WO2008063650A2

WO2008063650A2 - Systems of hydrogen and formic acid production in yeast

Info

Publication number: WO2008063650A2
Application number: PCT/US2007/024301
Authority: WO
Inventors: Pamela Silver; Caleb Kennedy; Patrick Boyle; David Savage
Original assignee: President And Fellows Of Harvard College
Priority date: 2006-11-20
Filing date: 2007-11-20
Publication date: 2008-05-29
Also published as: WO2008063650A3

Abstract

This invention relates to engineered yeast systems and to methods of using these yeast systems, alone or in combination with bacterial systems such as engineered bacterial systems, to generate hydrogen and formic acid.

Description

SYSTEMS OF HYDROGEN AND FORMIC ACID PRODUCTION IN YEAST

Related Applications

This patent application claims the benefit of U.S. Provisional Patent Application No. 60/860,269, filed November 20, 2006; U.S. Provisional Patent Application No. 60/919,658, filed March 23, 2007; and U.S. Provisional Patent Application No. 60/936,854, filed June 22, 2007; each of which is hereby incorporated by reference in its entirety.

Field of the Invention

This invention relates to engineered yeast systems and to methods of using these yeast systems, alone or in combination with bacterial systems, including engineered bacterial systems, to generate hydrogen and formic acid.

Background of the Invention

The most common industrial methods for producing hydrogen include steam reformation of natural gas, coal gasification, and splitting water with electricity typically generated from fossil fuels. These energy-intensive industrial processes release carbon dioxide and other greenhouse gases and pollutants as by-products.

Accordingly, there currently exists a need for cost-effective compositions, systems and methods of increasing production of hydrogen without negative side effects, such as pollution.

Summary of the Invention

This invention provides biological compositions, systems and methods for producing hydrogen and formic acid using an engineered yeast system, or a combination of an engineered yeast system and a bacterial system, such as an engineered bacterial system. The hydrogen produced is used in a variety of applications, including, for example, fuel cells. Fuel cells use hydrogen and oxygen to create electricity and effectively produce zero or near-zero emissions, with only water and heat as byproducts. They can be used in various applications, from portable devices to buildings to vehicles. The formic acid produced is used to generate hydrogen, or alternatively, the formic acid is used in a variety of commercial applications, such as an additive for livestock feed. Previous attempts to produce high yields of hydrogen using bacteria have faced problems. A major scientific hurdle in the production of hydrogen is that the compound NADH is the cell's major currency of reducing equivalents, catalyzing the reaction

NADH -> NAD⁺ + H⁺ + 2e The problem with using NADH is that this compound is not a strong enough as a reducing agent to efficiently produce hydrogen. The structures OfNAD⁺ and NADH are shown in Figures IA, IB and 1C. The engineered systems of the invention use engineered formic acid (formate) molecules as reducing agents.

According to a first aspect, the invention provides a fungal strain that has reduced or absent enzymatic activity of proteins formate dehydrogenase, glucose-6-phosphate dehydrogenase, alanine aminotransferase, and/or fumarase. For example, enzymatic activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99% or is undetectable as compared to the corresponding wild type strain. According to a second aspect, the invention provides a mixture of a fungal strain that produces formic acid and a bacterial strain that catalyzes the conversion of formic acid into hydrogen. The fungal strains provided herein are useful, e.g., to produce formic acid, and are also useful in combination with the mixed fungal and bacterial systems for producing biological hydrogen.

For example, the invention provides a strain of yeast, such as Saccharomyces cerevisiae, lacking the fungal formate dehydrogenase and also lacking glucose-6-phosphate dehydrogenase. For example, the engineered gene is characterized by 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 96% less, 97% less, 98% less, 99% less, two-fold lower, five-fold lower, 10-fold lower enzymatic activity as compared to the corresponding wild-type enzyme. The engineered yeast strains provided herein are designed to excrete formic acid. In some embodiments, the enzymatic activity for an enzyme selected from alanine aminotransferase, fumarase pyruvate decarboxylase and combinations thereof, is also reduced or absent in the yeast strain. For example, the engineered gene is characterized by 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 96% less, 97% less, 98% less, 99% less, two-fold lower, five-fold lower, 10-fold lower enzymatic activity as compared to the corresponding wild-type enzyme. For example, the genes encoding one or more of these enzymes has been disrupted. Alternatively or in addition, the yeast strain includes a constitutively active phosphoglycerate dehydrogenase. For example, a gene encoding phosphoglycerate dehydrogenase has been engineered to cause increased enzymatic activity in the yeast strain. For example, the engineered gene is characterized by 10% more, 20% more, 30% more, 40% more, 50% more, 60% more, 70% more, 75% more, 80% more, 85% more, 90% more, 95% more, 96% more, 97% more, 98% more, 99% more, two-fold higher, five-fold higher, 10-fold higher enzymatic activity as compared to the corresponding wild-type enzyme. In one embodiment, the yeast strain is a Saccharomyces strain.

The invention provides an isolated fungal cell that includes a mutated nucleic acid encoding a fungal formate dehydrogenase, wherein enzymatic activity of the fungal formate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified fungal formate dehydrogenase. The mutation is, for example, a knockout mutation. Other suitable mutations include, but are not limited to, one or more point mutations in the nucleotide sequence, a frameshift mutation, a deletion or insertion within the nucleotide sequence, the insertion of a stop codon within the sequence or any other truncation mutation. For example, enzymatic activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99% or is undetectable as compared to the corresponding wild type strain. In some embodiments, the fungal cell is a Saccharomyces cell, e.g., a Saccharomyces cerevisiae cell. Preferably, the fungal cells provided herein excrete formic acid. Alternatively or in addition, the fungal cell includes a mutated nucleic acid encoding an enzyme selected from glucose-6-phosphate dehydrogenase, alanine aminotransferase, fumarase, pyruvate decarboxylase, and combinations thereof, wherein enzymatic activity of the enzyme encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified corresponding enzyme. For example, enzymatic activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99% or is undetectable as compared to the corresponding wild type strain. The mutation is, for example, a knockout mutation. Other suitable mutations include, but are not limited to, one or more point mutations in the nucleotide sequence, a frameshift mutation, a deletion or insertion within the nucleotide sequence, the insertion of a stop codon within the sequence or any other truncation mutation.

Alternatively or in addition, the fungal cell includes a modified phosphoglycerate dehydrogenase, wherein enzymatic activity of the modified phosphoglycerate dehydrogenase is increased as compared to the enzymatic activity of an unmodified phosphoglycerate dehydrogenase. For example, enzymatic activity is increased by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 95%, 96%, 97%, 98%, 99%, two-fold, five-fold or ten-fold as compared to the corresponding wild type strain. In some embodiments, the modified phosphoglycerate dehydrogenase is constitutively active.

For example, the modified phosphoglycerate dehydrogenase is a heterologous phosphoglycerate dehydrogenase, such as a bacterial phosphoglycerate dehydrogenase. In some embodiments, the bacterial phosphoglycerate dehydrogenase is a fragment of an E. coli SerA protein. The bacterial phosphoglycerate dehydrogenase is, in some yeast cells, operably linked to a fungal promoter. For example, the fungal promoter is an inducible promoter or a constitutive promoter.

The mutated nucleic acids described herein are isolated nucleic acids. The term "isolated" nucleic acid molecule, as used herein, is a nucleic acid that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule can be substantially free of other cellular material, or culture medium, or of chemical precursors or other chemicals. The invention provides an isolated Saccharomyces cerevisiae cell that includes a mutated nucleic acid encoding fungal formate dehydrogenase, wherein enzymatic activity of the fungal formate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified fungal formate dehydrogenase, and wherein the Saccharomyces cerevisiae cell excretes formic acid. The mutation is, for example, a knockout mutation. Other suitable mutations include, but are not limited to, one or more point mutations in the nucleotide sequence, a frameshift mutation, a deletion or insertion within the nucleotide sequence, the insertion of a stop codon within the sequence or any other truncation mutation. For example, the mutated nucleic acid encoding fungal formate dehydrogenase is mutated fdhl gene, e.g., Yeast Open Reading Frame No. YOR388C, GenBank Accession No. Z75296, a mutated fdh2 gene, e.g., Yeast Open

Reading Frame No. YPL276W/YPL275W, GenBank Accession No. AY558055/Z73632, or both a mutated fdhl gene and a

gene. The mutation is, for example, a knockout mutation. Alternatively or in addition, the isolated Saccharomyces cerevisiae cell includes one or more mutated nucleic acids encoding an enzyme, wherein the mutated nucleic acid is selected from a mutated zwfl gene, e.g. Yeast Open Reading Frame No. YNL241C, GenBank Accession No. X57336, a mutated alt2 gene, e.g., Yeast Open Reading Frame No. YDRl 11 C, and a mutated/w/w/ gene, e.g. , Yeast Open Reading Frame No. YPL262W, a mutated p del gene, e.g., Yeast Open Reading Frame No. YLR044C, a mutated pdc5 gene, e.g., Yeast Open Reading Frame No. YLRl 34W, and a mutated pdcό gene, e.g., Yeast Open Reading Frame No. YGR087C, and wherein expression of the enzyme encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified corresponding enzyme. The mutation is, for example, a knockout mutation. Other suitable mutations include, but are not limited to, one or more point mutations in the nucleotide sequence, a frameshift mutation, a deletion or insertion within the nucleotide sequence, the insertion of a stop codon within the sequence or any other truncation mutation.

In some embodiments, the Saccharomyces cerevisiae cell also includes a modified phosphoglycerate dehydrogenase, wherein enzymatic activity of the modified phosphoglycerate dehydrogenase is increased as compared to the enzymatic activity of an unmodified phosphoglycerate dehydrogenase. The modified phosphoglycerate dehydrogenase is a bacterial phosphoglycerate dehydrogenase, for example, is a fragment of an E. coli SerA protein. In some embodiments, the bacterial phosphoglycerate dehydrogenase is operably linked to a fungal promoter, such as, for example, the CUPl promoter.

The invention also provides systems for producing biological hydrogen. These systems include any of the isolated fungal cells and strains described herein, and an isolated bacterial cell that includes a nucleic acid encoding a prokaryotic formate dehydrogenase. In some embodiments, the bacterial cell is Gram-negative. Alternatively or in addition, the bacterial cell is modified to overproduce formate dehydrogenase.

The invention also provides methods of producing formic acid by culturing the isolated fungal cells and strains described herein in a medium that comprises glucose. The glucose is, for example, an exogenous glucose source selected from the group consisting of glucose, fructose, sucrose, maltose, lactose, xylose, cellulose biomass, and a mixture thereof.

The invention also provides methods of producing biological hydrogen by (i) culturing a population of the fungal cells and/or strains described herein in a liquid medium under aerobic conditions; (ii) removing the population of fungal cells from the medium; (iii) introducing a population of bacterial cells into the medium, wherein the bacterial cells express formate dehydrogenase; (iv) incubating the population of bacterial cells in the medium under anaerobic conditions; and (v) collecting the gases from above the liquid medium. In some embodiments, the liquid medium includes glucose. The glucose is, for example, an exogenous glucose source selected from the group consisting of glucose, fructose, sucrose, maltose, lactose, xylose, cellulose biomass, and a mixture thereof.

Thus, the compositions, systems and methods provided herein combine the hydrogen-producing capabilities of bacteria with the conversion capabilities of yeast to generate increased yields of biological hydrogen. The systems comprise an engineered yeast system, or a combination of an engineered yeast system and an engineered bacterial system.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Brief Description of the Figures

Figures 1 A-IC are illustrations depicting the structures of NADH and NAD⁺.

Figure 2 is an illustration depicting the method of monitoring and optimizing the metabolic models of the invention.

Figures 3 A and 3B are a series of graphs depicting the level of formic acid production in yeast strains with the knockout mutations fdhl fdh2 and fdhl fdh2 zwfl alt2 fuml. Figure 3A is a comparison of the level of formic acid production in a yeast strain with the knockout mutaύom fdhl fdh2 as compared to an otherwise essentially wild-type yeast strain, while Figure 3B is a comparison of the level of formic acid production in a yeast strain with the knockout mutations fdhl fdh2 as compared to a yeast strain with the knockout mutations fdhl fdh2 zwfl alt2fuml.

Figure 4 is an illustration depicting the formate pathway of the invention.

Figure 5 is a graph depicting the level of hydrogen production in a system combining fungal and bacterial metabolism.

Detailed Description of the Invention

The invention provides compositions, systems and methods for economically generating hydrogen and formic acid. Engineered yeast systems are used to drive the conversion of glucose into formic acid. Formic acid is then used to generate hydrogen, for example. Alternatively, formic acid is used directly for other commercial purposes, such as an additive to livestock feed.

The hydrogen produced is used in a variety of applications, including, for example, fuel cells. Fuel cells use hydrogen and oxygen to create electricity and effectively produce zero or near-zero emissions, with only water and heat as byproducts. They can be used in various applications, from portable devices to buildings to vehicles.

The fungal cells, fungal strains, methods and systems provided herein are based on the discovery that a fungal strain lacking formate dehydrogenase secretes formic acid. This fungal formate dehydrogenase is thought to have the metabolic function of using excess formate, which may come from the medium, as a source of reducing equivalents and catalyzing the reaction HCOOH + NAD(P)⁺ -> CO₂ + NAD(P)H + H⁺. This fungal formate dehydrogenase is not to be confused with the prokaryotic formate dehydrogenase H, which catalyzes the reaction HCOOH -> CO₂ + H₂. Naturally occurring fungi do not have a formate dehydrogenase H activity. As described in the Examples, a yeast strain that is mutated in fdhl and fdh2 secretes formic acid, while the parental strain does not. The secretion of formic acid is significantly enhanced by mutation of the gene encoding glucose- 6-phosphate dehydrogenase, which is termed zwfl in many fungal species. Yet further enhancement is achieved by mutating the genes for alanine aminotransferase, termed alt2 in S. cerevisiae, and/or fumarase, texmtάfuml in S. cerevisiae.

Without wishing to be bound by theory, the formic acid that is produced by the fungal cells and strains of the invention originates from metabolic pathways involving serine, glycine, and folic acid. Figure 4 schematically illustrates the relevant pathways. Based on this discover, it was found that reducing or elimination of pyruvate decarboxylase activity also improves yields of formic acid. In S. cerevisiae, the relevant genes aτepdcl, pdc5, and pdcό. Reduction of pyruvate decarboxylase is also advantageous because the amount of ethanol that is produced is reduced or eliminated. This is advantageous when using bacteria to convert formic acid into hydrogen, as described in more detail below, since high levels of ethanol may inhibit growth and metabolism of bacteria.

Introduction of a highly active form of phosphoglycerate dehydrogenase also enhances formic acid production. Phosphoglycerate dehydrogenase catalyzes the conversion of 3 -phosphoglycerate into 3-phospho-hydroxypyruvate, which is the first committed step in the production of serine, glycine, and related metabolites from the carbon flux of glycolysis. Depending on the fungal species that is used, this enzyme may be regulated at the level of transcription, mRNA stability, translation, enzyme activity (for example, allosteric regulation), or other modes of regulation. For example, in S. cerevisiae, this enzyme, which corresponds to the Ser3 product, is regulated both transcriptionally and allosterically. One specific method for introducing such an enzyme activity, for example, is to express a fragment of the phosphoglycerate dehydrogenase protein from E. coli or another bacterium in a fungus, in which the protein fragment lacks the amino-terminal domain that mediates allosteric regulation. For example, a fragment of the SerA protein of E. coli is used. For example, the fragment of the SerA protein of E. coli lacks 5, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids from the amino terminal of the mature protein. The bacterially-derived DNA sequences encoding phosphoglycerate dehydrogenase are preferably expressed under the control of a fungal constitutive promoter or an inducible promoter whose regulation is preferably unrelated to amino acid metabolism, such as the CUPl promoter.

An engineered fungal strain is constructed according to standard techniques, for example using the techniques described, for example, in Goldstein, A.L., and McCusker, J.H. 1999: Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15 , 1541-1553; Goldstein, A.L., Pan, X., and McCusker, J.H. 1999: Heterologous URA3MX cassettes for gene replacement in Saccharomyces cerevisiae. Yeast 15, 507-511; Gϋldener, U., Heck, S., Fiedler, T., Beinhauer, J., and Hegemann, J.H. 1996: A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research 24, 2519-2524; Gϋldener, U., Heinisch, J., Kδhler G.J., Voss, D., and Hegemann, J.H. 2002: A second set of loxP marker cassettes for Cre- mediated multiple gene knockouts in budding yeast. Nucleic Acids Research 30, e23; and Overkamp, K. M., Kotter, P., van der Hoek, R., Schoondermark-Stolk, S., Luttic, M. A. H., van Dijken, J. P., and Pronk, J. T. Functional analysis of structural genes for NAD⁺- dependent formate dehydrogenase in Saccharomyces cerevisiae. Yeast 19, 509-520, the contents of each of these references is hereby incorporated by reference in their entirety. The resulting strain is then tested for formic acid production, for example using a kit from R-Biopharm (Darmstadt, Germany; catalogue number 10979732035).

While many of the specific details of genetic engineering steps are described above and in the Examples with respect to the yeast Saccharomyces cerevisiae and E. coli, it will be apparent to those skilled in the art of metabolic engineering that other fungal species may be used, such as Schizosaccharomyces pombe, K. lactis, Pischia pastoris, Aspergillus oryzae, Aspergillus nidulans, Neurospora crassa, or a basidiomycete. For a number of fungi, the genomes have been sequenced and methods for mutation of genomic sequences and introduction of exogenous DNA are known. Some fungi naturally lack certain genes, such as fdh genes, in which case the engineering steps may be easier. The invention also provides systems, methods and compositions for producing hydrogen from formic acid that has been produced by an engineered fungal strain of the invention. In one embodiment, a bacterial strain expressing a formate dehydrogenase is used to convert the formic acid into hydrogen and carbon dioxide. For example, a formate- producing yeast strain may be grown in an appropriate medium such as unbuffered yeast synthetic complete medium with 2% glucose for about 20 hours at 30 degrees C. It is generally preferable to grow the yeast in aerobic conditions. The yeast cells are then removed from the culture by filtration. In parallel, a culture of a bacterial strain that can express formate dehydrogenase is grown and then the bacteria are concentrated by centrifugation. The bacterial pellet is resuspended in the yeast culture medium and incubated anaerobically overnight at 37 degrees C or an optimal temperature for growth of the bacterium used. Hydrogen gas is obtained from the head space over the liquid culture.

In another embodiment, formic acid is used to produce increased yields of hydrogen using a symbiotic system. In the symbiotic systems of the invention, the engineered yeast produce formic acid using the artificial metabolic pathways described above, and engineered bacteria use their naturally occurring formate dehydrogenase systems to produce hydrogen. For example, E. coli may be used in combination with the engineered yeast of the invention, with the engineered yeast producing formic acid and the E. coli converting the formic acid into hydrogen and carbon dioxide. Depending on the needs of the user, it may be preferable to use an E. coli that has an active Fdh-H system and mutated Fdh-N and Fdh-0 systems. It may also be preferable to use an E. coli strain with mutations that block the direct use of glucose.

Synthetic systems that incorporate elements from more than one organism have been studied, (see e.g., Basu S et al. (2005) A synthetic multicellular system for programmed pattern formation. Nature 434:1130-1134; and Chen M-T, and Weiss R (2005) Artificial cell-cell communication in yeast Saccharomyces cerevisiae using signaling elements from Arabidopsis thaliana. Nature Biotechnology 12:1551-1555, each of which is hereby incorporated by reference in its entirety). Unlike other synthetic systems, the methods described herein use engineered yeast cells and bacterial cells, including engineered bacteria, to produce increased yields of hydrogen.

EXAMPLES

Example 1 : Monitoring Metabolic Fluxes in Artificial Metabolic Pathways

Computer-driven analysis of the metabolic fluxes in cells with artificial metabolic pathways is used to observe and determine the effect of the artificial pathways of the invention on the metabolism of the whole cell. Systems used to analyze the metabolic fluxes include the system of Duarte et al. (Duarte NC et al. (2004) Saccharomyces cerevisiae iND750, a fully compartmentalized genome-scale metabolic model. Genome Research. 14:1298-1309 which is hereby incorporated by reference in its entirety) and additional software created for this purpose. A number of criteria are observed, including cell viability, and modifications are made to the artificial metabolic pathway as necessary. The production of other metabolites, such as ethanol and acetate, is monitored in particular, as some of these metabolites have commercial value.

A schematic of the general approach to monitoring and optimizing the metabolic models is shown in Figure 2. Thus, this analytical system provides a method for evaluating a variety of metabolic modules in order to select those models that do not affect the metabolism of the cells for further optimization and evaluation.

Example 2. Identification of yeast mutations that enhance formic acid production.

The experiments and data presented herein were the first to demonstrate that the production of formic acid from yeast is enhanced by the introduction of mutations in the genes FDHl and FDH2. These genes encode proteins that are related in sequence to formate dehydrogenase proteins found in bacteria. A Saccharomyces cerevisiae strain CEN-PK containing gene knockouts of both FDHl and FDH2 was tested for formate production, and found to produce low levels of formate. In addition, the Saccharomyces cerevisiae strain 580a containing gene knockouts of both FDHl and FDH2 was constructed and was tested for formate production, and found to produce low levels of formate.

The metabolic flux model of Duarte et al. was modified and additional computer code was created to suggest knockout mutations that might be introduced to enhance formate production. The result of this analysis indicated that mutations in the genes ZWFl, ALT2, and FUMl would enhance formic acid production. Starting with the CEN-PK fdhl fdh2 strain described above, yeast strains containing various combinations of knockout mutations in zwfl, alt2, and fuml were constructed.

Serial gene replacement: The gene replacement strategy used herein is described in detail by Goldstein et al. (Yeast, vol. 15: 507-511 and 1541-1553 (1999)) and Gueldener et al. (Nucleic Acids Res. (1996) and Nucleic Acid Res. (2002), each of which is incorporated by reference in its entirety). Gene replacements were performed in a parent background to generate the fdhlΔ fdh2Δ deletions (Overkamp et al., Yeast 2002).

Deletion cassettes were amplified using a plasmid template, pUG6, {see Gueldener et al., Nucleic Acids Res., vol. 24:2519-24 (1996)) containing the KanMX gene encoding for kanamycin (Geneticin) resistance flanked by loxP recombination sites. Forward and reverse PCR primers were designed to amplify the loxP-KanMX-loxP fragment flanked by homologous DNA sequences that were specific to the gene being replaced. Linear PCR products were transformed using a standard lithium acetate protocol. Yeast transformants were selected on kanamycin-containing YEPD plates. Several clones (>5) were chosen for integration validation using single colony PCR. Two PCR reactions were assembled for each clone. First, a forward primer complementary to the 5' upstream (promoter) region of gene being replaced and a reverse primer complementary to the 5' coding sequence of the gene being replaced were used. This PCR reaction was designed to give a positive result if and only if the endogenous gene were still intact {i.e., had not been replaced by the loxP-KanMX-loxP cassette). Second, a forward primer (same as above) and a reverse primer complementary to the loxP-KanMX- loxP cassette were used. This PCR reaction was designed to give a positive result if and only if the endogenous gene has been replaced by loxP-KanMX-loxP. A single validated colony from above was transformed with the non-replicating plasmid pSH65 (see Gueldener et al., Nucleic Acids Res., vol 30: e23 (2002)) containing genes for phleomycin resistance and the Cre recombinase under transcriptional control of the GaW inducible promoter. Yeast transformants were selected on phleomycin-containing YEPD plates. Several clones (>20) were tested for loss of kanamycin resistance by replica plating. Kanamycin sensitive clones determined from above were tested for correct recombination and KanMX removal by single colony PCR. The forward primer was the same as that used above and the reverse primer was complementary to the 3' downstream (UTR) regions of the gene being replaced. Correct recombination and KanMX removal was assessed by observing the size of PCR product generated for each clone. A single kanamycin sensitive colony with validated Cre-mediated recombination was chosen for the next round of gene replacement (i.e., repeat of all steps above).

Using this procedure, a yeast strain comprising knockout mutations in fdhl, fdh2, zwfl, alt2, and fuml was constructed. To test for formic acid production, yeast strains with the knockout mutations fdhl fdh2 and fdhl fdh2 zwfl alt2fuml were grown in synthetic complete medium supplemented with 2% glucose in either aerobic or anaerobic conditions. Formic acid was measured using a kit from R-Biopharm (Darmstadt, Germany; catalogue number 10979732035), following the manufacturer's instructions. The results of a typical experiment are shown in Figures 3A and 3B. When an otherwise essentially wild-type yeast strain, such as the parental 580a strain or CEN-PK strain, was tested for formate production under the same conditions, formic acid was essentially undetectable.

Example 3. Enhancement of formate production by introduction of a constitutive phosphoglvcerate dehydrogenase expression construct.

To test whether expression of a constitutive form of phosphoglycerate dehydrogenase would enhance formate production, the following experiments were performed. First, a DNA fragment encoding part of the SerA protein was obtained from E. coli by PCR amplification according to standard techniques. The DNA sequence of the amplified product was: SerA DNA sequence:

>nucleotide sequence for D-3-phosphoglycerate dehydrogenase [EC: 1.1.1.95] serA

(E. coli): atggcaaaggtatcgctggagaaagacaagattaagtttctgctggtagaaggcgtgcac caaaaggcgctggaaagccttcgtgcagctggttacaccaacatcgaatttcacaaaggc gcgctggatgatgaacaattaaaagaatccatccgcgatgcccacttcatcggcctgcga tcccgtacccatctgactgaagacgtgatcaacgccgcagaaaaactggtcgctattggc tgtttctgtatcggaacaaaccaggttgatctggatgcggcggcaaagcgcgggatcccg gtatttaacgcaccgttctcaaatacgcgctctgttgcggagctggtgattggcgaactg ctgctgctattgcgcggcgtgccggaagccaatgctaaagcgcaccgtggcgtgtggaac aaactggcggcgggttcttttgaagcgcgcggcaaaaagctgggtatcatcggctacggt catattggtacgcaattgggcattctggctgaatcgctgggaatgtatgtttacttttat gatattgaaaataaactgccgctgggcaacgccactcaggtacagcatctttctgacctg ctgaatatgagcgatgtggtgagtctgcatgtaccagagaatccgtccaccaaaaatatg atgggcgcgaaagaaatttcactaatgaagcccggctcgctgctgattaatgcttcgcgc ggtactgtggtggatattccggcgctgtgtgatgcgctggcgagcaaacatctggcgggg gcggcaatcgacgtattcccgacggaaccggcgaccaatagcgatccatttacctctccg ctgtgtgaattcgacaacgtccttctgacgccacacattggcggttcgactcaggaagcg caggagaatatcggcctggaagttgcgggtaaattgatcaagtattctgacaatggctca acgctctctgcggtgaacttcccggaagtctcgctgccactgcacggtgggcgtcgtctg atgcacatccacgaaaaccgtccgggcgtgctaactgcgctgaacaaaatcttcgccgag cagggcgtcaacatcgccgcgcaatatctgcaaacttccgcccagatgggttatgtggtt attgatattgaagccgacgaagacgttgccgaaaaagcgctgcaggcaatgaaagctatt ccgggtaccattcgcgcccgtctgctgtactaa (SEQIDNO: 1) SerA protein sequence:

>amino acid sequence for D-3-phosphoglycerate dehydrogenase [EC:1.1.1.95] serA (E. coli)

MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGALDDEQLKESIRDAHFIGLR SRTHLTEDVINAAEKLVAIGCFCIGTNQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGEL LLLLRGVPEANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAESLGMYVYFY DIENKLPLGNATQVQHLSDLLNMSDWSLHVPENPSTKNMMGAKEISLMKPGSLLINASR GTWDIPALCDALASKHLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEA QENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHIHENRPGVLTALNKIFAE QGVNIAAQYLQTSAQMGYWIDIEADEDVAEKALQAMKAIPGTIRARLLY (SEQ ID NO: 2)

Forward Primer: 5' -CCT TAT CTA GAG CAA AGG TAT CGC TGG AG -3' (SEQ ID NO:3)

Reverse Primer: 5' - GGA ATC TGC AGC GGC CGC TAC TAG TTT AAG TAG AAT CCA AAC CCA ACA ATG GAT TTG GGA TTG GTT TAC CGT GGA TGT GCA TCA GAC GAC - 3' (SEQ ID NO: 4) This protein also had a V5 epitope tag for verification purposes. The resulting protein lacks the allosteric inhibition site of SerA. This coding sequence was placed under the control of the CUPl promoter and then inserted into the CEN plasmid pRS410 (Addgene, Cambridge MA; catalogue number 11258). The resulting plasmid was placed in the fdhlfdh2 and fdhl fdh2 zwfl alt2fuml strains described above, and the resulting strains tested for formic acid production. The table below shows typical results. The effect of the phosphoglycerate dehydrogenase expression construct was to enhance formate production about 3-fold.

Table 1:

Average of three biological replicates ± SD

Example 4. Production of hydrogen from a system combining fungal and bacterial metabolism.

To demonstrate production of hydrogen by a bacterium from medium generated by an engineered fungal strain of the invention, the following experiment was performed. 50 mL yeast cultures were grown in unbuffered synthetic complete media with 2% glucose for about 20 hours. The spent media was cleared by 2 micron filter sterilization and used to resuspend E. coli cell pellets processed as follows: 100 mL E. coli (K-12 strain MG1655) cultures were grown overnight in LB medium. Cultures were centrifuged and pellets resuspended in LB with 100 mM formic acid to induce expression of the hydrogenase complex. Induced cultures were grown anaerobically for an additional 4 hours after which they were centrifuged and pellets resuspended with the spent media obtained above. The E. coli cells resuspended in spent yeast media were incubated anaerobically about 18 hours. Head gas was measured by gas chromatography to quantify hydrogen production. Typical results of experiments such as the one described herein are shown in Figure 5. Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference.

Claims

What is claimed is:

1. An isolated fungal cell comprising a mutated nucleic acid encoding a fungal formate dehydrogenase and a mutated nucleic acid encoding glucose-6-phosphate dehydrogenase, wherein enzymatic activity of the fungal formate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified fungal formate dehydrogenase, and wherein enzymatic activity of the glucose-6-phosphate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified glucose-6-phosphate dehydrogenase.

2. The fungal cell of claim 1, wherein said cell excretes formic acid.

3. The fungal cell of claim 1 or claim 2 further comprising a mutated nucleic acid encoding alanine aminotransferase, wherein enzymatic activity of the alanine aminotransferase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified alanine aminotransferase.

4. The fungal cell of claim 1, claim 2 or claim 3 further comprising a mutated nucleic acid encoding fumarase, wherein enzymatic activity of the fumarase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified fumarase.

5. The fungal cell of any one of claims 1 to 4 further comprising a mutated nucleic acid encoding pyruvate decarboxylase, wherein enzymatic activity of the pyruvate decarboxylase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified pyruvate decarboxylase.

6. The fungal cell of any one of claims 1 to 5 further comprising a modified phosphoglycerate dehydrogenase, wherein enzymatic activity of the modified phosphoglycerate dehydrogenase is increased as compared to the enzymatic activity of an unmodified phosphoglycerate dehydrogenase.

7. The fungal cell of claim 6, wherein the modified phosphoglycerate dehydrogenase is constitutively active.

8. The fungal cell of claim 6, wherein the modified phosphoglycerate dehydrogenase is a heterologous phosphoglycerate dehydrogenase.

9. The fungal cell of claim 7, wherein the heterologous phosphoglycerate dehydrogenase is a bacterial phosphoglycerate dehydrogenase.

10 The fungal cell of claim 9, wherein the bacterial phosphoglycerate dehydrogenase is a fragment of an E. coli SerA protein.

11. The fungal cell of claim 9, wherein the bacterial phosphoglycerate dehydrogenase is operably linked to a fungal promoter.

12. The fungal cell of claim 11 , wherein the fungal promoter is an inducible promoter or a constitutive promoter.

13. The fungal cell of any one of claims 1 to 12, wherein the fungal cell is a Saccharomyces cell.

14. The fungal cell of claim 13, wherein the Saccharomyces cell is a Saccharomyces cerevisiae cell.

15. The fungal cell of any one of claims 3 to 14, wherein the fungal cell excretes formic acid.

16. An isolated Saccharomyces cerevisiae cell comprising a mutated nucleic acid encoding fungal formate dehydrogenase a mutated nucleic acid encoding glucose-6- phosphate dehydrogenase, wherein enzymatic activity of the fungal formate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified fungal formate dehydrogenase, and wherein enzymatic activity of the glucose- 6-phosphate dehydrogenase encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified glucose-6-phosphate dehydrogenase, and further wherein the Saccharomyces cerevisiae cell excretes formic acid.

17. The Saccharomyces cerevisiae cell of claim 16, wherein the mutated nucleic acid encoding fungal formate dehydrogenase is mutatedfdhl gene, a mutated fdh2 gene or both a mutated fdhl gene and a

gene and wherein the mutated nucleic acid encoding glucose-6-phosphate dehydrogenase is a mutated zwfl gene.

18. The Saccharomyces cerevisiae cell of claim 17 further comprising one or more mutated nucleic acids encoding an enzyme, wherein the mutated nucleic acid is selected from a mutated alt2 gene, and a mutatedywm/ gene, a mutated pdcl gene, a mutated pdc5 gene, and a mutated pdcό gene, and wherein expression of the enzyme encoded by the mutated nucleic acid is reduced as compared to the enzymatic activity of an unmodified corresponding enzyme.

19. The Saccharomyces cerevisiae cell of claim 17 further comprising a modified phosphoglycerate dehydrogenase, wherein enzymatic activity of the modified phosphoglycerate dehydrogenase is increased as compared to the enzymatic activity of an unmodified phosphoglycerate dehydrogenase.

20. The fungal cell of claim 19, wherein the modified phosphoglycerate dehydrogenase is a bacterial phosphoglycerate dehydrogenase.

21. The fungal cell of claim 20, wherein the bacterial phosphoglycerate dehydrogenase is a fragment of an E. coli SerA protein.

22. The fungal cell of claim 21, wherein the bacterial phosphoglycerate dehydrogenase is operably linked to a fungal promoter.

23. The fungal cell of claim 22, wherein the fungal promoter is the CUPl promoter.

24. A system for producing biological hydrogen, the system comprising: (a) an isolated yeast cell of any one of claims 1 to 15; and (b) an isolated bacterial cell comprising a nucleic acid encoding a prokaryotic formate dehydrogenase.

25. The system of claim 24, wherein the bacterial cell is Gram-negative.

26. The system of claim 24, wherein the bacterial cell is modified to overproduce formate dehydrogenase.

27. A method of producing formic acid, the method comprising culturing the isolated fungal cell of any one of claims 1 to 15 in a medium that comprises glucose.

28. The method of claim 27, wherein the glucose is an exogenous glucose source selected from the group consisting of glucose, fructose, sucrose, maltose, lactose, xylose, cellulose biomass, and a mixture thereof

29. A method of producing biological hydrogen comprising the steps of:

(a) culturing a population comprising the fungal cell of any one of claims 1 to 15 in a liquid medium under aerobic conditions;

(b) removing the population of fungal cells from the medium;

(c) introducing a population of bacterial cells into the medium, wherein the bacterial cells express formate dehydrogenase;

(d) incubating the population of bacterial cells in the medium under anaerobic conditions; and

(e) collecting the gases from above the liquid medium.

30. The method of claim 29, wherein the liquid medium in step (a) further comprises glucose.

31. The method of claim 30, wherein the glucose is an exogenous glucose source selected from the group consisting of glucose, fructose, sucrose, maltose, lactose, xylose, cellulose biomass, and a mixture thereof.