# Patent

## Title
Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors

## Abstract

The present invention provides for novel metabolic pathways to reduce or eliminate glycerol production and increase product formation. More specifically, the invention provides for a recombinant microorganism comprising a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source, such as lignocellulose, to a product, such as ethanol, wherein the one or more native and/or heterologous enzymes is activated, upregulated, or downregulated. The invention also provides for a recombinant microorganism comprising one or more heterologous enzymes that function to regulate glycerol synthesis and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated.

## Background
CROSS REFERENCE TO RELATED APPLICATION
The present invention application is a continuation of U.S. application Ser. No. 14/110,075, filed Mar. 21, 2014, which is a 371 National Stage Application of International Application No. PCT/US2012/032443, filed Apr. 5, 2012, which claims the benefit of U.S. Provisional Application No. 61/472,085. The entire contents of each are incorporated herein by reference in their entirety.


BACKGROUND OF THE INVENTION
Sequence Listing
This application contains a sequence listing which is submitted under 37 CFR §1.821(c) in an electronic form as the text file 177CRFSeqList.txt, created on Apr. 30, 2015, the size of which is 437,476 bytes and the content of which is incorporated by reference.
The conversion of biomass, such as corn, sugarcane or other energy crops, as well as simple sugars, to ethanol is routinely completed through the use of yeast fermentation. However, during yeast metabolism a major byproduct of fermentation is glycerol. Glycerol is formed during anaerobic growth as a way for the yeast to balance its redox state and regenerate NAD+ used as a cofactor during glycolysis. It has been shown that the function of glycerol is likely not as a metabolite itself but rather as an electron sink capturing electrons allowing further growth-linked metabolism to continue. As glycerol is a byproduct with low value, it can be an undesirable by-product of fermentation. It would be beneficial to reduce or eliminate this by-product and further direct more carbon towards desired end-products, such as ethanol.
Several strategies are available in the art for the conversion of glycerol to higher value products though biochemical or other means, but relatively little has been demonstrated for the removal or reduction of glycerol and improvement of overall sugar yield to ethanol or other desired end-products of metabolism. Through engineering of alternate pathways, potentially with the simultaneous reduction or deletion of the glycerol pathway, alternate or replacement electron acceptors for the regeneration of NAD+ can be used during yeast metabolism. Such alternate or replacement electron acceptors could be molecules such as formate or hydrogen.
The elimination of glycerol synthesis genes has been demonstrated but removal of this pathway completely blocked anaerobic growth of the yeast, preventing useful application during an industrial process. Ansell, R., et al., EMBO J. 16:2179-87 (1997); Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001); Guo, Z P., et al., Metab. Eng. 13:49-59 (2011). Other methods to bypass glycerol formation require the co-utilization of additional carbon sources, such as xylose or acetate, to serve as electron acceptors. Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996); Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010). By incorporating a formate pathway as an alternate electron acceptor, glycerol formation can be bypassed and ethanol yield can be increased. The engineering of a pyruvate formate lyase from E. coli, which is capable of converting pyruvate to formate, has been done to increase formate production. Waks, Z., and Silver, P. A., Appl. Env. Microbiol. 75:1867-1875 (2009). Formate engineering in Waks and Silver was done, however, to provide a source of formate in S. cerevisiae for the production of hydrogen by a secondary microorganism, E. coli. Waks and Silver did not combine formate production with the removal of glycerol formation, and the use of formate as an alternate electron acceptor for the reduction of glycerol was not proposed or evaluated. Thus, despite prior efforts to bypass and/or eliminate glycerol production, there exists a need for the engineering of alternate or replacement electron acceptors in a cell to direct more carbon towards desired end-products, such as ethanol.
The importance of engineering alternate or replacement electron acceptors is exemplified in the process of corn mash fermentation. About 16 billion gallons of corn-based ethanol are produced annually, so even small increases in ethanol yield, such as 5-10%, can translate into an extra billion or so gallons of ethanol over current yields. Ethanol production from corn mash typically results in glycerol yields ranging from 10-12 g/L. See Yang, R. D., et al., “Pilot plant studies of ethanol production from whole ground corn, corn flour, and starch,” Fuel Alcohol U.S.A., Feb. 13-16, 1982 (reported glycerol levels to be as high as 7.2% w/w of initial sugar consumed in normal corn mash fermentations or approximately 1.4 g/100 mL using 20% sugar). By reducing or eliminating the glycerol yield in the production of ethanol from corn and re-engineering metabolic processes, increased ethanol yields can be achieved. Additional benefits may be gained in the production of ethanol from corn. Corn mash is a nutrient rich medium, in some cases containing lipid and protein content that can be >3% of the total fermentation volume. As a result of the energy contained in these components, even higher ethanol yields may be achieved than what is predicted using, for example, pure sugar. The additional increases can come from the metabolism of lipids or amino acids in the corn mash medium. The recombinant cells and methods of the invention enable increasing ethanol yields from biomass fermentation by reducing or eliminating glycerol.
BRIEF SUMMARY OF THE INVENTION
The invention is generally directed to the reduction or removal of glycerol production in a host cell and to the engineering of an alternate electron acceptor for the regeneration of NAD+.
One aspect of the invention relates to a recombinant microorganism comprising: a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated. In some embodiments, the recombinant microorganism produces less glycerol than a control recombinant microorganism without deletion of said one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis. In some embodiments, the carbohydrate source is biomass. In some embodiments, the biomass comprises a lignocellulosic material selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof. In some embodiments, the biomass is corn mash or corn starch.
In particular aspects, the one or more native enzymes that function to produce glycerol are encoded by a gpd1 polynucleotide, a gpd2 polynucleotide, or both a gpd1 polynucleotide and a gpd2 polynucleotide. In certain embodiments, the recombinant microorganism further comprises a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide. In other aspects, the one or more native enzymes that function to produce glycerol are encoded by a gpp1 polynucleotide, a gpp2 polynucleotide, or both a gpp1 polynucleotide and a gpp2 polynucleotide.
In particular aspects, the one or more native enzymes that function to regulate glycerol synthesis are encoded by an fps1 polynucleotide.
In further aspects, the engineered metabolic pathways comprise conversion of pyruvate to acetyl-CoA and formate. In certain embodiments, pyruvate is converted to acetyl-CoA and formate by a pyruvate formate lyase (PFL). In some embodiments, the PFL is of prokaryotic or eukaryotic origin. In some embodiments, PFL is from one or more of a Bifidobacteria, an Escherichia, a Thermoanaerobacter, a Clostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or a Bacillus species. In some embodiments, PFL is from one or more of a Bacillus licheniformis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bifidobacterium adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis. In one embodiment, PFL is from a Bifidobacterium adolescentis. 
In additional aspects, the engineered metabolic pathways comprise conversion of acetyl-CoA to ethanol. In certain embodiments, acetyl-CoA is converted to acetaldehyde by an acetaldehyde dehydrogenase and acetaldehyde is converted to ethanol by an alcohol dehydrogenase. In certain embodiments, acetyl-CoA is converted to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase. In some embodiments, the acetaldehyde dehydrogenase, alcohol dehydrogenase, or bifuntional acetaldehyde/alcohol dehydrogenase is of prokaryotic or eukaryotic origin. In one embodiment, acetaldehyde dehydrogenase is from C. phytofermentans. In some embodiments, bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species. In some embodiments, bifunctional acetaldehyde/alcohol dehydrogenase is from Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis. In one embodiment, bifunctional acetaldehyde/alcohol dehydrogenase is from a Bifidobacterium adolescentis or Piromyces sp. E2.
In further aspects, the recombinant microorganism comprises a deletion of one or more native enzymes encoded by an fdh1 polynucleotide, an fdh2 polynucleotide, or both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the carbohydrate source for the recombinant microorganism is lignocellulose. In certain embodiments, the recombinant microorganism produces ethanol. In certain embodiments, the recombinant microorganism produces formate.
In certain embodiments, the recombinant microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis. In one embodiment, the recombinant microorganism is Saccharomyces cerevisiae. 
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpp1 polynucleotide and a gpp2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase. In further embodiments, one engineered metabolic pathway of the recombinant microorganism converts acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fps1 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase. In further embodiments, one engineered metabolic pathway of the recombinant microorganism converts acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fps1 polynucleotide and one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fps1 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide and a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and one or more native enzymes that function to regulate glycerol synthesis encoded by an fps1 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.
In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and one or more native enzymes that function to regulate glycerol synthesis encoded by an fps1 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide and a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.
In some embodiments, the deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis in the recombinant microorganism reduces glycerol formation by: more than about 10% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 20% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 30% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 40% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 50% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 60% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 70% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 80% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 90% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 95% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; or more than about 99% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis.
In some embodiments, the recombinant microorganism produces an amount of formate selected from: at least about 0.012 g/L in 24 hours; at least about 0.022 g/L in 48 hours; or at least about 2.5 g/L in 142 hours.
In some embodiments, the recombinant microorganism produces a formate yield selected from: at least about 0.05-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 0.1-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 0.5-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 1.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 75.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; or at least about 100-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.
In some embodiments, the recombinant microorganism produces an ethanol yield selected from: at least about 1% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 2% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 3% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 4% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 90% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 95% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; or at least about 99% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.
In some embodiments, the conversion of the carbohydrate source to ethanol by the recombinant microorganism, or the enzymes engineered therein, is under anaerobic conditions.
In some embodiments, the recombinant microorganism has an acetate uptake (g/L) under anaerobic conditions selected from: at least about 1% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; and at least about 90% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.
In some embodiments, the recombinant microorganism produces more ethanol at a slower glucose utilization rate compared to a recombinant microorganism without deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis, wherein the glucose utilization rate is selected from: at least about 1% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; and at least about 90% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.
Another aspect of the invention relates to a recombinant microorganism comprising: one or more heterologous enzymes that function to regulate glycerol synthesis, wherein said one or more heterologous enzymes is activated, upregulated or downregulated; and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated. In certain embodiments, the one or more heterologous enzymes that function to regulate glycerol synthesis are encoded by an fps1 polynucleotide. In one embodiment, the fps1 polynucleotide is from Escherichia coli. 
In some embodiments, one of the engineered metabolic pathways of the above recombinant microorganism comprises conversion of pyruvate to acetyl-CoA and formate. In certain embodiments, pyruvate is converted to acetyl-CoA and formate by a pyruvate formate lyase (PFL). In some embodiments, PFL is of prokaryotic or eukaryotic origin. In some embodiments, PFL is from one or more of a Bifidobacteria, an Escherichia, a Thermoanaerobacter, a Clostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or a Bacillus species. In some embodiments, the PFL is from one or more of a Bacillus licheniformis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bifidobacterium adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis. In one embodiment, PFL is from a Bifidobacterium adolescentis. 
In some embodiments, one of said engineered metabolic pathways of the above recombinant microorganism comprises conversion of acetyl-CoA to ethanol. In some embodiments, acetyl-CoA is converted to acetaldehyde by an acetaldehyde dehydrogenase and acetaldehyde is converted to ethanol by an alcohol dehydrogenase. In other embodiments, acetyl-CoA is converted to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase. In some embodiments, the acetaldehyde dehydrogenase, alcohol dehydrogenase, or bifuntional acetaldehyde/alcohol dehydrogenase is of prokaryotic or eukaryotic origin. In one embodiment, acetaldehyde dehydrogenase is from C. phytofermentans. In certain embodiments, the bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species. In some embodiments, the bifunctional acetaldehyde/alcohol dehydrogenase is from Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis. In one embodiment, the bifunctional acetaldehyde/alcohol dehydrogenase is from a Bifidobacterium adolescentis or Piromyces sp. E2.
In further aspects, the recombinant microorganism comprises a deletion of one or more native enzymes encoded by an fdh1 polynucleotide, an fdh2 polynucleotide, or both an fdh1 polynucleotide and an fdh2 polynucleotide.
In some embodiments, the recombinant microorganism produces ethanol. In other embodiments, the recombinant microorganism produces formate. In some embodiments, the recombinant microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaiyomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis. In one embodiment, the recombinant microorganism is Saccharomyces cerevisiae. 
In some embodiments, the recombinant microorganisms of the invention further comprise one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert xylose to xylulose-5-phosphate and/or arabinose to xylulose-5-phosphate, wherein the one or more native and/or heterologous enzymes are activated, upregulated or downregulated.
In some embodiments, the recombinant microorganisms of the invention further comprise one or more native and/or heterologous enzymes which encodes a saccharolytic enzyme, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes. In one aspect, the saccharolytic enzyme is an amylase, where the amylase is selected from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum or Arabidopsis thaliana. In another aspect, the saccharolytic enzyme is an amylase from S. fibuligera glucoamylase (glu-0111-CO).
Another aspect of the invention relates to a method for decreasing cellular glycerol comprising contacting biomass with a recombinant microorganism of the invention. A further aspect of the invention relates to a method for increasing cytosolic formate comprising contacting biomass with a recombinant microorganism of the invention. Another aspect of the invention relates to a process for converting biomass to ethanol comprising contacting biomass with a recombinant microorganism of the invention. In some embodiments, the biomass comprises lignocellulosic biomass. In some embodiments, the lignocellulosic biomass is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof. In some embodiments, the biomass is corn mash or corn starch.
In another aspect, the present invention also describes industrial yeast strains that express enzymes for the production of fuel ethanol from corn starch.



BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
FIG. 1 shows a schematic of high yield metabolism.
FIG. 2 depicts the glycolysis pathway.
FIG. 3 shows a schematic of the glycolysis/fermentation pathway.
FIG. 4 shows a map depicting location of primers used to make marked deletion of GPD1.
FIG. 5 shows a map depicting location of primers used to remove marker from GPD1 locus.
FIG. 6 shows a map depicting location of primers used to make marked deletion of GPD2.
FIG. 7 shows a map depicting location of primers used to remove marker from GPD2 locus.
FIG. 8 shows a map depicting location of primers used to make marked deletion of FDH1.
FIG. 9 shows a map depicting location of primers used to remove marker from FDH1 locus.
FIG. 10 shows a map depicting location of primers used to make marked deletion of FDH2.
FIG. 11 shows a map depicting location of primers used to remove marker from FDH2 locus.
FIG. 12 shows an alignment of PFL enzymes from various organisms.
FIG. 13 shows a graph of formate production over 48 hours.
FIG. 14 shows a graph of formate production at the end of 142 hours in microaerobic and anaerobic conditions.
FIG. 15 shows a graph of the growth of strains of the invention over 72 hours as measured by OD.
FIG. 16 shows a graph of glycerol production (g/L) of strains of the invention over 72 hours.
FIG. 17 shows a graph of ethanol production (g/L) of strains of the invention over 72 hours.
FIG. 18 shows a graph of glucose utilization (g/L) of strains of the invention over 72 hours.
FIG. 19 shows a graph of the growth of a strain of the invention over 142 hours as measured by OD.
FIG. 20 shows a graph of the relative growth rate (mOD/min) of strains of the invention.
FIG. 21 shows a graph of ethanol production (g/L) of strains of the invention after 50 hours of fermentation.
FIG. 22 shows a graph of ethanol production (g/L) of strains of the invention after 50 hours of fermentation.
FIG. 23 shows a graph of glycerol production (g/L) of strains of the invention after 50 hours of fermentation.
FIG. 24 shows a graph of glycerol production (g/L) of strains of the invention after 50 hours of fermentation.
FIG. 25 shows a graph of glucose utilization (g/L), glycerol production (g/L), and ethanol production (g/L) after 72 hours of fermentation.
FIG. 26 shows a diagram depicting integration of E. coli AADHs at the GPD1 locus.
FIG. 27 shows a diagram depicting integration of E. coli AADHs at the FCY1 locus
FIG. 28 shows a schematic diagram of a strategy for PCR construction and integration of KT-MX and NT-MX integration cassettes into both chromosomes of a target loci.
FIG. 29 shows a schematic diagram of strategy used to replace integrated KT-MX and NT-MX selection cassettes with a Mascoma Assembly on both chromosomes of a target loci.
FIG. 30 shows a molecular map and genotyping of MA0370 integrated at the FDH1 site of M3625.
FIG. 31 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0370 site.
FIG. 32 shows a molecular map and genotyping of MA0280 integrated at the FDH2 site of M3625.
FIG. 33 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0280 site.
FIG. 34 shows a molecular map and genotyping of MA0289 integrated at the GPD2 site of M3625.
FIG. 35 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0370 site.
FIG. 36 shows a molecular map and genotyping of MA0317 integrated at the FCY1 site of M3625.
FIG. 37 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0317 site.
FIG. 38 shows a graph depicting the results of a starch assay performed with strains M2390, M2519, M2691, M3498, and M3625.
FIG. 39 shows an anti-peptide Western blot analysis of cell extracts (PflA, PflB, AdhE) and aerobic culture supernatants (AE9).
FIG. 40 shows a graph depicting the results of a formate lyase assay performed with engineered strains M3465, M3625, M3679, M3680, and M2390.
FIG. 41 shows a graph depicting the results of an alcohol dehydrogenase assay performed with engineered strains M3465, M3625, M3679, and M3680.
FIG. 42 shows a graph depicting the results of a glucoamylase activity assay performed with engineered strains M3625 and M3680 using 50 μg/mL AE9 on corn starch at room temperature ( ̃25° C.).
FIG. 43A is a schematic showing insertion of promoters and terminators used to express, GPD2, and B. adolescentis pflA, pflB and adhE at the GPD1 locus in M3624 and M3515.
FIG. 43B is a schematic showing insertion of promoters and terminators used to express GPD1 and B. adolescentis pflA, pflB and adhE at the GPD2 locus in M3624 and M3515.
FIG. 43C is a schematic showing deletion of the FDH1 gene in M3624 and M3515.
FIG. 43D is a schematic showing insertions of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3624 and M3515.
FIG. 44A shows HPLC analysis of formate produced by glycerol reduction strains during fermentation of 28% solids corn mash.
FIG. 44B shows HPLC analysis of glycerol produced by glycerol reduction strains during fermentation of 28% solids corn mash.
FIG. 44C shows HPLC analysis of ethanol produced by glycerol reduction strains during fermentation of 28% solids corn mash.
FIG. 45A is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPD2 locus in M3465.
FIG. 45B is a schematic showing deletion of the FDH1 gene in M3465.
FIG. 45C is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3465.
FIG. 46A is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPD1 locus in M3469.
FIG. 46B is a schematic showing deletion of the FDH1 gene in M3469.
FIG. 46C is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3469.
FIG. 47 shows HPLC analysis of ethanol titers produced by glycerol reduction strains during fermentation of 30% solids corn mash.
FIG. 48 shows HPLC analysis of glycerol titers produced by glycerol reduction strains during fermentation of 30% solids corn mash.
FIG. 49 shows the reverse reaction catalyzed by Bifidobacterium adolecentis bifunctional alcohol dehydrogenase (AdhE) in which ethanol is converted to acetaldehyde.
FIG. 50 shows a diagram of the reaction for the conversion of acetaldehyde to acetyl CoA by AdhE.
FIG. 51 is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPP1 locus in TB655.
FIG. 52 is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPP2 locus in TB656.
FIG. 53 is a graph depicting decreased glycerol formation in strains TB655 and TB656 compared to strain M3297.
FIG. 54 is a graph depicting increased ethanol yield in strains TB655 and TB656 compared to strain M3297.
FIG. 55 is a graph depicting formate production in strains TB655, TB656, and M3297.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “heterologous” when used in reference to a polynucleotide, a gene, a polypeptide, or an enzyme refers to a polynucleotide, gene, polypeptide, or an enzyme not normally found in the host organism. “Heterologous” also 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, e.g., not in its natural location in the organism's genome. The heterologous polynucleotide or gene may be introduced into the host organism by, e.g., gene transfer. A heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
The term “heterologous polynucleotide” is intended to include a polynucleotide that encodes one or more polypeptides or portions or fragments of polypeptides. A heterologous polynucleotide may be derived from any source, e.g., eukaryotes, prokaryotes, viruses, or synthetic polynucleotide fragments.
The terms “promoter” or “surrogate promoter” is intended to include a polynucleotide that can transcriptionally control a gene-of-interest that it does not transcriptionally control in nature. In certain embodiments, the transcriptional control of a surrogate promoter results in an increase in expression of the gene-of-interest. In certain embodiments, a surrogate promoter is placed 5′ to the gene-of-interest. A surrogate promoter may be used to replace the natural promoter, or may be used in addition to the natural promoter. A surrogate promoter may be endogenous with regard to the host cell in which it is used, or it may be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used.
The terms “gene(s)” or “polynucleotide” or “polynucleotide sequence(s)” are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene may, for example, be in the form of linear DNA. In certain embodiments, the gene or polynucleotide is involved in at least one step in the bioconversion of biomass to, e.g., ethanol. Accordingly, the term is intended to include any gene encoding a polypeptide, such as the enzymes acetate kinase (ACK), phosphotransacetylase (PTA), lactate dehydrogenase (LDH), pyruvate formate lyase (PFL), aldehyde dehydrogenase (ADH) and/or alcohol dehydrogenase (ADH), acetyl-CoA transferase (ACS), acetaldehyde dehydrogenase (ACDH), acetaldehyde/alcohol dehydrogenase (AADH), glycerol-3-phosphate dehydrogenase (GPD), glycerol 3-phosphatase (GPP), acetyl-CoA synthetase, thiolase, CoA transferase, acetoacetate decarboxylase, alcohol acetyltransferase enzymes in the D-xylose pathway, such as xylose isomerase and xylulokinase, enzymes in the L-arabinose pathway, such as L-arabinose isomerase and L-ribulose-5-phosphate 4-epimerase. The term gene is also intended to cover all copies of a particular gene, e.g., all of the DNA sequences in a cell encoding a particular gene product.
The term “transcriptional control” is intended to include the ability to modulate gene expression at the level of transcription. In certain embodiments, transcription, and thus gene expression, is modulated by replacing or adding a surrogate promoter near the 5′ end of the coding region of a gene-of-interest, thereby resulting in altered gene expression. In certain embodiments, the transcriptional control of one or more genes is engineered to result in the optimal expression of such genes, e.g., in a desired ratio. The term also includes inducible transcriptional control as recognized in the art.
The term “expression” is intended to include the expression of a gene at least at the level of mRNA production.
The term “expression product” is intended to include the resultant product, e.g., a polypeptide, of an expressed gene.
The term “increased expression” is intended to include an alteration in gene expression at least at the level of increased mRNA production and, preferably, at the level of polypeptide expression. The term “increased production” is intended to include an increase in the amount of a polypeptide expressed, in the level of the enzymatic activity of the polypeptide, or a combination thereof, as compared to the native production of, or the enzymatic activity, of the polypeptide.
The terms “activity,” “activities,” “enzymatic activity,” and “enzymatic activities” are used interchangeably and are intended to include any functional activity normally attributed to a selected polypeptide when produced under favorable conditions. Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide. The polypeptide produced by a host cell and having enzymatic activity may be located in the intracellular space of the cell, cell-associated, secreted into the extracellular milieu, or a combination thereof. Techniques for determining total activity as compared to secreted activity are described herein and are known in the art.
The term “xylanolytic activity” is intended to include the ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses.
The term “arabinolytic activity” is intended to include the ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses.
The term “cellulolytic activity” is intended to include the ability to hydrolyze glycosidic linkages in oligohexoses and polyhexoses. Cellulolytic activity may also include the ability to depolymerize or debranch cellulose and hemicellulose.
As used herein, the term “lactate dehydrogenase” or “LDH” is intended to include the enzymes capable of converting pyruvate into lactate. It is understood that LDH can also catalyze the oxidation of hydroxybutyrate. LDH includes those enzymes that correspond to Enzyme Commission Number 1.1.1.27.
As used herein the term “alcohol dehydrogenase” or “ADH” is intended to include the enzymes capable of converting acetaldehyde into an alcohol, such as ethanol. ADH also includes the enzymes capable of converting acetone to isopropanol. ADH includes those enzymes that correspond to Enzyme Commission Number 1.1.1.1.
As used herein, the term “phosphotransacetylase” or “PTA” is intended to include the enzymes capable of converting acetyl-phosphate into acetyl-CoA. PTA includes those enzymes that correspond to Enzyme Commission Number 2.3.1.8.
As used herein, the term “acetate kinase” or “ACK” is intended to include the enzymes capable of converting acetate into acetyl-phosphate. ACK includes those enzymes that correspond to Enzyme Commission Number 2.7.2.1.
As used herein, the term “pyruvate formate lyase” or “PFL” is intended to include the enzymes capable of converting pyruvate into acetyl-CoA and formate. PFL includes those enzymes that correspond to Enzyme Commission Number 2.3.1.54.
As used herein, the term “formate dehydrogenase” or “FDH” is intended to include the enzymes capable of converting formate and NAD+ to NADH and CO2. FDH includes those enzymes that correspond to Enzyme Commission Number 1.2.1.2.
As used herein, the term “acetaldehyde dehydrogenase” or “ACDH” is intended to include the enzymes capable of converting acetyl-CoA to acetaldehyde. ACDH includes those enzymes that correspond to Enzyme Commission Number 1.2.1.3.
As used herein, the term “acetaldehyde/alcohol dehydrogenase” is intended to include the enzymes capable of converting acetyl-CoA to ethanol. Acetaldehyde/alcohol dehydrogenase includes those enzymes that correspond to Enzyme Commission Numbers 1.2.1.10 and 1.1.1.1.
As used herein, the term “glycerol-3-phosphate dehydrogenase” or “GPD” is intended to include the enzymes capable of converting dihydroxyacetone phosphate to glycerol-3-phosphate. GPD includes those enzymes that correspond to Enzyme Commission Number 1.1.1.8.
As used herein, the term “glycerol 3-phosphatase” or “GPP” is intended to include the enzymes capable of converting glycerol 3-phosphate to glycerol. GPP includes those enzymes that correspond to Enzyme Commission Number 3.1.3.21.
As used herein, the term “acetyl-CoA synthetase” or “ACS” is intended to include the enzymes capable of converting acetate to acetyl-CoA. Acetyl-CoA synthetase includes those enzymes that correspond to Enzyme Commission Number 6.2.1.1.
As used herein, the term “thiolase” is intended to include the enzymes capable of converting acetyl-CoA to acetoacetyl-CoA. Thiolase includes those enzymes that correspond to Enzyme Commission Number 2.3.1.9.
As used herein, the term “CoA transferase” is intended to include the enzymes capable of converting acetate and acetoacetyl-CoA to acetoacetate and acetyl-CoA. CoA transferase includes those enzymes that correspond to Enzyme Commission Number 2.8.3.8.
As used herein, the term “acetoacetate decarboxylase” is intended to include the enzymes capable of converting acetoacetate to acetone and carbon dioxide. Acetoacetate decarboxylase includes those enzymes that correspond to Enzyme Commission Number 4.1.1.4.
As used herein, the term “alcohol acetyltransferase” is intended to include the enzymes capable of converting acetyl-CoA and ethanol to ethyl acetate. Alcohol acetyltransferase includes those enzymes that correspond to Enzyme Commission Number 2.3.1.84.
The term “pyruvate decarboxylase activity” is intended to include the ability of a polypeptide to enzymatically convert pyruvate into acetaldehyde and carbon dioxide (e.g., “pyruvate decarboxylase” or “PDC”). Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide, comprising, e.g., the superior substrate affinity of the enzyme, thermostability, stability at different pHs, or a combination of these attributes. PDC includes those enzymes that correspond to Enzyme Commission Number 4.1.1.1.
The term “ethanologenic” is intended to include the ability of a microorganism to produce ethanol from a carbohydrate as a fermentation product. The term is intended to include, but is not limited to, naturally occurring ethanologenic organisms, ethanologenic organisms with naturally occurring or induced mutations, and ethanologenic organisms which have been genetically modified.
The terms “fermenting” and “fermentation” are intended to include the enzymatic process (e.g., cellular or acellular, e.g., a lysate or purified polypeptide mixture) by which ethanol is produced from a carbohydrate, in particular, as a product of fermentation.
The term “secreted” is intended to include the movement of polypeptides to the periplasmic space or extracellular milieu. The term “increased secretion” is intended to include situations in which a given polypeptide is secreted at an increased level (i.e., in excess of the naturally-occurring amount of secretion). In certain embodiments, the term “increased secretion” refers to an increase in secretion of a given polypeptide that is at least about 10% or at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, as compared to the naturally-occurring level of secretion.
The term “secretory polypeptide” is intended to include any polypeptide(s), alone or in combination with other polypeptides, that facilitate the transport of another polypeptide from the intracellular space of a cell to the extracellular milieu. In certain embodiments, the secretory polypeptide(s) encompass all the necessary secretory polypeptides sufficient to impart secretory activity to a Gram-negative or Gram-positive host cell or to a yeast host cell. Typically, secretory proteins are encoded in a single region or locus that may be isolated from one host cell and transferred to another host cell using genetic engineering. In certain embodiments, the secretory polypeptide(s) are derived from any bacterial cell having secretory activity or any yeast cell having secretory activity. In certain embodiments, the secretory polypeptide(s) are derived from a host cell having Type II secretory activity. In certain embodiments, the host cell is a thermophilic bacterial cell. In certain embodiments, the host cell is a yeast cell.
The term “derived from” is intended to include the isolation (in whole or in part) of a polynucleotide segment from an indicated source or the purification of a polypeptide from an indicated source. The term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from or based on a sequence associated with the indicated polynucleotide source.
The term “recombinant microorganism” or “recombinant host cell” is intended to include progeny or derivatives of the recombinant microorganisms of the invention. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny or derivatives may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
By “thermophilic” is meant an organism that thrives at a temperature of about 45° C. or higher.
By “mesophilic” is meant an organism that thrives at a temperature of about 20-45° C.
The term “organic acid” is art-recognized. “Organic acid,” as used herein, also includes certain organic solvents such as ethanol. The term “lactic acid” refers to the organic acid 2-hydroxypropionic acid in either the free acid or salt form. The salt form of lactic acid is referred to as “lactate” regardless of the neutralizing agent, i.e., calcium carbonate or ammonium hydroxide. The term “acetic acid” refers to the organic acid methanecarboxylic acid, also known as ethanoic acid, in either free acid or salt form. The salt form of acetic acid is referred to as “acetate.”
Certain embodiments of the present invention provide for the “insertion,” (e.g., the addition, integration, incorporation, or introduction) of certain genes or particular polynucleotide sequences within thermophilic or mesophilic microorganisms, which insertion of genes or particular polynucleotide sequences may be understood to encompass “genetic modification(s)” or “transformation(s)” such that the resulting strains of said thermophilic or mesophilic microorganisms may be understood to be “genetically modified” or “transformed.” In certain embodiments, strains may be of bacterial, fungal, or yeast origin.
Certain embodiments of the present invention provide for the “inactivation” or “deletion” of certain genes or particular polynucleotide sequences within thermophilic or mesophilic microorganisms, which “inactivation” or “deletion” of genes or particular polynucleotide sequences may be understood to encompass “genetic modification(s)” or “transformation(s)” such that the resulting strains of said thermophilic or mesophilic microorganisms may be understood to be “genetically modified” or “transformed.” In certain embodiments, strains may be of bacterial, fungal, or yeast origin.
The term “consolidated bioprocessing” or “CBP” refers to biomass processing schemes involving enzymatic or microbial hydrolysis that commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (amylases, cellulases, and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called CBP, which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.
The term “CBP organism” is intended to include microorganisms of the invention, e.g., microorganisms that have properties suitable for CBP.
In one aspect of the invention, the genes or particular polynucleotide sequences are inserted to activate the activity for which they encode, such as the expression of an enzyme. In certain embodiments, genes encoding enzymes in the metabolic production of ethanol, e.g., enzymes that metabolize pentose and/or hexose sugars, may be added to a mesophilic or thermophilic organism. In certain embodiments of the invention, the enzyme may confer the ability to metabolize a pentose sugar and be involved, for example, in the D-xylose pathway and/or L-arabinose pathway.
In one aspect of the invention, the genes or particular polynucleotide sequences are partially, substantially, or completely deleted, silenced, inactivated, or down-regulated in order to inactivate the activity for which they encode, such as the expression of an enzyme. Deletions provide maximum stability because there is no opportunity for a reverse mutation to restore function. Alternatively, genes can be partially, substantially, or completely deleted, silenced, inactivated, or down-regulated by insertion of nucleic acid sequences that disrupt the function and/or expression of the gene (e.g., P1 transduction or other methods known in the art). The terms “eliminate,” “elimination,” and “knockout” are used interchangeably with the terms “deletion,” “partial deletion,” “substantial deletion,” or “complete deletion.” In certain embodiments, strains of thermophilic or mesophilic microorganisms of interest may be engineered by site directed homologous recombination to knockout the production of organic acids. In still other embodiments, RNAi or antisense DNA (asDNA) may be used to partially, substantially, or completely silence, inactivate, or down-regulate a particular gene of interest.
In certain embodiments, the genes targeted for deletion or inactivation as described herein may be endogenous to the native strain of the microorganism, and may thus be understood to be referred to as “native gene(s)” or “endogenous gene(s).” An organism is in “a native state” if it has not been genetically engineered or otherwise manipulated by the hand of man in a manner that intentionally alters the genetic and/or phenotypic constitution of the organism. For example, wild-type organisms may be considered to be in a native state. In other embodiments, the gene(s) targeted for deletion or inactivation may be non-native to the organism.
Similarly, the enzymes of the invention as described herein can be endogenous to the native strain of the microorganism, and can thus be understood to be referred to as “native” or “endogenous.”
The term “upregulated” means increased in activity, e.g., increase in enzymatic activity of the enzyme as compared to activity in a native host organism.
The term “downregulated” means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in a native host organism.
The term “activated” means expressed or metabolically functional.
The term “adapted for growing” means selection of an organism for growth under conditions in which the organism does not otherwise grow or in which the organism grows slowly or minimally. Thus, an organism that is said to be adapted for growing under the selected condition, grows better than an organism that has not been adapted for growing under the selected conditions. Growth can be measured by any methods known in the art, including, but not limited to, measurement of optical density or specific growth rate.
The term “carbohydrate source” is intended to include any source of carbohydrate including, but not limited to, biomass or carbohydrates, such as a sugar or a sugar alcohol. “Carbohydrates” include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose, arabinose, or ribose), sugar derivatives (e.g., sorbitol, glycerol, galacturonic acid, rhamnose, xylitol), disaccharides (e.g., sucrose, cellobiose, maltose, or lactose), oligosaccharides (e.g., xylooligomers, cellodextrins, or maltodextrins), and polysaccharides (e.g., xylan, cellulose, starch, mannan, alginate, or pectin).
As used herein, an “amylolytic enzyme” can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis. The term “amylase” refers to an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (α-amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. All amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. Some amylases, such as γ-amylase (glucoamylase), also act on α-1,6-glycosidic bonds. Amylase enzymes include α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), and γ-amylase (EC 3.2.1.3). The α-amylases are calcium metalloenzymes, unable to function in the absence of calcium. By acting at random locations along the starch chain, α-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin. Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme and its optimum pH is about 6.7-7.0. Another form of amylase, β-amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Many microbes produce amylase to degrade extracellular starches. In addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, γ-amylase will cleave α(1-6) glycosidic linkages. Another amylolytic enzyme is alpha-glucosidase that acts on maltose and other short malto-oligosaccharides produced by alpha-, beta-, and gamma-amylases, converting them to glucose. Another amylolytic enzyme is pullulanase. Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan is regarded as a chain of maltotriose units linked by alpha-1,6-glycosidic bonds. Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase (Debranching enzyme). Another amylolytic enzyme, isopullulanase, hydrolyses pullulan to isopanose (6-alpha-maltosylglucose).
Isopullulanase (EC 3.2.1.57) is also known as pullulan 4-glucanohydrolase. An “amylase” can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis, including α-amylase, -amylase, glucoamylase, pullulanase, isopullulanase, and alpha-glucosidase.
As used herein, a “saccharolytic enzyme” can be any enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes.
Biomass
Biomass can include any type of biomass known in the art or described herein. For example, biomass can include, but is not limited to, starch, sugar, and lignocellulosic materials. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, or cane. The terms “lignocellulosic material,” “lignocellulosic substrate,” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and/or agricultural residues, forestry residues and/or forestry wastes, paper-production sludge and/or waste paper sludge, waste-water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants, and sugar-processing residues. The terms “hemicellulosics,” “hemicellulosic portions,” and “hemicellulosic fractions” mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan, and galactoglucomannan, inter alia), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan), and proteoglycans (e.g., arabinogalactan-protein, extensin, and proline-rich proteins).
In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, Agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.
Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making, and is typically removed from process wastewater in a primary clarifier. At a disposal cost of $30/wet ton, the cost of sludge disposal equates to $5/ton of paper that is produced for sale. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol. Processes provided by the present invention are widely applicable. Moreover, the saccharification and/or fermentation products may be used to produce ethanol or higher value added chemicals, such as organic acids, aromatics, esters, acetone and polymer intermediates.
Glycerol Reduction
Anaerobic growth conditions require the production of endogenouse electron acceptors, such as the coenzyme nicotinamide adenine dinucleotide (NAD+). In cellular redox reactions, the NAD+/NADH couple plays a vital role as a reservoir and carrier of reducing equivalents. Ansell, R., et al., EMBO J. 16:2179-87 (1997). Cellular glycerol production, which generates an NAD+, serves as a redox valve to remove excess reducing power during anaerobic fermentation in yeast. Glycerol production is, however, an energetically wasteful process that expends ATP and results in the loss of a reduced three-carbon compound. Ansell, R., et al., EMBO J. 16:2179-87 (1997). To generate glycerol from a starting glucose molecule, glycerol 3-phosphate dehydrogenase (GPD) reduces dihydroxyacetone phosphate to glycerol 3-phosphate and glycerol 3-phosphatase (GPP) dephosphorylates glycerol 3-phosphate to glycerol. Despite being energetically wasteful, glycerol production is a necessary metabolic process for anaerobic growth as deleting GPD activity completely inhibits growth under anaeroblic conditions. See Ansell, R., et al., EMBO J. 16:2179-87 (1997).
GPD is encoded by two isogenes, gpd1 and gpd2. GPD1 encodes the major isoform in anaerobically growing cells, while GPD2 is required for glycerol production in the absence of oxygen, which stimulates its expression. Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001). The first step in the conversion of dihydroxyacetone phosphate to glycerol by GPD is rate controlling. Guo, Z. P., et al., Metab. Eng. 13:49-59 (2011). GPP is also encoded by two isogenes, gpp1 and gpp2. The deletion of GPP genes arrests growth when shifted to anaerobic conditions, demonstrating that GPP is important for cellular tolerance to osmotic and anaerobic stress. See Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001).
Because glycerol is a major by-product of anaerobic production of ethanol, many efforts have been made to delete cellular production of glycerol. However, because of the reducing equivalents produced by glycerol synthesis, deletion of the glycerol synthesis pathway cannot be done without compensating for this valuable metabolic function. Attempts to delete glycerol production and engineer alternate electron acceptors have been made. Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996); Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010). Lidén and Medina both deleted the gpd1 and gpd2 genes and attempted to bypass glycerol formation using additional carbon sources. Lidén engineered a xylose reductase from Pichia stipitis into an S. cerevisiae gpd1/2 deletion strain. The xylose reductase activity facilitated the anaerobic growth of the glycerol-deleted strain in the presence of xylose. See Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996). Medina engineered an acetylaldehyde dehydrogenase, mhpF, from E. coli into an S. cerevisiae gpd1/2 deletion strain to convert acetyl-CoA to acetaldehyde. The acetylaldehyde dehydrogenase activity facilitated the anaerobic growth of the glycerol-deletion strain in the presence of acetic acid but not in the presence of glucose as the sole source of carbon. Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010); see also EP 2277989. Medina noted several issues with the mhpF-containing strain that needed to be addressed before implementing industrially, including significantly reduced growth and product formation rates than yeast comprising GPD1 and GPD2.
Additional attempts to redirect flux from glycerol to ethanol have included the engineering of a non-phosphorylating NADP+-dependent glyceraldehydes-3-phosphate dehydrogenase (GAPN) into yeast, either with or without the simultaneous knockout of GPD1. Bro, C., et al., Metab. Eng. 8:102-111 (2006); U.S. Patent Appl. Pub. No. US2006/0257983; Guo, Z. P., et al., Metab. Eng. 13:49-59 (2011). However, other cellular mechanisms exist to control the production and accumulation of glycerol, including glycerol exporters such as FPS1, that do not require the engineering of alternate NADP+/NADPH coupling or deletion of glycerol synthesis genes. Tamás, M. J., et al., Mol. Microbiol. 31:1087-1004 (1999).
FPS1 is a channel protein located in the plasma membrane that controls the accumulation and release of glycerol in yeast osmoregulation. Null mutants of this strain accumulate large amounts of intracellular glycerol, grow much slower than wild-type, and consume the sugar substrate at a slower rate. Tamás, M. J., et al., Mol. Microbiol. 31:1087-1004 (1999). Despite slower growth under anaerobic conditions, an fps1Δ strain can serve as an alternative to eliminating NAD+-dependant glycerol activity. An fps1Δ strain has reduced glycerol formation yet has a completely functional NAD+-dependant glycerol synthesis pathway. Alternatively, rather than deleting endogenous FPS1, constitutively active mutants of FPS1 or homologs from other organisms can be used to regulate glycerol synthesis while keep the NAD+-dependant glycerol activity intact. In embodiments of the invention that modulate FPS1, the recombinant host cells can still synthesize and retain glycerol and achieve improved robustness relative to strains that are unable to make glycerol.
An example FPS1 sequence from S. cerevisiae is shown below.
S. cerevisiae FPS1 (nucleotide; coding sequence underlined; SEQ ID NO: 1):







ttgacggcagttctcatagcatctcaaagcaatagcagtgcaaaagtaca


 


taaccgtaggaaggtacgcggtaggtatttgagttcgttggtggttatcc


 


tccgcaaggcgcttcggcggttatttgttgatagtcgaagaacaccaaaa


 


aaaatgctgttattgctttctccgtaaacaataaaacccggtagcgggat


 


aacgcggctgatgcttttatttaggaaggaatacttacattatcatgaga


 


acattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggca


 


gcaacggactgagggacggagagagttacggcataagaagtagtaggaga


 


gcagagtgtcataaagttatattattctcgtcctaaagtcaattagttct


 


gttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaa


 


gaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatg


 


tgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagat


 


ctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtga


 


atccggagacggcaagattgcccggccctttttgcggaaaagataaaaca


 


agatatattgcacttttccaccaagaaaaacaggaagtggattaaaaaat


 


caacaaagtataacgcctattgtcccaataagcgtcggttgttcttcttt


 


attattttaccaagtacgctcgagggtacattctaatgcattaaaagaca


 


tgagtaatcctcaaaaagctctaaacgactttctgtccagtgaatctgtt


 


catacacatgatagttctaggaaacaatctaataagcagtcatccgacga


 


aggacgctcttcatcacaaccttcacatcatcactctggtggtactaaca


 


acaataataacaataataataataataataacagtaacaacaacaacaac


 


ggcaacgatgggggaaatgatgacgactatgattatgaaatgcaagatta


 


tagaccttctccgcaaagtgcgcggcctactcccacgtatgttccacaat


 


attctgtagaaagtgggactgctttcccgattcaagaggttattcctagc


 


gcatacattaacacacaagatataaaccataaagataacggtccgccgag


 


tgcaagcagtaatagagcattcaggcctagagggcagaccacagtgtcgg


 


ccaacgtgcttaacattgaagatttttacaaaaatgcagacgatgcgcat


 


accatcccggagtcacatttatcgagaaggagaagtaggtcgagggctac


 


gagtaatgctgggcacagtgccaatacaggcgccacgaatggcaggacta


 


ctggtgcccaaactaatatggaaagcaatgaatcaccacgtaacgtcccc


 


attatggtgaagccaaagacattataccagaaccctcaaacacctacagt


 


cttgccctccacataccatccaattaataaatggtcttccgtcaaaaaca


 


cttatttgaaggaatttttagccgagtttatgggaacaatggttatgatt


 


attttcggtagtgctgttgtttgtcaggtcaatgttgctgggaaaataca


 


gcaggacaatttcaacgtggctttggataaccttaacgttaccgggtctt


 


ctgcagaaacgatagacgctatgaagagtttaacatccttggtttcatcc


 


gttgcgggcggtacctttgatgatgtggcattgggctgggctgctgccgt


 


ggtgatgggctatttctgcgctggtggtagtgccatctcaggtgctcatt


 


tgaatccgtctattacattagccaatttggtgtatagaggttttcccctg


 


aagaaagttccttattactttgctggacaattgatcggtgccttcacagg


 


cgctttgatcttgtttatttggtacaaaagggtgttacaagaggcatata


 


gcgattggtggatgaatgaaagtgttgcgggaatgttttgcgtttttcca


 


aagccttatctaagttcaggacggcaatttttttccgaatttttatgtgg


 


agctatgttacaagcaggaacatttgcgctgaccgatccttatacgtgtt


 


tgtcctctgatgttttcccattgatgatattattttgattttcattatca


 


atgcttccatggcttatcagacaggtacagcaatgaatttggctcgtgat


 


ctgggcccacgtcttgcactatatgcagttggatttgatcataaaatgct


 


ttgggtgcatcatcatcatttcttttgggttcccatggtaggcccattta


 


ttggtgcgttaatgggggggttggtttacgatgtctgtatttatcagggt


 


catgaatctccagtcaactggtctttaccagtttataaggaaatgattat


 


gagagcctggtttagaaggcctggttggaagaagagaaatagagcaagaa


 


gaacatcggacctgagtgacttctcatacaataacgatgatgatgaggaa


 


tttggagaaagaatggctcttcaaaagacaaagaccaagtcatctatttc


 


agacaacgaaaatgaagcaggagaaaagaaagtgcaatttaaatctgttc


 


agcgcggcaaaagaacgtttggtggtataccaacaattcttgaagaagaa


 


gattccattgaaactgcttcgctaggtgcgacgacgactgattctattgg


 


gttatccgacacatcatcagaagattcgcattatggtaatgctaagaagg


 


taacatgagaaaacagacaagaaaaagaaacaaataatatagactgatag


 


aaaaaaatactgcttactaccgccggtataatatatatatatatatatat


 


ttacatagatgattgcatagtgttttaaaaagctttcctaggttaagcta


 


tgaatcttcataacctaaccaactaaatatgaaaatactgacccatcgtc


 


ttaagtaagttgacatgaactcagcctggtcacctactatacatgatgta


 


tcgcatggatggaaagaataccaaacgctaccttccaggttaatgatagt


 


atccaaacctagttggaatttgccttgaacatcaagcagcgattcgatat


 


cagttgggagcatcaatttggtcattggaataccatctatgcttttctcc


 


tcccatattcgcaaaagtagtaagggctcgttatatacttttgaatatgt


 


aagatataattctatatgatttagtaatttattttctatacgctcagtat


 


ttttctgcagttgtcgagtaggtattaaacgcaaaagaagtccatccttt


 


tcatcattcaaatggacatcttggcaaagggcccagttatggaaaatctg


 


ggagtcatacaacgattgcagttggctatgccactcctggtaaggaatca


 


tcaagtctgataattctgtttatagccctttttttttttttttcatggtg


 


ttctcttctcattgcttttcaattttaagttcgttacctttcatatagag


 


tttcttaacagaaatttcacaacgaaaatataattaactacaggca


 






S. cerevisiae FPS1 (amino acid; SEQ ID NO:2):
Pyruvate Formate Lyase (PFL)
The conversion of the pyruvate to acetyl-CoA and formate is performed by pyruvate formate lyase (PFL). In E. coli, PFL is the primary enzyme responsible for the production of formate. PFL is a dimer of PflB that requires the activating enzyme PflAE, which is encoded by pflA, radical S-adenosylmethionine, and a single electron donor. See Waks, Z., and Silver, P. A., Appl. Env. Microbiol. 75:1867-1875 (2009). Waks and Silver engineered strains of S. cerevisiae to secrete formate by the addition of PFL and AdhE from E. coli and deletion of endogenous formate dehydrogenases and to produce hydrogen in a two-step process using E. coli. Waks and Silver, however, did not combine formate production with the removal of glycerol formation, and the use of formate as an alternate electron acceptor for the reduction of glycerol was not proposed or evaluated.
PFL enzymes for use in the recombinant host cells of the invention can come from a bacterial or eukaryotic source. Examples of bacterial PFL include, but are not limited to, Bacillus licheniformis DSM13, Bacillus licheniformis ATCC14580, Streptococcus thermophilus CNRZ1066, Streptococcus thermophilus LMG18311, Streptococcus thermophilus LMD-9, Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_2598), Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_3313), Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_2695), Lactobacillus plantarum JDM1 (Gene Accession No. JDM1_2087), Lactobacillus casei b123, Lactobacillus casei ATCC 334, Bifidobacterium adolescentis, Bifidobacterium longum NCC2705, Bifidobacterium longum DJO10A, Bifidobacterium animalis DSM 10140, Clostridium cellulolyticum, or Escherichia coli. Additional PFL enzymes may be from the PFL1 family, the RNR pfl superfamily, or the PFL2 superfamily.
pflA Sequences from Bacteria Include:

    
    
        Bacillus licheniformis DSM13 (nucleotide; SEQ ID NO:3):
        Bacillus licheniformis DSM13 (amino acid; SEQ ID NO:4):
        Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:5):
        Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:6):
        Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID N0:7):
        Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:8):
        Streptococcus thermophilus LMG18311 (nucleotide; SEQ ID N0:9):
        Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO:10):
        Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO:11):
        Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO:12):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_2596) (nucleotide; SEQ ID NO:13):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_2596) (amino acid; SEQ ID NO:14):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_3314) (nucleotide; SEQ ID NO:15):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_3314) (amino acid; SEQ ID NO:16):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2660) (nucleotide; SEQ ID NO:17):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2660) (amino acid; SEQ ID NO:18):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2085) (nucleotide; SEQ ID NO:19):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2085) (amino acid; SEQ ID NO:20):
        Lactobacillus casei b123 (nucleotide; SEQ ID NO:21):
        Lactobacillus casei b123 (amino acid; SEQ ID NO:22):
        Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:23):
        Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:24):
        Bifidobacterium adolescentis (nucleotide; SEQ ID NO:25):
        Bifidobacterium adolescentis (amino acid; SEQ ID NO:26):
        Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:27):
        Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:28):
        Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:29):
        Bifidobacterium longum DJO10A (amino acid; SEQ ID NO:30):
        Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:31):
        Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:32):
        Clostridium cellulolyticum (nucleotide; SEQ ID NO:33):
        Clostridium cellulolyticum (amino acid; SEQ ID NO:34):
        Escherichia coli (nucleotide; SEQ ID NO:35):
        Escherichia coli (amino acid; SEQ ID NO:36):
    
    


pflB sequences from bacteria include:

    
    
        Bacillus licheniformis DSM13 (nucleotide; SEQ ID NO:37):
        Bacillus licheniformis DSM13 (amino acid; SEQ ID NO:38):
        Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:39):
        Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:40):
        Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID NO:41):
        Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:42):
        Streptococcus thermophilus LMG18311 (nucleotide; SEQ ID NO:43):
        Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO:44):
        Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO:45):
        Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO:46):
        Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_2598) (nucleotide; SEQ ID NO:47):
        Lactobacillus plantarum WCFS1 (Gene Accession No. 1p_2598) (amino acid; SEQ ID NO:48):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_3313) (nucleotide; SEQ ID NO:49):
        Lactobacillus plantarum WCFS1 (Gene Accession No: 1p_3313) (amino acid; SEQ ID NO:50):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM12695) (nucleotide; SEQ ID NO:51):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM12695) (amino acid; SEQ ID NO:52):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2087) (nucleotide; SEQ ID NO:53):
        Lactobacillus plantarum JDM1 (Gene Accession No: JDM1_2087) (amino acid; SEQ ID NO:54):
        Lactobacillus casei b123 (nucleotide; SEQ ID NO:55):
        Lactobacillus casei b123 (amino acid; SEQ ID NO:56):
        Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:57):
        Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:58):
        Bifidobacterium adolescentis (nucleotide; SEQ ID NO:59):
        Bifidobacterium adolescentis (amino acid; SEQ ID NO:60):
        Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:61):
        Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:62):
        Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:63):
        Bifidobacterium longum DJO10A (amino acid; SEQ ID NO:64):
        Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:65):
        Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:66):
        Clostridium cellulolyticum (nucleotide; SEQ ID NO:67):
        Clostridium cellulolyticum (amino acid; SEQ ID NO:68):
        Escherichia coli (nucleotide; SEQ ID NO:69):
        Escherichia coli (amino acid; SEQ ID NO:70):
    
    


Examples of eukaryotic PFL include, but are not limited to, Chlamydomonas reinhardtii PflA1, Piromyces sp. E2, or Neocallimastix frontalis, Acetabularia acetabulum, Haematococcus pluvialis, Volvox carteri, Ostreococcus tauri, Ostreococcus lucimarinus, Micromonas pusilla, Micromonas sp., Porphyra haitanensis, and Cyanophora paradoxa), an opisthokont (Amoebidium parasiticum), an amoebozoan (Mastigamoeba balamuthi), a stramenopile (Thalassiosira pseudonana (2)) and a haptophyte (Prymnesium parvum), M. pusilla, Micromonas sp. O. tauri and O. lucimarinus) an amoebozoan (M. balamuthi), and a stramenopile (T. pseudonana). See Stairs, C. W., et al., “Eukaryotic pyruvate formate lyase and its activating enzyme were acquired laterally from a firmicute,” Mol. Biol. and Evol., published on-line on Feb. 3, 2011, at http://mbe.oxfordjournals.org/.
pflA sequences from eukaryotes include:

    
    
        Chlamydomonas reinhardtii PflA1 (nucleotide; SEQ ID N0:71):
        Chlamydomonas reinhardtii PflA1 (amino acid; SEQ ID N0:72):
        Neocallimastix frontalis (nucleotide; SEQ ID N0:73):
        Neocallimastix frontalis (amino acid; SEQ ID N0:74):
    
    


pfl1 sequences from eukaryotes include:

    
    
        Chlamydomonas reinhardtii PflA (nucleotide; SEQ ID N0:75):
        Chlamydomonas reinhardtii PflA (amino acid; SEQ ID N0:76):
        Piromyces sp. E2 (nucleotide; SEQ ID N0:77):
        Piromyces sp. E2 (amino acid; SEQ ID N0:78):
        Neocallimastix frontalis (nucleotide—partial CDS, missing start; SEQ ID NO:79):
        Neocallimastix frontalis (amino acid—partial CDS, missing start; SEQ ID NO:80):

Acetaldehyde/Alcohol Dehydrogenases

    
    


Engineering of acetaldehyde dehydrogenases, alcohol dehydrogenases, and/or bifunctional acetylaldehyde/alcohol dehydrogenases into a cell can increase the production of ethanol. However, because the production of ethanol is redox neutral, an acetaldehyde/alcohol dehydrogenase activity cannot serve as an alternative for the redox balancing that the production of glycerol provides to a cell in anaerobic metabolism. When Medina attempted to express an acetylaldehyde dehydrogenase, mhpF, from E. coli in an S. cerevisiae gpd1/2 deletion strain, the strain did not grow under anaerobic conditions in the presence of glucose as the sole source of carbon. Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010); see also EP 2277989. Rather, the anaerobic growth of the glycerol-deletion strain required the presence of acetic acid. However, an acetylaldehyde dehydrogenase has not been expressed in combination with PFL or with the recombinant host cells of the invention. Additionally, replacing the endogenous acetylaldehyde dehydrogenase activity with either an improved acetaldehyde dehydrogenase or using a bifunctional acetaldehyde/alcohol dehydrogenase (AADH) can positively affect the in vivo kinetics of the reaction providing for improved growth of the host strain.
AADH enzymes for use in the recombinant host cells of the invention can come from a bacterial or eukaryotic source. Examples of bacterial AADH include, but are not limited to, Clostridium phytofermentans, Escherichia coli, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bacteroides amylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroides suis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri (cattle only), Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus farciminis (swine only), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuterii, Leuconostoc mesenteroides, Pediococcus acidilacticii, Pediococcus pentosaceus, Propionibacterium acidpropionici (cattle only), Propionibacterium freudenreichii, Propionibacterium shermanii, Enterococcus cremoris, Enterococcus diacetylactis, Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, or Enterococcus thermophilus 
AdhE bacterial sequences include:

    
    
        Clostridium phytofermentans (nucleotide; SEQ ID NO:81):
        Clostridium phytofermentans (amino acid; SEQ ID NO:82):
        Escherichia coli (nucleotide; SEQ ID NO:83):
        Escherichia coli (amino acid; SEQ ID NO:84):
        Bifidobacterium adolescentis (amino acid; SEQ ID NO:100):
        Bacillus coagulans (amino acid; SEQ ID NO:101):
        Bacillus licheniformis (amino acid; SEQ ID NO: 102):
        Enterococcus faecium TX1330 (amino acid; SEQ ID NO:103):
    
    


Examples of eukaryotic AdhE include, but are not limited to, Chlamydomonas reinhardtii AdhE, Piromyces sp. E2, or Neocallimastix frontalis. 
AdhE sequences from eukaryotes include:

    
    
        Chlamydomonas reinhardtii AdhE (nucleotide; SEQ ID NO:85):
        Chlamydomonas reinhardtii AdhE (amino acid; SEQ ID NO:86):
        Piromyces sp. E2 (nucleotide; SEQ ID NO:87):
        Piromyces sp. E2 (amino acid; SEQ ID NO:88):

Consolidated Bioprocessing

    
    


Consolidated bioprocessing (CBP) is a processing strategy for cellulosic biomass that involves consolidating into a single process step four biologically-mediated events: enzyme production, hydrolysis, hexose fermentation, and pentose fermentation. Implementing this strategy requires development of microorganisms that both utilize cellulose, hemicellulosics, and other biomass components while also producing a product of interest at sufficiently high yield and concentrations. The feasibility of CBP is supported by kinetic and bioenergetic analysis. See van Walsum and Lynd (1998) Biotech. Bioeng. 58:316.
CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated saccharolytic enzyme production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with saccharolytic enzyme production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed saccharolytic systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring saccharolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-saccharolytic organisms that exhibit high product yields and titers to express a heterologous saccharolytic enzyme system enabling starch, cellulose, and, hemicellulose utilization.
Starch and Cellulose Degradation
The degradation of starch into component sugar units proceeds via amylolytic enzymes. Amylase is an example of an amylolytic enzyme that is present in human saliva, where it begins the chemical process of digestion. The pancreas also makes amylase (alpha amylase) to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylases. Amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds.
Several amylolytic enzymes are implicated in starch hydrolysis. Alpha-amylases (EC 3.2.1.1) (alternate names: 1,4-α-D-glucan glucanohydrolase; glycogenase) are calcium metalloenzymes, i.e., completely unable to function in the absence of calcium. By acting at random locations along the starch chain, alpha-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and “limit dextrin” from amylopectin. Because it can act anywhere on the substrate, alpha-amylase tends to be faster-acting than beta-amylase. Another form of amylase, beta-amylase (EC 3.2.1.2) (alternate names: 1,4-α-D-glucan maltohydrolase; glycogenase; saccharogen amylase) catalyzes the hydrolysis of the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names: Glucan 1,4-α-glucosidase; amyloglucosidase; Exo-1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase). In addition to cleaving the last α(1-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, gamma-amylase will cleave α(1-6) glycosidic linkages.
A fourth enzyme, alpha-glucosidase, acts on maltose and other short malto-oligosaccharides produced by alpha-, beta-, and gamma-amylases, converting them to glucose.
Three major types of enzymatic activities degrade native cellulose. The first type is endoglucanases (1,4-β-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (1,4-β-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-β-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure. The third type are β-glucosidases (β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units.
Even though yeast strains expressing enzymes for the production of fuel ethanol from whole grain or starch have been previously disclosed, the application has not been commercialized in the grain-based fuel ethanol industry, due to the relatively poor ability of the resulting strains to produce/tolerate high levels of ethanol. For example, U.S. Pat. No. 7,226,776 discloses that a polysaccharase enzyme expressing ethanologen can make ethanol directly from carbohydrate polymers, but the maximal ethanol titer demonstrated is 3.9 g/l. U.S. Pat. No. 5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages; however, no commercially relevant titers of ethanol are disclosed.
Heterologous Saccharolytic Enzymes
According to one aspect of the present invention, the expression of heterologous saccharolytic enzymes the recombinant microorganisms of the invention can be used advantageously to produce products such as ethanol from biomass sources. For example, cellulases from a variety of sources can be heterologously expressed to successfully increase efficiency of ethanol production. The saccharolytic enzymes can be from fungi, yeast, bacteria, plant, protozoan or termite sources. In some embodiments, the saccharolytic enzyme is from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum or Arabidopsis thaliana. 
In some embodiments, the cellulase for expression in the recombinant microorganisms of the invention is any cellulase disclosed in Table 4 or Table 7 in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein, or any cellulase suitable for expression in an appropriate host cell. In other embodiments, the amylase for expression in the recombinant microorganisms of the invention is any amylase such as alpha-amylases, beta-amylases, glucoamylases, alpha-glucosidases, pullulanase, or isopullulanase paralogues or orthologues, any amylase disclosed in Tables 15-19, preferably in Table 19, in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein, or any amylase suitable for expression in an appropriate host cell. In some embodiments of the invention, multiple saccharolytic enzymes from a single organism are co-expressed in the same recombinant microorganism. In some embodiments of the invention, multiple saccharolytic enzymes from different organisms are co-expressed in the same recombinant microorganism. In particular, saccharolytic enzymes from two, three, four, five, six, seven, eight, nine or more organisms can be co-expressed in the same recombinant microorganism. Similarly, the invention can encompass co-cultures of yeast strains, wherein the yeast strains express different saccharolytic enzymes. Co-cultures can include yeast strains expressing heterologous saccharolytic enzymes from the same organisms or from different organisms. Co-cultures can include yeast strains expressing saccharolytic enzymes from two, three, four, five, six, seven, eight, nine or more organisms.
Lignocellulases for expression in the recombinant microorganisms of the present invention include both endoglucanases and exoglucanases. Other lignocellulases for expression in the recombinant microorganisms of the invention include accesory enzymes which can act on the lignocellulosic material. The lignocellulases can be, for example, endoglucanases, glucosidases, cellobiohydrolases, xylanases, glucanases, xylosidases, xylan esterases, arabinofuranosidases, galactosidases, cellobiose phosphorylases, cellodextrin phosphorylases, mannanases, mannosidases, xyloglucanases, endoxylanases, glucuronidases, acetylxylanesterases, arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins, pectinases, and feruoyl esterases. In some embodiments, the lignocellulases of the invention can be any suitable enzyme for digesting the desired lignocellulosic material.
In certain embodiments of the invention, the lignocellulase can be an endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase, acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin, pectinase, and feruoyl esterase paralogue or orthologue. In particular embodiments, the lignocellulase is derived from any species named in Tables 4 and 7, in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein.
Xylose Metabolism
Xylose is a five-carbon monosaccharide that can be metabolized into useful products by a variety of organisms. There are two main pathways of xylose metabolism, each unique in the characteristic enzymes they utilize. One pathway is called the “Xylose Reductase-Xylitol Dehydrogenase” or XR-XDH pathway. Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the two main enzymes used in this method of xylose degradation. XR, encoded by the XYL1 gene, is responsible for the reduction of xylose to xylitol and is aided by cofactors NADH or NADPH. Xylitol is then oxidized to xylulose by XDH, which is expressed through the XYL2 gene, and accomplished exclusively with the cofactor NAD+. Because of the varying cofactors needed in this pathway and the degree to which they are available for usage, an imbalance can result in an overproduction of xylitol byproduct and an inefficient production of desirable ethanol. Varying expression of the XR and XDH enzyme levels have been tested in the laboratory in the attempt to optimize the efficiency of the xylose metabolism pathway.
The other pathway for xylose metabolism is called the “Xylose Isomerase” (XI) pathway. Enzyme XI is responsible for direct conversion of xylose into xylulose, and does not proceed via a xylitol intermediate. Both pathways create xylulose, although the enzymes utilized are different. After production of xylulose both the XR-XDH and XI pathways proceed through the enzyme xylulokinase (XK), encoded on gene XKS1, to further modify xylulose into xylulose-5-phosphate where it then enters the pentose phosphate pathway for further catabolism.
Studies on flux through the pentose phosphate pathway during xylose metabolism have revealed that limiting the speed of this step may be beneficial to the efficiency of fermentation to ethanol. Modifications to this flux that may improve ethanol production include a) lowering phosphoglucose isomerase activity, b) deleting the GND1 gene, and c) deleting the ZWF1 gene (Jeppsson et al., Appl. Environ. Microbiol. 68:1604-09 (2002)). Since the pentose phosphate pathway produces additional NADPH during metabolism, limiting this step will help to correct the already evident imbalance between NAD(P)H and NAD cofactors and reduce xylitol byproduct. Another experiment comparing the two xylose metabolizing pathways revealed that the XI pathway was best able to metabolize xylose to produce the greatest ethanol yield, while the XR-XDH pathway reached a much faster rate of ethanol production (Karhumaa et al., Microb Cell Fact. 2007 Feb. 5; 6:5). See also International Publication No. WO2006/009434, incorporated herein by reference in its entirety.
In some embodiments, the recombinant microorganisms of the invention have the ability to metabolize xylose using one or more of the above enzymes.
Arabinose Metabolism
Arabinose is a five-carbon monosaccharide that can be metabolized into useful products by a variety of organisms. L-Arabinose residues are found widely distributed among many heteropolysaccharides of different plant tissues, such as arabinans, arabinogalactans, xylans and arabinoxylans. Bacillus species in the soil participate in the early stages of plant material decomposition, and B. subtilis secretes three enzymes, an endo-arabanase and two arabinosidases, capable of releasing arabinosyl oligomers and L-arabinose from plant cell.
Three pathways for L-arabinose metabolism in microorganisms have been described. Many bacteria, including Escherichia coli, use arabinose isomerase (AraA; E.C. 5.3.1.4), ribulokinase (AraB; E.C. 2.7.1.16), and ribulose phosphate epimerase (AraD; E.C. 5.1.3.4) to sequentially convert L-arabinose to D-xylulose-5-phosphate through L-ribulose and L-ribulose 5-phosphate. See, e.g., Sa-Nogueira I, et al., Microbiology 143:957-69 (1997). The D-xylulose-5-phosphate then enters the pentose phosphate pathway for further catabolism. In the second pathway, L-arabinose is converted to L-2-keto-3-deoxyarabonate (L-KDA) by the consecutive action of enzymes arabinose dehydrogenase (ADH), arabinolactone (AL), and arabinonate dehydratase (AraC). See, e.g., Watanabe, S, et al., J. Biol. Chem. 281: 2612-2623 (2006). L-KDA can be further metabolized in two alternative pathways: 1) L-KDA conversion to 2-ketoglutarate via 2-ketoglutaric semialdehyde (KGSA) by L-KDA dehydratase and KGSA dehydrogenase or 2) L-KDA conversion to pyruvate and glycolaldehyde by L-KDA aldolase. In the third, fungal pathway, L-arabinose is converted to D-xylulose-5-phosphate through L-arabinitol, L-xylulose, and xylitol, by enzymes such as NAD(P)H-dependent aldose reductase (AR), L-arabinitol 4-dehydrogenase (ALDH), L-xylulose reductase (LXR), xylitol dehydrogenase (XyID), and xylulokinase (XyIB). These, and additional proteins involved in arabinose metabolism and regulation may be found at http://www.nmpdr.org/FIG/wiki/rest.cgi/NmpdrPlugin/SeedViewer?page=Subsystems; su bsystem=L-Arabinose_utilization, visited Mar. 21, 2011, which is incorporated by reference herein in its entirety.
AraC protein regulates expression of its own synthesis and the other genes of the Ara system. See Schleif, R., Trends Genet. 16(12):559-65 (2000). In the E. coli, the AraC protein positively and negatively regulates expression of the proteins required for the uptake and catabolism of the sugar L-arabinose. Homologs of AraC, such as regulatory proteins RhaR and RhaS of the rhamnose operon, have been identified that contain regions homologous to the DNA-binding domain of AraC (Leal, T. F. and de Sa-Nogueira, I., FEMS Microbiol Lett. 241(1):41-48 (2004)). Such arabinose regulatory proteins are referred to as the AraC/XylS family. See also, Mota, L. J., et al., Mol. Microbiol. 33(3):476-89 (1999); Mota, L. J., et al., J Bacteriol. 183(14):4190-201 (2001).
In E. coli, the transport of L-arabinose across the E. coli cytoplasmic membrane requires the expression of either the high-affinity transport operon, araFGH, a binding protein-dependent system on the low-affinity transport operon, araE, a proton symporter. Additional arabinose transporters include those identified from K. marxianus and P. guilliermondii, disclosed in U.S. Pat. No. 7,846,712, which is incorporated by reference herein.
In some embodiments, the recombinant microorganisms of the invention have the ability to metabolize arabinose using one or more of the above enzymes.
Microorganisms
The present invention includes multiple strategies for the development of microorganisms with the combination of substrate-utilization and product-formation properties required for CBP. The “native cellulolytic strategy” involves engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer. The “recombinant cellulolytic strategy” involves engineering natively non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system that enables cellulose utilization or hemicellulose utilization or both.
Many bacteria have the ability to ferment simple hexose sugars into a mixture of acidic and pH-neutral products via the process of glycolysis. The glycolytic pathway is abundant and comprises a series of enzymatic steps whereby a six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound pyruvate. This process results in the net generation of ATP (biological energy supply) and the reduced cofactor NADH.
Pyruvate is an important intermediary compound of metabolism. For example, under aerobic conditions pyruvate may be oxidized to acetyl coenzyme A (acetyl-CoA), which then enters the tricarboxylic acid cycle (TCA), which in turn generates synthetic precursors, CO2, and reduced cofactors. The cofactors are then oxidized by donating hydrogen equivalents, via a series of enzymatic steps, to oxygen resulting in the formation of water and ATP. This process of energy formation is known as oxidative phosphorylation.
Under anaerobic conditions (no available oxygen), fermentation occurs in which the degradation products of organic compounds serve as hydrogen donors and acceptors. Excess NADH from glycolysis is oxidized in reactions involving the reduction of organic substrates to products, such as lactate and ethanol. In addition, ATP is regenerated from the production of organic acids, such as acetate, in a process known as substrate level phosphorylation. Therefore, the fermentation products of glycolysis and pyruvate metabolism include a variety of organic acids, alcohols and CO2.
Most facultative anaerobes metabolize pyruvate aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate and acetyl-CoA. Acetyl-CoA is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (ACK) with the co-production of ATP, or reduced to ethanol via acetalaldehyde dehydrogenase (ACDH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re-oxidized to NAD by lactate dehydrogenase (LDH) during the reduction of pyruvate to lactate. NADH can also be re-oxidized by ACDH and ADH during the reduction of acetyl-CoA to ethanol, but this is a minor reaction in cells with a functional LDH.
Host Cells
Host cells useful in the present invention include any prokaryotic or eukaryotic cells; for example, microorganisms selected from bacterial, algal, and yeast cells. Among host cells thus suitable for the present invention are microorganisms, for example, of the genera Aeromonas, Aspergillus, Bacillus, Escherichia, Kluyveromyces, Pichia, Rhodococcus, Saccharomyces and Streptomyces. 
In some embodiments, the host cells are microorganisms. In one embodiment the microorganism is a yeast. According to the present invention the yeast host cell can be, for example, from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia. Yeast species as host cells may include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K lactis, K marxianus, or K. fragilis. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In one particular embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the yeast is a thermotolerant Saccharomyces cerevisiae. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
In some embodiments, the host cell is an oleaginous cell. The oleaginous host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. According to the present invention, the oleaginous host cell can be an oleaginous microalgae host cell. For example, the oleaginous microalgea host cell can be from the genera Thraustochytrium or Schizochytrium. Biodiesel could then be produced from the triglyceride produced by the oleaginous organisms using conventional lipid transesterification processes. In some particular embodiments, the oleaginous host cells can be induced to secrete synthesized lipids. Embodiments using oleaginous host cells are advantegeous because they can produce biodiesel from lignocellulosic feedstocks which, relative to oilseed substrates, are cheaper, can be grown more densely, show lower life cycle carbon dioxide emissions, and can be cultivated on marginal lands.
In some embodiments, the host cell is a thermotolerant host cell. Thermotolerant host cells can be particularly useful in simultaneous saccharification and fermentation processes by allowing externally produced cellulases and ethanol-producing host cells to perform optimally in similar temperature ranges.
Thermotolerant host cells can include, for example, Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenula polymorpha and Kluyveromyces host cells. In some embodiments, the thermotolerant cell is an S. cerevisiae strain, or other yeast strain, that has been adapted to grow in high temperatures, for example, by selection for growth at high temperatures in a cytostat.
In some particular embodiments, the host cell is a Kluyveromyces host cell. For example, the Kluyveromyces host cell can be a K. lactis, K. marxianus, K. blattae, K. phaffii, K. yarrowii, K. aestuarii, K. dobzhanskii, K. wickerhamii K. thermotolerans, or K. waltii host cell. In one embodiment, the host cell is a K. lactis, or K. marxianus host cell. In another embodiment, the host cell is a K. marxianus host cell.
In some embodiments, the thermotolerant host cell can grow at temperatures above about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C. or about 42° C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., or about 43° C., or about 44° C., or about 45° C., or about 50° C.
In some embodiments of the present invention, the thermotolerant host cell can grow at temperatures from about 30° C. to 60° C., about 30° C. to 55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to 55° C. or about 40° C. to 50° C. In some embodiments of the present invention, the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C. to 60° C., about 30° C. to 55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to 55° C. or about 40° C. to 50° C.
In some embodiments, the host cell has the ability to metabolize xylose. Detailed information regarding the development of the xylose-utilizing technology can be found in the following publications: Kuyper M et al. FEMS Yeast Res. 4: 655-64 (2004), Kuyper M et al. FEMS Yeast Res. 5:399-409 (2005), and Kuyper M et al. FEMS Yeast Res. 5:925-34 (2005), which are herein incorporated by reference in their entirety. For example, xylose-utilization can be accomplished in S. cerevisiae by heterologously expressing the xylose isomerase gene, XylA, e.g., from the anaerobic fungus Piromyces sp. E2, overexpressing five S. cerevisiae enzymes involved in the conversion of xylulose to glycolytic intermediates (xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase) and deleting the GRE3 gene encoding aldose reductase to minimise xylitol production.
In some embodiments, the host cell has the ability to metabolize arabinose. For example, arabinose-utilization can be accomplished by heterologously expressing, e.g., one or more of arabinose isomerase, ribulokinase, or ribulose phosphate epimerase.
The host cells can contain antibiotic markers or can contain no antibiotic markers.
In certain embodiments, the host cell is a microorganism that is a species of the genera Thermoanaerobacterium, Thermoanaerobacter, Clostridium, Geobacillus, Saccharococcus, Paenibacillus, Bacillus, Caldicellulosiruptor, Anaerocellum, or Anoxybacillus. In certain embodiments, the host cell is a bacterium selected from the group consisting of: Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter the rmohydrosulfuricus, Thermoanaerobacter ethanolicus, Thermoanaerobacter brocki, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium phytofermentans, Clostridium straminosolvens, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, and Anaerocellum thermophilum. In certain embodiments, the host cell is Clostridium thermocellum, Clostridium cellulolyticum, or Thermoanaerobacterium saccharolyticum. 
Codon Optimized Polynucleotides
The polynucleotides encoding heterologous cellulases can be codon-optimized. As used herein the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism.
The CAI of codon optimized sequences of the present invention corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0. A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of “As” or “Ts” (e.g., runs greater than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can be removed from the sequences if these are known to effect transcription negatively. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes. Examples of such restriction enzyme sites include PacI, AscI, BamHI, BglII, EcoRI and XhoI. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with “second best” codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized.
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 Ter
TGA Ter



TTG Leu (L)
TCG Ser (S)
TAG Ter
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 http://www.kazusa.or.jp/codon/ (visited Feb. 28, 2011), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000,” 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. The table 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
hundred
















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. Codon-optimized coding regions can be designed by various different methods.
In one method, a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 2 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG.
In another method, the actual frequencies of the codons are distributed randomly throughout the coding sequence. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 2 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.
These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method; however, the sequence always encodes the same polypeptide.
When using the methods above, the term “about” is used precisely to account for fractional percentages of codon frequencies for a given amino acid. As used herein, “about” is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less. Using again the example of the frequency of usage of leucine in human genes for a hypothetical polypeptide having 62 leucine residues, the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or “about 5,” i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 UUG codons or “about 8,” i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or “about 8,” i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or “about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or “about 4,” i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or “about 25,” i.e., 24, 25, or 26 CUG codons.
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 VectorNII 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, e.g., the “backtranslation” function at www.entelechon.com/2008/10/backtranslation-tool/(visited Feb. 28, 2011) and the “backtranseq” function available at emboss.bioinformatics.nl/cgi-bin/emboss/backtranseq (visited Feb. 28, 2011). 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.
A number of options are available for synthesizing codon optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence is synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO® vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.
In additional embodiments, a full-length polypeptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire polypeptide, and then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide are made from the original codon-optimized coding region. As would be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advantage of this approach is that synthesizing codon-optimized nucleic acid fragments encoding each fragment, variant, and derivative of a given polypeptide, although routine, would be time consuming and would result in significant expense.
Transposons
To select for foreign DNA that has entered a host it is preferable that the DNA be stably maintained in the organism of interest. With regard to plasmids, there are two processes by which this can occur. One is through the use of replicative plasmids. These plasmids have origins of replication that are recognized by the host and allow the plasmids to replicate as stable, autonomous, extrachromosomal elements that are partitioned during cell division into daughter cells. The second process occurs through the integration of a plasmid onto the chromosome. This predominately happens by homologous recombination and results in the insertion of the entire plasmid, or parts of the plasmid, into the host chromosome. Thus, the plasmid and selectable marker(s) are replicated as an integral piece of the chromosome and segregated into daughter cells. Therefore, to ascertain if plasmid DNA is entering a cell during a transformation event through the use of selectable markers requires the use of a replicative plasmid or the ability to recombine the plasmid onto the chromosome. These qualifiers cannot always be met, especially when handling organisms that do not have a suite of genetic tools.
One way to avoid issues regarding plasmid-associated markers is through the use of transposons. A transposon is a mobile DNA element, defined by mosaic DNA sequences that are recognized by enzymatic machinery referred to as a transposase. The function of the transposase is to randomly insert the transposon DNA into host or target DNA. A selectable marker can be cloned onto a transposon by standard genetic engineering. The resulting DNA fragment can be coupled to the transposase machinery in an in vitro reaction and the complex can be introduced into target cells by electroporation. Stable insertion of the marker onto the chromosome requires only the function of the transposase machinery and alleviates the need for homologous recombination or replicative plasmids.
The random nature associated with the integration of transposons has the added advantage of acting as a form of mutagenesis. Libraries can be created that comprise amalgamations of transposon mutants. These libraries can be used in screens or selections to produce mutants with desired phenotypes. For instance, a transposon library of a CBP organism could be screened for the ability to produce more ethanol, or less lactic acid and/or more acetate.
Native Cellulolytic Strategy
Naturally occurring cellulolytic microorganisms are starting points for CBP organism development via the native strategy. Anaerobes and facultative anaerobes are of particular interest. The primary objective is to engineer the metabolization of biomass to solvents, including but not limited to, acetone, isopropanol, ethyl acetate, or ethanol. Metabolic engineering of mixed-acid fermentations in relation to, for example, ethanol production, has been successful in the case of mesophilic, non-cellulolytic, enteric bacteria. Recent developments in suitable gene-transfer techniques allow for this type of work to be undertaken with cellulolytic bacteria.
Recombinant Cellulolytic Strategy
Non-cellulolytic microorganisms with desired product-formation properties are starting points for CBP organism development by the recombinant cellulolytic strategy. The primary objective of such developments is to engineer a heterologous cellulase system that enables growth and fermentation on pretreated lignocellulose. The heterologous production of cellulases has been pursued primarily with bacterial hosts producing ethanol at high yield (engineered strains of E. coli, Klebsiella oxytoca, and Zymomonas mobilis) and the yeast Saccharomyces cerevisiae. Cellulase expression in strains of K. oxytoca resulted in increased hydrolysis yields—but not growth without added cellulase—for microcrystalline cellulose, and anaerobic growth on amorphous cellulose. Although dozens of saccharolytic enzymes have been functionally expressed in S. cerevisiae, anaerobic growth on cellulose as the result of such expression has not been definitively demonstrated.
Aspects of the present invention relate to the use of thermophilic or mesophilic microorganisms as hosts for modification via the native cellulolytic strategy. Their potential in process applications in biotechnology stems from their ability to grow at relatively high temperatures with attendant high metabolic rates, production of physically and chemically stable enzymes, and elevated yields of end products. Major groups of thermophilic bacteria include eubacteria and archaebacteria. Thermophilic eubacteria include: phototropic bacteria, such as cyanobacteria, purple bacteria, and green bacteria; Gram-positive bacteria, such as Bacillus, Clostridium, Lactic acid bacteria, and Actinomyces; and other eubacteria, such as Thiobacillus, Spirochete, Desulfotomaculum, Gram-negative aerobes, Gram-negative anaerobes, and Thermotoga. Within archaebacteria are considered Methanogens, extreme thermophiles (an art-recognized term), and Thermoplasma. In certain embodiments, the present invention relates to Gram-negative organotrophic thermophiles of the genera Thermus, Gram-positive eubacteria, such as genera Clostridium, and also which comprise both rods and cocci, genera in group of eubacteria, such as Thermosipho and Thermotoga, genera of Archaebacteria, such as Thermococcus, Thermoproteus (rod-shaped), Thermofilum (rod-shaped), Pyrodictium, Acidianus, Sulfolobus, Pyrobaculum, Pyrococcus, Thermodiscus, Staphylothermus, Desulfurococcus, Archaeoglobus, and Methanopyrus. Some examples of thermophilic or mesophilic (including bacteria, procaryotic microorganism, and fungi), which may be suitable for the present invention include, but are not limited to: Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium the rmocellulaseum, Clostridium phytofermentans, Clostridium straminosolvens, Thermoanaerobacterium thermosaccarolyticum, Thermoanaerobacterium saccharolyticum, Thermobacteroides acetoethylicus, Thermoanaerobium brockii, Methanobacterium thermoautotrophicum, Anaerocellum thermophilium, Pyrodictium occultum, Thermoproteus neutrophilus, Thermofilum librum, Thermothrix thioparus, Desulfovibrio thermophilus, Thermoplasma acidophilum, Hydrogenomonas thermophilus, Thermomicrobium roseum, Thermus flavas, Thermus ruber, Pyrococcus jitriosus, Thermus aquaticus, Thermus thermophilus, Chloroflexus aurantiacus, Thermococcus litoralis, Pyrodictium abyssi, Bacillus stearothermophilus, Cyanidium caldarium, Mastigocladus laminosus, Chlamydothrix calidissima, Chlamydothrix penicillata, Thiothrix carnea, Phormidium tenuissimum, Phormidium geysericola, Phormidium subterraneum, Phormidium bijahensi, Oscillatoria filiformis, Synechococcus lividus, Chloroflexus aurantiacus, Pyrodictium brockii, Thiobacillus thiooxidans, Sulfolobus acidocaldarius, Thiobacillus thermophilica, Bacillus stearothermophilus, Cercosukifer hamathensis, Vahlkampfia reichi, Cyclidium citrullus, Dactylaria gallopava, Synechococcus lividus, Synechococcus elongatus, Synechococcus minervae, Synechocystis aquatilus, Aphanocapsa thermalis, Oscillatoria terebriformis, Oscillatoria amphibia, Oscillatoria germinata, Oscillatoria okenii, Phormidium laminosum, Phormidium parparasiens, Symploca thermalis, Bacillus acidocaldarias, Bacillus coagulans, Bacillus thermocatenalatus, Bacillus licheniformis, Bacillus pamilas, Bacillus macerans, Bacillus circulans, Bacillus laterosporus, Bacillus brevis, Bacillus subtilis, Bacillus sphaericus, Desulfotomaculum nigrificans, Streptococcus thermophilus, Lactobacillus thermophilus, Lactobacillus bulgaricus, Bifidobacterium thermophilum, Streptomyces fragmentosporus, Streptomyces the rmonitrificans, Streptomyces thermovulgaris, Pseudonocardia thermophila, Thermoactinomyces vulgaris, Thermoactinomyces sacchari, Thermoactinomyces candidas, Thermomonospora curvata, Thermomonospora viridis, Thermomonospora citrina, Microbispora thermodiastatica, Microbispora aerata, Microbispora bispora, Actinobifida dichotomica, Actinobifida chromogena, Micropolyspora caesia, Micropolyspora faeni, Micropolyspora cectivugida, Micropolyspora cabrobrunea, Micropolyspora thermovirida, Micropolyspora viridinigra, Methanobacterium thermoautothropicum, Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, variants thereof, and/or progeny thereof.
In particular embodiments, the present invention relates to thermophilic bacteria selected from the group consisting of Clostridium cellulolyticum, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum. 
In certain embodiments, the present invention relates to thermophilic bacteria selected from the group consisting of Fervidobacterium gondwanense, Clostridium thermolacticum, Moorella sp., and Rhodothermus marinus. 
In certain embodiments, the present invention relates to thermophilic bacteria of the genera Thermoanaerobacterium or Thermoanaerobacter, including, but not limited to, species selected from the group consisting of: Thermoanaerobacterium thermosulfurigenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus, Thermoanaerobacter brockii, variants thereof, and progeny thereof.
In certain embodiments, the present invention relates to microorganisms of the genera Geobacillus, Saccharococcus, Paenibacillus, Bacillus, and Anoxybacillus, including, but not limited to, species selected from the group consisting of: Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof, and progeny thereof.
In certain embodiments, the present invention relates to mesophilic bacteria selected from the group consisting of Saccharophagus degradans; Flavobacterium johnsoniae; Fibrobacter succinogenes; Clostridium hungatei; Clostridium phytofermentans; Clostridium cellulolyticum; Clostridium aldrichii; Clostridium termitididis; Acetivibrio cellulolyticus; Acetivibrio ethanolgignens; Acetivibrio multivorans; Bacteroides cellulosolvens; and Alkalibacter saccharofomentans, variants thereof and progeny thereof.
Organism Development Via the Native Cellulolytic Strategy
One approach to organism development for CBP begins with organisms that naturally utilize cellulose, hemicellulose and/or other biomass components, which are then genetically engineered to enhance product yield and tolerance. For example, Clostridium thermocellum is a thermophilic bacterium that has among the highest rates of cellulose utilization reported. Other organisms of interest are xylose-utilizing thermophiles such as Thermoanaerobacterium saccharolyticum and Thermoanaerobacterium the thermosaccharolyticum. Organic acid production may be responsible for the low concentrations of produced ethanol generally associated with these organisms. Thus, one objective is to eliminate production of acetic and lactic acid in these organisms via metabolic engineering. Substantial efforts have been devoted to developing gene transfer systems for the above-described target organisms and multiple C. thermocellum isolates from nature have been characterized. See McLaughlin et al. (2002) Environ. Sci. Technol. 36:2122. Metabolic engineering of thermophilic, saccharolytic bacteria is an active area of interest, and knockout of lactate dehydrogenase in T. saccharolyticum has recently been reported. See Desai et al. (2004) Appl. Microbiol. Biotechnol. 65:600. Knockout of acetate kinase and phosphotransacetylase in this organism is also possible.
Organism Development Via the Recombinant Cellulolytic Strategy
An alternative approach to organism development for CBP involves conferring the ability to grow on lignocellulosic materials to microorganisms that naturally have high product yield and tolerance via expression of a heterologous cellulasic system and perhaps other features. For example, Saccharomyces cerevisiae has been engineered to express over two dozen different saccharolytic enzymes. See Lynd et al. (2002) Microbiol. Mol. Biol. Rev. 66:506.
Whereas cellulosic hydrolysis has been approached in the literature primarily in the context of an enzymatically-oriented intellectual paradigm, the CBP processing strategy requires that cellulosic hydrolysis be viewed in terms of a microbial paradigm. This microbial paradigm naturally leads to an emphasis on different fundamental issues, organisms, cellulasic systems, and applied milestones compared to those of the enzymatic paradigm. In this context, C. thermocellum has been a model organism because of its high growth rate on cellulose together with its potential utility for CBP.
In certain embodiments, organisms useful in the present invention may be applicable to the process known as simultaneous saccharification and fermentation (SSF), which is intended to include the use of said microorganisms and/or one or more recombinant hosts (or extracts thereof, including purified or unpurified extracts) for the contemporaneous degradation or depolymerization of a complex sugar (i.e., cellulosic biomass) and bioconversion of that sugar residue into ethanol by fermentation.
Ethanol Production
According to the present invention, a recombinant microorganism can be used to produce ethanol from biomass, which is referred to herein as lignocellulosic material, lignocellulosic substrate, or cellulosic biomass. Methods of producing ethanol can be accomplished, for example, by contacting the biomass with a recombinant microorganism as described herein, and as described in commonly owned U.S. Patent Application Publication No. 2011/0189744 A1, U.S. Patent Application Publication No. 2011/0312054 A1, U.S. Patent Application Publication No. 2012/0003701, International Appl. No. PCT/US2009/065571, International Appl. No. PCT/US2009/069443, International Appl. No. PCT/US2009/064128, International Appl. No. PCT/IB2009/005881, and PCT/US2009/065571, the contents of each are incorporated by reference herein.
Numerous cellulosic substrates can be used in accordance with the present invention. Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.
It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.
In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a recombinant microorganism of the invention. In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a co-culture comprising yeast cells expressing heterologous cellulases.
In some embodiments, the invention is directed to a method for fermenting cellulose. Such methods can be accomplished, for example, by culturing a host cell or co-culture in a medium that contains insoluble cellulose to allow saccharification and fermentation of the cellulose.
The production of ethanol can, according to the present invention, be performed at temperatures of at least about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., or about 43° C., or about 44° C., or about 45° C., or about 50° C. In some embodiments of the present invention, the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C. to 60° C., about 30° C. to 55° C., about 30° C. to 50° C., about 40° C. to 60° C., about 40° C. to 55° C. or about 40° C. to 50° C.
In some embodiments, methods of producing ethanol can comprise contacting a cellulosic substrate with a recombinant microorganism or co-culture of the invention and additionally contacting the cellulosic substrate with externally produced cellulase enzymes. Exemplary externally produced cellulase enzymes are commercially available and are known to those of skill in the art.
In some embodiments, the methods comprise producing ethanol at a particular rate. For example, in some embodiments, ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter.
In some embodiments, the host cells of the present invention can produce ethanol at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter more than a control strain (e.g., a wild-type strain) and grown under the same conditions. In some embodiments, the ethanol can be produced in the absence of any externally added cellulases.
Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays. Methods of determining ethanol production are within the scope of those skilled in the art from the teachings herein. The U.S. Department of Energy (DOE) provides a method for calculating theoretical ethanol yield. Accordingly, if the weight percentages are known of C6 sugars (i.e., glucan, galactan, mannan), the theoretical yield of ethanol in gallons per dry ton of total C6 polymers can be determined by applying a conversion factor as follows:

(1.11 pounds of C6 sugar/pound of polymeric sugar)×(0.51 pounds of ethanol/pound of sugar)×(2000 pounds of ethanol/ton of C6 polymeric sugar)×(1 gallon of ethanol/6.55 pounds of ethanol)×( 1/100%), wherein the factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as the specific gravity of ethanol at 20° C.

And if the weight percentages are known of C5 sugars (i.e., xylan, arabinan), the theoretical yield of ethanol in gallons per dry ton of total C5 polymers can be determined by applying a conversion factor as follows:

(1.136 pounds of C5 sugar/pound of C5 polymeric sugar)×(0.51 pounds of ethanol/pound of sugar)×(2000 pounds of ethanol/ton of C5 polymeric sugar)×(1 gallon of ethanol/6.55 pounds of ethanol)×( 1/100%), wherein the factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as the specific gravity of ethanol at 20° C.

It follows that by adding the theoretical yield of ethanol in gallons per dry ton of the total C6 polymers to the theoretical yield of ethanol in gallons per dry ton of the total C5 polymers gives the total theoretical yield of ethanol in gallons per dry ton of feedstock.
Applying this analysis, the DOE provides the following examples of theoretical yield of ethanol in gallons per dry ton of feedstock: corn grain, 124.4; corn stover, 113.0; rice straw, 109.9; cotton gin trash, 56.8; forest thinnings, 81.5; harwood sawdust, 100.8; bagasse, 111.5; and mixed paper, 116.2. It is important to note that these are theoretical yields. The DOE warns that depending on the nature of the feedstock and the process employed, actual yield could be anywhere from 60% to 90% of theoretical, and further states that “achieving high yield may be costly, however, so lower yield processes may often be more cost effective.” (Ibid.)
EXEMPLIFICATION
The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
Example 1
Improving Ethanol Yield Through Engineering of Alternate Electron Acceptors
The present Example describes pathways to reduce or eliminate glycerol by engineering alternate electron acceptors in a yeast cell. Glycerol is an undesired by-product of sugar metabolism during anaerobic growth in yeast. The amount of glycerol produced during anaeroblic growth on glucose has been empirically determined by Medina, V G, et al., Appl. Env. Microbiol. 76:190-95 (2010):

56 mmol glucose→1 g biomass+88 mmol ethanol+95 mmol CO2+11 mmol glycerol+1.7 mmol acetate

Assuming glycerol production is primarily for the regeneration of NAD+ for the continuation of glycolysis, a half reaction for glycerol production is (Medina, V G, et al., Appl. Env. Microbiol. 76:190-95 (2010)):

0.5 glucose+NADH+H++ATP→glycerol+NAD++ADP+Pi 

The following pathways describe engineering an alternate electron acceptor for glycerol in the above half reaction, engineering an increase in ethanol yield during anaerobic growth on glucose by using improved enzyme activities for converting glucose to ethanol, and/or deleting endogenous glycerol-producing or glycerol-regulating genes.
1.1 Engineering of a Formate Pathway in Yeast
Production of formate from glucose can provide similar reducing equivalents as glycerol, as shown in the following half reaction:

0.5 glucose+NADH+H++ADP+Pi→formate+NAD++ATP+ethanol

In addition to balancing the redox constraints of the cell, this pathway provides increased ATP yield and results in an overall anaerobic growth equation of:

56 mmol glucose→1 g biomass+99 mmol ethanol+95 mmol CO2+11 mmol formate+1.7 mmol acetate

Engineering in a formate pathway as an alternate electron acceptor to glycerol results in an increase of 12.5% in the theoretical yield of ethanol. Enzymes than can be targeted to engineer such an increase include pyruvate formate lyase (PFL) and formate dehydrogenase (FDH). See FIG. 1.
1.1.1 Expression of PFL
The conversion of pyruvate to acetyl-CoA and formate is performed by PFL. Thus, to produce formate in yeast, a PFL can be expressed. PFLs are common in bacteria from a variety of sources. Vigorous hydrogen producing bacteria, such as from a clostridium, thermoanaerobacterium, or other anaerobic bacteria will likely result in an increased productivity. Examples of PFL include, but are not limited, Bacillus licheniformis, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium adolescentis, Clostridium cellulolyticum, Escherichia coli, Chlamydomonas reinhardtii PflA, Piromyces sp. E2, or Neocallimastix frontalis. See Example 4 and Table 1 below.
1.1.2 Deletion of FDH
To prevent yeast from converting formate to CO2 and NADH, endogeneous FDH genes can be deleted or downregulated. Deleting or downregulating fdh1, fdh2, or both genes can enhance the redox balance and ethanol yield of the recombinant microorganisms of the invention.
1.2 Improving Conversion of Acetyl-CoA to Ethanol
To improve the conversion of acetyl-CoA to ethanol, endogenous yeast genes can be replaced or complimented with either an improved acetaldehyde dehydrogenase (e.g., from C. phytofermentans or other source) to convert acetyl-CoA to acetaldehyde, or a bifunctional acetaldehyde/alcohol dehydrogenase (AADH) to convert acetyl-CoA to acetaldehyde and acetaldehyde to ethanol. By engineering in one or more such enzymes, the in vivo kinetics of the conversion of acetyl-CoA to ethanol can be increased, providing for improved growth of the host strain. The bi-functional alcohol/aldehyde dehydrogenase can come from a variety of microbial sources, including but not limited to E. coli, C. acetobutylicum, T. saccharolyticum, C. thermocellum, C. phytofermentans, Piromyces SP E2, or Bifidobacterium adolescentis. 
1.3 Deletion or Downregulation of Glycerol Pathway
Deleting or altering expression of glycerol formation genes will reduce or block endogenous production of glycerol and may enhance acetate uptake. Deletion of gpd1, gpd2, or both genes and/or deletion of gpp1, gpp2, or both genes may be used to eliminate glycerol formation and enhance ethanol yield. However, the complete elimination of glycerol may not be practical for an industrial process. See Guo, Z P., et al., Metab. Eng. 13:49-59 (2011). Thus, rather than the complete removal of any one, all, or some combination of these glycerol formation genes, one or more of these genes can be altered or downregulated to reduce glycerol formation and enhance ethanol yield.
Example 2
Deletion or Downregulation of Glycerol-Regulating Gene FPS1 to Improve Ethanol Yield
Instead of, or in addition to, downregulating glycerol production through deletion or alteration of glycerol-forming genes, glycerol production can be downregulated by deletion or alteration of a glycerol-regulating gene. FPS1 is an aquaglyceroporin responsible for efflux of glycerol. An fps1Δ strain has reduced glycerol formation yet has a completely functional NAD+-dependant glycerol synthesis pathway. In addition to deletion of FPS1, constitutively active mutants of FPS1 or homologs from other organisms can be expressed to alter glycerol production. Because such FPS1 deletion or alteration strains can still synthesize and retain glycerol, improved robustness may be observed relative to strains that are unable to make glycerol.
Null mutants of an fps1Δ strain grow much slower anaerobically than wild-type due to intracellular glycerol accumulation. Tamás, M. J., et al., Molecular Microbiol. 31(4):1087-1104 (1999). However, preliminary data indicates that expression of a B. adolescentis bifunctional AADH in conjunction with B. adolescentis PFL in an fps1Δ strain can enable anaerobic growth of fps1Δ strain (see Example 7 and FIGS. 22 and 24). Additionally, significantly improved osmotic tolerance was also observed when FPS1 was deleted in glycerol mutant strains containing ADH and PFL alone. Increased resistance to osmotic stress was determined by observation of improved growth of the fps1Δ mutant on several different high osmolarity media including 1M sodium chloride, 1M sorbitol, and 1M xylitol. The fps1Δ mutant was created by marker recycle resulting in a deletion of a large region of the FPS1 coding sequence (sequence of native and deletion below).
Sequence of FPS1 locus (coding sequence is underlined; SEQ ID NO:104):







aacgcggctgatgcttttatttaggaaggaatacttacattatcatgaga


 


acattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggca


 


gcaacggactgagggacggagagagttacggcataagaagtagtaggaga


 


gcagagtgtcataaagttatattattctcgtcctaaagtcaattagttct


 


gttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaa


 


gaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatg


 


tgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagat


 


ctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtga


 


atccggagacggcaagattgcccggcccttttgcggaaaagataaaacaa


 


gatatattgcactttttccaccaagaaaaacaggaagtggattaaaaaat


 


caacaaagtataacgcctattgtcccaataagcgtcggttgttatcttta


 


ttattttaccaagtacgctcgagggtacattctaatgcattaaaagacat


 


gagtaatcctcaaaaagctctaaacgactactgtccagtgaatctgttca


 


tacacatgatagttctaggaaacaatctaataagcagtcatccgacgaag


 


gacgctcttcatcacaaccttcacatcatcactctggtggtactaacaac


 


aataataacaataataataataataataacagtaacaacaacaacaacgg


 


caacgatgggggaaatgatgacgactatgattatgaaatgcaagattata


 


gaccttctccgcaaagtgcgcggcctactcccacgtatgttccacaatat


 


tctgtagaaagtgggactgctttcccgattcaagaggttattcctagcgc


 


atacattaacacacaagatataaaccataaagataacggtccgccgagtg


 


caagcagtaatagagcattcaggcctagagggcagaccacagtgtcggcc


 


aacgtgcttaacattgaagatttttacaaaaatgcagacgatgcgcatac


 


catcccggagtcacatttatcgagaaggagaagtaggtcgagggctacga


 


gtaatgctgggcacagtgccaatacaggcgccacgaatggcaggactact


 


ggtgcccaaactaatatggaaagcaatgaatcaccacgtaacgtccccat


 


tatggtgaagccaaagacattataccagaaccctcaaacacctacagtca


 


gccctccacataccatccaattaataaatggtcttccgtcaaaaacactt


 


atttgaaggaatattagccgagtttatgggaacaatggttatgattatat


 


cggtagtgctgttgtagtcaggtcaatgttgctgggaaaatacagcagga


 


caatttcaacgtggctttggataaccttaacgttaccgggtcttctgcag


 


aaacgatagacgctatgaagagtttaacatccttggtttcatccgttgcg


 


ggcggtaccatgatgatgtggcattgggctgggctgctgccgtggtgatg


 


ggctatttactgcgctggtggtagtgccatctcaggtgctcatttgaatc


 


cgtctattacattagccaatttggtgtatagaggttttcccctgaagaaa


 


gttccttattactttgctggacaattgatcggtgccacacaggcgctttg


 


atcttgtttatttggtacaaaagggtgttacaagaggcatatagcgattg


 


gtggatgaatgaaagtgttgcgggaatgttttgcgtttttccaaagcctt


 


atctaagacaggacggcaatttttttccgaatttttatgtggagctatgt


 


tacaagcaggaacatttgcgctgaccgatccttatacgtgtttgtcctct


 


gatgttttcccattgatgatgtttattttgattttcattatcaatgcttc


 


catggcttatcagacaggtacagcaatgaatttggctcgtgatctgggcc


 


cacgtcttgcactatatgcagttggatttgatcataaaatgctttgggtg


 


catcatcatcatttcttttgggttcccatggtaggcccatttattggtgc


 


gttaatgggggggttggtttacgatgtctgtatttatcagggtcatgaat


 


ctccagtcaactggtctttaccagtttataaggaaatgattatgagagcc


 


tggtttagaaggcctggttggaagaagagaaatagagcaagaagaacatc


 


ggacctgagtgacttctcatacaataacgatgatgatgaggaatttggag


 


aaagaatggctcttcaaaagacaaagaccaagtcatctatttcagacaac


 


gaaaatgaagcaggagaaaagaaagtgcaatttaaatctgttcagcgcgg


 


caaaagaacgtttggtggtataccaacaattcttgaagaagaagattcca


 


ttgaaactgcttcgctaggtgcgacgacgactgattctattgggttatcc


 


gacacatcatcagaagattcgcattatggtaatgctaagaaggtaacatg


 


agaaaacagacaagaaaaagaaacaaataatatagactgatagaaaaaaa


 


tactgcttactaccgccggtataatatatatatatatatatatttacata


 


gatgattgcatagtgttttaaaaagctttcctaggttaagctatgaatct


 


tcataacctaaccaactaaatatgaaaatactgacccatcgtcttaagta


 


agttgacatgaactcagcctggtcacctactatacatgatgtatcgcatg


 


gatggaaagaataccaaacgctaccttccaggttaatgatagtatccaaa


 


cctagaggaatttgccagaacatcaagcagcgattcgatatcagttggga


 


gcatcaatttggtcattggaataccatctatgcttactcctcccatattc


 


gcaaaagtagtaagggctcgttatatacttttgaatatgtaagatataat


 


tctatatgatttagtaatttattttctatacgctcagtatttactgcagt


 


tgtcgagtaggtattaaacgcaaaagaagtccatccttttcatcattcaa


 


atggacatcttggcaaagggcccagttatggaaaatctgggagtcataca


 


acgattgcagttggctatgccactcctggtaaggaatcatcaagtctgat


 


aattctgttttttagccatttttttttttttttcatg






Sequence of fps1Δ mutation (part of the fps1 coding sequence was not deleted (underlined) and the region that was deleted is represented by a Δ; SEQ ID NO:105):







aacgcggctgatgcttttatttaggaaggaatacttacattatcatgaga


 


acattgtcaagggcattctgatacgggccttccatcgcaagaaaaaggca


 


gcaacggactgagggacggagagagttacggcataagaagtagtaggaga


 


gcagagtgtcataaagttatattattctcgtcctaaagtcaattagttct


 


gttgcgcttgacaatatatgtcgtgtaataccgtcccttagcagaagaaa


 


gaaagacggatccatatatgttaaaatgcttcagagatgtttctttaatg


 


tgccgtccaacaaaggtatcttctgtagcttcctctattttcgatcagat


 


ctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtga


 


atccggagacggcaagattgcccggccctttttgcggaaaagataaaaca


 


agatatattgcactttttccaccaagaaaaacaggaagtggattaaaaaa


 


tcaacaaagtataacgcctattgtcccaataagcgtcggttgttcttctt


 


tattattttaccaagtacgctcgagggtacattctaatgcattaaaagac


 


Δgattcgcattatggtaatgctaagaaggtaacatgagaaaacagacaag


 


aaaaagaaacaaataatatagactgatagaaaaaaatactgcttactacc


 


gccggtataatatatatatatatatatatttacatagatgattgcatagt


 


gttttaaaaagctttcctaggttaagctatgaatcttcataacctaacca


 


actaaatatgaaaatactgacccatcgtcttaagtaagttgacatgaact


 


cagcctggtcacctactatacatgatgtatcgcatggatggaaagaatac


 


caaacgctaccttccaggttaatgatagtatccaaacctagttggaattt


 


gccttgaacatcaagcagcgattcgatatcagttgggagcatcaatttgg


 


tcattggaataccatctatgcttttctcctcccatattcgcaaaagtagt


 


aagggctcgttatatacttttgaatatgtaagatataattctatatgatt


 


tagtaatttattttctatacgctcagtatttttctgcagttgtcgagtag


 


gtattaaacgcaaaagaagtccatccttttcatcattcaaatggacatct


 


tggcaaagggcccagttatggaaaatctgggagtcatacaacgattgcag


 


ttggctatgccactcctggtaaggaatcatcaagtctgataattctgttt


 


tttagccctttttttttttttttcatg


 






Example 3
Generating Yeast Strains with a Deleted or Downregulated Glycerol-Production Pathway
To create yeast strains with altered glycerol production, endogenous glycerol-producing or regulating genes can either be deleted or downregulated, by generating the following genetic backgrounds:














Haploid Strains
Diploid Strains


















Glycerol
gpd1Δ gpd2Δ fdh1Δ fdh2Δ
gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ


Elimination
fdh1Δ fdh2Δ fps1Δ
fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ


Background
gpd1Δ gpd2Δ fdh1Δ fdh2Δ
fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ



fps1Δ
fps1Δ/fps1Δ



gpd1Δ gpd2Δ fps1Δ
gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ



gpd1Δ gpd2Δ
fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ




fps1Δ/fps1Δ




gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ




fps1Δ/fps1Δ




gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ


Glycerol
gpd1Δ gpd2Δ::GPD1 fdh1Δ
gpd1Δ/gpd1Δ


Reduction
fdh2Δ
gpd2Δ/gpd2Δ::GPD1/GPD1


Background
gpd1Δ gpd2Δ::GPD1
fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ



gpd1Δ gpd2Δ::GPD1
gpd1Δ/gpd1Δ



fdh1Δfdh2Δ fps1Δ
gpd2Δ/gpd2Δ::GPD1/GPD1



gpd1Δ gpd2Δ::GPD1 fps1Δ
gpd1Δ/gpd1Δ



gpd1Δ: GPD2 gpd2Δ
gpd2Δ/gpd2Δ::GPD1/GPD1



gpd1::GPD2 gpd2Δ
fdh1Δ/fdh1Δ fdh2Δ/fdh2Δ



fdh1Δfdh2Δ
fps1Δ/fps1Δ




gpd1Δ/gpd1Δ




gpd2Δ/gpd2Δ::GPD1/GPD1




fps1Δ/fps1Δ




gpd1Δ/gpd1Δ::GPD2/GPD2




gpd2Δ/gpd2Δ




gpd1Δ/gpd1Δ::GPD2/GPD2




gpd2Δ/gpd2Δ fdh1Δ/fdh1Δ




fdh2Δ/fdh2Δ









Strains in the glycerol elimination background were created by deleting one or more of the following genes: gpd1, gpd2, fdh1, fdh2, and/or fps1. Strains in the glycerol reduction background have been created by deleting one or more of the following genes: gpd1, gpd2, fdh1, fdh2, and/or fps1, and by expressing GPD1 under the control of the gpd2 promoter (designated gpd2Δ::GPD1). These strains in which GPD1 is expressed from the gpd2 promoter make a smaller amount of glycerol relative to a wild-type strain.
3.1 Generation of Glycerol-Elimination Strain gpd1Δ gpd2Δ fdh1Δ fdh2Δ
To produce glycerol-elimination strain gpd1Δ gpd2Δ fdh1Δ fdh2Δ, the following methods were used. All genetic modifications were generated using positive selections to insert genetic elements and negative selections to remove genetic elements. See FIGS. 4-11. The genetic elements were amplified by PCR and transformed into host strains, followed by selection and screening for the desired modification. The sequence of native gpd1, gpd2, fdh1, fdh2 from S. cerevisiae, and resulting loci following deletion, are listed below.
Sequence of GPD1 locus (coding sequence is underlined; SEQ ID NO:89):







tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaaga


 


cagggtcaatgagactgttgtcctcctactgtccctatgtctctggccga


 


tcacgcgccattgtccctcagaaacaaatcaaacacccacaccccgggca


 


cccaaagtccccacccacaccaccaatacgtaaacggggcgccccctgca


 


ggccctcctgcgcgcggcctcccgccttgcttctctccccttccttttct


 


ttttccagttttccctattttgtccctttttccgcacaacaagtatcaga


 


atgggttcatcaaatctatccaacctaattcgcacgtagactggcttggt


 


attggcagtttcgtagttatatatatactaccatgagtgaaactgttacg


 


ttaccttaaattctttctccctttaattttcttttatcttactctcctac


 


ataagacatcaagaaacaattgtatattgtacaccccccccctccacaaa


 


cacaaatattgataatataaagatgtctgctgctgctgatagattaaact


 


taacttccggccacttgaatgctggtagaaagagaagttcctcttctgtt


 


tctttgaaggctgccgaaaagcctttcaaggttactgtgattggatctgg


 


taactggggtactactattgccaaggtggttgccgaaaattgtaagggat


 


acccagaagttttcgctccaatagtacaaatgtgggtgttcgaagaagag


 


atcaatggtgaaaaattgactgaaatcataaatactagacatcaaaacgt


 


gaaatacttgcctggcatcactctacccgacaatttggttgctaatccag


 


acttgattgattcagtcaaggatgtcgacatcatcgttttcaacattcca


 


catcaatttttgccccgtatctgtagccaattgaaaggtcatgttgattc


 


acacgtcagagctatctcctgtctaaagggttttgaagttggtgctaaag


 


gtgtccaattgctatcctcttacatcactgaggaactaggtattcaatgt


 


ggtgctctatctggtgctaacattgccaccgaagtcgctcaagaacactg


 


gtctgaaacaacagttgcttaccacattccaaaggatttcagaggcgagg


 


gcaaggacgtcgaccataaggttctaaaggccttgttccacagaccttac


 


ttccacgttagtgtcatcgaagatgttgctggtatctccatctgtggtgc


 


tttgaagaacgttgttgccttaggttgtggtttcgtcgaaggtctaggct


 


ggggtaacaacgcttctgctgccatccaaagagtcggtttgggtgagatc


 


atcagattcggtcaaatgtttttcccagaatctagagaagaaacatacta


 


ccaagagtctgctggtgttgctgatttgatcaccacctgcgctggtggta


 


gaaacgtcaaggttgctaggctaatggctacttctggtaaggacgcctgg


 


gaatgtgaaaaggagttgttgaatggccaatccgctcaaggtttaattac


 


ctgcaaagaagttcacgaatggttggaaacatgtggctctgtcgaagact


 


tcccattatttgaagccgtataccaaatcgtttacaacaactacccaatg


 


aagaacctgccggacatgattgaagaattagatctacatgaagattagat


 


ttattggagaaagataacatatcatactttcccccacttttttcgaggct


 


cttctatatcatattcataaattagcattatgtcatttctcataactact


 


ttatcacgttagaaattacttattattattaaattaatacaaaatttagt


 


aaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaat


 


cctatagagcaaaaggattttccattataatattagctgtacacctcttc


 


cgcattttttgagggtggttacaacaccactcattcagaggctgtcggca


 


cagttgcttctagcatctggcgtccgtatgtatgggtgtattttaaataa


 


taaacaaagtgccacaccttcaccaattatgtctttaagaaatggacaag


 


ttccaaagagcttgcccaaggctcgacaaggatgtactttggaatatcta


 


tattcaagtacgtggcgcgcatatgtttgagtgtgcacacaataaaggtt






Sequence of gpd1 Δ mutation (part of the gpd1 coding sequence was not deleted (underlined) and the region that was deleted is represented by a Δ; SEQ ID NO:90):







tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaaga


 


cagggtcaatgagactgttgtcctcctactgtccctatgtctctggccga


 


tcacgcgccattgtccctcagaaacaaatcaaacacccacaccccgggca


 


cccaaagtccccacccacaccaccaatacgtaaacggggcgccccctgca


 


ggccctcctgcgcgcggcctcccgccttgcttctctccccttccttttct


 


ttttccagttttccctattttgtccctttttccgcacaacaagtatcaga


 


atgggttcatcaaatctatccaacctaattcgcacgtagactggcttggt


 


attggcagtttcgtagttatatatatactaccatgagtgaaactgttacg


 


ttaccttaaattaaattctttccctttaattttcttttatcttactctcc


 


tacataagacatcaagaaacaattgtatattgtacaccccccccctccac


 


aaacacaaatattgataatataaagatgtctgctgctgctgatagΔtcta


 


catgaagattagatttattggagaaagataacatatcatactttccccca


 


cttttttcgaggctcttctatatcatattcataaattagcattatgtcat


 


ttctcataactactttatcacgttagaaattacttattattattaaatta


 


atacaaaatttagtaaccaaataaatataaataaatatgtatatttaaat


 


tttaaaaaaaaaatcctatagagcaaaaggattttccattataatattag


 


ctgtacacctcttccgcattttttgagggtggttacaacaccactcattc


 


agaggctgtcggcacagttgcttctagcatctggcgtccgtatgtatggg


 


tgtattttaaataataaacaaagtgccacaccttcaccaattatgtcttt


 


aagaaatggacaagttccaaagagcttgcccaaggctcgacaaggatgta


 


ctttggaatatctatattcaagtacgtggcgcgcatatgtttgagtgtgc


 


acacaataaaggtt






Sequence of GPD2 locus (coding sequence is underlined; SEQ ID NO:91):







atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattac


 


cgagtttgttttccttcacatgatgaagaaggtttgagtatgctcgaaac


 


aataagacgacgatggctctgccattgttatattacgcttttgcggcgag


 


gtgccgatgggttgctgaggggaagagtgtttagcttacggacctattgc


 


cattgttattccgattaatctattgttcagcagctcttctctaccctgtc


 


attctagtattttttttttttttttttggttttacttttttttcttcttg


 


cctttttttcttgttactttttttctagttttttttccttccactaagct


 


ttttccttgatttatccttgggttcttctttctactcctttagatttttt


 


ttttatatattaatttttaagtttatgtattttggtagattcaattctct


 


ttccctttccttttccttcgctccccttccttatcaatgcttgctgtcag


 


aagattaacaagatacacattccttaagcgaacgcatccggtgttatata


 


ctcgtcgtgcatataaaattttgccttcaagatctactttcctaagaaga


 


tcattattacaaacacaactgcactcaaagatgactgctcatactaatat


 


caaacagcacaaacactgtcatgaggaccatcctatcagaagatcggact


 


ctgccgtgtcaattgtacatttgaaacgtgcgcccttcaaggttacagtg


 


attggttctggtaactgggggaccaccatcgccaaagtcattgcggaaaa


 


cacagaattgcattcccatatcttcgagccagaggtgagaatgtgggttt


 


ttgatgaaaagatcggcgacgaaaatctgacggatatcataaatacaaga


 


caccagaacgttaaatatctacccaatattgacctgccccataatctagt


 


ggccgatcctgatcttttactccatcaagggtgctgacatccttgttttc


 


aacatccctcatcaatttttaccaaacatagtcaaacaattgcaaggcca


 


cgtggcccctcatgtaagggccatctcgtgtctaaaagggttcgagttgg


 


gctccaagggtgtgcaattgctatcctcctatgttactgatgagttagga


 


atccaatgtggcgcactatctggtgcaaacttggcaccggaagtggccaa


 


ggagcattggtccgaaaccaccgtggcttaccaactaccaaaggattatc


 


aaggtgatggcaaggatgtagatcataagattttgaaattgctgttccac


 


agaccttacttccacgtcaatgtcatcgatgatgttgctggtatatccat


 


tgccggtgccttgaagaacgtcgtggcacttgcatgtggtttcgtagaag


 


gtatgggatggggtaacaatgcctccgcagccattcaaaggctgggttta


 


ggtgaaattatcaagttcggtagaatgtttttcccagaatccaaagtcga


 


gacctactatcaagaatccgctggtgttgcagatctgatcaccacctgct


 


caggcggtagaaacgtcaaggttgccacatacatggccaagaccggtaag


 


tcagccttggaagcagaaaaggaattgcttaacggtcaatccgcccaagg


 


gataatcacatgcagagaagttcacgagtggctacaaacatgtgagttga


 


cccaagaattcccattattcgaggcagtctaccagatagtctacaacaac


 


gtccgcatggaagacctaccggagatgattgaagagctagacatcgatga


 


cgaatagacactctccccccccctccccctctgatctttcctgttgcctc


 


tttttcccccaaccaatttatcattatacacaagttctacaactactact


 


agtaacattactacagttattataattttctattctctttttctttaaga


 


atctatcattaacgttaatttctatatatacataactaccattatacacg


 


ctattatcgtttacatatcacatcaccgttaatgaaagatacgacaccct


 


gtacactaacacaattaaataatcgccataaccttttctgttatctatag


 


ccataaagctgtttcttcgagctttttcactgcagtaattctccacatgg


 


gcccagccactgagataagagcgctatgttagtcactactgacggctctc


 


cagtcatttatgtgattttttagtgactcatgtcgcatttggcccgtttt


 


tttccgctgtcgcaacctatttccattaacggtgccgtatggaagagtca


 


tttaaaggcaggagagagagattactcatcttcattggatcagattgatg


 


actgcgtacggcagat






Sequence of gpd2Δ mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:92):







atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattac


 


cgagtttgttttccttcacatgatgaagaaggtttgagtatgctcgaaac


 


aataagacgacgatggctctgccattgttatattacgcttttgcggcgag


 


gtgccgatgggttgctgaggggaagagtgtttagcttacggacctattgc


 


cattgttattccgattaatctattgttcagcagctcttctctaccagtca


 


ttctagtattttttttttttttttttggttttacttttttttcttcttgc


 


ctttttttcttgttactttttttctagttttttttccttccactaagctt


 


tttccttgatttatccttgggttcttctttctactcctttagattttttt


 


tttatatattaatttttaagtttatgtattttggtagattcaattctctt


 


tccctttccttttccttcgctccccttccttatcΔctctgatctttcctg


 


ttgcctctttttcccccaaccaatttatcattatacacaagttctacaac


 


tactactagtaacattactacagttattataattttctattctctttttc


 


tttaagaatctatcattaacgttaatttctatatatacataactaccatt


 


atacacgctattatcgtttacatatcacatcaccgttaatgaaagatacg


 


acaccctgtacactaacacaattaaataatcgccataaccttttctgtta


 


tctatagcccttaaagctgtttcttcgagctttttcactgcagtaattct


 


ccacatgggcccagccactgagataagagcgctatgttagtcactactga


 


cggctctccagtcatttatgtgattttttagtgactcatgtcgcatttgg


 


cccgtttttttccgctgtcgcaacctatttccattaacggtgccgtatgg


 


aagagtcatttaaaggcaggagagagagattactcatcttcattggatca


 


gattgatgactgcgtacggcagat






Sequence of FDH1 locus (coding sequence is underlined; SEQ ID NO:93):







tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattat


 


cgctaacgatcaagaggaactgagaccttattagttgtctttgttggcgt


 


aacataaatttcttaggaaaagagaaaattatctcgaaggcaaaaataaa


 


ccaagcctcgagtttaatggttttctaaaaaacactttaaaaacagatcg


 


ccataaaaggagaagctccgtaggagaccgttttcgaaacctatgtagaa


 


ataaagggaaagctccaacggtttggataaatctttagaagcatagagtt


 


tatacaacattcagtacgaaatgtactctcgaaacgttctcttttcacgg


 


tgcttagtagcagaaaaaagtgtcggaaattacctattagtcaccactcg


 


aggataggcttgaaagagagttttaaccccaacttttctattttgcactt


 


gtttggctatggtttaaaacattctgtttggaccaacagcccaagcggct


 


tatcccttttctttttacccttataatcgggaatttccttactaggaagg


 


caccgatactagaactccgaatgaaaaagacatgccagtaataaaactat


 


tttgatgttatgcggaatatactattcttggattattcactgttaactaa


 


aagttggagaaatcactctgcactgtcaatcattgaaaaaaagaacatat


 


aaaagggcacaaaattgagtcttttttaatgagttcttgctgaggaaagt


 


ttagttaatatatcatttacgtaaaatatgcatattcttgtattgtgctt


 


tttttattcattttaagcaggaacaatttacaagtattgcaacgctaatc


 


aaatcaaaataacagctgaaaattaatatgtcgaagggaaaggttttgct


 


ggttctttacgaaggtggtaagcatgctgaagagcaggaaaagttattgg


 


ggtgtattgaaaatgaacttggtatcagaaatttcattgaagaacaggga


 


tacgagttggttactaccattgacaaggaccctgagccaacctcaacggt


 


agacagggagttgaaagacgctgaaattgtcattactacgccctttttcc


 


ccgcctacatctcgagaaacaggattgcagaagctcctaacctgaagctc


 


tgtgtaaccgctggcgtcggttcagaccatgtcgatttagaagctgcaaa


 


tgaacggaaaatcacggtcaccgaagttactggttctaacgtcgtttctg


 


tcgcagagcacgttatggccacaattttggttttgataagaaactataat


 


ggtggtcatcaacaagcaattaatggtgagtgggatattgccggcgtggc


 


taaaaatgagtatgatctggaagacaaaataatttcaacggtaggtgccg


 


gtagaattggatatagggttctggaaagattggtcgcatttaatccgaag


 


aagttactgtactacgactaccaggaactacctgcggaagcaatcaatag


 


attgaacgaggccagcaagcttttcaatggcagaggtgatattgttcaga


 


gagtagagaaattggaggatatggttgctcagtcagatgttgttaccatc


 


aactgtccattgcacaaggactcaaggggtttattcaataaaaagcttat


 


ttcccacatgaaagatggtgcatacttggtgaataccgctagaggtgcta


 


tttgtgtcgcagaagatgttgccgaggcagtcaagtctggtaaattggct


 


ggctatggtggtgatgtctgggataagcaaccagcaccaaaagaccatcc


 


ctggaggactatggacaataaggaccacgtgggaaacgcaatgactgttc


 


atatcagtggcacatctctggatgctcaaaagaggtacgctcagggagta


 


aagaacatcctaaatagttacttttccaaaaagtttgattaccgtccaca


 


ggatattattgtgcagaatggttcttatgccaccagagcttatggacaga


 


agaaataagagtgattatgagtatttgtgagcagaagttttccggtctcc


 


ttttgttcttgttttggcgtattctccactattcgtccatagcacattta


 


taccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaa


 


atagaagagcggtttattaatatcaaataattgaaactgctgatatggta


 


gctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacgg


 


tagcggttcaaataaagaggaaaagtctttctcccttactgtttttctgg


 


aatttggctcgtcgttaataacagaactaaagatacagtaaaaggagaga


 


tcgcaatcaacttcattaattgtaacagtagcataatcacaactgatcat


 


ctacactataaacagtttttatttctaattatgggcgcctggccggctca


 


aacattgtgcttttaagactccaaaagtatctgctgcagaaaagagccat


 


ataatgttaagtgttcagggataggttatcgcttactacttcaaacgttt


 


cgaaggaaagccagggaagcctatatctgattccctgtttcataatccaa


 


tgcagccactagcttataattatttgaactatttgtcgaacatcacagta


 


ataaaatccccagaaagttccacttgctgcatattggcacctgttgattc


 


actctccatcacttttttgttagccgcccagcctagaaagtctttaaata


 


catctgaaatttttttttttttaacagtgcacccgtgcatcatacctcat


 


gcaaggtacctttttttctcaaaggtattgtcttccattgaagtggcact


 


atggcatgatgaaccctgagcatttctgaattcaacagaaccaaattgtc


 


cagaaataaatctgtccgacatgaattatgaaactttttttcaattaagt


 


gaagagaattttgcagcgtcttaccattattttgacccattggtcgcatg


 


tttgcgctttgacttcgagaaccatgttaaagcttacttgtacgacaacc


 


aatgaagtatattacggcagtttttttggactgggtcaaaaaaagtgttg


 


cataatcaaatcaggaacacattaaaatgttgtaaaatttgtcttagtat


 


cacctgagtggttattcattacgtacta






Sequence of fdh1Δ mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:94):







tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattat


 


cgctaacgatcaagaggaactgagaccttattagttgtctttgttggcgt


 


aacataaatttcttaggaaaagagaaaattatctcgaaggcaaaaataaa


 


ccaagcctcgagtttaatggttttctaaaaaacactttaaaaacagatcg


 


ccataaaaggagaagctccgtaggagaccgttttcgaaacctatgtagaa


 


ataaagggaaagctccaacggtttggataaatctttagaagcatagagtt


 


tatacaacattcagtacgaaatgtactctcgaaacgttctatttcacggt


 


gcttagtagcagaaaaaagtgtcggaaattacctattttgtcaccactcg


 


aggataggcttgaaagagagttttaaccccaacttttctattttgcactt


 


gtttggctatggtttaaaacattctgtttggaccaacagcccaagcggct


 


tatcccttttctttttttcccttataatcgggaatttccttactaggaag


 


gcaccgatactagaactccgaatgaaaaagacatgccagtaataaaacta


 


ttttgatgttatgcggaatatactattcttggattattcactgttaacta


 


aaagttggagaaatcactctgcactgtcaΔtggcagtaaacgatcttcac


 


ggtagcggttcaaataaagaggaaaagtctttctcccttactgtttttct


 


ggaatttggctcgtcgttaataacagaactaaagatacagtaaaaggaga


 


gatcgcaatcaacttcattaattgtaacagtagcataatcacaactgatc


 


atctacactataaacagtttttatttctaattatgggcgcctggccggct


 


caaacattgtgcttttaagactccaaaagtatctgctgcagaaaagagcc


 


atataatgttaagtgttcagggataggttatcgcttactacttcaaacgt


 


ttcgaaggaaagccagggaagcctatatctgattccctgtttcataatcc


 


aatgcagccactagcttataattatttgaactatagtcgaacatcacagt


 


aataaaatccccagaaagttccacttgctgcatattggcacctgttgatt


 


cactctccatcacttttttgttagccgcccagcctagaaagtctttaaat


 


acatctgaaatttttttttttttaacagtgcacccgtgcatcatacctca


 


tgcaaggtacctttttttctcaaaggtattgtcttccattgaagtggcac


 


tatggcatgatgaaccctgagcatactgaattcaacagaaccaaattgtc


 


cagaaataaatctgtccgacatgaattatgaaactttttttcaattaagt


 


gaagagaattttgcagcgtcttaccattattttgacccattggtcgcatg


 


tttgcgctttgacttcgagaaccatgttaaagcttacttgtacgacaacc


 


aatgaagtatattacggcagtttttttggactgggtcaaaaaaagtgttg


 


cataatcaaatcaggaacacattaaaatgttgtaaaatttgtcttagtat


 


cacctgagtggttattcattacgtacta


 






Sequence of FDH2 locus (coding sequence is underlined; SEQ ID NO:95):







tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacct


 


cgatgaaagagaaactggaatgatagatctctttttctcaaaatttcgtt


 


aatatgtaataataaggttcctgatgtaatttgtttttgtacaaattatt


 


ttagattctggaggttcaaataaaatatatattacagccaacgattaggg


 


gggacaagacttgattacacatttttcgttggtaacttgactcttttatg


 


aaaagaaaacattaagttgaaggtgcacgcttgaggcgctcctttttcat


 


ggtgcttagcagcagatgaaagtgtcagaagttacctattttgtcaccat


 


ttgagaataagcttgaaagaaagttgtaaccccaacttttctatcttgca


 


cttgtttggaccaacagccaaacggcttatcccttttcttttcccttata


 


atcgggaatttccttactaggaaggcaccgatactataactccgaatgaa


 


aaagacatgccagtaataaaaataattgatgttatgcggaatatactatt


 


cttggattattcactgttaactaaaagttggagaaatcactctgcactgt


 


caatcattgaaaaaaagaacatataaaagggcacaaaatcgagtcttttt


 


taatgagttcttgctgaggaaaatttagttaatatatcatttacataaaa


 


catgcatattattgtgttgtactttctttattcattttaagcaggaataa


 


ttacaagtattgcaacgctaatcaaatcgaaataacagctgaaaattaat


 


atgtcgaagggaaaggttttgctggttctttatgaaggtggtaagcatgc


 


tgaagagcaggaaaagttattggggtgtattgaaaatgaacttggtatca


 


gaaatttcattgaagaacagggatacgagttggttactaccattgacaag


 


gaccctgagccaacctcaacggtagacagggagttgaaagacgctgaaat


 


tgtcattactacgccctttttccccgcctacatctcgagaaacaggattg


 


cagaagctcctaacctgaagctctgtgtaaccgctggcgtcggttcagac


 


catgtcgatttagaagctgcaaatgaacggaaaatcacggtcaccgaagt


 


tactggttctaacgtcgtttctgtcgcagagcacgttatggccacaattt


 


tggttttgataagaaactataatggtggtcatcaataagcaattaatggt


 


gagtgggatattgccggcgtggctaaaaaatgagtatgatctggaagaca


 


aaataatttcaacggtaggtgccggtagaattggatatagggttctggaa


 


agattggtcgcatttaatccgaagaagttactgtactacgactaccagga


 


actacctgcggaagcaatcaatagattgaacgaggccagcaagcttttca


 


atggcagaggtgatattgttcagagagtagagaaattggaggatatggtt


 


gctcagtcagatgttgttaccatcaactgtccattgcacaaggactcaag


 


gggtttattcaataaaaagcttatttcccacatgaaagatggtgcatact


 


tggtgaataccgctagaggtgctatttgtgtcgcagaagatgttgccgag


 


gcagtcaagtctggtaaattggctggctatggtggtgatgtctgggataa


 


gcaaccagcaccaaaagaccatccctggaggactatggacaataaggacc


 


acgtgggaaacgcaatgactgttcatatcagtggcacatctctgcatgct


 


caaaagaggtacgctcagggagtaaagaacatcctaaatagttacttttc


 


caaaaagtttgattaccgtccacaggatattattgtgcagaatggttctt


 


atgccaccagagcttatggacagaagaaataagagtgattatgagtattt


 


gtgagcagaagttttccggtctccttttgttcttgttttggcgtattctc


 


cactattcgtccatagcacatttataccttagctaaatattttgtaaagc


 


aaaattttcgttatctcttaaaaaatagaagagcggtttattaatatcaa


 


ataattgaaactgctgatatggtagctatatacaaaatctgctgtcaaaa


 


tttggcagtaaacgatcttcacggtagcggttcaaataaagaggaaaagt


 


ccttctcccttactgtttttctggaatttggctcgtcgttaataacagaa


 


ctaaagatacagtaaaaggagagatcgcaatcaacttcattaattgtaac


 


agtagcataatcacaactggttatctgcgttatagacaattcttactcac


 


aatgatgggcgcttagttggctgtaaacgtcgctttttaaaactccgaaa


 


agttaccgctacagaaaaaaaccataaatgtatgctagttgcgcagagag


 


gtttagggtccaaaatttactaccctgtcgctcactacagcgactgtccc


 


gaattaagcccgaagagacgcagaactgttgtatgaacctcatgaaacca


 


ctgatcttgaagatttagaccttcagaatcgttttcaattagaagtatac


 


aagaagtctttgtacaataatgtcaagacagagctctgaattatagttca


 


gccttgttattttttttt






Sequence of fdh2Δ mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:96):







tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacct


 


cgatgaaagagaaactggaatgatagatctctttttctcaaaatttcgtt


 


aatatgtaataataaggttcctgatgtaatttgtttttgtacaaattatt


 


ttagattctggaggttcaaataaaatatatattacagccaacgattaggg


 


gggacaagacttgattacacatttttcgttggtaacttgactcttttatg


 


aaaagaaaacattaagttgaaggtgcacgcttgaggcgctcctttttcat


 


ggtgcttagcagcagatgaaagtgtcagaagttacctattttgtcaccat


 


ttgagaataagcttgaaagaaagttgtaaccccaacttttctatcttgca


 


cttgtttggaccaacagccaaacggcttatcccttttcttttcccttata


 


atcgggaatttccttactaggaaggcaccgatactataactccgaatgaa


 


aaagacatgccagtaataaaaataattgatgttatgcggaatatactatt


 


cttggattattcactgttaactaaaagttggagaaatcactctgcactgt


 


caatcattgaaaaaaagaacatataaaagggcacaaaatcgagtcttttt


 


taatgagttcttgctgaggaaaatttagttaatatatcatttacataaaa


 


catgcatattattgtgttgtactttctttattcattttaagcaggaataa


 


ttacaagtattgcaacgctaatcaaatcgaaataacagctgaaaattaat


 


Δtaagagtgattatgagtatttgtgagcagaagttttccggtctcctttt


 


gttcttgttttggcgtattctccactattcgtccatagcacatttatacc


 


ttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatag


 


aagagcggtttattaatatcaaataattgaaactgctgatatggtagcta


 


tatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggtagc


 


ggttcaaataaagaggaaaagtccttctcccttactgtttttctggaatt


 


tggctcgtcgttaataacagaactaaagatacagtaaaaggagagatcgc


 


aatcaacttcattaattgtaacagtagcataatcacaactggttatctgc


 


gttatagacaattcttactcacaatgatgggcgcttagttggctgtaaac


 


gtcgctttttaaaactccgaaaagttaccgctacagaaaaaaaccataaa


 


tgtatgctagttgcgcagagaggtttagggtccaaaatttactaccctgt


 


cgctcactacagcgactgtcccgaattaagcccgaagagacgcagaactg


 


ttgtatgaacctcatgaaaccactgatcttgaagatttagaccttcagaa


 


tcgttttcaattagaagtatacaagaagtctttgtacaataatgtcaaga


 


cagagctctgaattatagttcagccttgttattttttttt






3.2 Generation of Glycerol-Reduced Strain Comprising gpd2Δ::GPD1
Glycerol-reduction strain gpd2Δ::GPD1, was constructed as described above. The sequence of gpd1Δ/gpd1Δ gpd2Δ/gpd2Δ::GPD1/GPD1 is provided below.
Sequence of GPD1 at GPD2 locus (inserted GPD1 is underlined; SEQ ID NO:97):







agtaactgtgacgatatcaactctttttttattatgtaataagcaaacaa


 


gcacgaatggggaaagcctatgtgcaatcaccaaggtcgtcccttttttc


 


ccatttgctaatttagaatttaaagaaaccaaaagaatgaagaaagaaaa


 


caaatactagccctaaccctgacttcgtttctatgataataccctgcttt


 


aatgaacggtatgccctagggtatatctcactctgtacgttacaaactcc


 


ggttattttatcggaacatccgagcacccgcgccttcctcaacccaggca


 


ccgcccccaggtaaccgtgcgcgatgagctaatcctgagccatcacccac


 


cccacccgttgatgacagcaattcgggagggcgaaaaataaaaactggag


 


caaggaattaccatcaccgtcaccatcaccatcatatcgccttagcctct


 


agccatagccatcatgcaagcgtgtatcttctaagattcagtcatcatca


 


ttaccgagtttgttttccttcacatgatgaagaaggtttgagtatgctcg


 


aaacaataagacgacgatggctctgccattgttatattacgcttttgcgg


 


cgaggtgccgatgggttgctgaggggaagagtgtttagcttacggaccta


 


ttgccattgttattccgattaatctattgttcagcagctcttctctaccc


 


tgtcattctagtatttttttttttttttttttggttttacttttttttct


 


tcttgcctttttttcttgttactttttttctagttttttttccttccact


 


aagctttttccttgatttatccttgggttcttctttctactcctttagat


 


tttttttttatatattaattttaagtttatgtattttggtagattcaatt


 


ctctttccctttccttttccttcgctccccttccttatcaatgtctgctg


 


ctgctgatagattaaacttaacttccggccacttgaatgctggtagaaag


 


agaagttcctcttctgtttctttgaaggctgccgaaaagcctttcaaggt


 


tactgtgattggatctggtaactggggtactactattgccaaggtggttg


 


ccgaaaattgtaagggatacccagaagttttcgctccaatagtacaaatg


 


tgggtgttcgaagaagagatcaatggtgaaaaattgactgaaatcataaa


 


tactagacatcaaaacgtgaaatacttgcctggcatcactctacccgaca


 


atttggttgctaatccagacttgattgattcagtcaaggatgtcgacatc


 


atcgttttcaacattccacatcaatttttgccccgtatctgtagccaatt


 


gaaaggtcatgttgattcacacgtcagagctatctcctgtctaaagggtt


 


ttgaagttggtgctaaaggtgtccaattgctatcctcttacatcactgag


 


gaactaggtattcaatgtggtgctctatctggtgctaacattgccaccga


 


agtcgctcaagaacactggtctgaaacaacagttgcttaccacattccaa


 


aggatttcagaggcgagggcaaggacgtcgaccataaggttctaaaggcc


 


ttgttccacagaccttacttccacgttagtgtcatcgaagatgttgctgg


 


tatctccatctgtggtgctttgaagaacgttgagccttaggttgtggttt


 


cgtcgaaggtctaggctggggtaacaacgcttctgctgccatccaaagag


 


tcggtttgggtgagatcatcagattcggtcaaatgtttttcccagaatct


 


agagaagaaacatactaccaagagtctgctggtgttgctgatttgatcac


 


cacctgcgctggtggtagaaacgtcaaggttgctaggctaatggctactt


 


ctggtaaggacgcctgggaatgtgaaaaggagttgttgaatggccaatcc


 


gctcaaggtttaattacctgcaaagaagttcacgaatggttggaaacatg


 


tggctctgtcgaagacttcccattatttgaagccgtataccaaatcgttt


 


acaacaactacccaatgaagaacctgccggacatgattgaagaattagat


 


ctacatgaagattagacactctccccccccctccccctctgatattcctg


 


ttgcctctttttccccccaaccaatttatcattatacacaagttctacaa


 


ctactactagtaacattactacagttattataattttctattctcttttt


 


cttaagaatctatcattaacgttaatttctatatatacataactaccatt


 


atacacgctattatcgtttacatatcacatcaccgttaatgaaagatacg


 


acaccctgtacactaacacaattaaataatcgccataaccttttctgtta


 


tctatagcccttaaagctgtttcttcgagctttttcactgcagtaattct


 


ccacatgggcccagccactgagataagagcgctatgttagtcactactga


 


cggctctccagtcatttatgtgattttttagtgactcatgtcgcatttgg


 


cccgtttttttccgctgtcgcaacctatttccattaacggtgccgtatgg


 


aagagtcatttaaaggcaggagagagagattactcatcttcattggatca


 


gattgatgactgcgtacggcagatagtgtaatctgagcagttgcgagacc


 


cagactggcactgtctcaatagtatattaatgggcatacattcgtactcc


 


cttgttcttgcccacagttctctctctctttacttcttgtatcttgtctc


 


cccattgtgcagcgataaggaacattgttctaatatacacggatacaaaa


 


gaaatacacat






Example 4
Cloning and Characterization of PFL and AADH Enzymes
To identify PFL enzymes for use in the strains of the invention, several PFL enzymes were identified for cloning and functional analysis. See Table 1. Functionality was determined by plasmid based expression of each PFL in the fcyΔ::ADHE gpd1Δ . . . . ADHE gpd2Δfdh1Δfdh2Δ (M2158) background. FIG. 22 shows fermentation performance in 20% corn mash. A PFL was determined to be functional based on the presence or absence of a yield increase over M2085. The C. cellulolyticum PFL was determined to be non-functional based on data shown in FIG. 13. The strain listed as M1992 +pMU2481 is M2085 plus a plasmid expressing the C. cellulolyticum PFL. This strain does not appear to make formate.







TABLE 1







Analysis of PFL Enzymes









Organism
Functional
SEQ ID NOs:





Bacillus licheniformis ATCC_14580
nd
 6 and 40


Streptococcus thermophilus LMD_9
nd
12 and 46


Lactobacillus plantarum WCFS1
nd
16 and 50


(lp_3314 and lp_3313)


Lactobacillus casei ATCC_334
yes
24 and 58


Bifidobacterium adolescentis
yes
26 and 60


Clostridium cellulolyticum
no
34 and 68


Escherichia coli
yes
36 and 70


Chlamydomonas reinhardtii PflA
yes
72 and 76


Piromyces sp. E2
yes
78


Neocallimastix frontalis
yes
74 and 80









As shown in Example 7, eight of nine PFL enzymes that were tested can enable the glycerol elimination and glycerol reduction technologies described herein. An alignment of six of these PFL enzymes is shown in FIG. 12. Four of the residues are absent in C. cellulolyticum yet conserved among the other PFL enzymes (indicated with asterisks in FIG. 12). There is an insertion of 18 amino acids at position 640 of the B. adolescentis PFL, which is not present in the other PFL enzymes. The eukaryotic PFLs (Piromyces and Chlamydomonas) have an N-terminal extension which has been reported to be involved in mitochondrial targeting. Deletion of this sequence may improve the performance of these enzymes in S. cerevisiae. These differences may provide insights into identifying additional PFL enzymes for use in the strains of the invention.
To identify AADH enzymes for use in the strains of the invention, several AADH enzymes were identified for cloning and functional analysis. See Table 2. Functionality was determined though analysis of the data listed in Table 3 below and shown in FIG. 20. An AADH enzyme was determined to be functional if a strain containing the genotype gpd1Δgpd2Δ plus a given AADH, had a faster anaerobic growth rate than strain gpd1Δgpd2Δ (FIG. 20) and there was evidence for acetate consumption (Table 3).







TABLE 2







Analysis of AADH Enzymes











Organism
Functional
SEQ ID NO:















Escherichia coli
yes
84



Clostridium phytofermentans
yes
82



Chlamydomonas reinhardtii
yes
86



Piromyces sp. E2
yes
88



Bifidobacterium adolescentis
yes
100










When glycerol deletion strains are grown anaerobically, they are not capable of growth or fermentation and cannot consume sugar during glycolysis. However, if these glycerol deletion strains are complemented with an AADH, the strains are able to grow with the supplementation of acetate in the media. FIG. 20 shows the growth rates of the parental strain, the glycerol deletion strain, and four glycerol deletion strains expressing AADHs from Escherichia coli (Eco), Clostridium phytofermentans (Cph), Chlamydomonas reinhardtii (Chl), and Piromyces sp. E2 (Pir). As shown in FIG. 20, all four genes can restore growth levels above the glycerol deletion strain (as noted by the dotted line) indicating a functional AADH.
The product yields and conversion of acetate by the strains above, as well as additional strains, are shown in Table 3. The glycerol deletion strain was unable to consume sugar or produce ethanol. The parent strain produced glycerol and ethanol but was unable to convert the acetate in the media, initially present at  ̃2 g/L, giving an ethanol yield of 0.41 g/g glucose, consistent with anaerobic ethanol yields. The glycerol deletion strains complemented with AADHs, however, were able to consume glucose and produce ethanol without producing glycerol, or the glycerol production was significantly decreased compared to the parent strain (Chl AADH). See Table 3. In these glycerol deletion mutants, the acetate levels were also reduced, resulting in higher ethanol yields (calculated as grams ethanol produced per gram consumed glucose) than was achieved by the parent strain.







TABLE 3







Product Yields and Acetate Conversion of


Glycerol Deletion Strains Expressing AADH














Acetate

Ethanol
Growth


Strain
Glycerola
Uptakea
Ethanola
Yieldb
rate (hr−1)















Parent M139
1.37
0.14
10.41
0.42
0.27


Δgpd1Δgpd2
0.00
0.00
0.00
0.00
0.01


M2032


Eco AADH
0.00
0.62
11.39
0.46
0.17


M1991


Cph AADH
0.00
0.66
11.21
0.45
0.18


M1991


Chl AADH
0.00
0.32
9.04
0.47
0.04


M1991


Pir AADH
0.00
0.68
11.17
0.45
0.17


M1991


Bad AADH
0.00
0.67
10.95
0.44
0.18


M1991


Eco mhpF
0.00
0.03
0.50
0.02
0.06


M1991


Cph ADH
0.00
0.60
11.29
0.45
0.19


(1428)


M1991


Cph ADH
0.00
0.74
11.22
0.45
0.20


(2642)


M1991


Tsac AADH
0.00
0.59
11.89
0.48
0.16


M1991





agrams per liter


bgram ethanol produced per gram sugar consumed






Example 5
Expression of PFL and AADH and Detection of Formate
To examine the expression of formate in a yeast strain of the invention, E. coli PFL was cloned and expressed in an FDH deletion strain. Strain M1992+pMU2483 has deletions of FDH1 and FDH2 and a plasmid expressing the E. coli PflA and PflB. This strain was constructed by transforming strain M1992 (fdh1Δfdh2Δ) with plasmids expressing either C. cellulolyticum PFL (pMU2481) or E. coli PFL (pMU2483).
The strains were grown in YNB medium buffered with HEPES at pH 6.5, and formate was measured using a formate detection kit from Megazymes (Cat. No. K-FORM), according to manufacturer's specifications. As shown in FIG. 13, approximately 0.0125 g/L and 0.023 g/L formate was measured after 24 hours and 48 hours of growth, respectively. Similar results have been achieved by overexpressing an E. coli PFL in S. cerevesiae. Waks and Silver, Appl. Env. Microbiol. 75:1867-75 (2009).
PFL was also co-expressed in a strain expressing AADH. See FIG. 14. M2085 is a background strain with the genotype gpd1 Δgpd2Δfdh1Δfdh2Δ and M2032 has the genotype gpd1 Δgpd2Δ. Both of these strains are unable to grow anaerobically even in the presence of acetate. M2158 was created by integrating multiple copies of E. coli AADHs at the GPD1 and FCY1 locus in the M2085 background. The integration schemes are shown in FIGS. 26 and 27 and the corresponding nucleotide sequences for M2158 are listed below. One copy of AADH is driven by the native GPD1 promoter. See FIG. 26. The second copy is oriented in the reverse direction and is driven by the phosphofructokinase promoter. See FIG. 26. For AADH integration in FCY1, one copy is driven by the ENO1 promoter and a second copy is driven by the PFK1 promoter. See FIG. 27. M2182 was created by transforming a vector expressing the B. adolescentis PFLs into the M2158 background.
Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQ ID NO:98):







tagattcttttcgaatttgtggtgaagataggaaagttggtacagttctc


 


catcaattttccatattttgctaaaaactcccttgcatgtctctttgcat


 


tcatttctcctgtatacgggttcaacacatcaatcgaattttgcaaagtt


 


gtctccatttctagaagactttcatcgggaataaaaaattcatatccatt


 


attcaaaaacgataatgatccctcgtacttacctgtgtaattggatattt


 


tataccatacttcaaaaatatccttggcctcacttctggtaggatacctt


 


tcgccatgtctgccaatcatttgaacttgcgttaatctacaaccttcagg


 


aatatcagtgggtataccgtagttagcgggaaaggagaaatatggcgcag


 


accctccaagaaagggaaacagactcttctgagagccaattagttcaata


 


tccgcaaaacttctgagtgggatggagagtgccttagataatagaacacc


 


taaacaaatggcaaaaataacgggcttcaccattgttcctgtatggtgta


 


ttagaacatagctgaaaatacttctgcctcaaaaaagtgttaaaaaaaag


 


aggcattatatagaggtaaagcctacaggcgcaagataacacatcaccgc


 


tctcccccctctcatgaaaagtcatcgctaaagaggaacactgaaggttc


 


ccgtaggttgtctttggcacaaggtagtacatggtaaaaactcaggatgg


 


aataattcaaattcaccaatttcaacgtcccttgtttaaaaagaaaagaa


 


tttttctctttaaggtagcactaatgcattatcgatgatgtaaccattca


 


cacaggttatttagcttttgatccttgaaccattaattaacccagaaata


 


gaaattacccaagtggggctctccaacacaatgagaggaaaggtgacttt


 


ttaagggggccagaccctgttaaaaacctttgatggctatgtaataatag


 


taaattaagtgcaaacatgtaagaaagattctcggtaacgaccatacaaa


 


tattgggcgtgtggcgtagtcggtagcgcgctcccttagcatgggagagg


 


tctccggttcgattccggactcgtccaaattattttttactttccgcggt


 


gccgagatgcagacgtggccaactgtgtctgccgtcgcaaaatgatttga


 


attttgcgtcgcgcacgtttctcacgtacataataagtattttcatacag


 


actagcaagacgaggtggtcaaaatagaagcgtcctatgttttacagtac


 


aagacagtccatactgaaatgacaacgtacttgacttttcagtattttct


 


ttttctcacagtctggttatttttgaaagcgcacgaaatatatgtaggca


 


agcattttctgagtctgctgacctctaaaattaatgctattgtgcacctt


 


agtaacccaaggcaggacagttaccttgcgtggtgttactatggccggaa


 


gcccgaaagagttatcgttactccgattattttgtacagctgatgggacc


 


ttgccgtcttcattttttttttttttcacctatagagccgggcagagctg


 


cccggcttaactaagggccggaaaaaaaacggaaaaaagaaagccaagcg


 


tgtagacgtagtataacagtatatctgacacgcacgtgatgaccacgtaa


 


tcgcatcgcccctcacctctcacctctcaccgctgactcagcttcactaa


 


aaaggaaaatatatactattcccaggcaaggtgacagcggtccccgtctc


 


ctccacaaaggcctctcctggggtttgagcaagtctaagtttacgtagca


 


taaaaattctcggattgcgtcaaataataaaaaaagtaaccccacttcta


 


cttctacatcggaaaaacattccattcacatatcgtctttggcctatctt


 


gttttgtcctcggtagatcaggtcagtacaaacgcaacacgaaagaacaa


 


aaaaagaagaaaacagaaggccaagacagggtcaatgagactgttgtcct


 


cctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaa


 


caaatcaaacacccacaccccgggcacccaaagtccccacccacaccacc


 


aatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcctcccg


 


ccttgcttctctccccttccttttctttttccagttttccctattttgtc


 


cctttttccgcacaacaagtatcagaatgggttcatcaaatctatccaac


 


ctaattcgcacgtagactggcttggtattggcagtttcgtagttatatat


 


atactaccatgagtgaaactgttacgttaccttaaattctttctcccttt


 


aattttcttttatcttactctcctacataagacatcaagaaacaattgta


 


tattgtacaccccccccctccacaaacacaaatattgataatataaagat


 


ggctgttactaatgtcgctgaacttaacgcactcgtagagcgtgtaaaaa


 


aagcccagcgtgaatatgccagtttcactcaagagcaagtagacaaaatc


 


ttccgcgccgccgctctggctgctgcagatgctcgaatcccactcgcgaa


 


aatggccgttgccgaatccggcatgggtatcgtcgaagataaagtgatca


 


aaaaccactttgcttctgaatatatctacaacgcctataaagatgaaaaa


 


acctgtggtgttctgtctgaagacgacacttttggtaccatcactatcgc


 


tgaaccaatcggtattatttgcggtatcgttccgaccactaacccgactt


 


caactgctatcttcaaatcgctgatcagtctgaagacccgtaacgccatt


 


atcttctccccgcacccgcgtgcaaaagatgccaccaacaaagcggctga


 


tatcgttctgcaggctgctatcgctgccggtgctccgaaagatctgatcg


 


gctggatcgatcaaccttctgttgaactgtctaacgcactgatgcaccac


 


ccagacatcaacctgatcctcgcgactggtggtccgggcatggttaaagc


 


cgcatacagctccggtaaaccagctatcggtgtaggcgcgggcaacactc


 


cagttgttatcgatgaaactgctgatatcaaacgtgcagttgcatctgta


 


ctgatgtccaaaaccttcgacaacggcgtaatctgtgcttctgaacagtc


 


tgttgttgttgttgactctgtttatgacgctgtacgtgaacgttttgcaa


 


cccacggcggctatctgttgcagggtaaagagctgaaagctgttcaggat


 


gttatcctgaaaaacggtgcgctgaacgcggctatcgttggtcagccagc


 


ctataaaattgctgaactggcaggcttctctgtaccagaaaacaccaaga


 


ttctgatcggtgaagtgaccgttgttgatgaaagcgaaccgttcgcacat


 


gaaaaactgtccccgactctggcaatgtaccgcgctaaagatttcgaaga


 


cgcggtagaaaaagcagagaaactggttgctatgggcggtatcggtcata


 


cctcttgcctgtacactgaccaggataaccaaccggctcgcgtttcttac


 


ttcggtcagaaaatgaaaacggcgcgtatcctgattaacaccccagcgtc


 


tcagggtggtatcggtgacctgtataacttcaaactcgcaccttccctga


 


ctctgggttgtggttcttggggtggtaactccatctctgaaaacgttggt


 


ccgaaacacctgatcaacaagaaaaccgttgctaagcgagctgaaaacat


 


gttgtggcacaaacttccgaaatctatctacttccgccgtggctccctgc


 


caatcgcgctggatgaagtgattactgatggccacaaacgtgcgctcatc


 


gtgactgaccgcttcctgttcaacaatggttatgctgatcagatcacttc


 


cgtactgaaagcagcaggcgttgaaactgaagtcttcttcgaagtagaag


 


cggacccgaccctgagcatcgttcgtaaaggtgcagaactggcaaactcc


 


ttcaaaccagacgtgattatcgcgctgggtggtggaccccgatggacgcc


 


gcgaagatcatgtgggttatgtacgaacatccggaaactcacttcgaaga


 


gctggcgctgcgctttatggatatccgtaaacgtatctacaagttcccga


 


aaatgggcgtgaaagcgaaaatgatcgctgtcaccaccacttctggtaca


 


ggttctgaagtcactccgtttgcggttgtaactgacgacgctactggtca


 


gaaatatccgctggcagactatgcgctgactccggatatggcgattgtcg


 


acgccaacctggttatggacatgccgaagtccctgtgtgctttcggtggt


 


ctggacgcagtaactcacgccatggaagcttatgtttctgtactggcatc


 


tgagttctctgatggtcaggctctgcaggcactgaaactgctgaaagaat


 


atctgccagcgtcctaccacgaagggtctaaaaatccggtagcgcgtgaa


 


cgtgttcacagtgcagcgactatcgcgggtatcgcgtttgcgaacgcctt


 


cctgggtgtatgtcactcaatggcgcacaaactgggttcccagttccata


 


ttccgcacggtctggcaaacgccctgctgatttgtaacgttattcgctac


 


aatgcgaacgacaacccgaccaagcagactgcattcagccagtatgaccg


 


tccgcaggctcgccgtcgttatgctgaaattgccgaccacttgggtctga


 


gcgcaccgggcgaccgtactgctgctaagatcgagaaactgctggcatgg


 


ctggaaacgctgaaagctgaactgggtattccgaaatctatccgtgaagc


 


tggcgttcaggaagcagacttcctggcgaacgtggataaactgtctgaag


 


atgcattcgatgaccagtgcaccggcgctaacccgcgttacccgctgatc


 


tccgagctgaaacagattctgctggatacctactacggtcgtgattatgt


 


agaaggtgaaactgcagcgaagaaagaagctgctccggctaaagctgaga


 


aaaaagcgaaaaaatccgcttaagtcgagagcttttgattaagccttcta


 


gtccaaaaaacacgtttttttgtcatttatttcattttcttagaatagtt


 


tagtttattcattttatagtcacgaatgttttatgattctatatagggtt


 


gcaaacaagcatttttcattttatgttaaaacaatttcaggtttaccttt


 


tattctgcttgtggtgacgcgtgtatccgcccgctcttttggtcacccat


 


gtatttaattgcataaataattcttaaaagtggagctagtctatttctat


 


ttacatacctctcatttctcatttcctcctaatgtgtcaatgatcatatt


 


cttaactggaccgatcttattcgtcagattcaaaccaaaagttcttaggg


 


ctaccacaggaggaaaattagtgtgatataatttaaataatttatccgcc


 


attcctaatagaacgttgttcgacggatatctttctgcccaaaagggttc


 


taagctcaatgaagagccaatgtctaaacctcgttacattgaaaatacag


 


taaatggttccaccattattatgttggtcttgtttagtatggccgatcgg


 


cgtgtgttttgtttgcaccttttatatagtagaagaatatttgtataatt


 


cttattagtactgcaacctaaccactaattatcaacaattattggattat


 


ataaaggaggtaaattgccggattaaaatcaaatatcattcatcaacaag


 


tattcatattgtcggcatatttttacatgcggtgtaagtatttggatcgt


 


attcttatagtgtcaatacctcgaagcagcgtttcaagtaccagacgtat


 


gtaggaactttttaacgtcgagtccgtaagatttgatcagtattaaaaaa


 


atctagataaatgagtggtacaaataaaaacatcattaaaaatcgttaaa


 


taaaaaagtatgaagatcatctattaaagtattagtagccattagcctta


 


aaaaaatcagtgctagtttaagtataatctcgggcgcgccggccgaggcg


 


gttaagcggattttttcgcttttttctcagctttagccggagcagcttct


 


ttcttcgctgcagtttcaccttctacataatcacgaccgtagtaggtatc


 


cagcagaatctgtttcagctcggagatcagcgggtaacgcgggttagcgc


 


cggtgcactggtcatcgaatgcatcttcagacagtttatccacgttcgcc


 


aggaagtctgcttcctgaacgccagcttcacggatagatttcggaatacc


 


cagttcagctttcagcgtttccagccatgccagcagtttctcgatcttag


 


cagcagtacggtcgcccggtgcgctcagacccaagtggtcggcaatttca


 


gcataacgacggcgagcctgcggacggtcatactggctgaatgcagtctg


 


cttggtcgggttgtcgttcgcattgtagcgaataacgttacaaatcagca


 


gggcgtttgccagaccgtgcggaatatggaactgggaacccagtttgtgc


 


gccattgagtgacatacacccaggaaggcgttcgcaaacgcgatacccgc


 


gatagtcgctgcactgtgaacacgttcacgcgctaccggatttttagacc


 


cttcgtggtaggacgctggcagatattctttcagcagtttcagtgcctgc


 


agagcctgaccatcagagaactcagatgccagtacagaaacataagcttc


 


catggcgtgagttactgcgtccagaccaccgaaagcacacagggacttcg


 


gcatgtccataaccaggttggcgtcgacaatcgccatatccggagtcagc


 


gcatagtctgccagcggatatttctgaccagtagcgtcgtcagttacaac


 


cgcaaacggagtgacttcagaacctgtaccagaagtggtggtgacagcga


 


tcattttcgctttcacgcccattttcgggaacttgtagatacgtttacgg


 


atatccataaagcgcagcgccagctcttcgaagtgagtttccggatgttc


 


gtacataacccacatgatcttcgcggcgtccatcggggaaccaccaccca


 


gcgcgataatcacgtctggtttgaaggagtttgccagttctgcaccttta


 


cgaacgatgctcagggtcgggtccgcttctacttcgaagaagacttcagt


 


ttcaacgcctgctgctttcagtacggaagtgatctgatcagcataaccat


 


tgttgaacaggaagcggtcagtcacgatgagcgcacgtttgtggccatca


 


gtaatcacttcatccagcgcgattggcagggagccacggcggaagtagat


 


agatttcggaagtttgtgccacaacatgttttcagctcgcttagcaacgg


 


ttttcttgttgatcaggtgtttcggaccaacgttttcagagatggagtta


 


ccaccccaagaaccacaacccagagtcagggaaggtgcgagtttgaagtt


 


atacaggtcaccgataccaccctgagacgctggggtgttaatcaggatac


 


gcgccgttttcattttctgaccgaagtaagaaacgcgagccggttggtta


 


tcctggtcagtgtacaggcaagaggtatgaccgataccgcccatagcaac


 


cagtttctctgctttttctaccgcgtcttcgaaatctttagcgcggtaca


 


ttgccagagtcggggacagtttttcatgtgcgaacggttcgctttcatca


 


acaacggtcacttcaccgatcagaatcttggtgttttctggtacagagaa


 


gcctgccagttcagcaattttataggctggctgaccaacgatagccgcgt


 


tcagcgcaccgtttttcaggataacatcctgaacagctttcagctcttta


 


ccctgcaacagatagccgccgtgggttgcaaaacgttcacgtacagcgtc


 


ataaacagagtcaacaacaacaacagactgttcagaagcacagattacgc


 


cgttgtcgaaggttttggacatcagtacagatgcaactgcacgtttgata


 


tcagcagtttcatcgataacaactggagtgttgcccgcgcctacaccgat


 


agctggtttaccggagctgtatgcggctttaaccatgcccggaccaccag


 


tcgcgaggatcaggttgatgtctgggtggtgcatcagtgcgttagacagt


 


tcaacagaaggttgatcgatccagccgatcagatctttcggagcaccggc


 


agcgatagcagcctgcagaacgatatcagccgctttgttggtggcatctt


 


ttgcacgcgggtgcggggagaagataatggcgttacgggtcttcagactg


 


atcagcgatttgaagatagcagttgaagtcgggttagtggtcggaacgat


 


accgcaaataataccgattggttcagcgatagtgatggtaccaaaagtgt


 


cgtcttcagacagaacaccacaggttttttcatctttataggcgttgtag


 


atatattcagaagcaaagtggtttttgatcactttatcttcgacgatacc


 


catgccggattcggcaacggccattttcgcgagtgggattcgagcatctg


 


cagcagccagagcggcggcgcggaagattttgtctacttgctcttgagtg


 


aaactggcatattcacgctgggctttttttacacgctctacgagtgcgtt


 


aagttcagcgacattagtaacagccataattcttaattaactttgatatg


 


attttgtttcagattttttatataaaagctttcccaaatagtgctaaagt


 


gaacttagattttttggtacctgtttcgaaattaaaaatagaaaaatttc


 


tctccctatattgttattcttacttcaaatttgtttatcgtttatttact


 


aggcgagacttgagtagacgacaatccaaatagaattaacagattttatt


 


ggtagaaagcaataatattctttagatggttgagaataaagaagtaaaaa


 


aaccagtaaagagaaaaagaaaaggaagaaaattaaagaaaaaggatgat


 


tacacaagaagataataaaaaaactcctttattaagagcggaagaattta


 


ataatgaagatgggaataagcaaaacaaaaacaaagaagggaaaaaaaat


 


aaaaaatcgtatttatttatttaaaaaatcatgttgatgacgacaatgga


 


aaaaaaaaaccgatttcactttctcatccttatatttttcaaaggttgat


 


gcaagtcgatctcaaatcggataacgctgccaactgggaaattccgcaat


 


tccgcaagaaaaaaaaaaatgtgaaaacgtgattgcattttttacaggtc


 


ctaaaggatttagcccacatatcaagagggtggcagtaattgcactgatt


 


aagcattcgtcagcattaggcgaatgtgtgcatgaatattgccagtgtgc


 


tcgatattagagagtacattgaagaatattgtaccggattatgtacaata


 


actttgttaatgagatattaattttcttttttactagccgctatcccatg


 


cacgatgctaaatttcaagaagaaactgagatttaaaaaattagtggaag


 


ctgataaaacggactataatggtgtatggattgaggaatctcgacatgtt


 


tttccatcgttttcaacgatgactgtaacccgtagattgaaccaggcatg


 


ccaaagttagttagatcagggtaaaaattatagatgaggtatttattgga


 


gaaagataacatatcatactttcccccacttttttcgaggctcttctata


 


tcatattcataaattagcattatgtcatttctcataactactttatcacg


 


ttagaaattacttattattattaaattaatacaaaatttagtaaccaaat


 


aaatataaataaatatgtatatttaaattttaaaaaaaaaatcctataga


 


gcaaaaggattttccattataatattagctgtacacctatccgcattttt


 


tgagggtggttacaacaccactcattcagaggctgtcggcacagttgctt


 


ctagcatctggcgtccgtatgtatgggtgtattttaaataataaacaaag


 


tgccacaccttcaccaattatgtctttaagaaatggacaagttccaaaga


 


gcttgcccaaggctcgacaaggatgtactttggaatatctatattcaagt


 


acgtggcgcgcatatgtttgagtgtgcacacaataaaggtttttagatat


 


tttgcggcgtcctaagaaaataaggggtttcttaaaaaataacaatagca


 


aacaaagttccttacgatgatttcagatgtgaatagcatggtcatgatga


 


gtatatacgtttttataaataattaaaagttttcctcttgtctgtttttt


 


tgttggctcgtggttgttctcgaaaaaggagagttttcattttcgaaata


 


ggtgattatcatcatgttgttatcaccccacgacgaagataatacggagc


 


tcaccgttttctttttttttcctttggctgaaatttcccaccagaacaaa


 


cgtgacaaaattatctttgaatccaaagtagcttatatatatacgtagaa


 


gtgtttcgagacacacatccaaatacgaggttgttcaatttaaacccaag


 


aatacataaaaaaaatatagatatattaacttagtaaacaatgactgcaa


 


gcacaccatccaatgtcatgacattgttcttgttaaggcatggacaaagt


 


gaattgaatcacgagaatatattctgtggttggattgacgctaagctaac


 


cgaaaaaggtaaagaacaagctcgtcattctgccgagctaatcgaacaat


 


attgtaaagctaataatttgagattaccccagattggttacacctcacgt


 


ttaattaggacccaacagaccatagaaacgatgtgtgaagaatttaagtt


 


aaagccacaactgcaggttgtttacgactttaataaaatcaaacttggag


 


acgaatttggcagtgatgacaaggataatatgaaaatcccgattatcaaa


 


cttggaggctaaatgaacgtcattacggttcctggcagggccagaggaaa


 


ccgaatgttttaaaagaatatggtaaggataaatatatgttcattaggag


 


agattacgagggtaagccaccacctgtagatcttgaccgtgagatgattc


 


aacaagaaaatgagaagggctcttctactgggtacgaattcaaggagcca


 


aacagacaaataaaatatgaattggaatgcagcaatcatgacattgtatt


 


accggattccgaatctcttcgtgaagtggtttatagattgaatccattct


 


acaaaatgtcatattaaaattagccaatcaatatgatgaatcttcatgcc


 


tgattgtgggccatggaagttcagtgagatcgctactgaaaattctggag


 


ggtatatcagatgatgacatcaagaatgttgatattccaaatggtatccc


 


cttagtcgttgaattagataagaataatggtcttaagtttatcagaaaat


 


tctacctagatcctgaatctgctaagatcaatgctgagaaagtccgtaat


 


gagggtttcataaaaaatccttaaggggggtaagtatataatataattgt


 


gtattttccgaagtataatgaaaaccaatagaaaacttattataagtcca


 


atgaggtactttaaaaatgtgatatttataagaacattcctgaatgcaga


 


tatatgatatatattgtaaatatatatagatgtgtatatgtatttccatt


 


ttgtgtgaggttttcttcttttatctcctatataatttgtaaccttaatt


 


aacccatgacataaccaatattagcctttgcaaattttgtaacttcttga


 


cgttgttctaacgacaaatcttcatgcttcgattttatatgccttgttaa


 


agcatccagtctcgaaaacgtcttctgatagccctcagatccaagaattt


 


ttatacactccgagcaacggaagacaatcttccttttagcgtgaatggta


 


ttttggtgtctcgttaaatcataggaccttgaaaattgggcaccacacgg


 


ttcatttgtaatgagattcattatctgacacgtaaatatttcgttattac


 


cgtcagaagatgacgtatgggccgatggtgatgcagtcgaaggtttcgaa


 


ttcgaatttgtagatgaatgtgaagataagtgcttc






Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQ ID NO:99):







cagaattggtgatattcttcattatcccctcgacatctttacttttgatc


 


agctttgtgtatagcgggatatccgattggaacttggcttcagcaacaaa


 


cttgccaaagtggattctcctactcaagctttgcaaacattctatatctc


 


tagtggcaacagaaccgaagttattcttatcatcaccatctcttttcgaa


 


attaatggtataatcttttcaatataaacttatttattttatcattgtaa


 


ttaacttctggggcataaggcgccaaaatttgtgggtagttaatgctcgg


 


taagaatgatttctgaatcttgtcaggaaagaagggagtttcatcaggtg


 


attcgaatcttctgatgcgagaatgcgcaatttcaagatttgaaagagcc


 


caatccaagaaagatcctttaaaattcggaatttctaaacctggatggtt


 


tgcctcataaactgaaggacatgtggcgaaatgcgacctctcaataaatt


 


tgaagatgatcgaatcctccattctaactaattcatctctaatattttgt


 


agatttaaaacagtttctggttttgtgaaatccatatcttataccaattt


 


tatgcaggatgctgagtgctatttgttagcaaaacggaatcgatgtttca


 


acttttcaatactttttttcttcttttcttcgaacttcgaagattataga


 


gatccattgaaaatttacgataaaggaaatgcaacacgaagtttgaaaaa


 


aagttgatattgaaaaaaaaaaaaaaagagaaccaaaaaaaaattaaaaa


 


acgtgaaaacgatggtttaacaacttttttcgaatttggtatacgtggaa


 


aaacgaatgtatagatgcatttttaaagaatatatataaaatttagtaat


 


tgtattccgcgagcggcgcaataggtgatttcatttagctgctttcagtt


 


tgcgtatcatttcttcattgatcttggcttctctatctaaatcctctttc


 


gacttgtaaagtccccaagttctaaaccataagaaccgcctcaatctgga


 


aaatttgtcagtatcaagaccataattcgtgtatgactgaatcaaatgta


 


atccactttcgtcatgagtaaattcggccttgctcagagactcctggatt


 


ttggctaacaacgcagtcccttcgatgcatatagctaggccacaaattat


 


gccaataacggtccatgggttgatgttttcttgaattctttcgtttttca


 


tgctatttgcgtcttcccaagtcccagcgttccagtattcatactgcgcg


 


ttagagtggtagccataagagccggcatattggtaattttcagtattaac


 


gttagaacgtggtgaatacgatgtggtccagccttgcctcgttgtgtcat


 


atacgatctttttctttgggtcacaaagaatatcatatgcttgagagatg


 


actttaaatctatgtagtttttcgcttgatgttagcagcagcggtgattt


 


actatcactgttggtaaccttttctgagctaaatatttgaatgttatcgg


 


aatggtcagggtggtacaattttacataacgatgatattttttttttaac


 


gacttcttgtccagtttaggatttccagatccggcctttggaatgccaaa


 


aatatcatagggagttggatctgccaactcaggccattgttcatccctta


 


tcgtaagttttctattgccatttttatcgttcgctgtagcatacttagct


 


ataaaagtgatttgtgggggacacttttctacacatgataagtgccactt


 


gaataaaaatgggtatacgaacttatggtgtagcataacaaatatattgc


 


aagtagtgacctatggtgtgtagatatacgtacagttagttacgagccta


 


aagacacaacgtgtttgttaattatactgtcgctgtaatatcttctcttc


 


cattatcaccggtcattccttgcaggggcggtagtacccggagaccctga


 


acttttctttttttttttgcgaaattaaaaagttcattttcaattcgaca


 


atgagatctacaagccattgttttatgttgatgagagccagcttaaagag


 


ttaaaaatttcatagctactagtcttctaggcgggttatctactgatccg


 


agcttccactaggatagcacccaaacacctgcatatttggacgaccttta


 


cttacaccaccaaaaaccactttcgcctctcccgcccctgataacgtcca


 


ctaattgagcgattacctgagcggtcctcttttgtttgcagcatgagact


 


tgcatactgcaaatcgtaagtagcaacgtctcaaggtcaaaactgtatgg


 


aaaccttgtcacctcacttaattctagctagcctaccctgcaagtcaaga


 


ggtctccgtgattcctagccacctcaaggtatgcctctccccggaaactg


 


tggccttttctggcacacatgatctccacgatttcaacatataaatagct


 


tttgataatggcaatattaatcaaatttattttacttctttcttgtaaca


 


tctctcttgtaatcccttattccttctagctatttttcataaaaaaccaa


 


gcaactgcttatcaacacacaaacactaaatcaaaatggctgttactaat


 


gtcgctgaacttaacgcactcgtagagcgtgtaaaaaaagcccagcgtga


 


atatgccagtttcactcaagagcaagtagacaaaatcttccgcgccgccg


 


ctctggctgctgcagatgctcgaatcccactcgcgaaaatggccgttgcc


 


gaatccggcatgggtatcgtcgaagataaagtgatcaaaaaccactttgc


 


ttctgaatatatctacaacgcctataaagatgaaaaaacctgtggtgttc


 


tgtctgaagacgacacttttggtaccatcactatcgctgaaccaatcggt


 


attatttgcggtatcgttccgaccactaacccgacttcaactgctatctt


 


caaatcgctgatcagtctgaagacccgtaacgccattatcttctccccgc


 


acccgcgtgcaaaagatgccaccaacaaagcggctgatatcgttctgcag


 


gctgctatcgctgccggtgctccgaaagatctgatcggctggatcgatca


 


accttctgttgaactgtctaacgcactgatgcaccacccagacatcaacc


 


tgatcctcgcgactggtggtccgggcatggttaaagccgcatacagctcc


 


ggtaaaccagctatcggtgtaggcgcgggcaacactccagttgttatcga


 


tgaaactgctgatatcaaacgtgcagttgcatctgtactgatgtccaaaa


 


ccttcgacaacggcgtaatctgtgcttctgaacagtctgttgttgttgtt


 


gactctgtttatgacgctgtacgtgaacgttttgcaacccacggcggcta


 


tctgttgcagggtaaagagctgaaagctgttcaggatgttatcctgaaaa


 


acggtgcgctgaacgcggctatcgttggtcagccagcctataaaattgct


 


gaactggcaggcttctctgtaccagaaaacaccaagattctgatcggtga


 


agtgaccgttgttgatgaaagcgaaccgttcgcacatgaaaaactgtccc


 


cgactctggcaatgtaccgcgctaaagatttcgaagacgcggtagaaaaa


 


gcagagaaactggttgctatgggcggtatcggtcatacctcttgcctgta


 


cactgaccaggataaccaaccggctcgcgtttcttacttcggtcagaaaa


 


tgaaaacggcgcgtatcctgattaacaccccagcgtctcagggtggtatc


 


ggtgacctgtataacttcaaactcgcaccttccctgactctgggttgtgg


 


ttcttggggtggtaactccatctctgaaaacgttggtccgaaacacctga


 


tcaacaagaaaaccgttgctaagcgagctgaaaacatgttgtggcacaaa


 


cttccgaaatctatctacttccgccgtggctccctgccaatcgcgctgga


 


tgaagtgattactgatggccacaaacgtgcgctcatcgtgactgaccgct


 


tcctgttcaacaatggttatgctgatcagatcacttccgtactgaaagca


 


gcaggcgttgaaactgaagtcttcttcgaagtagaagcggacccgaccct


 


gagcatcgttcgtaaaggtgcagaactggcaaactccttcaaaccagacg


 


tgattatcgcgctgggtggtggttccccgatggacgccgcgaagatcatg


 


tgggttatgtacgaacatccggaaactcacttcgaagagctggcgctgcg


 


ctttatggatatccgtaaacgtatctacaagttcccgaaaatgggcgtga


 


aagcgaaaatgatcgctgtcaccaccacttctggtacaggttctgaagtc


 


actccgtttgcggttgtaactgacgacgctactggtcagaaatatccgct


 


ggcagactatgcgctgactccggatatggcgattgtcgacgccaacctgg


 


ttatggacatgccgaagtccctgtgtgctttcggtggtctggacgcagta


 


actcacgccatggaagcttatgtttctgtactggcatctgagttctctga


 


tggtcaggctctgcaggcactgaaactgctgaaagaatatctgccagcgt


 


cctaccacgaagggtctaaaaatccggtagcgcgtgaacgtgttcacagt


 


gcagcgactatcgcgggtatcgcgtttgcgaacgccttcctgggtgtatg


 


tcactcaatggcgcacaaactgggttcccagttccatattccgcacggtc


 


tggcaaacgccctgctgatttgtaacgttattcgctacaatgcgaacgac


 


aacccgaccaagcagactgcattcagccagtatgaccgtccgcaggctcg


 


ccgtcgttatgctgaaattgccgaccacttgggtctgagcgcaccgggcg


 


accgtactgctgctaagatcgagaaactgctggcatggctggaaacgctg


 


aaagctgaactgggtattccgaaatctatccgtgaagctggcgttcagga


 


agcagacttcctggcgaacgtggataaactgtctgaagatgcattcgatg


 


accagtgcaccggcgctaacccgcgttacccgctgatctccgagctgaaa


 


cagattctgctggatacctactacggtcgtgattatgtagaaggtgaaac


 


tgcagcgaagaaagaagctgctccggctaaagctgagaaaaaagcgaaaa


 


aatccgcttaagtcgagagcttttgattaagccttctagtccaaaaaaca


 


cgtttttttgtcatttatttcattttcttagaatagtttagtttattcat


 


tttatagtcacgaatgttttatgattctatatagggttgcaaacaagcat


 


ttttcattttatgttaaaacaatttcaggtttaccttttattctgcttgt


 


ggtgacgcgtgtatccgcccgctcttttggtcacccatgtatttaattgc


 


ataaataattcttaaaagtggagctagtctatttctatttacatacctct


 


catttctcatttcctcctaatgtgtcaatgatcatattcttaactggacc


 


gatcttattcgtcagattcaaaccaaaagttcttagggctaccacaggag


 


gaaaattagtgtgatataatttaaataatttatccgccattcctaataga


 


acgttgttcgacggatatctttctgcccaaaagggttctaagctcaatga


 


agagccaatgtctaaacctcgttacattgaaaatacagtaaatggttcca


 


ccattattatgttggtcttgtttagtatggccgatcggcgtgtgttttgt


 


ttgcaccattttatatagtagaagaatatttgtcttaattcttattagta


 


ctgcaacctaaccactaattatcaacaattattggattatataaaggagg


 


taaattgccggattaaaatcaaatatcattcatcaacaagtattcatatt


 


gtcggcatatttttacatgcggtgtaagtatttggatcgtattcttatag


 


tgtcaatacctcgaagcagcgtttcaagtaccagacgtatgtaggaactt


 


tttaacgtcgagtccgtaagatttgatcagtattaaaaaaatctagataa


 


atgagtggtacaaataaaaacatcattaaaaatcgttaaataaaaaagta


 


tgaagatcatctattaaagtattagtagccattagccttaaaaaaatcag


 


tgctagtttaagtataatctcgggcgcgccggccgaggcggttaagcgga


 


ttttttcgcttttttctcagctttagccggagcagcttctttcttcgctg


 


cagtttcaccttctacataatcacgaccgtagtaggtatccagcagaatc


 


tgtttcagctcggagatcagcgggtaacgcgggttagcgccggtgcactg


 


gtcatcgaatgcatcttcagacagtttatccacgttcgccaggaagtctg


 


cttcctgaacgccagcttcacggatagatttcggaatacccagttcagct


 


ttcagcgtttccagccatgccagcagtttctcgatcttagcagcagtacg


 


gtcgcccggtgcgctcagacccaagtggtcggcaatttcagcataacgac


 


ggcgagcctgcggacggtcatactggctgaatgcagtctgcttggtcggg


 


ttgtcgttcgcattgtagcgaataacgttacaaatcagcagggcgtttgc


 


cagaccgtgcggaatatggaactgggaacccagtttgtgcgccattgagt


 


gacatacacccaggaaggcgttcgcaaacgcgatacccgcgatagtcgct


 


gcactgtgaacacgttcacgcgctaccggatttttagacccttcgtggta


 


ggacgctggcagatattctttcagcagtttcagtgcctgcagagcctgac


 


catcagagaactcagatgccagtacagaaacataagcttccatggcgtga


 


gttactgcgtccagaccaccgaaagcacacagggacttcggcatgtccat


 


aaccaggttggcgtcgacaatcgccatatccggagtcagcgcatagtctg


 


ccagcggatatttctgaccagtagcgtcgtcagttacaaccgcaaacgga


 


gtgacttcagaacctgtaccagaagtggtggtgacagcgatcattttcgc


 


tttcacgcccattttcgggaacttgtagatacgtttacggatatccataa


 


agcgcagcgccagctcttcgaagtgagtttccggatgttcgtacataacc


 


cacatgatcttcgcggcgtccatcggggaaccaccacccagcgcgataat


 


cacgtctggtttgaaggagtttgccagttctgcacctttacgaacgatgc


 


tcagggtcgggtccgcttctacttcgaagaagacttcagtttcaacgcct


 


gctgctttcagtacggaagtgatctgatcagcataaccattgttgaacag


 


gaagcggtcagtcacgatgagcgcacgtttgtggccatcagtaatcactt


 


catccagcgcgattggcagggagccacggcggaagtagatagatttcgga


 


agtttgtgccacaacatgttttcagctcgcttagcaacggttttcttgtt


 


gatcaggtgtttcggaccaacgttttcagagatggagttaccaccccaag


 


aaccacaacccagagtcagggaaggtgcgagtttgaagttatacaggtca


 


ccgataccaccctgagacgctggggtgttaatcaggatacgcgccgtttt


 


cattttctgaccgaagtaagaaacgcgagccggttggttatcctggtcag


 


tgtacaggcaagaggtatgaccgataccgcccatagcaaccagtttctct


 


gcatttctaccgcgtcttcgaaatctttagcgcggtacattgccagagtc


 


ggggacagtttttcatgtgcgaacggttcgctttcatcaacaacggtcac


 


ttcaccgatcagaatcttggtgttttctggtacagagaagcctgccagtt


 


cagcaattttataggctggctgaccaacgatagccgcgttcagcgcaccg


 


tttttcaggataacatcctgaacagctttcagctctttaccctgcaacag


 


atagccgccgtgggttgcaaaacgttcacgtacagcgtcataaacagagt


 


caacaacaacaacagactgttcagaagcacagattacgccgttgtcgaag


 


gttttggacatcagtacagatgcaactgcacgtttgatatcagcagtttc


 


atcgataacaactggagtgttgcccgcgcctacaccgatagctggtttac


 


cggagctgtatgcggctttaaccatgcccggaccaccagtcgcgaggatc


 


aggttgatgtctgggtggtgcatcagtgcgttagacagttcaacagaagg


 


ttgatcgatccagccgatcagatctttcggagcaccggcagcgatagcag


 


cctgcagaacgatatcagccgctttgttggtggcatcttttgcacgcggg


 


tgcggggagaagataatggcgttacgggtcttcagactgatcagcgattt


 


gaagatagcagttgaagtcgggttagtggtcggaacgataccgcaaataa


 


taccgattggttcagcgatagtgatggtaccaaaagtgtcgtcttcagac


 


agaacaccacaggttttttcatctttataggcgttgtagatatattcaga


 


agcaaagtggtttttgatcactttatcttcgacgatacccatgccggatt


 


cggcaacggccattttcgcgagtgggattcgagcatctgcagcagccaga


 


gcggcggcgcggaagattttgtctacttgctcttgagtgaaactggcata


 


ttcacgctgggctttttttacacgctctacgagtgcgttaagttcagcga


 


cattagtaacagccataattcttaattaactttgatatgattttgtttca


 


gattttttatataaaagctttcccaaatagtgctaaagtgaacttagatt


 


ttttggtacctgtttcgaaattaaaaatagaaaaatttctctccctatat


 


tgttattcttacttcaaatttgtttatcgtttatttactaggcgagactt


 


gagtagacgacaatccaaatagaattaacagattttattggtagaaagca


 


ataatattctttagatggttgagaataaagaagtaaaaaaaccagtaaag


 


agaaaaagaaaaggaagaaaattaaagaaaaaggatgattacacaagaag


 


ataataaaaaaactcctttattaagagcggaagaatttaataatgaagat


 


gggaataagcaaaacaaaaacaaagaagggaaaaaaaataaaaaatcgta


 


tttatttatttaaaaaatcatgttgatgacgacaatggaaaaaaaaaacc


 


gatttcactttctcatccttatatttttcaaaggttgatgcaagtcgatc


 


tcaaatcggataacgctgccaactgggaaattccgcaattccgcaagaaa


 


aaaaaaaatgtgaaaacgtgattgcattttttacaggtcctaaaggattt


 


agcccacatatcaagagggtggcagtaattgcactgattaagcattcgtc


 


agcattaggcgaatgtgtgcatgaatattgccagtgtgctcgatattaga


 


gagtacattgaagaatattgtaccggattatgtacaataactttgttaat


 


gagatattaattttcttttttactagccgctatcccatgcacgatgctaa


 


atttcaagaagaaactgagatttaaaaaattagtggaagctgataaaacg


 


gactataatggtgtatggattgaggaatctcgacatgtttttccatcgtt


 


ttcaacgatgactgtaacccgtagattgaaccaggcatgccaaagttagt


 


tagatcagggtaaaaattatagatgaggtttaattaaacaagcacgcagc


 


acgctgtatttacgtatttaattttatatatttgtgcatacactactagg


 


gaagacttgaaaaaaacctaggaaatgaaaaaacgacacaggaagtcccg


 


tatttactattttttccttccttttgatggggcagggcggaaatagagga


 


taggataagcctactgcttagctgtttccgtctctacttcggtagttgtc


 


tcaattgtcgtttcagtattacctttagagccgctagacgatggttgagc


 


tatttgttgagggaaaactaagttcatgtaacacacgcataacccgatta


 


aactcatgaatagcttgattgcaggaggctggtccattggagatggtgcc


 


ttattttccttataggcaacgatgatgtcttcgtcggtgttcaggtagta


 


gtgtacactctgaatcagggagaaccaggcaatgaacttgttcctcaaga


 


aaatagcggccataggcatggattggttaaccacaccagatatgcttggt


 


gtggcagaatatagtccttttggtggcgcaattttcttgtacctgtggta


 


gaaagggagcggttgaactgttagtatatattggcaatatcagcaaattt


 


gaaagaaaattgtcggtgaaaaacatacgaaacacaaaggtcgggccttg


 


caacgttattcaaagtcattgtttagttgaggaggtagcagcggagtata


 


tgtattccttttttttgcctatggatgttgtaccatgcccattctgctca


 


agcttttgttaaaattatttttcagtattttttcttccatgttgcgcgtt


 


acgagaacagaagcgacagataaccgcaatcatacaactagcgctactgc


 


ggggtgtaaaaagcacaagaactaagccaagatcacaacagttatcgata


 


aaatagcagtgtttgcatggccattgagaaggacaacattggcgtgcgcg


 


ccaatgttgtctcaccatgtagctccaaacgagttgtaagagacggaccg


 


ctcacgcttccgaagcggtcagaaaacgcttcccagtatgcagttgacct


 


acattcaacctgcaaatattgctttgcttcaagaaatgattacacagacg


 


tctattttcttctacataatgcacgaaacttgggcatttagtcatgtagc


 


cgcctagcgagcctgggtgccgtcctatctcctttgttcgtgcaaagaga


 


caggaacacacactgcgttctcttgcggccggtctggcggactcaggggt


 


gcggcgtttgcttaaccggagggaataataaaatcggggtgacgcaagta


 


tgaagtcatgtgtgcttagcaattacgtagagggattagaaataatagtg


 


tgcggttatcggaaccggctcttgttcccgtttagagcaacccaggtgca


 


ggcgtactttaaagtattttctttcttttttttcctgctacttacgctag


 


gagctgccgcagctgcaaagccgacgtcggagaggcaggtgatcttcggc


 


tcggccgacaaatcccctggatatcattggcctgtcgaggtatcggccgc


 


gtggaactaccgggaattactatgcaaaacaattggaaatctggtaggaa


 


aaccttgttctagaacttggcgattgctgacaaagaagaaaagggcctat


 


tgttgctgcctcttttgttgttcttcctcgtattgtcttgccggtgttct


 


ttgtgtcttttgtgtgtaggttcttactattatagtgctctttgctatta


 


tattttcttcgttttcactttgcgtaatgtaacggtcttaaacaaagttt


 


tttttttttcgctcttgcattttccttttctgctctatcttatttgctaa


 


ttgtagtttcagaagttttacttaaatatagcactattttccagttttaa


 


tgtttcttctcattgctttcttttataattttcgcatataattatacatt


 


tacggtgtcttaactctccctcttcacccctcattattccagaaaatact


 


aatacttcttcacacaaaagaacgcagttagacaatcaacaatgaatcct


 


aaatcctctacacctaagattccaagacccaagaacgcatttattctgtt


 


cagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtg


 


tggaaataccccataattcaaacatttctaaaattattggtacgaagtgg


 


aagggcttacaaccggaagataaggcacactgggaaaatctagcggagaa


 


ggagaaactagaacatgaaaggaagtatcctgaatacaaatacaagccgg


 


taagaaagtctaagaagaagcaactacttttgaaggaaatcgagcaacag


 


cagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgca


 


attacaacagccctttaacaacaatatagttcttatgaaaagagcacatt


 


ctctttcaccatcttcctcggtgtcaagctcgaacagctatcagttccaa


 


ttgaacaatgatcttaagaggttgcctattccttctgttaatacttctaa


 


ctatatggtctccagatctttaagtggactacctttgacgcatgataaga


 


cggcaagagacctaccacagctgtcatctcaactaaattctattccatat


 


tactcagctccacacgacccttcaacgagacatcattacctcaacgtcgc


 


tcaagctcaaccaagggctaactcgacccctcaattgccctttatttcat


 


ccattatcaacaacagcagtcaaacaccggtaactacaactaccacatcc


 


acaacaactgcgacatcttctcctgggaaattctcctcttctccgaactc


 


ctctgtactggagaacaacagattaaacagtatcaacaattcaaatcaat


 


atttacctccccctctattaccttctctgcaagattttcaactggatcag


 


taccagcagctaaagcagatgggaccaacttatattgtcaaaccactgtc


 


tcacaccaggaacaatctattgtccacaactacccctacgcatcatcaca


 


ttcctcatataccaaaccaaaacattcctctacatcaaattataaactca


 


agcaacactgaggtcaccgctaaaactagcctagtttctccgaaatgatt


 


ttttttttccatttcttctttccgttatattatattatactatattccct


 


ttaactaaaaatttatgcatttggctcctgtttaataaaagtttaaatc






These strains were grown in YPD containing 50 g/L glucose under anaerobic and microaerobic conditions, and formate was measured over 142 hours. As shown in FIG. 14, at the end of 142 hours, strains containing the heterologous PFL and AADH made more formate than the wildtype strain M139.
Example 6
Expression of PFL and AADH and Detection of Ethanol
The purpose of this Example is to determine whether formate production can confer anaerobic growth on fdh, gpd, and/or fps deletion strains. Yeast strains containing an fdh1Δfdh2Δgpd1Δgpd2Δ genetic background (M2025) were transformed with vectors expressing PflA/B cassettes from C. cellulolyticum (TB274) and E. coli (TB275). Each of these strains also contained a second construct expressing the E. coli AdhE. YPD medium was prepared and added to hungate tubes, oxygen was purged with nitrogen, and the tubes were autoclaved for 20 minutes. A pre-culture of TB274 and TB275 was prepared overnight in YPD medium containing antibiotics which select for maintenance of both plasmids. A pre-culture of M1901, the parent strain of M2025, and M2025 itself were prepared in YPD and included as positive and negative controls, respectively. A strain referred to as TB267 was created which contains only the bifunctional ADH plasmid. This strain was prepared in YPD plus antibiotic to select for the plasmid. This strain controls for the potential effect of ADH or other electron acceptors that may be present in YPD medium.
All strains were inoculated to final OD's of about 0.05 or below. The OD of each culture was measured at 0, 24, 48, and 72 hours (FIG. 15), and samples were prepared and submitted for HPLC determination of metabolite levels. As expected, the M1901 strain grew fairly quickly, consuming all the sugar substrate by 24 hours. The M2025 and TB267 strains, which are unable to make glycerol, did not show a significant increase in OD during this experiment. The TB274 and TB275 strains, which express both PFL and ADH, were able to grow after a 24 hour lag time. See FIG. 15. These data indicate that the introduced metabolic pathway in TB274 and TB275 does not block cellular growth.
The production of glycerol in these strains is shown in FIG. 16. The anaerobic growth of M1901 was accompanied by glycerol production as expected. A trace amount of glycerol was observed in the PflA/B containing strains TB274 and TB275, but this was at or below the level of the negative controls which did not grow. See FIG. 16. These data, in conjunction with the OD data, indicate that the expression of PflA/B and AdhE allowed for anaerobic growth of M2025 without associated glycerol production.
The production of ethanol and glucose concentration are shown in FIGS. 17 and 18, respectively. Both M1901 and TB274 consumed all the sugar, but about 5 g/L glucose remained in the TB275 fermentation. See FIG. 18. TB274 had an 11% increase in ethanol yield in comparison to M1901. See FIG. 17. The increase in yield was higher than expected and likely came partially at the expense of biomass, although this was not determined.
Strains containing PFL and AADH were compared to other strains engineered to express AADH. A description of these strains appears in Table 4.







TABLE 4







Genetic Backgrounds for PFL Expression










Strain Name
Genetic Background







M139
wt control



M2085
gdp1Δ gpd2Δ fdh1Δ fdh2Δ



M2158
gdp1Δ gpd2Δ fdh1Δ fdh2Δ + AADH



M2182
gdp1Δ gpd2Δ fdh1Δ fdh2Δ + AADH and PFL










These five strains were tested in nitrogen purged bottles. As shown in FIG. 19, strain M2182 which has both PFL and AADH expression cassettes in a gdp1Δ gpd2Δ fdh1Δfdh2Δ background, had a faster growth rate and reached a higher OD than M2158, which contains only the bifunctional ADH activity and did not appear to grow at all. See FIG. 19. These data show that a strain expressing both PFL and AADH activities grows better than the other engineered functionalities. Similar improvements over these other engineered functionalities are also observed in corn mash fermentations (see Example 7 below).
Example 7
Production of Ethanol from Corn Mash
The purpose of this experiment was to determine whether PFLs cloned from the organisms listed below in Table 5 could provide for increased ethanol yield when used in fermentation of 20% corn mash. A total of nine PFLs have been tested for function in yeast. Of these, only the C. cellulolyticum PFL had no positive effect on growth of glycerol synthesis mutants in corn mash fermentations. Additionally, no formate was observed in formate assays when using a strain containing C. cellulolyticum PFL. This strain was not tested for performance on corn mash fermentation.
Eight PFLs were tested for functionality in strain M2158 which has the E. coli AADH integrated on the chromosome of a gpd1Δ gpd2Δ fdh1Δ fdh2Δ background (M2085) or M2275, which is identical to M2158 except that it also has the gpd2Δ::GPD1 glycerol reduction mutation. Two separate corn mash fermentation experiments were performed using 20% solids in a baffled shake flask using the strains listed in Table 5. Performance of the strains was evaluated by HPLC analysis of metabolites.







TABLE 5







Genetic Backgrounds for Corn Mash Fermentations










Strain Name
Genetic Background







M139
wt control



M2085
gpd1Δ gpd2Δ fdh1Δ fdh2Δ



M2158
integrated AADH



M2180
M2158 + B. lichenformis PFL



M2181
M2158 + L. planatarum PFL



M2182
M2158 + B. adolescentis PFL



M2183
M2158 + S. thermophilus PFL



M2184
M2158 + E. coli PFL



M2321
M2158 + L. casei PFL



M2322
M2158 + C. reinhardtii PFL



M2323
M2158 + Piromyces PFL



M2324
M2158 + E. coli PFL



M2275
M2158 + gpdΔ::GPD1



M2326
M2275 + B. adolescentis PFL










As shown in FIGS. 21 and 22, the addition of PFL improves the ethanol yield of strains containing only AADH. FIGS. 23 and 24 show that these strains do not make glycerol as expected. The approximately 4 g/L glycerol observed is already in the industrial mash substrate used for this experiment. FIG. 25 demonstrates a 9% increase in ethanol yield with strain M2326, which is a glycerol reduction strain.
Example 8
The following example demonstrates the creation of the Saccharomyces cerevisiae strain M3625. The genotype of strain M3625 is: Δgpd2::B. adolescentis pflA/pflB/adhE Δfdh1Δfdh2::B. adolescentis pflA/pflB/adhE fcy1Δ::S. fibuligera glucoamylase (glu-0111-CO). Strain M2390 is referred to as Ethanol Red (new) from LaSaffre (pahc.com/Phibro/Performance-Products/Catalog/23/Ethanol-Red.html).
The genetic modification techniques utilized to develop Saccharomyces cerevisiae strain M3625 relied upon directed integration to insert the genes for Bifidobacterium adolescentis pflA, pflB, AdhE and S. fibuligera glu-0111-CO at specific and known sites within the yeast chromosome. The directed integration approach creates transgenic strains with integration events that are stable and easy to characterize. Chromosomal integration, by its very nature, reduces the probability of any mobilization of the heterologous DNA and enhances strain stability relative to other approaches.
The MX cassettes are the most commonly used engineering tool when an insertion or deletion of a genetic element is desired at a given chromosomal loci (Wach A, et al., Yeast 10(13):1793-1808 (1994)). A recyclable MX cassette contains one or more markers which enable both dominant and negative selection (Goldstein, A. L. and McCusker, J. H., Yeast 15:1541-1553 (1999); Ito-Harashima, S. and McCusker, J. H., Yeast 21:53-61 (2004)). The dominant marker enables selection for the modification and the counter selectable marker enables subsequent removal of the marker system via Cre-Lox mediated recombination (Güldener, Ulrich, et al., Nucleic Acids Research (1996) 24 (13) 2519-2524) or recombination between duplicated homologous regions flanking the cassette. Since the markers are removed, they can be reused during subsequent engineering steps and ensures no undesirable foreign genetic material remains in the strain.
To create stable homozygous integrations in M3625, two new HSV-thymidine kinase (TDK) based MX cassettes were developed. Expression of thymidine kinase in S. cerevisiae results in sensitivity to the compound fluoro-deoxyuracil (FUDR). The cellular toxicity of FUDR is dependent on the presence of two enzymes involved in pyrimidine metabolism: thymidine kinase (Tdk) and thymidilate synthetase (ThyA). Tdk converts FUDR to fluoro-dUMP (F-dUMP) which is a covalent inhibitor of ThyA and the basis for counter selection in a variety of eukaryotic organisms (Czako, M., and L. Marton, (1994) Plant Physiol 104:1067-1071; Gardiner, D. M., and B. J. Howlett, (2004) Curr Genet 45:249-255; Khang, C. H., et al., (2005) Fungal Genet Biol 42:483-492; Szybalski, W. (1992) Bioessays 14:495-500).
The HSV-TDK expression cassette was independently fused to two commonly used dominant selectable markers which confer resistance to the drugs G418 (Kan) or nourseothricin (Nat) (Goldstein, A. L. and McCusker, J. H., Yeast 15:1541-1553 (1999)). Transformation of both double expression cassettes, referred to as KT-MX and NT-MX, enables positive selection for integration into both chromosomes as illustrated in FIG. 28A. The transformed deletion assembly contains four PCR products, a 5′flank(p1) which is homologous upstream of the target site, KT-MX cassette(p2), NT-MX cassette(p3), and a 3′ flank(p4) homologous downstream of the target site. Each component is amplified individually using primers which create homologous overlapping extensions of each PCR product. See Tables 7 and 8. The bent dashed lines in FIG. 28A represent homology between the KT/NT-MX cassettes and the 5′ flank and the bent solid lines represent homology with the 3′ flank. For each round of engineering, PCR amplicons of upstream and downstream regions flanking the target site are designed to contain homologous tails for both the KT-MX and NT-MX cassettes. Both the flanks and the markers are transformed followed by selection on YPD medium containing both G418 and Nat. See FIG. 28B. FIG. 28B shows a schematic of the chromosome after replacement of the target site with KT-MX and NT-MX.
After each engineering step taken in the construction of M3625, all markers are subsequently deleted and/or replaced with a desired expression cassette (Mascoma Assembly) resulting in a strain free of antibiotic markers (FIG. 29). FIG. 29 demonstrates that the transformed Mascoma Assembly contains a quantity of PCR products which is dependent on the desired engineering event (pX), a 5′flank(p1) which homologous upstream of the target site and a 3′ flank (p4) homologous downstream of the target site. Each component is amplified individually using primers which create homologous overlapping extensions. The overlapping bent lines in FIG. 29 represent homology at the end of those PCR products. FIG. 29B shows a schematic of a chromosome following selection on FUDR and replacement of genetic markers with the Mascoma assembly. Confirmation of marker removal was evaluated by southern blot, PCR and dilution plating onto selective medium as described below.
Four loci were modified during the construction of M3625. The integration procedure strategy described above was used at the FDH1, GPD1 and GPD2 loci using the Mascoma Assemblies listed in Table 6. Detailed molecular maps depicting the components of each Mascoma Assembly are provided in FIGS. 30-37.







TABLE 6







Genetic modifications contained in M3625.










Target Locus
Locus Modification
Cassette ID
Cassette Description





FDH1
Clean Deletion
MA0370
Clean Deletion of





FDH1


FDH2
Replaced with
MA0280
2 copies of pflA/B



expression cassette

and 4 copies of adhE


GPD2
Replaced with
MA0289
2 copies of pflA/B



expression cassette

and 4 copies of adhE


FCY1
Replaced with
MA0317
Four copies of



expression cassette

Glucoamylase
















TABLE 7







Primers used for the creation of strain M3625.











PCR Product 1;
PCR Product 2;
PCR Product 3;


Target Locus
Primer Pair
Primer Pair
Primer Pair





GPD1
GPD1 5′ Flank;
pAGTEF-kan/nat-
GPD1 3′ Flank;



X11824/X15546
pHXT2-TDK;
X15547/X11829




X15380/X15382


GPD2
GPD2 5′ Flank;
pAGTEF-kan/nat-
GPD2 3′ flank;



X11816/X15548
pHXT2-TDK;
X15549/X11821




X15380/X15382


FDH2
FDH2 5′ Flank;
pAGTEF-kan/nat-
FDH2 3′ flank;



X16096/X15554
pHXT2-TDK;
X15555/X11845




X15380/X15382


FDH1
FHD1 5′ Flank;
pAGTEF-kan/nat-
FDH1 3′ flank;



X15559/X15550
pHXT2-TDK;
X15552/X15553




X15380/X15382
















TABLE 8







Sequences of Primers used for creation of strain M3625











SEQ ID


Primer
Sequence
NO





X11824
aagcctacaggcgcaagataacacatcac
110


 


X15546
ggacgaggcaagctaaacagatctctagacctactttatattatcaatatttgtgtttg
111


 


X15380
taggtctagagatctgtttagcttgc
112


 


X15382
gagactacatgatagtccaaaga
113


 


X15547
ccgtttcttttctttggactatcatgtagtctcatttattggagaaagataacatatca
114


 


X11829
ctcagcattgatcttagcagattcaggatctaggt
115


 


X11816
gcagtcatcaggatcgtaggagataagca
116


 


X15548
ggacgaggcaagctaaacagatctctagacctatgataaggaaggggagcg
117



aaggaaaa


 


X15549
ccgtttcttttctttggactatcatgtagtctcctctgatctttcctgttgcctctttt
118


 


X11821
tcacaagagtgtgcagaaataggaggtgga
119


 


X16096
catggtgcttagcagcagatgaaagtgtca
120


 


X15554
ggacgaggcaagctaaacagatctctagacctaattaattttcagctgttatttcgatt
121


 


X15555
ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag
122


 


X11845
ttacttgtgaaactgtctccgctatgtcag
123


 


X15559
ggaaggcaccgatactagaactccg
124


 


X15550
gggacgaggcaagctaaacagatctctagacctaattaattttcagctgttattttgat
125


 


X15552
ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag
126


 


X15553
accagcgtctggtggacaaacggccttcaac
127









Genotyping and Sequencing of MA0370
To confirm that FDH1 was deleted after insertion of MA370, PCR products were amplified from M2390 and M3625 genomic DNA using primers X17826 and X16944. The expected results are listed in Table 10 and the sequences of the primers used are listed in Table 11. A molecular map depicting the MA0370 integration site is shown in FIG. 30. The molecular map depicts the location of flanks used to remove the KT-MX and NT-MX markers and the position of primers used for genotyping. See FIG. 30 (5′ flank, S. cerevisiae FDH1 upstream flanking region; 3′ flank—S. cerevisiae FDH1 downstream flanking region. Region AA—amplified and sequenced chromosomal DNA region). Primer pair X15556/X15871 was used for the FDH1 5′ Flank and primer pair X15870/X15553 was used for the FHD1 3′ Flank to create the assembly shown in FIG. 30. Sequences for the primers for assembly used are found in Table 9. An agarose gel image showing PCR products used to determine genotype is shown in FIG. 31 (lane 1: 1 KB ladder; lane 2: M2390 (X17826/X16944); lane 3: M3625 (X17826/X16944)) (see Table 11).







TABLE 9







Primers used to create the MA0370 integration site.









Primer
Sequence
SEQ ID NO





X15556
ccactcgaggataggcttgaaaga
128


 


X15870
ctaatcaaatcaaaataacagctgaaaattaatgagtgattatgagta
129



tttgtgagcag


 


X15871
aaaacttctgctcacaaatactcataatcactcattaattttcagctgttattt
130



tgatt


 


X15553
accagcgtctggtggacaaacggccttcaac
127









In order to determine the exact DNA sequence of the M3625 MA0370 site, region AA was amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.







TABLE 10







Primers and summary of results of MA0370 genotyping.














Expected
Correct Size


Lane
Template DNA
Primers
size(bp)
Observed





1
1 KB ladder
N/A
N/A
N/A


2
M2390
X17826/X16944
4386 bp
yes


3
M3625
X17826/X16944
3237
yes
















TABLE 11







Sequence of primers used for MA0370 genotyping.











Primer
Sequence
SEQ ID NO







X17826
tcgctaacgatcaagaggaactg
152



 



X16944
tacacgtgcatttggacctatc
153










Genotyping and Sequencing of MA0280
To confirm that MA280 was inserted at the FDH2 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 13. Sequences of the primers used for genotyping and sequencing MA0280 are listed in Table 14. A molecular map depicting the MA0280 integration site is shown in FIG. 32. The molecular map depicts the location of flanks used to replace the KT-MX and NT-MX markers and insert the MA0280 expression cassette. The position of primers used for genotyping for genotyping are indicated on the map. See FIG. 32 (Feature description on map of MA0280 site of the M3625 strain; FDH2 5′ flank—S. cerevisiae FDH2 upstream flanking region; PFK1p—S. cerevisiae PFK1 gene promoter; ADHE—Bifidobacterium adolescentis ADHE coding gene; HXT2t-S. cerevisiae HXT2 gene terminator; ENO1p—S. cerevisiae ENO1 gene promoter; PFLB—Bifidobacterium adolescentis PFLB coding gene; ENO1t—S. cerevisiae ENO1 gene terminator; ADH1p—S. cerevisiae ADH1 gene promoter; PFLA—Bifidobacterium adolescentis PFLA coding gene; PDC1t—S. cerevisiae PDC1 gene terminator; FBA1t—S. cerevisiae FBA1 gene terminator; TPI1p—S. cerevisiae TPI1 gene promoter; FDH2 3′ flank—S. cerevisiae FDH2 downstream flanking region. Regions BA-BE, amplified and sequenced chromosomal DNA regions). Primer pair X16096/X17243 was used for the FDH2 5′ Flank, primer pair X16738/X16620 was used for pPFK1-ADH-HXT2, primer pair X16621/X13208 was used for pENO1-PFL-ENO1t, primer pair X13209/X17242 was used for pADH1-PFL-PDC1t, primer pair X17241/X16744 was used for pTPI-ADH-FBA1trc, and primer pair X17244/X11845 was used for the FDH2 5′ Flank to create the assembly shown in FIG. 32. Sequences for the primers used to create the assembly shown in FIG. 32 are found in Table 12. An agarose gel image showing PCR products used to genotype and sequence the MA0280 site is shown in FIG. 33 (lane 1: 1 KB ladder; lane 2: M3625 (17413/15810); lane 3: M3625 (17834/14554); lane 4: M3625 (16291/15229); lane 5: M3625 (16503/11317); lane 6: (16241/16946) lane 7: 1 KB ladder) (see Table 14).







TABLE 12







Primers used to create the MA0280 integration site.









Primer
Sequence
SEQ ID NO





X16096
catggtgcttagcagcagatgaaagtgtca
120


 


X17243
tagttagatcagggtaaaaattatagatgaggtattaattttcagct
131



gttatttcgatt


 


X16738
ctaatcaaatcgaaataacagctgaaaattaatacctcatctataat
132



ttttaccctgat


 


X16620
tcggatcagtagataacccgcctagaagactaggttacattgaaa
133



atacagtaaatggt


 


X16621
tggtggaaccatttactgtattttcaatgtaacctagtcttctaggcg
134



ggttatctact


 


X13208
ccgaaatattccacggtttagaaaaaaatcggaggtttagacattg
135



gctcttcattgag


 


X13209
aagctcaatgaagagccaatgtctaaacctccgatttttttctaaac
136



cgtggaatattt


 


X17242
acatcatcttttaacttgaatttattctctagctttcaatcattggagc
137



aatcatttta


 


X17241
gtccatgtaaaatgattgctccaatgattgaaagctagagaataa
138



attcaagttaaaag


 


X16744
aaaaacttctgctcacaaatactcataatcactcctacttattcccttc
139



gagattatatc


 


X17244
gttcctagatataatctcgaagggaataagtaggagtgattatgagta
140



tttgtgagcag


 


X11845
ttacttgtgaaactgtctccgctatgtcag
141









In order to determine exact DNA sequence of the M3625 MA0280 site, regions BA-BE were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.







TABLE 13







Primers and summary of results of MA0280 genotyping.














Expected
Correct Size


Lane
Template DNA
Primers
size(bp)
Observed





1
1 KB ladder
N/A
N/A
N/A


2
M3625
17413/15810
5567
Yes


3
M3625
17834/14554
3686
Yes


4
M3625
16291/15229
2569
Yes


5
M3625
16503/11317
4352
yes


6
M3625
16241/16946
2478
yes


7
1 KB ladder
N/A
N/A
N/A
















TABLE 14







Sequence of primers used for genotyping MA0280.









Primer
Sequence
SEQ ID NO





17413
ggattcttcgagagctaaga
154


 


15810
gacttgcagggtaggctagctagaatt
155


 


17834
gctgcttcgaggtattgaca
156


 


14554
ggctcttcattgagcttagaaccc
157


 


16291
aactggaccgatcttattcgt
158


 


15229
agtccactgcggagtcatttcaaag
159


 


16503
ctgccagcgaattcgactctgcaat
160


 


11317
cagtcgctgtagtgagcgacagggtagtaa
161


 


16241
ctttgcattagcatgcgta
162


 


16946
taggtcgagaccagaatgcatgt
163









Genotyping and Sequencing of MA0289
To confirm that MA0289 was inserted at the GPD2 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 16. Sequences for the primers used for genotyping MA0289 are listed in Table 17. A molecular map depicting the MA0289 integration site is shown in FIG. 34. The molecular map depicts the location of flanks used to replace the KT-MX and NT-MX markers and insert the MA0280 expression cassette. The position of primers used for genotyping are indicated on the map. See FIG. 34 (Feature description on map of MA0280 site of the M3625 strain; GPD2 5′ flank—S. cerevisiae GPD2 upstream flanking region; ADHE—Bifidobacterium adolescentis ADHE coding gene; HXT2t—S. cerevisiae HXT2 gene terminator; PDC1t—S. cerevisiae PDC1 gene terminator; PFLA—Bifidobacterium adolescentis PFLA coding gene; ADH1p—S. cerevisiae ADH1 gene promoter; ENO1t—S. cerevisiae ENO1 gene terminator; PFLB—Bifidobacterium adolescentis PFLB coding gene; ENO1p—S. cerevisiae ENO1 gene promoter; FBA1t—S. cerevisiae FBA1 gene terminator; TPI1p—S. cerevisiae TPI1 gene promoter; GPD2 3′ flank—S. cerevisiae GPD2 downstream flanking region; Regions CA-CF—amplified and sequenced chromosomal DNA regions). Primer pair X15473/X17460 was used for the GPD2 5′ Flank, primer pair X17459/X17289 was used for ADH-HXT2, primer pair X17290/X13209 was used for pADH1-PFL-PDC1trc, primer pair X13208/X15735 was used for pENO1-PFL-ENO1trc, primer pair X15736/X17457 was used for pTPI-ADH-FBA1trc, and primer pair X17458/X15476 was used for the GPD2 3′ Flank to create the assembly shown in FIG. 34. Sequences for the primers used to create the assembly shown in FIG. 34 are found in Table 15. An agarose gel image showing PCR products used to genotype and sequence the MA0280 site is shown in FIG. 35 (lane 1: 1 KB ladder; lane 2: M3625 (17413/15810); lane 3: M3625 (17834/14554); lane 4: M3625 (16291/15229); lane 5: M3625 (16503/11317); lane 6: (16241/16946); lane 7: 1 KB ladder).







TABLE 15







Primers used to create the MA0289 integration site.









Primer
Sequence
SEQ ID NO





X15473
agtcatcaggatcgtaggagataagc
142


 


X17460
agaagataatatttttatataattatattaatcctaatcttcatgtag
143



atctaattctt


 


X17459
cctttccttttccttcgctccccttccttatcaatggcagacgcaa
144



agaagaaggaaga


 


X17289
gtccatgtaaaatgattgctccaatgattgaaagttacattgaaa
145



atacagtaaatggt


 


X17290
tggtggaaccatttactgtattttcaatgtaactttcaatcattgga
146



gcaatcatttta


 


X13208
ccgaaatattccacggtttagaaaaaaatcggaggtttagacattg
135



gctcttcattgag


 


X13209
aagctcaatgaagagccaatgtctaaacctccgatttttttctaaac
136



cgtggaatattt


 


X15735
catcttttaacttgaatttattctctagcctagtcttctaggcgggttat
147



ctactgat


 


X15736
agataacccgcctagaagactaggctagagaataaattcaagtta
148



aaagatgatgttga


 


X17457
tgggggaaaaagaggcaacaggaaagatcagagctacttattc
149



ccttcgagattatatc


 


X17458
gttcctagatataatctcgaagggaataagtagctctgatctttcct
150



gttgcctcttttt


 


X15476
gtagatctgcccagaatgatgacgtt
151









In order to determine exact DNA sequence of the M3625 MA0289 site, regions CA-CF were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.







TABLE 16







Primers and summary of results of MA0289 genotyping.














Expected
Correct Size


Lane
Template DNA
Primers
size(bp)
Observed





1
1 KB ladder
N/A
N/A
N/A


2
M3625
16939/16940
2477
Yes


3
M3625
16807/14567
3831
Yes


4
M3625
17834/14557
2478
Yes


5
M3625
16640/14552
3978
yes


6
M3625
17586/16806
4789
yes


7
1 KB ladder
N/A
N/A
N/A
















TABLE 17







Sequence of primers used for genotyping MA0289.









Primer
Sequence
SEQ ID NO





16939
atgctgatgcatgtccacaaag
164


 


16940
ccttatcagtcaattgaggaaag
165


 


16807
gcgatgagctaatcctgagccat
166


 


14567
tggttccaccattattatgttggt
167


 


17834
gctgcttcgaggtattgaca
168


 


14557
ctaaaccgtggaatatttcggatat
169


 


16640
cctcatcagctctggaacaacga
170


 


14552
gatccgagcttccactaggatagc
171


 


17586
gcagtatgcaagtctcatgctg
172


 


16806
gaacttgcaggcaccgatcttca
173









Genotyping and Sequencing of MA0317
To confirm that MA0317 was inserted at the FCY1 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 19. Sequences for the primers used to genotype MA0317 are listed in Table 20. A molecular map depicting the MA0317 integration site is shown in FIG. 36. The molecular map depicts the location of flanks used to replace the FCY1 gene with MA0317 and the position or primers used for genotyping. See FIG. 36 (Feature description on the map of MA0371 site of M3625; FCY1 5′ flank—S. cerevisiae FCY1 upstream flanking region; ENO1p—S. cerevisiae ENO1 gene promoter; AE9 —S. fibuligera glu 0111 coding gene; ENO1t—S. cerevisiae ENO1 gene terminator; PDC1t—S. cerevisiae PDC1 terminator; ADH1p—S. cerevisiae ADH1 gene promoter; FCY1 3′ flank—S. cerevisiae FCY1 downstream flanking region; Regions FA-FE, amplified and sequenced chromosomal DNA regions). Primer pair X18868/X18844 was used for the FCY 5′ Flank, primer pair X18845/X15464 was used for pENO-AE9-ENO1t, primer pair X15465/X11750 was used for pADH1-AE9-PDC1t, and primer pair X15479/X18869 was used for the FCY 3′ Flank to create the assembly shown in FIG. 36. Sequences for the primers used to create the assembly shown in FIG. 36 are found in Table 18. An agarose gel image showing PCR products used to determine genotype is shown in FIG. 37 (lane 1: 1 KB ladder; lane 2: M3625 (13092/17586); lane 3: M3625 (10871/14554); lane 4: M3625 (16291/17887); lane 5: M3625 (16640/16509); lane 6: M3625 (13246/13095); lane 7: 1 KB ladder).







TABLE 18







Primers used to create the MA0317 integration site.









Primer
Sequence
SEQ ID NO





X18868
gccaaagtggattctcctactcaagctttgc
184


 


X18844
tcggatcagtagataacccgcctagaagactagtagctatgaaattt
185



ttaactctttaa


 


X18845
agccagcttaaagagttaaaaatttcatagctactagtcttctaggcg
186



ggttatctact


 


X15464
gtccatgtaaaatgattgctccaatgattgaaagaggtttagacattg
187



gctcttcattg


 


X15465
ctaagctcaatgaagagccaatgtctaaacctctttcaatcattggag
188



caatcatttta


 


X11750
ataaaattaaatacgtaaatacagcgtgctgcgtgctcgatttttttcta
189



aaccgtgga


 


X15479
agcacgcagcacgctgtatttacgta
190


 


X18869
agatcctgtggtagtgctgtctgaacagaa
191









In order to determine exact DNA sequence of the M3625 MA0317 site, regions FA-FE were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.







TABLE 19







Primers and summary of results of MA0317 genotyping.














Expected
Correct Size


Lane
Template DNA
Primers
size(bp)
Observed





1
1 KB ladder
N/A
N/A
N/A


2
M3625
13092/17586
2368
yes


3
M3625
10871/14554
2966
yes


4
M3625
16291/17887
2778
yes


5
M3625
16640/16509
1334
yes


6
M3625
13246/13095
2863
yes


7
1 KB ladder
N/A
N/A
N/A
















TABLE 20







Sequence of primers used for genotyping MA0317.









Primer
Sequence
SEQ ID NO





13092
ccacaccatagacttcagccttcttag
174


 


17586
gcagtatgcaagtctcatgctg
175


 


10871
cgttcgctgtagcatacttagctat
176


 


14554
ggctcttcattgagcttagaaccc
177


 


16291
aactggaccgatcttattcgt
178


 


17887
actgcctcattgatggtggta
179


 


16640
cctcatcagctctggaacaacga
180


 


16509
gtatgattgcggttatctgtcgc
181


 


13246
cctatggatgttgtaccatgcc
182


 


13095
ccaatatcttgcagtccatcctcgtcgc
183









FIG. 38 shows the results of starch assay demonstrating starch degrading activity in M3625. The assay was performed as described in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein in its entirety.
Western Blot Protein Detection:
Anti-PflA, anti-PflB, anti-GA (AE9) and anti-AdhE antibodies:
In an effort to detect the presence of and help characterize a number of enzymes engineered into the yeast strain, polyclonal antibodies were produced in rabbits at Lampire Biological Products, Pipersville, Pa., against synthesized peptides with sequence similarity to the engineered proteins. Table 11 depicts the peptides that were used as immunogens for the rabbits:







TABLE 21







Immunogens used for antibody production









Protein
Immunogen
SEQ ID NO





Sf GA (AE9)
intact purified protein
106



DNKNRYKINGNYKAGC



NSGKKHIVESPQLSSRGGC



CDHIDDNGQLTEEINRYTG


 


Ba pflA
CQNPDTWKMRDGKPVYYE
107



GLTSSEENVENVAKIC


 


Ba pflB
WEGFTEGNWQKDIDVRDC
108



KQRDKDSIPYRNDFTECPEC



CNTITPDGLGRDEEEERIGN


 


Ba AdhE
DAKKKEEPTKPTPEEKLC
109



CKNLGVNPGKTPEEGVEN



CGSYGGNSVSGVNQAVN









For all of the synthesized peptides a terminal Cys was added for conjugation. Both the peptides and the purified GA protein were conjugated to KLH prior to injection into the rabbit. A 50 day protocol was used for antibody production with ELISA monitoring of the various bleeds against the immunogen. After testing these polyclonal antibodies in a Western blot against the lysate from the engineered yeast strains, serum from the positive rabbits was purified using a Protein G column. The purified antibodies were dialyzed into PBS, concentration was determined by absorbance at 280 nm and the antibodies were used for further evaluation of the strains. Upon evaluation by SDS-PAGE, the antibodies appeared to be >90% pure.
Antibodies raised against the synthesized peptides were used in Western blot detection of each engineered protein in cell extracts and culture supernatants as described below.
Strain Growth Conditions:
Cells were plated from freezer stock on YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) agar for 48 hours and used to inoculate 25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) in a 50 mL culture tube. Cells were grown aerobically for 8 hours at 35° C. with shaking at 250 rpm, then 1 mL was removed to inoculate a sealed, CO2 purged serum bottle containing 50 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) with 7 mg/L ergosterol, 289 mg/L ethanol and 544 mg/L Tween 80. These cultures were then grown anaerobically overnight ( ̃16 h) at 35° C. with shaking at 250 rpm. Cells were harvested by centrifugation and washed with 25 mL deionized water. The resulting wet cell pellets were used for Western blot detection of PflA, PflB and AdhE.
Aerobic cultures used to inoculate the serum bottles were returned to the shaking incubator for an additional 40 hours. At the end of incubation, cells were pelleted by centrifugation and the supernatant was recovered and concentrated  ̃10× using a 10 kDa molecular weight cut-off (MWCO) filter membrane. The resulting concentrates were used for Western blot detection of extracellular AE9 glucoamylase.
Cell Lysis and Sample Preparation:
For Western blots of PflB and AdhE, cells were homogenized by mechanical disruption with 0.5 mm diameter beads and agitation at 4800 rpm in a bead beater. 100 μL of wet cells were added to homogenization buffer containing 1 mM phenylmethanesulfonylfluoride (PMSF), 2 mM dithiothreitol (DTT) and 1% dimethyl sulfoxide (DMSO) in 100 mM sodium phosphate buffer pH 7.4. Cells were agitated for 6 cycles of 10 seconds each, cooling on ice between cycles. Cell debris was pelleted by centrifugation and supernatant was recovered. 15 μL of the resulting supernatant was added to 15 μl, 2× concentrated SDS-PAGE sample buffer with 50 mM DTT and loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.
For Western blot detection of PflA, cells were lysed by adding 40 μL wet cells to 40 μL 2× concentrated SDS-PAGE sample buffer with 50 mM DTT. The mixture was then incubated at room temperature for 30 minutes, followed by heating at 100° C. for 2 minutes. Cells were pelleted by centrifugation and 30 μL of the supernatant was loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.
For AE9 analysis, 15 μL of concentrated aerobic culture supernatant was added to 15 μL 2× concentrated SDS-PAGE sample buffer with 50 mM DTT and loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.
Following gel electrophoresis, proteins were transferred to a polyvinylidine fluoride (PVDF) membrane and blocked overnight with Tris buffered saline (TBS; 10 mM Tris, 150 mM sodium chloride pH 7.5) containing 2% weight by volume (w/v) bovine serum albumin (BSA). The blocking solution was then removed, and primary peptide antibodies were diluted to approximately 2 μg/mL in Tris buffered saline with Tween 20 (TBST; TBS with 0.1% v/v Tween 20) and added to each membrane. After a 1 hour incubation, the primary antibody was discarded and the membrane was washed for 3 periods of 5 minutes each in 10 mM Tris, 500 mM sodium chloride, 0.1% Tween 20 pH 7.5 (THST). The secondary antibody, goat anti-rabbit with horseradish peroxidase label, was diluted 1:7500 in TBST, added to the blot and incubated for 1 hour. The secondary antibody was then discarded and the blot was again washed with THST for 3 periods of 5 minutes each. The wash solution was then discarded, enhanced chemiluminescence (ECL) substrate was added, and the blot was read by a series of composite exposures using a gel imaging camera.
As shown in FIG. 39, for anti-PflA, PflB and AdhE primary antibodies, bands of approximately the correct molecular weight were detected in each experimental strain, whereas no band was detected in the background control strain (M2390). For anti-AE9, bands were detected in strains engineered to express the protein (M3625 and M3680) but were absent in other strains. See FIG. 39. There appeared to be two distinct bands for PflB, which may indicate oxygenic cleavage of the protein due to aerobic cell lysis conditions.
Pyruvate Formate Lyase Activity Assay:
Pyruvate formate lyase (PflB) is activated in the absence of oxygen by Pfl activase (PflA) and catalyzes the reaction of pyruvate and CoA to formate and acetyl-CoA. The activity of PflB was measured in cell extracts by measuring formate production when extracts were added to a reaction mixture containing pyruvate, CoA and DTT.
Strain Growth Conditions:
Cells were plated from freezer stock on YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agar for 48 hours and used to inoculate 25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) in a 50 mL culture tube. Cells were grown aerobically for 8 hours at 35° C. with shaking at 250 rpm, then 1 mL was removed to inoculate a sealed, CO2 purged serum bottle containing 50 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) with 7 mg/L ergosterol, 289 mg/L ethanol and 544 mg/L Tween 80. These cultures were then grown anaerobically overnight ( ̃16 h) at 35° C. with shaking at 250 rpm. Cells were harvested by centrifugation and washed with 25 mL deionized water in an anaerobic chamber.
Cell Lysis and Sample Preparation:
Cells were homogenized in an anaerobic chamber by mechanical disruption with 0.5 mm diameter beads and agitation at 4800 rpm in a bead beater. 1004 of wet cells were added to homogenization buffer containing 1 mM PMSF, 2 mM DTT and 1% DMSO in 100 mM sodium phosphate buffer pH 7.4. Cells were agitated for 6 cycles of 10 seconds each, cooling on ice between cycles. Cell debris was pelleted by centrifugation at 14,100×g for 10 minutes and supernatant was recovered and clarified by filtration through a 0.22 μm filter membrane. The resulting extract was used directly in the activity assay.
Pfl Activity Assay:
A 2× concentrated assay substrate mixture consisted of 20 mM sodium pyruvate, 0.11 mM CoA and 20 mM DTT. Reagents were weighed out, brought into an anaerobic chamber and added to 10 mL of 100 mM sodium phosphate buffer pH 7.4 which had been thoroughly degassed. 1004 of cell extract was added to 100 μL of the concentrated assay mixture and incubated at ambient temperature ( ̃29° C.) for 30 minutes. Samples were then removed from the anaerobic chamber and heated in a heating block at 100° C. for 90 seconds followed by cooling on ice to precipitate protein. Precipitate was removed by centrifugation at 15,000×g for 10 minutes. The resulting supernatant was analyzed for formate concentration using the formic acid assay kit available from Megazyme International Ireland, Bray, Co. Wicklow, Ireland.
Remaining cell extracts were diluted 1:8 in 100 mM sodium phosphate buffer pH 7.4 and assayed for total protein content using the BCA total protein determination method. Formate concentrations of the Pfl assay samples were normalized to the total protein concentration of the sample.
As shown in FIG. 40, experimental strains with engineered Pfl activity (M3465, M3625, M3679, and M3680) showed significantly higher amounts of formate present after incubation with the reaction mixture than the background control strain (M2390).
Alcohol Dehydrogenase E (AdhE) Enzymatic Activity Assays
AdhE is an intracellular bi-functional enzyme catalyzing the formation of ethanol from acetyl-CoA by way of acetaldehyde as an intermediate. This is accomplished by an acetaldehyde dehydrogenase activity and an alcohol dehydrogenase activity working in series. Saccharomyces cerevisiae strains have native alcohol dehydrogenase (Adh) activity; the intent of these activity assays is to show that Adh activity is retained by the engineered strains, and an additional acetaldehyde dehydrogenase activity (from AdhE) is present.
Alcohol Dehydrogenase Activity:
As mentioned above, Bifidobacterium adolecentis bifunctional alcohol dehydrogenase (AdhE) has 2 primary functions. One function is the conversion of acetaldehyde to ethanol. This reversible reaction utilizes NADH as a cofactor. In order to evaluate the presence of this enzyme and ensure that it has the desired activity, an assay was developed to evaluate the reverse reaction in which ethanol is converted to acetaldehyde. See FIG. 49. The rate of the reaction is monitored by NADH absorbance at 340 nm.
Strain Growth Conditions:
Strains were patched from freezer stock onto a YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agar plate and incubated overnight at 35° C. From that plate, 50 mL shake tubes with 25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) were inoculated and incubated at 35° C., 250 rpm overnight. The cultures were centrifuged at 5000 rpm×5 min at 4 C, washed with deionized (DI) water and centrifuged at 5000 rpm×5 min at 4 C, washed a second time with DI water and centrifuged at 5000 rpm×5 min at 4 C, and then put on ice.
Cell Lysis and Sample Preparation:
100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5 mm Tubes along with 500 μL 100 mM Na2PO4, 2.5 mM MgCl2, 0.5 mM CaCl2 pH 7.4 buffer and 6 μL 100 mM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0 m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2 mL tubes. 1 μL of New England Biolabs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37° C. for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22 μm filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm. 504 of sample was pulled and diluted with 450 μL 100 mM Na2PO4 pH 7.4 in separate sample tubes and then placed on ice.
Alcohol Dehydrogenase Activity Assay:
The assay used to determine alcohol dehydrogenase activity of AdhE was adapted from the method of Vallee, B. L. and Hoch, F. L., Proc Natl Acad Sci USA (1955) 41(6): 327-338. 100 μL 0.1M Na4P2O7 pH 9.6 buffer, 32 μL 2M ethanol, and 1.66 μL 0.025M NAD+ were added to each well in a 96 well plate. Once the lysate was added to the reaction mixture, the total volume of reaction was equivalent to 153.66 μL resulting in final concentrations of 65 mM Na4P2O7 pH 9.6 buffer, 416.5 mM ethanol, and 0.27 mM NAD+. To begin the reaction, 204, of 1:10 diluted lysate was pipetted into each well and the absorbance at 340 nm was observed and recorded over 1.7 min using Spectramax M2 and Softmax software. Each sample was done in duplicate to ensure reproducibility. Thermo Scientific's BCA Protein Assay Kit was used to measure total protein concentration from the lysate generated. This data was used to normalize the data generated from the actual reaction during analysis.







TABLE 22







Data for alcohol dehydrogenase activity assay













Average activity
Standard

%
p-



(μmol NADH/min/mg)
Deviation
% CV
Change
value
















M2390
599
15.6
2.6




M3465
659
16.2
2.5
10
0.1942


M3625
999.5
132
13.2
66.9
0.0108


M3679
755
82
10.9
26
0.0603


M3680
698
29.7
4.3
16.5
0.0996





Alcohol dehydrogenase activities of engineered strains. p-value was based on a one-tailed T-test.






The background strain, M2390, performed as expected in this assay. Although it did not have AdhE engineered into its genome, it still expressed wild-type alcohol dehydrogenase and thus was active in the alcohol dehydrogenase assay. Other strains with AdhE engineered into their genomes should have expressed the bi-functional enzyme and should have been more active given the total protein concentration was equal in each sample used in the assay. With a p-value of <0.05, M3625 demonstrated a statistically significant higher activity than the background strain. However, the other strains have a p-value>0.05 indicating that they are within error of the background strain even though there was an increase in activity as shown by the % change over the background activity. See Table 22. After normalizing the protein concentrations, a graphical representation of the data shows that each strain was more active than the background strain M2390 during a 1.7 minute reaction period. FIG. 41 shows the activity of each strain plotted in μmol NADH/mg total protein vs. time during a 1.7 minute reaction.
Based on these results, the assay showed alcohol dehydrogenase activity in all strains. However, M2390 is less active and slower at converting NAD+ to NADH than the other strains indicating that the engineered AdhE is present in each strain and it appears to be functioning properly.
Acetaldehyde Dehydrogenase Activity:
The second activity of AdhE is the reversible reaction converting acetaldehyde to acetyl coenzyme A. This activity is not native to Saccharomyces cerevisiae strains, and should only be present in the engineered strains. In order to evaluate the presence of this enzyme and ensure that it has the desired activity, an assay was developed to measure the conversion of acetaldehyde to acetyl CoA by AdhE. The rate of the reaction is monitored by NADH absorbance at 340 nm. A diagram of the reaction is provided in FIG. 50.
Strain Growth Conditions:
Strains were patched from a freezer stock onto a YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agar plate and incubated overnight at 35° C. From that plate, 50 mL shake tubes with 25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) were inoculated and incubated at 35° C., 250 rpm overnight. The cultures were centrifuged at 5000 rpm×5 min at 4° C., washed with DI water and centrifuged at 5000 rpm×5 min at 4 C, washed a second time with DI water and centrifuged at 5000 rpm×5 min at 4° C., and then put on ice.
Cell Lysis and Sample Preparation:
100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5 mm Tubes along with 5004, 100 mM Na2PO4, 2.5 mM MgCl2, 0.5 mM CaCl2 pH 7.4 buffer and 6 μL 100 mM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0 m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2 mL tubes. 1 μL of New England Biolabs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37° C. for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22 μm filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm using the Eppendorf centrifuge 5424. 50 μL of sample was pulled and diluted with 450 μL 100 mM Na2PO4 pH 7.4 in separate sample tubes and then were placed on ice.
Acetaldehyde Dehydrogenase Activity Assay:
800 μL 50 mM Na4P2O7 pH 9.6, 50 μL 0.025M NAD+, 50 μL 1M acetaldehyde, and 50 μL 1:10 diluted lysate were added to a Plastibrand micro UV-cuvette. The cuvette was placed into a Shimadzu UV-1700 set to read absorbance at 340 nm. 50 μL of 2 mM CoA were pipetted into the cuvette which was then mixed by gently pipetting the contents of the cuvette and the absorbance was monitored for 5 minutes. The resulting final concentrations of each reagent were 40 mM Na4P2O7 pH 9.6, 1.25 mM NAD+, 50 mM acetaldehyde, and 0.1 mM CoA. Each sample was done in duplicate to ensure reproducibility. Thermo Scientific's BCA Protein Assay Kit was used to measure total protein concentration from the lysate generated. This data was used to normalize the data generated from the actual reaction during analysis.
Data for alcohol dehydrogenase (acetaldehyde dehydrogenase activity) assay is shown in Table 23. Note the lysate used in this assay was the same lysate used in the alcohol dehydrogenase assay detailed in the previous section.







TABLE 23







Acetaldehyde Dehydrogenase Activity












Average activity
Standard

p-



(μmol NADH/min/mg)
Deviation
% CV
value















M2390
0
0
0.0



M3465
163
24
14.7
0.0054


M3625
115
7.07
6.2
0.0009


M3679
106
9.97
9.4
0.0022


M3680
177
7.07
4.0
0.0004





Acetaldehyde dehydrogenase activity. p-value was based on a one-tailed T-test.






The background strain, M2390, performed as expected in this assay. The wild-type strain should have no acetaldehyde dehydrogenase activity, as demonstrated by this assay. The other strains with AdhE engineered into their genomes should have expressed the protein and had acetaldehyde dehydrogenase activity. This activity was observed in all of the engineered strains (M3465, M3625, M3679, and M3680) with minimal error and a p-value of <0.05.
Formate Dehydrogenase Activity
In strains M3465, M3625, M3679, and M3680 formate dehydrogenase was knocked out of the genome in the hopes to balance redox with the various engineering steps that were undertaken. The background strain, M2390, should have the gene intact. To ensure that the native Saccharomyces cerevisiae formate dehydrogenase gene was removed, an enzymatic assay was developed. Formate dehydrogenase catalyzes the conversion of formate to carbon dioxide at the expense of NAD+.




Enzymatic activity can be monitored by measuring NADH formation at 340 nm.
Strain Growth Conditions:
M2390, M3465, M3625, M3679, and M3680 were patched from a freezer stock onto a YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agar plate and incubated overnight at 35° C. From that plate, 50 mL shake tubes with 25 mL YPD+24 mM Sodium Formate were inoculated and incubated at 35° C., 250 rpm overnight (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose). The cultures were centrifuged at 5000 rpm×5 min at 4° C., washed with DI water and centrifuged at 5000 rpm×5 min at 4° C., washed a second time with DI water and centrifuged at 5000 rpm×5 min at 4 C, and then put on ice.
Cell Lysis and Sample Preparation:
100 μL of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5 mm Tubes along with 500 μL 100 mM Na2PO4, 2.5 mM MgCl2, 0.5 mM CaCl2 pH 7.4 buffer and 6 μL 100 mM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0 m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2 mL tubes. 1 μL of New England Biolabs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37° C. for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22 μm filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm using the Eppendorf centrifuge 5424.
Formate Dehydrogenase Activity Assay:
800 μL 62.5 mM K2PO4 pH 7.0, 50 μL 40 mM NAD+, and 50 uL 1M Sodium Formate were added to a Plastibrand micro UV-cuvette. The cuvette was placed into a Shimadzu UV-1700 set to read absorbance at 340 nm and blanked. 100 μL of undiluted lysate sample were pipetted into the cuvette which was then mixed by gently pipetting the contents of the cuvette and the absorbance was monitored for 2.5 minutes. The resulting final concentrations of each reagent were 50 mM Potassium Phosphate, 2 mM NAD+, and 0.05M Sodium Formate. Each sample was done in duplicate to ensure reproducibility.







TABLE 24







Fdh activity of engineered strains











Average μmol
Standard




NADH/min
Deviation
% CV
















M3631
0.03
0.00269
0.865



M2390
0.008
0
0



M3465
0
0
0



M3625
0
0
0



M3679
0
0
0



M3680
0
0
0










As shown in Table 24, the FDH knockout strains (M3465, M3625, M3679, and M3680) did not exhibit any formate dehydrogenase activity. The background strain, M2390, had minimal activity. The positive control strain, M3631, which overexpresses FDH was active and produced a significant amount of NADH that was observed and recorded.
AE9 glucoamylase activity assay:
Saccharomycopsis fibuligera GLU1 glucoamylase (AE9) produces glucose from starch.
Extracellular AE9 glucoamylase activity on raw corn starch was assayed to determine the presence of glucoamylase activity in aerobic culture supernatants of engineered strains. Cells were grown aerobically, removed by centrifugation, and the resulting supernatant was assayed for activity and compared to supernatant from strain M2390, which does not contain AE9.
Cell Growth Conditions:
Cells were plated on YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L dextrose) agar for 48 hours and used to inoculate 25 mL YPD (20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose) in a 50 mL culture tube. Cells were grown aerobically for 48 hours at 35° C. with shaking at 250 rpm. After 48 hours, cells were removed via centrifugation and the supernatant was recovered.
Sample Preparation:
The recovered aerobic culture supernatant was clarified by filtration through a 0.22 μm filter membrane and concentrated  ̃10× using a 10 kDa molecular weight cut-off filter. The retained concentrate was then analyzed for AE9 concentration via a phenyl reverse phase (phenyl-RP) HPLC method developed in-house using purified AE9 as a standard. Samples were diluted to an AE9 concentration of 50 μg/mL and used directly in the activity assay.
Glucoamylase Activity Assay:
A 2.2% (weight by volume) corn starch solution was made up in 50 mM sodium acetate buffer pH 5.0. In a 96-well assay plate, 504 of supernatant (adjusted to 50 μg/mL AE9 concentration) was added to 450 μL 2.2% starch. The plate was incubated at room temperature without shaking, and 50 μL of sample was taken at 1, 2, 5, 10, 30, 120 and 210 minutes. Wells were mixed by pipette aspiration after initial enzyme addition, as well as at each sampling thereafter. Samples were analyzed via 3,5-dinitrosalicylic acid (DNS) method to determine reducing sugars.
As shown in FIG. 42, the aerobic culture supernatants of M3625 and M3680 showed similar activity on raw corn starch, as measured by DNS analysis (Somogyi, M., Notes on Sugar Determination, JBC (200)45 (1952)). Amylolytic activity of M2390 supernatant was negligible in this assay.
The above data show that pflA, pflB and AdhE are present in strain M3625 and have the proper activity. Fdh activity, seen in the background strain as well as in the positive control when fdh was overexpressed, was not present in the engineered strains indicating that this gene was knocked out successfully.
Example 9
The following example demonstrates the ethanol yield of the Saccharomyces cerevisiae strain M3624. The genotype of strain M3624 is: Δgpd1::GPD2-B. adolescentis pflA/pFlB/adhEΔgpd2::GPD1-B. adolescentis pflA/pflB/adhE Δfdh1 Δfdh2::B. adolescentis pflA/pflB/adhE. Strain M3624 was created according to the same methods employed above in Example 8. Detailed molecular maps for strain M3624 are shown in FIGS. 43A-D. FIG. 43A shows insertion at the GPD1 locus; GPD2 expressed from the GPD1 promoter; PFK2t-PFK2 terminator; HXT2t-HXT2 terminator; pADH1-ADH1 promoter; PDC1 term-PDC1 terminator; FBA1 term-FBA1 terminator; pTPI1-TPI1 promoter; Scer ENO1 ter-ENO1 terminator. FIG. 43B shows insertion at the GPD2 locus; GPD1 expressed from the GPD2 promoter; TDH3 term-TDH3 terminator; pPFK1-PFK1 promoter; HXT2t-HXT2 terminator; PDC1 term-PDC1 terminator; pADH1-ADH1 promoter; S.cer ENO1 ter-ENO1 terminator; FBA1 term-FBA1 terminator; pTPI1-TPI1 promoter. FIG. 43C shows deletion of the FDH1 gene; flanking regions to create deletion of FDH1. FIG. 43D shows insertion at the FDH2 locus; pPFK1-PFK1 promoter, S.cer ENO1 ter-ENO1 terminator; pADH1-ADh1 promoter; FBA1 term-FBA1 terminator; PDC1 term-PDC1 terminator; pTPI-TPI1 promoter.
The data shown in FIG. 44 demonstrates that a 3.4% ethanol yield increase is obtained through reduction of glycerol and production of formate. M2390 is the control strain, M3515 and M3624 are engineered with the genotype Δgpd1::GPD2-B. adolescenits pflA/pflB/adhE Δgpd2::GPD1-B. adolescentis pflA/PFlB/adhE fdh1Δfdh2Δ::B. adolescentis pflA/pflB/adhE. M3027 is engineered with the genotype Δgpd1 Δgpd2::GPD1-B. adolescentis pflA/PFlB/adhE fdh1Δfdh2Δ::B. adolescentis pflA/pflB/adhE. Panel A shows measurement of formate concentration, panel B shows measurement of glycerol concentration, and panel C shows measurement of ethanol concentration.
Both M3515 and M3624 have been engineered at 4 separate loci. The GPD1 gene is expressed from the GPD2 promoter and the GPD2 gene is expressed from the GPD1 promoter, the FDH1 and FDH2 genes have been deleted. Additionally, the B. adolescentis pflA, pflB and adhE genes are expressed as shown in FIGS. 43A, B, and D.
Example 10
The following example demonstrates the ethanol yield of the Saccharomyces cerevisiae strains M3465 and M3469. The genotype of strain M3465 is: Δgpd2::B. adolescentis pflA/pflB/adhE Δfdh1Δ fdh2::B. adolescentis pflA/pflB/adhE. The genotype of strain M3469 is: Δgpd1::B. adolescentis pflA/pflB/adhE fdh1Δ fdh2Δ::B. adolescentis pflA/pflB/adhE. Strains M3465 and M3469 were created according to the same methods employed above in Example 8. Detailed molecular maps of strains M3465 and M3469 are shown in FIGS. 45 A-C and 46 A-C, respectively. FIG. 45A shows insertion at the GPD2 locus; pPFK1-PFK1 promoter; HXT2t-HXT2 terminator; PDC1 term-PDC1 terminator; pADH1-ADH1 promoter; S.cer ENOL ter-ENOL terminator; FBA1 term-FBA1 terminator; pTPI1-TPI1 promoter. FIG. 45B shows deletion of the FDH1 gene; flanking regions to create deletion of FDH1. FIG. 45C shows insertion at the FDH2 locus; pPFK1-PFK1 promoter; S.cer ENO1 ter-ENO1 terminator; pADH1-ADh1 promoter; FBA1 term-FBA1 terminator; PDC1 term-PDC1 terminator; pTPI-TPI1 promoter. FIG. 46A shows insertion at the GPD1 locus; pPFK1-PFK1 promoter; HXT2t-HXT2 terminator; PDC1 term-PDC1 terminator; pADH1-ADH1 promoter; S.cer ENO1 ter-ENO1 terminator; FBA1 term-FBA1 terminator; pTPI1-TPI1 promoter. FIG. 46B shows deletion of the FDH1 gene; flanking regions to create deletion of FDH1. FIG. 46C shows insertion at the FDH2 locus; pPFK1-PFK1 promoter, S.cer ENO1 ter-ENO1 terminator; pADH1-ADh1 promoter; FBA1 term-FBA1 terminator; PDC1 term-PDC1 terminator; pTPI-TPI1 promoter.
This example demonstrates that the ethanol yield increase is dependent on the level of glycerol reduction. Fermentation of 30% solids corn mash by M3465, which contains a deletion of the GPD2, FDH1 and FDH2 genes and expression of B. adolescentis pflA, pflB and adhE genes from the GPD2 and FDH2 loci, results in a 1.5% increase in ethanol titer. As shown in FIG. 47, fermentation of 30% solids corn mash by M3469, which contains a deletion of the GPD1, FDH1 and FDH2 genes and expression of B. adolescentis pflA, pflB and adhE genes from the GPD1 and FDH2 loci, results in a 2.5% increase in ethanol titer. M2390 is the control parent strain. As shown in FIG. 48, fermentation of corn mash by M3465 and M3469 results in  ̃15% lower glycerol and 30% lower glycerol levels respectively. M2390, represented in FIGS. 47 and 48, is the control strain.
Example 11
An alternative way to reduce glycerol formation is through deletion of the glycerol-3-phosphate phosphatase (GPP) genes. Saccharomyces contains two copies of these genes, GPP1 and GPP2. The data below demonstrates that expression of B. adolescentis pflA, pflB and adhE in backgrounds containing deletions of FDH1, FDH2 and either GPP1 or GPP2 results in decreased glycerol formation (FIG. 53; strain comparison, min buff medium, glucose 40 g/L, anaerobic fermentations, 35° C.-72 hr.) and increased ethanol yield (FIG. 54; strain comparison, min buff medium, glucose 40 g/L, anaerobic fermentations, 35° C.-72 hr.). Production of formate was also observed. See FIG. 55 (strain comparison, min buff medium, glucose 40 g/L, anaerobic fermentations, 35° C.-72 hr.).
The strains engineered to measure the glycerol formation, ethanol yield, and formate production were the Saccharomyces cerevisiae strains M3297, TB655, and TB656. Strains M3297, TB655, and TB656 were created according to the same methods employed above in Example 8. The genotype of strain M3297 is: Δfdh1Δfdh2::pflA/pflB/adhE. This strain contains only deletion in the FDH genes plus expression fo pflA, pflB and AdhE. The genotype of strain TB655 is: Δfdh1Δfdh2::pflA/pflB/adhEΔgpp1::pflA/pflB/adhE. This strain contains deletion in the FDH genes, expression of pflA, pflB and AdhE, and deletion of GPP1. See FIG. 51. The genotype of strain TB656 is: Δfdh1Δfdh2::pflA/pflB/adhEΔgpp2::pflA/pflB/adhE. This strain contains deletion in the FDH genes, expression of pflA, pflB and AdhE and deletion of GPP1. See FIG. 52.
The amount of ethanol, glycerol, and formate produced by strains TB655 and TB656 was measured using the methods described above. Compared to the control strain M3297, strains TB655 and TB656 demonstrated statistically significant changes in the amount of ethanol, glycerol, and formate produced. Relative to strain M3297, strain TB655 (gpp1 mutant) demonstrated a 1.3% increase in ethanol titer, 10% reduction in glycerol, and 100% more formate produced, whereas strain TB656 (gpp2 mutant) demonstrated a 0.95% increase in ethanol titer, 6.1% reduction in glycerol formation, and 100% more formate produced. These results demonstrate the novel combination of GPP mutation with a metabolic engineering solution to balance redox during anaerobic growth.
INCORPORATION BY REFERENCE
All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## Claims

1. A recombinant yeast comprising:
(a) a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis, wherein said one or more enzymes is encoded by a gpd1, gpd2, gpp1, gpp2 or fps1 polynucleotide; and
(b) one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein one of said metabolic pathways comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase, wherein one of said metabolic pathways comprises conversion of acetyl-CoA to ethanol by an acetaldehyde dehydrogenase, alcohol dehydrogenase, or a bifunctional acetaldehyde/alcohol dehydrogenase, and wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated.

2. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

3. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpp1 polynucleotide and a gpp2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

4. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fps1 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

5. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fps1 polynucleotide and said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide, wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

6. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.

7. The recombinant yeast of claim 6, further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

8. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to produce glycerol is encoded by both a gpd1 polynucleotide and a gpd2 polynucleotide and said one or more native enzymes that function to regulate glycerol synthesis is encoded by an fsp1 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpd1 polynucleotide operably linked to a native gpd2 promoter polynucleotide.

9. The recombinant yeast of claim 8, further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

10. The recombinant yeast of claim 2, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

11. The recombinant yeast of claim 3, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

12. The recombinant yeast of claim 4, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

13. The recombinant yeast of claim 5, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

14. The recombinant yeast of claim 6, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

15. The recombinant yeast of claim 7, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

16. The recombinant yeast of claim 8, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

17. The recombinant yeast of claim 9, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme.

18. The recombinant yeast of claim 1, wherein said one or more native enzymes that function to produce glycerol is encoded by a gpd2 polynucleotide, and wherein one of said engineered metabolic pathways comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme, and further comprising a deletion of one or more native enzymes encoded by both an fdh1 polynucleotide and an fdh2 polynucleotide.

19. The recombinant yeast of claim 1, wherein said yeast is a member of a genus selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.

20. The recombinant yeast of claim 1, wherein said yeast is a member of a species selected from the group consisting of S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lac tis, K. marxianus, and K. fragilis.

21. The recombinant yeast of claim 1, wherein said yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorphs, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymmphus, Schizosaccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis.

22. The recombinant yeast of claim 1, wherein said yeast is Saccharomyces cerevisiae.

23. The recombinant yeast of claim 1, wherein said pyruvate formate lyase is from one or more of a Bijidobacteria, an Escherichia, a Thennoanaerobacter, a Clostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or a Bacillus species.

24. The recombinant yeast of claim 1, wherein said pyruvate formate lyase is from one or more of a Bacillus lichenifonnis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bijidobacteriwn adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis.

25. The recombinant yeast of claim 1, wherein said pyruvate formate lyase is from a Bifidobacterium adolescentis.

26. The recombinant yeast of claim 1, wherein said bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species.

27. The recombinant yeast of claim 1, wherein said bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis.

28. The recombinant yeast of claim 1, wherein said bifunctional acetaldehyde/alcohol dehydrogenase is from a Bifidobacterium adolescentis or Piromyces sp. E2.

29. The recombinant yeast of claim 1, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme, and wherein said saccharolytic enzyme is from a microorganism selected from the group consisting of H. grisea, T aurantiacus, T emersonii, T reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Arabidopsis thaliana, and S. fibuligera.

30. The recombinant yeast of claim 1, wherein said yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorphs, Phaffia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymmphus, Schizosaccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis, wherein said pyruvate formate lyase is from one or more of a Bacillus lichenifonnis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bijidobacteriwn adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis, and wherein said bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis.

31. The recombinant yeast of claim 30, wherein one of said engineered metabolic pathways comprises conversion of a carbohydrate source to one or more sugar units by a saccharolytic enzyme, and wherein said saccharolytic enzyme is from a microorganism selected from the group consisting of H. grisea, T aurantiacus, T emersonii, T reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Arabidopsis thaliana, and S. fibuligera.