1
0.9
1.1


12.92
3.9
3.4
3.4
3.6


18.27
5.5
4.9
5.0
5.5


21.05
6.84
7.4
6.6
7.6


39.97
15.3
18.2
15.6
10.9


42.36
19.1
21.4
17.2
7.0


45.02
19.4
21.3
18.9
13.4


60.92
25.1
26.5
14.4
10.9


66.32
20.1
26.2
16.2
11.1


69.01
24.1
25.6
16.1
12.5


85.15
22
23.8
16.7
11.5
















TABLE 11







Thiamine concentration in culture medium











Time (h)
F_140
F_141
F_142
F_143














0.39
107
22
10
0


12.92
105
19
9
0


18.27
100
16
7
0


21.05
96
14
5
0


39.97
71
0
0
0


42.36
58
0
0
0


45.02
53
0
0
0


60.92
30
0
0
0


66.32
24
0
0
0


69.01
24
0
0
0


85.15
22
0
0
0









Example 6
Nicotinic Acid Increases Isobutanol Production
The effect of nicotinic acid on isobutanol production was analyzed in shake flask experiments. Cultivations were performed under both aerobic and anaerobic conditions in the presence of water (negative control), vitamin solution (positive control), yeast extract peptone (YEP, positive control), or nicotinic acid (NA) with strain Saccharomyces cerevisiae isobutanol producing NYLA84 strain (construction described elsewhere herein).
Vitamin stock solution contained biotin, 50 mg/L; Ca-panthotenate, 1 g/L; nicotinic acid, 1 g/L; myo-inositol, 25 g/l; thiamine.HCl, 1 g/L; pyridoxine hydrochloride, 1 g/L; p-aminobenzoic acid, 0.2 g/L; riboflavin, 0.2 g/L; and folic acid, 2 mg/L. One liter (1 L) of ergosterol and Tween 80 stock solution contain 10 g of ergosterol dissolved in 500 mL 100% ethanol solution and 500 mL Tween 80.
Experiments were carried out in 125 mL shake flasks with 20 mL shake flask medium. Composition of the base medium was (per L of water): 6.7 g YNB without amino acids (Difco 0919-15-3); 2.8 g Yeast Synthetic Drop-out Medium Supplement without histidine, leucine, tryptophan, and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 0.8 mL of ergosterol and Tween 80 stock solution.
OD at λ=600 nm was determined in a HEλIOS α spectrophotometer (Thermo Electron Corporation, USA). If biomass concentration of the sample exceeded the linear range of the spectrophotometer, sample was diluted with water. Glucose and fermentation by-product analysis were determined by a Waters HPLC utilizing a Bio-Rad Aminex HPX-87H column with diode array (DA) operated at 210 nm and a refractive index (RI) detector at 50° C. Chromatographic separation was achieved using 0.01N H2SO4 as the mobile phase with a flow rate of 0.6 mL/min and a column temperature of 40° C.
OD at λ=600 nm was determined in a HEλIOS α spectrophotometer (Thermo Electron Corporation, USA). If biomass concentration of the sample exceeded the linear range of the spectrophotometer, sample was diluted with water. Glucose and fermentation by-product analysis were determined by HPLC utilizing a Bio-Rad Aminex HPX-87H column with diode array (DA) at 210 nm and refractive index (RI) detector operated at 50° C. Chromatographic separation was achieved using 0.01 M H2SO4 as the mobile phase with a flow rate of 0.08 mL/min and a column temperature of 60° C.
Seed cultures were started in 125 mL shake flasks (open cups, 10 mL medium inoculated with 1 mL from a frozen vial). The flasks were incubated at 260 rpm and 30° C. After 24 hours OD in the flask was about 1.0. Ten (10) mL of the seed was inoculated into 2 L shake flask with 135 mL of the medium. Fifty (50) mL of 1M MES buffer was added and 14.3 mL of 50% (w/w) glucose solution.
When OD at λ=600 nm in the 2 L flask reached about 1.0, 18 mL of this culture broth was distributed in 125 mL shake flasks. Two (2) mL of (a) bidestilled water, (b) nicotinic acid stock solution (with 1 g/L nicotinic acid in water), (c) 2 ml of YEP stock solution (with 10 g/l yeast extract (YE) and 20 g/L peptone), and (d) 20 μL of vitamin stock solution in 1.980 mL of bidest water were added to the cultures. The aerobic shake flasks with air permeable lid were incubated at 30° C. and 260 rpm. The anaerobic shake flasks were started in the anaerobic hood and incubated as well at 30° C. and 260 rpm. Initial OD at λ=600 nm in the cultures were about 0.700. Aerobic experiments were analyzed after 25.8 h of the process, anaerobic data after 24.2 h. Generated biomass (delta OD), consumed glucose (delta glucose), and produced isobutanol (delta isobutanol) were determined and are shown in FIG. 9.
Negative control cultivations exhibited growth, consumed glucose, and produced isobutanol under aerobic as well as anaerobic conditions. However, experiments supplemented with NA showed increased isobutanol production (FIG. 9A), increased consumption of glucose (FIG. 9B) as well as increased biomass formation (FIG. 9C) as compared to the negative control experiments. Increase was significantly more pronounced under anaerobic than under aerobic conditions. Positive controls containing either a mixture of vitamins or YEP showed improved production of isobutanol, consumption of glucose and production of biomass, indicating additional beneficial effects of other media compounds and/or vitamins.
Example 7
Medium Concentrations of Nicotinic Acid for Isobutanol Production in Aerobic Shake Flask Cultivations
The strain used in this Example was the isobutanol producing NYLA 84 strain, described elsewhere herein. Vitamin stock solution contained biotin, 50 mg/L; Ca-panthotenate, 1 g/L; nicotinic acid, 1 g/L; myo-inositol, 25 g/l; thiamine.HCl, 1 g/L; pyridoxine hydrochloride, 1 g/L; p-aminobenzoic acid, 0.2 g/L; riboflavin, 0.2 g/L; and folic acid, 2 mg/L. One liter (1 L) of ergosterol and Tween 80 stock solution contains 10 g of ergosterol dissolved in 500 mL 100% ethanol solution and 500 mL Tween 80. Composition of the base medium was (per L of water): 6.7 g YNB without amino acids (Difco 0919-15-3); 2.8 g Yeast Synthetic Drop-out Medium Supplement without histidine, leucine, tryptophan, and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 5 g ethanol; 3 g glucose; 0.8 mL of Ergosterol & Tween 80 stock solution.
OD at λ=600 nm was determined in a HEλIOS α spectrophotometer (Thermo Electron Corporation, USA). If biomass concentration of the sample exceeded the linear range of the spectrophotometer, sample was diluted with water. Glucose and fermentation by-product analysis were determined by a Waters HPLC utilizing a Bio-Rad Aminex HPX-87H column with diode array (DA) operated at 210 nm and a refractive index (RI) detector at 50° C. Chromatographic separation was achieved using 0.01N H2SO4 as the mobile phase with a flow rate of 0.6 mL/min and a column temperature of 40° C.
Several seed cultures inoculated each with a 1 mL glycerol frozen stock vial were grown in 10 mL base medium for 24 h. At the start of the range finding experiment, seed cultures were pooled and 16 mL each of the pooled seed culture were distributed into 125 mL shake flasks, 2 mL of 1 M MES buffer added, and additionally 2 mL of (a) bidestilled water, (b-d) nicotinic acid (NA) stock solutions with 60 mg/L, 600 mg/L and 1000 mg/L of NA dissolved in bidestilled water, respectively, and (e) YE stock solution with 200 g/l YE. The aerobic shake flasks with air permeable lid were incubated at 30° C. and 260 rpm. Initial OD at λ=600 nm in the cultures was 0.764. Biomass and isobutanol produced were analyzed at 2.00 h, 18.75 h, and 24.5 h of the process and results are shown in FIG. 11.
Biomass and isobutanol were produced in the control culture without NA. However, NA supplemented cultures with NA concentrations as low as 6 mg/L showed significantly increased biomass and isobutanol titers at all measurement points. Positive control containing YE showed increased titers of biomass and isobutanol, indicating additional beneficial effects of other YE components.
Example 8
Nicotinic Acid Increases Isobutanol Production
The effect of nicotinic acid on isobutanol production was analyzed in shake flask experiments. Cultivations were performed under both aerobic and anaerobic conditions in the presence of water (negative control), vitamin solution (positive control), yeast extract peptone (YEP, positive control), or nicotinic acid (NA) with strain Saccharomyces cerevisiae isobutanol producing NYLA84 strain (construction described elsewhere herein).
Vitamin stock solution contained biotin, 50 mg/L; Ca-panthotenate, 1 g/L; nicotinic acid, 1 g/L; myo-inositol, 25 g/l; thiamine.HCl, 1 g/L; pyridoxine hydrochloride, 1 g/L; p-aminobenzoic acid, 0.2 g/L; riboflavin, 0.2 g/L; and folic acid, 2 mg/L. One liter (1 L) of ergosterol and Tween 80 stock solution contain 10 g of ergosterol dissolved in 500 mL 100% ethanol solution and 500 mL Tween 80.
Experiments were carried out in 125 mL shake flasks with 20 mL shake flask medium. Composition of the base medium was (per L of water): 6.7 g YNB without amino acids (Difco 0919-15-3); 2.8 g Yeast Synthetic Drop-out Medium Supplement without histidine, leucine, tryptophan and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 0.8 mL of ergosterol and Tween 80 stock solution.
OD at λ=600 nm was determined in a HEλIOS α spectrophotometer (Thermo Electron Corporation, USA). If biomass concentration of the sample exceeded the linear range of the spectrophotometer, sample was diluted with water. Glucose and fermentation by-product analysis were determined by a Waters HPLC utilizing a Bio-Rad Aminex HPX-87H column with diode array (DA) operated at 210 nm and a refractive index (RI) detector at 50° C. Chromatographic separation was achieved using 0.01N H2SO4 as the mobile phase with a flow rate of 0.6 mL/min and a column temperature of 40° C.
Seed cultures were started in 125 mL shake flasks (open cups, 10 mL medium inoculated with 1 mL from a frozen vial). The flasks were incubated at 260 rpm and 30° C. After 24 hours OD in the flask was about 1.0. Ten (10) mL of the seed was inoculated into 2 L shake flask with 135 mL of the medium. Fifty (50) mL of 1M MES buffer was added and 14.3 mL of 50% (w/w) glucose solution.
When OD at λ=600 nm in the 2 L flask reached about 1.0, 18 mL of this culture broth was distributed in 125 mL shake flasks. Two (2) mL of (a) bidest. water, (b) nicotinic acid stock solution (with 1 g/L nicotinic acid in water), (c) 2 ml of YEP stock solution (with 10 g/l YE and 20 g/L peptone), and (d) 20 μL of vitamin stock solution in 1.980 mL of bidest water were added to the cultures. The aerobic shake flasks with air permeable lid were incubated at 30° C. and 260 rpm. The anaerobic shake flasks were started in the anaerobic hood and incubated as well at 30° C. and 260 rpm. Initial OD at λ=600 nm in the cultures were about 0.500. Experiments were sampled after 6.3 and 20.8 h. Generated biomass (delta OD), consumed glucose (delta glucose), and produced isobutanol (delta isobutanol) were determined and are shown in FIG. 10.
Negative control cultivations exhibited growth, consumed glucose, and produced isobutanol under aerobic as well as anaerobic conditions. However, experiments supplemented with NA showed increased isobutanol production and both conditions (FIG. 10A+D), increased consumption of glucose (FIG. 103B+E) as well as increased biomass formation (FIG. 10C+F) as compared to the negative control experiments. The increase was significantly more pronounced under anaerobic than under aerobic conditions. Positive controls containing either a mixture of vitamins or YEP showed improved production of isobutanol, consumption of glucose and production of biomass, indicating additional beneficial effects of other media compounds and/or vitamins.
Example 9
Nicotinic Acid Supplementation Improves Isobutanol Production in Bioreactors
Based on the findings in shake flasks experiments, bioreactor cultivations were performed with the BP1063 isobutanol producing strain (construction described herein) with addition of water (control) and initial nicotinic acid concentrations of 100 mg/L, 20 mg/L and 5 mg/L.
One liter (1 L) of inoculum medium contained: 6.7 g, Yeast Nitrogen Base w/o amino acids (Difco 0919-15-3); 2.8 g, Yeast Synthetic Drop-out Medium Supplement Without histidine, leucine, tryptophan and uracil (Sigma Y2001); 20 mL of 1% (w/v) L-leucine; 4 mL of 1% (w/v) L-tryptophan; 1 mL of ergosterol and Tween 80 solution; 3 g of ethanol; 3 g of glucose. Preparation of ergosterol and Tween 80 solution: for 10 mL of the solution, 100 mg of ergosterol was dissolve in 5 mL 100% EtOH and 5 mL Tween 80. It was heated at 70° C. for 10 minutes.
A 125 mL shake flask was inoculated directly from a frozen vial by pipetting the whole vial culture (approx. 1 ml) into 10 mL of the inoculum medium. The flask was incubated at 260 rpm and 30° C. The strain was grown overnight until OD about 1.0. At this point four 2 L baffled shake flasks with 90 mL of the inoculum medium were inoculated from the overnight culture. The 2 L flasks were incubated at 260 rpm and 30° C. When OD in the flasks reached about 1.0, 10 mL of 1M MES buffer and about 100 mL of oleyl alcohol were added to the shake flasks. Twenty-four (24) hours after this point the oleyl alcohol was removed, the cultures were pooled together and each fermentor was inoculated with 80 mL of the inoculum.
One liter (1 L) of bioreactor medium was prepared with: Salts: ammonium sulfate 10.0 g, potassium phosphate monobasic 5.33 g, magnesium sulfate heptahydrate 2.0 g, zinc sulfate heptahydrate, 0.47 g. Vitamins: biotin (D−) 0.40 mg, Ca D(+) panthotenate 8.00 mg, myo-inositol 200.00 mg, pyridoxol hydrochloride 8.00 mg, p-aminobenzoic acid 1.60 mg, riboflavin 1.60 mg, folic acid 0.02 mg, thiamine 30.0 mg. Amino acids: yeast synthetic drop-out medium supplement without histidine, leucine, tryptophan and uracil (Sigma Y2001) 2.8 g, 1% (w/v) L-leucine 20 mL, 1% (w/v) L-tryptophan 4 mL. Trace elements: EDTA (Titriplex 1117) 99.38 mg, zinc sulphate heptahydrate 29.81 mg, manganese chloride dehydrate 5.57 mg, cobalt(II)chloride hexahydrate 1.99 mg, copper(II)sulphate pentahydrate 1.99 mg, Di-sodium molybdenum dehydrate 2.65 mg, calcium chloride dehydrate 29.81 mg, iron sulphate heptahydrate 19.88 mg, boric acid, 6.63 mg, potassium iodide 0.66 mg. Ergosterol and Tween 80 solution 1 mL. Glucose 20 g.
Experiments were carried out in a 2 L BIOSTAT B-DCU Tween2 L bioreactors from Sartorius (Germany). The fermentors are connected to mass-spec from Thermo Electron Corporation (USA). Directly after inoculation with 80 mL of the inoculum, the volume in fermentors was about 800 mL, dissolved oxygen tension (DOT) was controlled at 10%, pH was controlled at 5.25, aeration was controlled at 0.5 L/min, 0.8 L of oleyl alcohol was added. Oleyl alcohol was used in order to extract isobutanol from culture broth. In this way toxicity of isobutanol was lowered in the culture broth. During the whole fermentation, glucose was kept at about 20 g/L by feeding glucose solution (50% w/w).
OD at λ=600 nm was determined in a HEλIOS a spectrophotometer (Thermo Electron Corporation, USA). If biomass concentration of the sample exceeded the linear range of the spectrophotometer, sample was diluted with water. Glucose and fermentation by-product analysis were determined by HPLC utilizing a Shodex® SH-1011 column with a Shodex® SH-G guard column (both available from Waters Corporation, Milford, Mass.), with diode array (DA) and refractive index (RI) detector. Chromatographic separation was achieved using 0.01 N H2SO4 as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C.
Isobutanol was measured in oleyl alcohol, culture broth, and off-gas samples by GC method, HPLC method, and mass-spectrometry, respectively. The methods were described above. Analysis for glucose and fermentation by-product composition is also described above.
For analysis of nicotinamid and nicotinic acid concentrations, samples were prepared by centrifugation of the fermentation and filtered using 0.2 micron filters. An reversed phase HPLC method using a Zorbax SB-C8, 4.6×250 mm, 5 micron column was established. Mobile phase A was 0.05% TFA in H2O and B was methanol. Eluent gradient is shown above. Flow rate was 1.0 mL/min. Injection volume was 10 pt. Run time was 24 minutes. Column temperature and sample temperature were 30° C. and 10° C., respectively. A PDA detector was used to monitor wavelengths 210 nm and 260 nm. 260 nm is the preferred wavelength to quantify nicotinamid and nicotinic acid in fermentation samples, while the 210 nm signal was used for verification.
Time courses of isobutanol, biomass and NA concentrations in aqueous phase are shown in FIG. 12. It can be seen that supplementation of the bioreactor cultivations with NA concentrations of as low as 5 mg/L lead to improved final isobutanol titers in the aqueous phase. High consumption of NA in excess of 20 mg/L until the end of the process in the 100 mg/L supplemented culture indicates high demand of the isobutanol producing cells for NA.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

## Claims

1. A commercial method for producing isobutanol comprising:
a. providing a production culture comprising recombinant cells, wherein the recombinant cells comprise an engineered isobutanol biosynthetic pathway, wherein the engineered isobutanol biosynthetic pathway comprises heterologous polynucleotides encoding polypeptides that catalyze the following substrate to product conversions of the pathway:
a) pyruvate to acetolactate, catalyzed by an acetolactate synthase;
b) acetolactate to 2,3-dihydroxyisovalerate, catalyzed by an acetohydroxy acid reductoisomerase;
c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, catalyzed by a acetohydroxy acid dehydratase;
d) a-ketoisovalerate to isobutyraldehyde, catalyzed by a branched-chain keto acid decarboxylase; and
e) isobutyraldehyde to isobutanol, catalyzed by a branched-chain alcohol dehydrogenase,
wherein the recombinant cells comprise at least one genetic modification that eliminates pyruvate decarboxylase activity,
and wherein the recombinant cells are capable of producing isobutanol, and production media comprising
1. thiamine or a biosynthetic precursor thereof;
2. optionally nicotinic acid, nicotinamid, or a biosynthetic precursor of NAD; and
3. a production feed derived from biomass comprising a fermentable carbon source;



wherein the production media contains less than 1 g/L of multi-component media additives; and
b. contacting the production culture with the production media in a fermentation vessel to form a production broth under conditions whereby isobutanol is produced.

2. The method of claim 1, wherein the production media is substantially free of multi-component media additives.

3. The method of claim 1, wherein the production broth contains at least about 5 mg/L thiamine or a biosynthetic precursor thereof.

4. The method of claim 1, wherein the thiamine or a biosynthetic precursor thereof is present in an amount sufficient to provide at least about 30% of the rate, titer, or specific productivity observed in media that contains about 10 g/L yeast extract.

5. The method of claim 1, wherein the recombinant cells are yeast.

6. The method of claim 5, wherein the yeast is crabtree positive or crabtree negative.

7. The method of claim 1, wherein the recombinant cell comprises a modification of an endogenous gene encoding a thiamine pyrophosphate-dependent (TPP) gene.

8. The method of claim 7, wherein the TPP-dependent gene is a pyruvate decarboxylase gene.

9. The production broth of claim 1, wherein the broth comprises isobutanol.