WO2015013295A1 - Recombinant production of chemicals from methane or methanol - Google Patents

Abstract

Methods for producing valuable chemicals using recombinant organisms that utilize methane or methanol as a carbon source as well as methods for creating cells useful in such methods.

Description

RECOMBINANT PRODUCTION OF CHEMICALS FROM

METHANE OR METHANOL

SUMMARY OF THE INVENTION

[0001] The present invention provides methods for producing valuable chemicals using recombinant organisms that utilize methane or methanol as a carbon source as well as methods for creating cells useful in such methods. In various embodiments, the methods utilize cells that naturally catabolize single-carbon compounds for energy or biomass formation and have been modified using recombinant DNA technology such that this catabolism produces valuable chemicals. The invention provides a number of microorganisms for converting methane or methanol compounds into higher value chemicals via central metabolic pathways and metabolites. In various embodiments the metabolites that connect methane or methanol to higher value compounds include acetyl-CoA, pyruvate, intermediates of glycolysis, intermediates of gluconeogenesis, amino acids, fatty acids, and in any other compound previously known to be produced via cellular processes. In various embodiments, the microorganisms utilized in the methods of the invention are naturally occurring and can catabolize and grow on methane or methanol. In other embodiments, recombinant cells that have been genetically modified to utilize methane or methanol are utilized in the methods of the invention; the invention also provides such cells.

[0002] Methods for isolation and culturing of newly identified organisms that can catabolize methane or methanol are also provided. In some embodiments, the microorganism catabolizes methanol but not methane; in other embodiments, the microorganism catabolizes methane but not methanol, and in other embodiments the microorganism catabolizes both methane and methanol. Regardless of the intermediates involved in catabolism, all metabolic pathways of the host cells provided by or utilized in the methods of the invention eventually assimilate the carbon derived from methane or methanol to support energy and biomass formation. The present invention also provides chemical production methods in which these microorganisms are cultured under conditions that result in the production of the desired chemicals. Also provided are methods for making recombinant cells that result in production of specific target chemicals, including in one embodiment, malonic acid. DETAILED DESCRIPTION

[0003] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0004] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like.

[0005] The term "accession number", and similar terms such as "protein accession number", "UniProtID", "genelD", "gene accession number" refer to designations given to specific proteins or genes. These identifiers describe a gene or enzyme sequence in publicly accessible databases, such as NCBI.

[0006] Amino acids in a protein coding sequence are identified herein by the following abbreviations and symbols. Specific amino acids are identified by a single- letter abbreviation, as follows: A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G is glycine, H is histidine, L is leucine, I is isoleucine, K is lysine, M is methionine, F is phenylalanine, P is proline, S is serine, T is threonine, W is tryptophan, Y is tyrosine, and V is valine. A dash (-) in a consensus sequence indicates that there is no amino acid at the specified position. A plus (+) in a consensus sequence indicates any amino acid may be present at the specified position. Thus, a plus in a consensus sequence herein indicates a position at which the amino acid is generally non-conserved; a homologous enzyme sequence, when aligned with the consensus sequence, can have any amino acid at the indicated "+" position. At positions in a consensus sequence where one of a subset of amino acids can be present, the following abbreviations are used: B represents that one of the amino acids R, K, or H is present at the indicated position; J represents that one of the amino acids D or E is present at the indicated position; O represents that one of the amino acids I, L, or V is present at the indicated position; U represents that one of the amino acids S or T is present at the indicated position; and represents that one of the amino acids A, D, R, H, K, S, T, N, Q, or Y (or a subset of those amino acids) is present at the indicated position. Illustrative subsets of i include i is A, D, K, S, T, N, or Y and Xi is S or N. Specific amino acids in a protein coding sequence are identified by their respective single-letter abbreviation followed by the amino acid position in the protein coding sequence where 1 corresponds to the amino acid (typically methionine) at the N-terminus of the protein. For example, E124 in S. cerevisiae wild type EHD3 refers to the glutamic acid at position 124 from the EHD3 N-terminal methionine {i.e., Ml). Amino acid substitutions {i.e., point mutations) are indicated by identifying the mutated {i.e., progeny) amino acid after the single-letter code and number in the parental protein coding sequence; for example, E124A in S. cerevisiae EHD3 refers to substitution of alanine for glutamic acid at position 124 in the EHD3 protein coding sequence. The mutation may also be identified in parentheticals, for example EHD3 (E124A). Multiple point mutations in the protein coding sequence are separated by a backslash (/); for example, EHD3 E124A/Y125A indicates that mutations E124A and Y125A are both present in the EHD3 protein coding sequence. The number of mutations introduced into some examples has been annotated by a dash followed by the number of mutations, preceding the parenthetical identification of the mutation (e.g. A5W8H3-1 (E95Q)). The Uniprot IDs with and without the dash and number are used interchangeably herein (i.e. A5W8H3-1 (E95Q) = A5W8H3 (E95Q)).

[0007] As used herein, the term "express", when used in connection with a nucleic acid encoding an enzyme or an enzyme itself in a cell, means that the enzyme, which may be an endogenous or exogenous (heterologous) enzyme, is produced in the cell. The term "overexpress", in these contexts, means that the enzyme is produced at a higher level, i.e., enzyme levels are increased, as compared to the wild- type, in the case of an endogenous enzyme. Those skilled in the art appreciate that overexpression of an enzyme can be achieved by increasing the strength or changing the type of the promoter used to drive expression of a coding sequence, increasing the strength of the ribosome binding site or Kozak sequence, increasing the stability of the mRNA transcript, altering the codon usage, increasing the stability of the enzyme, and the like.

[0008] The terms "expression vector" or "vector" refer to a nucleic acid and/or a composition comprising a nucleic acid that can be introduced into a host cell, e.g., by transduction, transformation, or infection, such that the cell then produces ("expresses") nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell, that are contained in or encoded by the nucleic acid so introduced. Thus, an "expression vector" contains nucleic acids (ordinarily DNA) to be expressed by the host cell. Optionally, the expression vector can be contained in materials to aid in achieving entry of the nucleic acid into the host cell, such as the materials associated with a virus, liposome, protein coating, or the like. Expression vectors suitable for use in various aspects and embodiments of the present invention include those into which a nucleic acid sequence can be, or has been, inserted, along with any preferred or required operational elements. Thus, an expression vector can be transferred into a host cell and, typically, replicated therein (although, one can also employ, in some embodiments, non-replicable vectors that provide for "transient" expression). In some embodiments, an expression vector that integrates into chromosomal, mitochondrial, or plastid DNA is employed. In other embodiments, an expression vector that replicates extrachromasomally is employed. Typical expression vectors include plasmids, and expression vectors typically contain the operational elements required for transcription of a nucleic acid in the vector. Such plasmids, as well as other expression vectors, are described herein or are well known to those of ordinary skill in the art.

[0009] The terms "ferment", "fermentative", and "fermentation" are used herein to describe culturing microbes under conditions to produce useful chemicals, including but not limited to conditions under which microbial growth, be it aerobic or anaerobic, occurs.

[0010] The term "heterologous" as used herein refers to a material that is non- native to a cell. For example, a nucleic acid is heterologous to a cell, and so is a "heterologous nucleic acid" with respect to that cell, if at least one of the following is true: (a) the nucleic acid is not naturally found in that cell (that is, it is an "exogenous" nucleic acid); (b) the nucleic acid is naturally found in a given host cell (that is, "endogenous to"), but the nucleic acid or the RNA or protein resulting from transcription and translation of this nucleic acid is produced or present in the host cell in an unnatural (e.g., greater or lesser than naturally present) amount; (c) the nucleic acid comprises a nucleotide sequence that encodes a protein endogenous to a host cell but differs in sequence from the endogenous nucleotide sequence that encodes that same protein (having the same or substantially the same amino acid sequence), typically resulting in the protein being produced in a greater amount in the cell, or in the case of an enzyme, producing a mutant version possessing altered (e.g. higher or lower or different) activity; and/or (d) the nucleic acid comprises two or more nucleotide sequences that are not found in the same relationship to each other in the cell. As another example, a protein is heterologous to a host cell if it is produced by translation of RNA or the corresponding RNA is produced by transcription of a heterologous nucleic acid; a protein is also heterologous to a host cell if it is a mutated version of an endogenous protein, and the mutation was introduced by genetic engineering.

[0011] The term "homologous", as well as variations thereof, such as

"homology", refers to the similarity of a nucleic acid or amino acid sequence, typically in the context of a coding sequence for a gene or the amino acid sequence of a protein. Homology searches can be employed using a known amino acid or coding sequence (the "reference sequence") for a useful protein to identify homologous coding sequences or proteins that have similar sequences and thus are likely to perform the same useful function as the protein defined by the reference sequence. As will be appreciated by those of skill in the art, a protein having greater than 90% identity to a reference protein as determined by, for example and without limitation, a BLAST (blast.ncbi.nlm.nih.gov) search is highly likely to carry out the identical biochemical reaction as the reference protein. In some cases, two enzymes having greater than 20% identity will carry out identical biochemical reactions, and the higher the identity, i.e., 40% or 80% identity, the more likely the two proteins have the same or similar function. As will be appreciated by those skilled in the art, homologous enzymes can be identified by BLAST searching.

[0012] The terms "host cell" and "host microorganism" are used interchangeably herein to refer to a living cell that can be (or has been) transformed via insertion of an expression vector. A host microorganism or cell as described herein may be a prokaryotic cell (e.g. , a microorganism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0013] The terms "isolated" or "pure" refer to material that is substantially, e.g. greater than 50% or greater than 75%, or essentially, e.g. greater than 90%, 95%, 98% or 99%, free of components that normally accompany it in its native state, e.g. the state in which it is naturally found or the state in which it exists when it is first produced.

[0014] A carboxylic acid as described herein can be a salt, acid, base, or derivative depending on the structure, pH, and ions present. The terms "malonate" and "malonic acid" are used interchangeably herein. Malonic acid is also called propanedioic acid (C₃H₄0₄; CAS# 141-82-2).

[0015] As used herein, the term "nucleic acid" and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and to polyribonucleotides (containing D-ribose). "Nucleic acid" can also refer to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970). A "nucleic acid" may also be referred to herein with respect to its sequence, the order in which different nucleotides occur in the nucleic acid, as the sequence of nucleotides in a nucleic acid typically defines its biological activity, e.g., as in the sequence of a coding region, the nucleic acid in a gene composed of a promoter and coding region, which encodes the product of a gene, which may be an RNA, e.g. a rRNA, tRNA, or mRNA, or a protein (where a gene encodes a protein, both the mRNA and the protein are "gene products" of that gene).

[0016] The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, ribosome-binding site, and transcription terminator) and a second nucleic acid sequence, the coding sequence or coding region, wherein the expression control sequence directs or otherwise regulates transcription and/or translation of the coding sequence.

[0017] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0018] As used herein, "recombinant" refers to the alteration of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or RNA in a cell or virus or expression vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination. Recombinant can also refer to manipulation of DNA or RNA in a cell or virus by random or directed mutagenesis. A "recombinant" cell or nucleic acid can typically be described with reference to how it differs from a naturally occurring counterpart (the "wild-type"). In addition, any reference to a cell or nucleic acid that has been "engineered" or "modified" and variations of those terms, is intended to refer to a recombinant cell or nucleic acid.

[0019] The terms "transduce", "transform", "transfect", and variations thereof as used herein refers to the introduction of one or more nucleic acids into a cell. For practical purposes, the nucleic acid must be stably maintained or replicated by the cell for a sufficient period of time to enable the function(s) or product(s) it encodes to be expressed for the cell to be referred to as "transduced", "transformed", or "transfected". As will be appreciated by those of skill in the art, stable maintenance or replication of a nucleic acid may take place either by incorporation of the sequence of nucleic acids into the cellular chromosomal DNA, e.g., the genome, as occurs by chromosomal integration, or by replication extrachromosomally, as occurs with a freely-replicating plasmid. A virus can be stably maintained or replicated when it is "infective": when it transduces a host microorganism, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.

[0020] This invention provides methods for producing chemicals using host cells, which may be recombinant host cells that utilize methane or methanol as a carbon source as well as methods for creating new cells useful in such methods. A wide variety of organisms are provided by or utilized in the methods of the invention, and these include organisms that naturally catabolize methanol or methane or both. This invention provides a variety of organisms that do not naturally catabolize methanol or methane but have been modified to do so. The invention can be practiced with cells that are prokaryotic or eukaryotic and have previously been identified to catabolize methanol or methane or have been modified to do so. The invention also provides for methods for isolating cells from the environment that catabolize methanol or methane. In various embodiments, the invention utilizes and/or provides cells from the genera listed in the natural occurring organisms section below. In various embodiments, the invention utilizes and/or provides cells from the genera Saccharomyces, Escherichia, Pichia, Issatchenkia, Aspergillus, and Yarrowia. The invention utilizes and/or provides cells that catabolize methanol or methane and have been modified to produce organic acids, fatty acids, chemicals, fuels, and enzyme products. The invention also provides recombinant host cells and nucleic acids that are used for the catabolism of methanol and/or methane. The invention provides recombinant host cells and nucleic acids, such as expression vectors that are transformed into cells, for the production of organic acids, fatty acids, chemicals, fuels and enzyme products from methanol or methane. Also provided by the invention are methods for production of the products at commercial scale.

Naturally occurring organisms that consume methanol

[0021] In various embodiments, the invention utilizes cells that naturally catabolize methanol, including prokaryotic and eukaryotic host cells. Suitable cells include cells from the following genera: Amycolatopsis, Brevibacillus, Burkholderia, Candida, Candidatus Methylomirabilis, Hyphomicrobium, Komagataella, Methanomonas, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Ogataea, Paracoccus, Pichia, Pseudomonas, Rhodobacter, Rhodococcus, and Hansenula. The invention provides methods for culturing these cells in a medium that comprises minerals, vitamins, a nitrogen source, methanol, and optionally another carbon source.

[0022] In one embodiment, the cells utilized in practice of the invention belong to the genus Hansenula, including but not limited to cells of the species H. polymorpha. Different strains of H. polymorpha, including NRRL Y-5445, NRRL Y- 7560, NRRL Y-1798, can be obtained from culture collections such as the USDA ARS NRRL culture collection or can be otherwise isolated and identified. Hansenula polymorpha is also referred to Ogataea angusta or Ogataea polymorpha. Suitable media for culturing Hansenula cells contain mineral salts, vitamins, a nitrogen source and methanol. In some embodiments the media is composed of 5 g/L ammonium sulfate, lg/L monopotassium phosphate, 0.5 g/L magnesium sulfate, 0.1 g/L sodium chloride, 0.1 g/L calcium chloride, 2 mg/L inositol, 0.5 mg/L boric acid, 0.4 mg/L calcium pentothenate, 0.4 mg/L niacin, 0.4 mg/L pyridoxine hydrochloride, 0.4 mg/L thiamine HC1, 0.4 mg/L zinc sulfate, 0.4 mg/L manganese sulfate, 0.2 mg/L p- aminobenzoic acid, 0.2 mg/L riboflavin, 0.2 mg/L sodium molybdate, 0.2 mg/L ferric chloride, 0.1 mg/L Potassium iodide, 40 μg/L copper sulfate, 2 μg/L folic acid and 2 μg/L biotin. In some embodiments, the media is supplemented with 10 mg/L adenine, 50 mg/L L-arginine HC1, 80 mg/L L-aspartic acid, 20 mg/L L-histidine HC1, 50 mg/L L-isoleucine, 100 mg/L L- leucine, 50 mg/L L-lysine HC1, 20 mg/L methionine, 50 mg/L L-phenylalanine, 100 mg/L L-threonine, 50 mg/L L-tryptophan, 50 mg/L L- tyrosine and 140 mg/L L-valine. The concentration of methanol in the media can range from 0.5-1% (vol/vol). Optimal pH for growth is between 4.5 and 5.5, but process culture conditions may result in a range in pH from 2.5 to 5.5. Optimal temperature for growth is between 35°C and 45°C, but optimal process culture conditions may utilize a range in temperature between 25°C and 50°C. In some embodiments, glucose is used in addition to methanol as a carbon source and added to media at concentrations ranging from 0.5-30% (wt/vol). In shake flasks, these cells can be aerated via shaking at 100-400 rpm.

[0023] In another embodiment, the cells utilized in practice of the invention the genus Komagataella, including but not limited to cells of the species K. pastoris (also referred to as Pichia pastoris). Suitable media for culturing Komagataella cells contain mineral salts, vitamins, a nitrogen source and methanol. In some embodiments the media is composed of similar components to those described for Hansenula. In some embodiments, the media is supplemented with similar components to those described for Hansenula. The concentration of methanol in the media can range from 0.5-1% (vol/vol). Optimal pH is between 4.5 and 6.5, but process culture conditions may result in a range of pH from 2.5 to 6.5. Optimal temperature is between 25°C and 35°C, but optimal process culture conditions may utilize a range of temperature between 25°C and 50°C. In some embodiments, glucose is used in addition to methanol as a carbon source and added to media at concentrations ranging from 0.5- 5% (wt/vol). In shake flasks, these cells can be aerated via shaking at 100-400 rpm.

[0024] In another embodiment, the cells utilized in practice of the invention belong to the genus Pichia. including but not limited to cells of the species, P. methanolica. Suitable media for culturing Pichia cells contain mineral salts, vitamins, a nitrogen source and methanol. In some embodiments the media is composed of similar components as described for Hansenula. In some embodiments, two-fold concentrated media is used to provide faster growth rates. In some embodiments, the media is supplemented with similar supplements as described for Hansenula. The concentration of methanol in the media can range from 0.5-5% (vol/vol). Optimal pH for growth is between 5.5 and 6.5, but process culture conditions may result in a range in pH from 2.5 to 5.5. Optimal temperature for growth is between 28°C and 35°C, but process culture conditions may utilize a range of temperature between 25°C and 50°C. In some embodiments glucose, is used in addition to methanol as a carbon source and added to media at concentrations ranging from 0.5-2% (wt/vol). In shake flasks, these cells can be aerated via shaking at 100-400 rpm.

[0025] In another embodiment, the cells utilized in practice of the invention belong to the genus Candida, including but not limited to cells of the species C. boidinii, or C. methanolovescens. Suitable media for culturing Candida cells contain mineral salts, vitamins, a nitrogen source, and methanol. In some embodiments the media is composed of similar components as those described for Hansenula. The concentration of methanol in the media can range from 0.5-5% (vol/vol). Optimal pH is between 4.0 and 5.0, but process culture conditions may result in a range in pH from 2.5 to 6.5. Optimal temperature for growth is between 28°C and 32°C, but process culture conditions may utilize a range in temperature between 25°C and 50°C. In some embodiments, glucose is used in addition to methanol as a carbon source and added to media at concentrations ranging from 0.5-2% (wt/vol). In shake flasks, these cells can be aerated via shaking, e.g. at 100-400 rpm. For concentrations of methanol above 2%, some cells may begin to exhibit slow growth, decreased catabolism, and other phenotypes related to methanol toxicity. One skilled in the art will appreciate that this toxicity can be addressed by genetic or fermentation process changes. Briefly, genes can be modified to increase the concentration of methanol that a cell can tolerate. Fermentation process conditions can be manipulated to increase methanol concentration with minimized toxicity. Such methods include pulse-feeding, where a bolus of methanol is added and so initially present at high concentration, but the cells are present at a concentration high enough to catabolize it quickly, thereby minimizing toxic effects. One skilled in the art will appreciate that high cell concentrations can be achieved by extending the biomass formation phase of a fermentation.

[0026] The host cells described above are eukaryotic cells, but the methods of the invention can be practiced with prokaryotic cells that catabolize methanol. In some instances, prokaryotic cells that catabolize methane, such as those described below, are also able to catabolize methanol and so can be used in accordance with the invention. Suitable media for culturing such prokaryotic cells is described below, and is suitable for use in the methods of the invention for catabolizing methanol into useful products. Such media may contain methanol in addition to methane or can alternatively contain methanol but no methane. Methanol concentrations in media suitable for use with prokaryotic cells will range from 0.5-2% or higher (e.g. 5% or higher). Any prokaryotic cell that can utilize methanol is useful in accordance with the present invention.

[0027] In other embodiments, including those with either prokaryotic or eukaryotic cells, a strain that has been mutagenized and optimized for higher methanol tolerance is generated, and the use of such strains allows for higher concentrations of methanol in the fermentation media. For example, cells tolerant to methanol concentrations greater than 1% (vol/vol) can be produced and utilized in the methods of the invention. In some embodiments, the cells are tolerant to methanol concentrations in the range of 2% to 10% (vol/vol) or higher.

[0028] In many commercial embodiments, fermentations of any of the above cells will be run at volumes of 10L, 100L, 300L, 1000L, lOkL, 25kL, 50kL, lOOkL, 200kL, 400kL, 500kL, 1ML or larger scale. Methanol is delivered to the fermentation vessel by suitable methods of delivering a liquid feed including pumping to the top or bottom of the fermenter. Typical fermentations that utilize only sugar often do not require large amounts of oxygen. Using methanol as a carbon source requires addition of oxygen for a high yielding, efficient process. To add oxygen to such large culture volumes, the invention provides, in one embodiment, the use of a sparger to deliver a controlled mixture of gases including oxygen. In some embodiments the oxygen is a component of air. In some embodiments, the oxygen is mixed with carbon dioxide. In some embodiments, the oxygen concentration in the fermenter vessel is 1% (v/v), 5% (v/v), 10% (v/v), 20% (v/v), 33% (v/v), 40% (v/v), 50% (v/v) and greater. In some embodiments air is sparged into the fermentation vessel. In some embodiments, a mixture of two gases is sparged into the fermentation vessel and is composed of oxygen, and carbon dioxide. In some embodiments the mixture of gases has a 1: 1 concentration in the fermentation vessel. In some embodiments, oxygen is delivered to the bottom of the fermentation vessel and at an effective pressure such that it is a liquid. As the fermentation process vessel size increases, the cost of gas mixtures that is sparged into the fermenter becomes increasingly expensive. In some embodiments the gases are recycled after passing through a fermentation vessel. In some embodiments, unused oxygen, and/or carbon dioxide from the fermentation is recycled. In some embodiments, the gas is recycled with pressure swing adsorption. In other embodiments, the gas is recycled with selectively permeable gas membranes. Recycling gas can decrease the cost of producing chemicals from methanol or methane.

[0029] In all of the above embodiments, sugar or glycerol, referred to as carbon sources, can also be present in the media as a carbon source in addition to methanol, and in such embodiments, the concentration of carbon source in the media can range from, for example and without limitation, 0.5-60% (wt/vol). In some embodiments the sugar includes glucose, sucrose, fructose, xylose, arabinose, or others.

Naturally occurring organisms that consume methane

[0030] In various embodiments, the invention utilizes cells that catabolize methane, including prokaryotic and eukaryotic host cells. The cells provided naturally oxidize methane into methanol and then further catabolize the methanol for biomass or energy production through a variety of metabolic pathways. The invention is practiced with a wide variety of host cells including host cells provided by the invention are from the following genera: Amycolatopsis, Brevibacillus, Burkholderia, Candidatus Methylomirabilis, Hyphomicrobium, Methanomonas, Methyl obacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Paracoccus, Pseudomonas, Rhodobacter, and Rhodococcus. The invention provides methods for culturing these cells in a medium that comprises minerals, vitamins, a nitrogen source, methane, and optionally another carbon source. Also provided by the invention are methods for isolating new cells that catabolize methane.

[0031] In one embodiment, the cells utilized in practice of the invention belong to the genus Pseudomonas, including but not limited to cells of the P. methanica. Suitable media for culturing Pseudomonas cells contain mineral salts, vitamins, a nitrogen source and methane. In some embodiments the media is composed of 2 g/L sodium nitrate, 0.2 g/L magnesium sulfate heptahydrate, 0.04 g/L potassium chloride, 0.015 g/L calcium chloride, 0.21 g/L disodium phoshpate, 0.09 g/L sodium phosphate, 1 mg/L iron sulfate heptahydrate, 5 ug/L copper sulfate pentahydrate, 10 ug/L boric acid, 10 ug/L manganese sulfate pentahydrate, 70 ug/L zinc sulfate heptahydrate and 10 ug/L molybdenum trioxide. The base nutrient media is formulated as a liquid or as solid 2% agar plates. The carbon source, methane, is provided by growth in a dessicator, or rubber stoppered flask. Atmospheric gas is first evacuated from the growth vessel and then methane and atmospheric gas are added back in suitable ratios. In some embodiments, a 1:1 mixture of methane:air is added to the growth vessel to 1 atmosphere. In other embodiments, the ratio of methane to air or methane to oxygen is adjusted to result in more optimal growth. After every 24h of growth, the vessel's atmosphere is refreshed with a methane:air mixture. Optimal pH for growth is between 6.0 and 8.0, but process conditions may result in a range in pH from 2.5 to 8.0. Optimal temperature is between 28°C and 35°C, but process culture conditions may utilize a range in temperature between 25°C and 45°C.

[0032] In one embodiment, the cells utilized in practice of the invention belong to the genus Methanomonas, including but not limited to the cells of the species M. methanooxidans. Suitable media for culturing Methanomonas cells contain mineral salts, vitamins, a nitrogen source and methane. In some embodiments the media is composed of components described for Pseudomonas. The base nutrient media is formulated as liquid or solid 2% agar plates. The carbon source, methane, is provided by growth in a dessicator, or rubber stoppered flask. Atmospheric gas is first evacuated from the growth vessel and then methane and atmospheric gas are added back in suitable ratios. In some embodiments, a 1:1 mixture of methane:air is added to the growth vessel to 1 atmosphere. In other embodiments, the ratio of methane to air or methane to oxygen are adjusted to result in more optimal growth. After every 24h of growth, the vessel's atmosphere is refreshed with a methane:air mixture. Optimal pH for growth is between 6.0 and 8.0, but process conditions may result in a range in pH from 2.5 to 8.0. Optimal temperature is between 28°C and 35°C, but process culture conditions may utilize a range in temperature between 25 °C and 45 °C.

[0033] In another embodiment, the cells utilized in practice of the invention belong to the genus Methylobacterium, including but not limited to cells of the species, M. extorquens AMI. Suitable media for culturing Methylobacterium cells contain minerals salts, vitamins, a nitrogen source and methane. In some embodiments the media is composed of 1.62 g/L ammonium chloride, 0.2 g/L magnesium sulfate, 2.21 g/L potassium phosphate, 1.25 g/L sodium phosphate dihydrate, 15 mg/L sodium ethylenediaminetetraacetic acid dihydrate, 4.5 mg/L zinc sulfate heptahydrate, 0.3 mg/L cobalt chloride hexahydrate, 1 mg/L manganese chloride tetrahydrate, 1 mg/L boric acid, 2.5 mg/L calcium chloride, 0.4 mg/L sodium molybdenum tetraoxide dihydrate, 3 mg/L iron sulfate heptahydrate, and 0.3 mg/L copper sulfate pentahydrate. The base nutrient media is formulated as liquid or solid 2% agar plates. The carbon source, methane, is provided by growth in a dessicator, or rubber stoppered flask. Atmospheric gas is first evacuated from the growth vessel and then methane and atmospheric gas are added back in suitable ratios. In some embodiments, a 1: 1 mixture of methane:air is added to the growth vessel to 1 atmosphere. In other embodiments, the ratio of methane to air or methane to oxygen are adjusted to result in more optimal growth. After every 24h of growth, the vessel's atmosphere is refreshed with a methane:air mixture. Optimal pH for growth is between 6.0 and 8.0, but process conditions may result in a range in pH from 2.5 to 8.0. Optimal temperature is between 28°C and 35°C, but process culture conditions may utilize a range in temperature between 25 °C and 45 °C.

[0034] In another embodiment, the cells utilized in practice of the invention belong to the genus Methylococcus, including but not limited to cells of the species M. capsulatus. Suitable media and growth conditions for culturing Methylococcus cells are similar if not identical to those described for Methylobacterium above.

[0035] In another embodiment, the cells utilized in practice of the invention belong to the genus Methylosinus, including but not limited to cells of the species, M. sporium or M. trichosporium. Suitable media and growth conditions for culturing Methylosinus cells are similar if not identical to those described for Methylobacterium above.

[0036] In some embodiments, media for culturing methane catabolizing cells include a variety of copper concentrations. In some embodiments, inclusion of copper in the media results in inhibiting methane oxidation from soluble methane monooxygenases and eliminates non-specific, global oxidation of other alkanes to increase process yield for a given product. In another embodiment and in the presence of copper, methane oxidation occurs via a particulate methane monooxygenase, which exhibits less non-specific oxidation. In another embodiment of the invention, media formulations reduce copper concentrations such that methane oxidation proceeds through the soluble monooxygenase and increases overall process productivity and rates of methane catabolism. In some embodiments, non-specific oxidation of other alkanes is desired.

[0037] In many commercial embodiments, fermentations of any of the above cells will be run at volumes of 10L, 100L, 300L, 1000L, lOkL, 25kL, 50kL, lOOkL, 200kL, 400kL, 500kL, 1ML or larger scale. To add methane to such large culture volumes, the invention provides, in one embodiment, the use of a sparger to deliver a controlled mixture of gases including methane. In some embodiments the methane is mixed with air. In some embodiments, the methane is mixed with oxygen. In some embodiments the methane is mixed with oxygen and carbon dioxide. In some embodiments, the methane concentration in the fermenter vessel is 1% (v/v), 5% (v/v), 10% (v/v), 20% (v/v), 33% (v/v), 40% (v/v), 50% (v/v) and greater. In some embodiments oxygen concentration in the fermenter vessel is % (v/v), 5% (v/v), 10% (v/v), 20% (v/v), 33% (v/v), 40% (v/v), 50% (v/v) and greater. In some embodiments a mixture of methane and oxygen are sparged into the fermentation vessel. In some embodiments methane and air are sparged into the fermentation vessel. In some embodiments, a mixture of three gases is sparged into the fermentation vessel and is composed of methane, oxygen, and carbon dioxide. In some embodiments the mixture of gases has a 1: 1: 1 concentration in the fermentation vessel. In some embodiments, methane is delivered to the bottom of the fermentation vessel and at an effective pressure such that it is a liquid. As the fermentation process vessel size increases, the cost of gas mixtures that is sparged into the fermenter becomes increasingly expensive. In some embodiments the gases are recycled after passing through a fermentation vessel. In some embodiments, unused methane, oxygen, and/or carbon dioxide from the fermentation is recycled. In some embodiments, the gas is recycled with pressure swing adsorption. In other embodiments, the gas is recycled with selectively permeable gas membranes. Recycling gas can decrease the cost of producing chemicals from methanol or methane.

[0038] In all of the above embodiments, methanol can also be present in the media as a carbon source in addition to methane, and in such embodiments, the concentration of methanol in the media can range from, for example and without limitation, 0.1-2% (vol/vol). In all of the above embodiments, sugar or glycerol can also be present in the media as a carbon source in addition to methane (or methane and methanol), and in such embodiments, the concentration of sugar in the media can range from, for example and without limitation, 0.5-60% (wt/vol). In some embodiments the sugar includes glucose, sucrose, fructose, xylose, arabinose, or others. In shake flasks, these cells can be aerated via shaking, e.g. at 100-400 rpm. Modified cells for methanol catabolism

[0039] This invention provides recombinant host cells and methods for producing chemicals using them. These recombinant host cells have been genetically engineered to utilize methanol as a carbon source or to utilize it more efficiently than the wild type host cells from which they are derived. A wide variety of organisms are provided by and utilized in the methods of the invention; these include organisms that cannot naturally catabolize the single carbon substrate, methanol, but have been modified to do so. The invention can be practiced with cells that are prokaryotic, eukaryotic or otherwise naturally occurring. In some embodiments, cells provided by the invention naturally catabolize methanol but do so via suboptimal pathways and have been modified to catabolize methanol via more optimal pathways. In various embodiments, the invention provides cells from the genera Amycolatopsis, Aspergillus, Bacillus, Brevibacillus, Burkholderia Candida, Candidatus Clostridium, Corynebacterium, Escherichia, Hansenula, Hyphomicrobium, Issatchenkia, Kluyveromyces, Komagataella, Lactobacillus, Manheimia, Methanomonas, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylomirabilis, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Ogataea, Paracoccus, Pichia, Pseudomonas, Rhodobacter, Rhodococcus, Saccharomyces, Schizosaccharomyces, and Yarrowia. The cells provided by the invention are modified to possess one or more of the genes required for catabolizing methanol. Provided by the invention are four general pathways for methanol catabolism: 1) methanol oxidation to formaldehyde for energy production, 2) formaldehyde oxidation to formate for energy production, 3) formate oxidation to carbon dioxide for energy production, and 4) formaldehyde assimilation for biomass formation. Generally, host cells of the invention express at least 2 of 4 different biochemical pathways to enable methanol catabolism. In all of these cells there is a pathway to oxidize methanol to formaldehyde. Formaldehyde can be further catabolized via oxidation or assimilation. Formaldehyde oxidation leads to the production of NADH/NADPH for energy production. In some embodiments, formaldehyde is oxidized to formate. In these embodiments, it is further oxidized to carbon dioxide via a formate dehydrogenase and produces NADH for energy production. In some embodiments, formaldehyde assimilation directs formaldehyde into central metabolites used for biomass production. During formaldehyde assimilation, energy is produced. In some embodiments, the cells provided by the invention are modified to contain enzymes that convert methanol into formaldehyde and formaldehyde further into biomass or energy production for the cells. Also provided by the invention are genes (and their corresponding enzymes) and expression vectors containing them that can be transformed into cells and result in catabolism of methanol. Also provided by the invention are methods for production of a variety of useful products at commercial scale.

Methanol oxidation to formaldehyde

[0040] Provided by the invention are cells that have been modified to oxidize methanol to formaldehyde via one of two pathways. In one embodiment, cells contain a heterologous methanol dehydrogenase that oxidizes methanol to formaldehyde and produces NADH. In another embodiment, cells contain a heterologous methanol oxidase that oxidizes methanol to formaldehyde and hydrogen peroxide. In these embodiments, the cells contain a heterologous catalase that converts hydrogen peroxide into water and oxygen. In some embodiments, cells that naturally catabolize methanol with a methanol oxidase have been modified to contain a methanol dehydrogenase. Pathways and genes for oxidizing methanol into formaldehyde utilized in the recombinant host cells of the invention are described in more detail below.

Methanol oxidation to formaldehyde via methanol dehydrogenases

[0041] The present invention provides modified host cells that catabolize methanol and methods for culturing such cells. In one embodiment, the cells are modified to contain an NAD-dependent alcohol dehydrogenase (EC. 1.1.1.244) that converts methanol into formaldehyde. Example 7 and 9 below describes multiple genetic modifications of S. cerevisiae cells to express genes that enable it to catabolize methanol. One of the genes expressed is a methanol dehydrogenase (AN A42952) from Bacillus methanolica. Example 8 below describes Pichia kudriavzevii cells that have been modified to express genes that enable them to catabolize methanol. One of the genes expressed is a methanol dehydrogenase enzyme from B. methanolica. The invention provides a wide variety of host cells and genes suitable for modifying a cell to catabolize methanol in addition to those shown in the examples. Suitable host cells that can be modified as described include those listed previously. Example 7 below demonstrates the use of the promoter TEF1, which is appended to the 5' end of the methanol dehydrogenase (AN A42952) gene, to drive expression in S. cerevisiae. Example 8 below demonstrates the use of the TEF1 promoter, which is appended to the 5' end of the methanol dehydrogenase gene, to drive expression in P. kudriavzevii. A variety of other promoters can be used in accordance with the invention to drive expression of genes in S. cerevisiae and P. kudriavzevii. Suitable promoters include, without limitation, the PGK1 and TDH3 promoters.

[0042] Suitable NAD-dependent alcohol dehydrogenases that convert methanol into formaldehyde include EIJ78800.1, EIJ83424.1, Q2NGI3.1, P42327.1, EDZ12794.1, AAL00968.1, EGW40324.1, ABM09061.1, ABK36659.1, EER41381.1, EKE58762.1, EMP12409.1, EMT62998.1, WP_011773405.1, WP_005325714.1, WP_009616336.1, WP_010634498.1, YP_856044.1, YP_946457.1, EEQ93101.1, AGM43456.1, YP_008042803.1, EGD44968.1, EKD15160.1, Q2NGV2.1, EEN88363.1, WP_003942095.1, AAM98772.1, EJO15902.1, P31005.3, Q2NEN0.1, P36234.2, Q07511.2, 013437.1, Q2NFL8.1, YP_448353.1, YP_447794.1, YP_447905.1, YP_448089.1, YP_448327.1, YP_448340.1, EU80893.1, ADJ47941.1, AF079652.1, WP_013227992.1, YP_006552597.1, YP_003768343.1, AEK44841.1, YP_005534298.1, and A42952 among others.

[0043] Methods for detecting modified cells that catabolize methanol are provided by the invention. In Example 1, clonal isolates of the modified S. cerevisiae cells are isolated by and grown in accordance with the methods of the invention. After growth, cells are separated from fermentation broth. The fermentation broth can be analyzed for catabolism of methanol by high pressure liquid chromatography (HPLC), using an ion-exchange resin (eg. Aminex HPX-87H, BioRAD, Hercules, CA), a column temperature of 50°C, and a mobile phase of 5 mM sulfuric acid and flow rate of 0.7 ml/min. A standard of methanol (Sigma) is injected onto the HPLC and the corresponding peak is detected by RI detection at -17 min. Fermentation broth from modified S. cerevisiae cells containing the methanol dehydrogenase enzyme is analyzed and shown to catabolize methanol, whereas wildtype S. cerevisiae cells catabolize no methanol. When the suitable genes and enzymes for catabolism of methanol are present in the host cell, the cell is grown with methods and media in accordance with the invention, and methanol is catabolized. Further analysis of S. cerevisiae cells modified to catabolize methanol by measuring biomass formation via OD 600nm demonstrates greater biomass production compared to the wildtype control cells.

[0044] In one embodiment, the invention provides modified host cells that catabolize methanol via a nicotinoprotein methanol dehydrogenase (EC. 1.1.99.37). Provided by the invention are Saccharomyces cerevisiae cells that are modified to contain an alcohol dehydrogenase (AN WP_003897664.1) from Methylobacterium smegmatis and are able to catabolize methanol. The invention provides Pichia kudriavzevii cells that have been modified to contain an alcohol dehydrogenase enzyme from M. smegmatis and are able to catabolize methanol. However the invention provides a variety of host cells and enzymes as follows.

[0045] Suitable nicotinoprotein methanol dehydrogenase enzymes include

WP_006360532.1, WP_006368341.1, WP_006368508.1, WP_006553557.1 WP_006865796.1, WP_007240717.1, WP .007315923.1, WP_008355702.1 WP .009154061.1, WP_009678089.1, WP_010229649.1, WP_010242003.1 WP_010591966.1, WP .013809089.1, WP_014361145.1, WP_014671061.1 WP_014805764.1, WP_015746107.1, WP_006932857.1, YP_118435.1 YP_705992.1, YP_006570807.1, YP_006668667.1, WP_005173993.1 WP .010842989.1, CCW10366.1, YP_890461.1, YP_004495541.1, CAM01686.1 AFM20522.1, AEV73570.1, EME62476.1, EHR50676.1, EON32465.1 YP_001104611.1, YP_006442847.1, YP_005000785.1, ABM 16248.1, ACV77191.1 ACY21185.1, YP_956254.1, YP_003200180.1, YP_003273078.1, Q53062.2 EMP12409.1, EGD44968.1, ELQ86070.1, WP_003897664.1, EEN88363.1 WP_003942095.1 among others.

[0046] In one embodiment, the invention provides modified host cells that catabolize methanol via a cytochrome C_L-dependent methanol dehydrogenase (EC. 1.1.2.7). The invention provides Saccharomyces cerevisiae cells that have been modified to contain an alcohol dehydrogenase (AN YP_003070571.1 and YP_003070568.1) from Methylobacterium extorquens DM4 and are able to catabolize methanol. The invention provides below provides Pichia kudriavzevii cells that have been modified to contain an alcohol dehydrogenase enzyme from M. extorquens and are able to catabolize methanol. However the invention provides a variety of host cells and enzymes as follows.

[0047] Suitable cytochrome c-dependent methanol dehydrogenase enzymes include P14775.1, CAX26756.1, CCB63722.1, CCE25108.1, ACS42166.1, AAU92932.1, YP_113284.1, YP .002965443.1, YP .003070568.1, YP .004674298.1, YP_004918691.1, BAA23275.1, AAF43728.1, ABE77339.1, CBE67231.1, YP .003205076.1, ACB32199.1 among others.

[0048] Suitable sources for methanol dehydrogenases are Amycolatopsis methanolica, Mycobacterium gastri MB 19, Rhodococcus rhodochrous LMD 89.129, Rhodococcus erythropolis DSM 1069, and Mycobacterium sp. DSM 3803, MethylobaciUus flagellatus, Methylobacterium extorquens AMI, Methylobacterium organophilum, Methylomonas sp. J, Methylophaga thalassica, Methylophilus methylotrophus W3A1, Paracoccus denitrificans, and Paracoccus versutus.

Methanol oxidation to formaldehyde via methanol oxidases

[0049] The present invention provides modified host cells that catabolize methanol via an alcohol oxidase (EC. 1.1.3.13) that converts methanol into formaldehyde and hydrogen peroxide. In these embodiments, modified cells contain a heterologous catalase that converts hydrogen peroxide into water and oxygen. The invention provides S. cerevisiae cells that have been modified to contain an alcohol oxidase (AN AAA34321.1) and catalase enzyme (AN BAB69893.1) from Candida boidinii and are able to catabolize methanol. The invention provides Pichia kudriavzevii cells that have been modified to contain an alcohol oxidase and catalase enzyme from Candida boidinii and are able to catabolize methanol. However the invention provides a variety of host cells and enzymes as follows.

[0050] Suitable alcohol oxidase enzymes that convert methanol into formaldehyde include EIJ79022.1, EIJ79690.1, ELJ82004.1, ELJ82005.1, EIJ82370.1, ELJ82371.1, EIJ82924.1, ELJ84348.1, ACL41485.1, P81156.1, NP_566729.1, YP_002489574.1, AEE76762.1, EIJ78359.1, EIJ78393.1, ELJ79443.1, EIJ81633.1, ELJ78360.1, EIJ79444.1, EIJ81632.1, EIJ82043.1, EIJ77587.1, ELJ78292.1, ELJ78367.1, EIJ82955.1, 3Q9T_A, 3Q9T_B, 3Q9T_C, 4AAH_B, 4AAH_D, 4AAH_A, 4AAH_C, 2JBV_A, 2JBV_B, ELR66099.1, ELR66572.1, EJD40360.1, EJD41743.1, EJD50220.1, WP_007463330.1, WP_007464972.1, ABI14440.1, EJU01918.1, EOD81270.1, WP_002535908.1, AAA34321.1, AFO55203.1, Q00922.1, P04842.1, P04841.1 among others.

[0051] Suitable enzymes that convert hydrogen peroxide into water and oxygen include WP_008574782.1, WP_009492216.1, WP_009550547.1,

WP_013512707.1 WP_013964265.1 WP_014035677.1 WP_014813351.1 WP_015766802.1 WP_007917260.1 WP_007919761.1 WP_003599145.1 WP_006081102.1 WP_006085349.1 WP_011268380.1 WP_011336028.1 WP_011348926.1 WP_011548133.1 WP_011796211.1 WP_011982131.1 WP_012004841.1 WP_012313921.1 WP_012551601.1 WP_012768725.1 WP_015885989.1 WP_005989373.1 WP_005994599.1 WP_006086507.1 WP 006452102.1 WP 007681311.1 WP 007731213.1 WP_007805430.1 WP 007854113.1 WP_007895272.1, WP_007930032.1, WP_007937270.1,

WP_007950986.1 WP_007963762.1, WP_008010502.1, WP_008069984.1, WP_008084630.1 WP_008109957.1, WP_008115964.1, WP_009128618.1, WP_013551748.1 WP_013594560.1, WP_007909771.1, WP_006079727.1, WP 008576465.1 CAB56850.1, CAA38588.1 among others.

[0052] Suitable sources of the enzymes, methanol oxidase and catalase include Candida boidinii, Candida methanolovescens, Komagataella pastoris, Ogataea angusta, Pichia metahnolica, among others.

[0053] Once methanol is oxidized to formaldehyde as described above, formaldehyde is further oxidized into formate and then carbon dioxide or is assimilated into central metabolic precursors. The invention provides modified cells that direct formaldehyde to these separate metabolic pathways, as described below. Formaldehyde oxidation to formate

[0054] The formaldehyde oxidation pathway to formate includes 5 possible pathways including thiol-independent oxidation, glutathione-dependent oxidation, mycothiol-dependent oxidation, tetrahydrofolate-dependent oxidation, and H4MPT- dependent oxidation. In another embodiment and in a 6th pathway, RuMP cyclic oxidation, indirectly oxidizes formaldehyde after it is assimilated into a hexulose 6- phosphate intermediate. In some embodiments and to provide energy for cell growth on methanol and eliminate the highly toxic formaldehyde intermediate metabolite, host cells are engineered to oxidize formaldehyde to carbon dioxide and in turn produce NADH/NAD(P)H co-factors that can be used for production of ATP in central metabolic pathways or as reducing equivalents for biosynthetic pathways. Provided by the invention are recombinant cells that utilize one or more of 6 suitable oxidation routes for formaldehyde oxidation to formate.

Thiol-independent formaldehyde oxidation

[0055] Natural cells that oxidize formaldehyde via the thiol-independent pathway, contain a formaldehyde dehydrogenase that produces formate, NADH and 2 H+. Suitable cells containing this pathway include Pseudomonas putida. The present invention provides modified host cells that oxidize formaldehyde via a thiol- independent oxidation pathway containing a heterologous formaldehyde dehydrogenase enzyme (FDH). Example 7 below provides Saccharomyces cerevisiae cells that have been modified to contain multiple enzymes useful in catabolizing methanol, including a FDH (AN YP_008111392.1) from Pseudomonas putida. These recombinant cells are able to oxidize formaldehyde to formate. However the invention provides a variety of host cells and enzymes in addition to the cells exemplified.

[0056] Suitable thiol-independent formaldehyde dehydrogenases include

YP .947616.1, YP .990317.1, YP_001061825.1, YP_001079157.1, YP_001074774.1 YP_001100315.1, YP_001108655.1, YP .001351520.1, YP_001853994.1 YP_002234932.1, YP_002443394.1, YP_002784270.1, YP_002875140.1 YP .004753733.1, YP_005978120.1, YP_005984383.1, YP_006264311.1 YP_006277264.1, YP_006326331.1, YP_006556791.1, YP_007598452.1 YP_004720850.1, NP_521614.1, YP_105240.1, YP_262781.1, NP_254108.1 NP_354566.2, YP_001024816.1, YP_003898523.1, YP_004278825.1 YP_004571417.1, YP_005189801.1, YP_005369175.1, AGK49647.1 YP .007921814.1, CAH38002.1, AFY22196.1, YP_110566.1, P46154.3 YP_770646.1, YP_007032001.1, AAN65959.1, AFR28906.1, AGL87404.1 YP .006661944.1, YP_008002908.1, NP_742495.1, BAN52208.1, YP_008111392.1 AEV65401.1 among others.

RuMP cyclic oxidation of formaldehyde to carbon dioxide

[0057] Natural cells that oxidize formaldehyde via the ribulose 5- monophosphate (RuMP) cyclic oxidation pathway begin with two precursors, formaldehyde and RuMP. The RuMP and formaldehyde are first converted into hexulose 6-phosphate (H6P) with the enzyme hexulose 6-phosphate synthase. H6P is then converted into fructose 6-phosphate (F6P) with the enzyme 6-phospho-3- hexuloisomerase. F6P is then converted glucose 6-phosphate (G6P) via an isomerase. G6P is converted into 6-phospho D-glucono-l,5-lactone with the enzyme glucose 6- phosphate dehydrogenase which produces NADPH + H+. 6-phospho D-glucono-1,5- lactone is converted into 6-phospho D-gluconate, which is finally converted back into the starting substrate, RuMP, while producing NADH and carbon dioxide with the enzyme NAD+ dependent 6-phosphogluconate dehydrogenase. Suitable sources for RuMP cyclic oxidation of formaldehyde include Brevibacillus brevis SI, Methylobacillus flagellatus, Methylomonas aminofaciens 77a, and Mycobacterium gastri MN19. Suitable genes include hps, rmpA, rmpB, zwf, and gndA. The present invention provides modified host cells that oxidize formaldehyde via a RuMP cyclic oxidation pathway. Example 7 below provides Saccharomyces cerevisiae cells that are modified to contain an HPS (AN ABJ63600.1) and HI (AN WP_003552753.1) from Brevibacillus brevis SI and are able to oxidize formaldehyde into carbon dioxide. Example 8 below provides Pichia kudriavzevii cells that have been modified to contain an HPS (AN ABJ63600.1), an HI (AN WP_003552753.1), a 6-phosphogluconate dehydrogenase from Brevibacillus brevis SI and are able to catabolize methanol. While examples 7 and 8 below provide cells that have been modified to contain 2 of the 6 enzymatic activities required for a complete RuMP cyclic oxidation pathway, one skilled in the art will appreciate that some of the enzymes native to the RuMP cyclic oxidation pathway exist naturally in hosts that do not naturally catabolize methanol or formaldehyde. In many embodiments, cells modified to catabolize methanol and formaldehyde naturally contains enzymes required for RuMP cyclic oxidation including a phosphohexose isomerase, a glucose- 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and a 6-phosphogluconate dehydrogenase. In these embodiments only the enzymes not present in the modified cells must be expressed. However the invention provides a variety of host cells and enzymes as follows.

[0058] Suitable HPS enzymes include YP_115430.1, YP_176845.1

YP_185502.1, YP .301709.1, YP .301716.1, YP .302238.1, YP_544362.1 YP_809790.1, YP_001055461.1, YP_002429149.1, YP_002559440.1 YP_003485610.1, YP_003502426.1, YP_005707964.1, YP_005855367.1 YP_005858493.1, YP_006194711.1, YP_006470384.1, YP_006709388.1 YP_006700692.1, YP_006042078.1, YP_007969207.1, YP_187812.1 YP_003872970.1, YP_004479862.1, YP_004732614.1, YP_005398998.1 YP_005399644.1, EAR68750.1, EDJ89466.1, EAV47244.1, EIM05259.1 ABN07165.1, EKC82856.1, ELK44699.1, CCE75044.1, EON80240.1, EON82083.1 EON85721.1, WP_015489823.1, YP_007685384.1, NP_461682.1, YP_152642.1 YP_153252.1, ABN07618.1, AAM30911.1, NP_371094.1, YP_833183.1 NP_577949.1, YP_040023.1 among others.

[0059] Suitable HI enzymes include WP_006215287.1, WP_009166436.1

YP_416017.1, YP_709135.1, YP_006938326.1, YP_006938819.1, YP_006544334.1 YP_006544816.1, YP_006628530.1, YP_006709389.1, YP_007394192.1 YP_007491001.1, YP_007685385.1, YP_007867634.1, YP_008050389.1 EPC12662.1, EPC13144.1, EPC13509.1, EPC17633.1, EPC18642.1, EPC20316.1 EPC20344.1, EPC23618.1, EPC25770.1, EPC35016.1, EPC35045.1, EPC36952.1 EPC38837.1, WP_016383230.1, WP_016384253.1, CCW20899.1, WP_004607138.1 WP_009535380.1, AGP27571.1, YP_003852311.1, YP_005741297.1 YP .008167735.1, AGP67227.1, AGP71929.1, WP .020613988.1, EHP85623.1, AFJ02830.1, AFI85301.1, AEF96627.1, YP_004370221.1, ADK83279.1, ADN34938.1, AEB09040.1, YP_003805873.1, YP_003893376.1.

Glutathione dependent formaldehyde oxidation

[0060] Natural cells that oxidize formaldehyde into formate via the glutathione-dependent pathway utilize formaldehyde and glutathione as precursors that are first converted into S-hydroxymethylglutathione via a S-hydroxglutathione synthase. S-hydroxyglutathione is then converted into S-formylglutathione via the enzyme glutathione-dependent formaldehyde dehydrogenase and produces H+ and NAD(P)H. S-formylglutathione is converted into formate, releasing H+ and glutathione via the enzyme S-formylglutathione hydrolase. The present invention provides recombinant host cells that oxidize formaldehyde via a glutathione dependent oxidation pathway containing at a minimum the enzymes formaldehyde dehydrogenase (FDH) and S-formylglutathione hydrolase (FGH). The invention provides Saccharomyces cerevisiae cells that have been modified to contain an FDH (AN AAC44551.1) and FGH (AN AAC44554.1) from Paracoccus denitrificans and are able to catabolize formaldehyde into formate. The invention provides Pichia kudriavzevii cells that have been modified to contain FDH (AN AAC44551.1) and FGH (AN AAC44554.1) from Paracoccus denitrificans and are able to catabolize formaldehyde into formate. However the invention provides a variety of host cells and enzymes as follows.

[0061] Suitable FDH enzymes include ELZ96420.1, EMA06490.1,

EMA10473.1, EMA10775.1, EMA27812.1, EMA37918.1, EMA44468.1, EMA49083.1, EMA52447.1, EMA53947.1, AFY22196.1, YP_003736860.1, YP_007032001.1, YP_007227278.1, BAA04743.1, BAC16635.1, AEH35491.1, YP_004595370.1, EHB85873.1, YP_001265707.1, YP_003204070.1, YP_003394714.1, YP_005060061.1, YP .002487096.1, YP_003396210.1, YP_001583515.1, YP_004183484.1, YP_004216923.1, AAF54571.1, NP_524310.1, YP_001363686.1, YP_001666600.1, YP_001751724.1, YP_001859509.1, YP_001888795.1, YP_002489735.1, YP_003084575.1, YP_003389378.1, YP_003607370.1, YP_004333498.1, YP_004476269.1, NP_864907.1, AEW04442.1, YP_005256114.1, ACY58243.1, ACY62296.1, ADE64279.1, AEL74298.1, AAM86220.1, CAC85637.1 among others. [0062] Suitable FGH enzymes include YP_004783602.1, YP_003908080.1,

YP_004088867.1, YP_004154193.1, YP_004212160.1, YP_004229374.1, YP_004314447.1, YP_004489577.1, YP_004499969.1, YP_004504921.1, YP_004548550.1, YP_004556575.1, YP_005277288.1, YP_006024383.1, NP_181684.1, ADN76261.1, ADN76469.1, AFY33211.1, AFY38220.1, AFY41281.1, AFZ08250.1, AFZ30585.1, AFZ56972.1, AFZ45429.1, YP_007048431.1, YP_007066045.1, YP_007071054.1, YP_007116666.1, YP_007169643.1, YP_007127745.1, YP_007155882.1, YP_003913335.1, YP_003913543.1, AEC09995.1, ABV92222.1, AEH90380.1, AFZ13038.1, YP_001531823.1, YP_007142548.1, YP_004614474.1, AFY67916.1, AFY89519.1, AFZ00218.1, AFZ35486.1, YP_007093388.1, YP_007110968.1, YP_007132452.1, YP_007136190.1, WP_007868628.1, WP_008122233.1 among others.

[0063] Suitable S-(hydroxymethyl)- glutathione synthases include

YP_002157578.1, D1ZK87.1, Q51669.3, Q0CMY6.1, Q0V314.1, B8NDP1.1, A2QBH6.1, P0CL54.1, E3QRY8.1, C8VDQ3.1, E3S405.1, C9S7Z6.1, B6QNA1.1, B2ACV0.2, C7Z147.2, Q89GX9.1, Q92WX6.1, Q98LU4.2, A5EP16.1, A4YZ37.1, Q8P5F3.1, Q8PPF1.1, Q4UYL9.1, Q07HI5.1, Q21D57.1, Q3BXJ3.1, A1AXY8.1, B0RNW7.1, B2W9N9.1, B6HE56.1 among others.

[0064] Suitable formate dehydrogenases include WP_012072999.1,

WP. _.012320147 1, WP. .012452393 1, WP. .012856603 1, WP. .012857829 1,

WP. .007737698 1, WP. _.008405584 1, WP. .007944946 1, WP. _.007951404 1,

WP. _.007993242 1, WP. _.008146874 1, WP. _.011624442 1, WP. _.011627522 1,

WP. _.012090497 1, WP. .011635549 1, WP. .007832886 1, WP. .007861298 1,

WP. .007902320 1, WP. .007941169 1, WP. .007967016 1, WP. _.007970082 1,

WP. _.007983787 1, WP. .008013281 1, WP. .008018690 1, WP. .008050101 1,

WP. .008056679 1, WP. .008072169 1, WP. .008082740 1, WP. .008096735 1,

WP. _.007914857 1, WP. _.011903237 1, WP. .012090500 1, WP. .009549340 1,

WP. .012418780 1, WP. _.012550127 1, WP. .002683313 1, WP. .013886220 1,

WP. .013908415 1, WP. .011629030 1, WP. .012527783 1, WP. .012592300 1,

WP. _.012764954 1, WP. _.012770347 1, WP. _.015854202 1, WP. .007176168 1,

WP. .014238293 1, WP. _.012017144 1, WP. .012493218 1, WP. _.012511992 1,

WP_012769165.1 among others.

[0065] Suitable sources for these pathway enzymes include Methylobacterium extorquens AMI, Methylosinus trichosporium, Methylosinus trichosporium OB3b, Moraxella, Mycobacterium vaccae, Escherichia coli K-12 substr. MG1655, Homo sapiens, Paracoccus denitrificans, Paracoccus versutus, Rhodobacter sphaeroides and Saccharomyces cerevisiae S288c. Suitable genes are gfa, frmA, SFA1, flhA, adhl, frmB, yeiG, fghA, fdhlA, fdhlB, andfdh.

Mycothiol-dependent formaldehyde oxidation

[0066] Natural cells that catabolize formaldehyde into formate utilize formaldehyde and mycothiol as precursors that are converted into S- hydroxymethylmycothiol, which is converted into S-formylmycothiol via the enzyme mycothyiol-dependent formaldehyde dehydrogenase and produces H+ and NADH. S- formylmycothiol is converted into formate, releasing mycothiol and H+. The present invention provides modified host cells that oxidize formaldehyde via a mycothiol- dependent oxidation pathway containing at a minimum the enzymes formaldehyde dehydrogenase (FDH). Example 7 below provides Saccharomyces cerevisiae cells that have been modified to contain an FDH (AN AAC44551.1) from Mycobacterium tuberculosis and are able to catabolize formaldehyde to formate. However the invention provides a variety of host cells and enzymes as follows.

[0067] Suitable mycothiol dependent formaldehyde dehydrogenases include

ACY98017.1, ADB31528.1, ADB75512.1, ADB77300.1, ADW06553.1 ADG96617.1, ADG98911.1, ADL46786.1, ADP80806.1, ADP81795.1 ADU10609.1, AEH09234.1, AEM85230.1, AEM88079.1, AEN09039.1 EGE45461.1, ADD41110.1, YP_002489517.1, YP_003101495.1, YP_003102324.1 YP_003200733.1, YP_003300055.1, YP_003380327.1, YP_003409883.1

YP_003411671.1 YP_003510203.1, YP_003657448.1, YP_003659742.1 YP_003836362.1 YP_003114870.1, YP_003271336.1, YP_004016676.1 YP_004017665.1 YP_004084760.1, YP_004583155.1, YP_004815510.1 YP 004818359.1 YP_004926070.1, EHB53720.1, EHI82325.1, ADB75524.1 ADG77049.1, AEA22904.1, AEM84190.1, YP_003409895.1, YP_003645388.1 YP_004330757.1, YP_004814470.1, ADH67817.1, YP_003680323.1 among others. Tetrahydrofolate dependent formaldehyde oxidation to formate

[0068] In some embodiments, formaldehyde is oxidized to formate in a tetrahydrofolate-dependent pathway. Formaldehyde and tetrahydrofolate spontaneously react to form 5,10-methylenetetrahydrofolate. 5,10- methylenetetrahydrofolate is then converted into 5,10-methenetetrahydrofolate with the enzyme NADP-dependent methylenetetrahydrofolate dehydrogenase and produces NADPH. 5,10-methenetetrahydrofolate is converted into 10-formyl-tetrahydrofolate with the enzyme methylenetetrahydrofolate cyclohydrolase. 10-formyl- tetrahydrofolate is finally converted into formate releasing tetrahydrofolate and producing ATP with the enzyme formate tetrahydrofolate ligase. Formate can proceed through oxidation as previously described. Suitable sources for this pathway include Hyphomicrobium zavarzinii ZV580, Methylobacterium extorquens AMI. Suitable genes include fo ID, mtdA, fchA, ftfL.

Η4ΜΡΊ "-dependent formaldehyde oxidation to formate

[0069] In another embodiment, formaldehyde oxidation proceeds through a tetrahydromethanopterin dependent pathway similar to the tetrahydrofolate pathway. Suitable sources for this pathway include Burkholderia xenovorans LB400, Hyphomicrobium zavarzinii ZV580, Methylobacterium extorquens AMI. Formate oxidation to carbon dioxide

[0070] In the embodiments described above where formaldehyde is catabolized to formate, the invention provides a pathway for further oxidation of formate into carbon dioxide. In some embodiments, formate is further oxidized to carbon dioxide and produces NADH and 2H+ with the enzyme formate dehydrogenase. Suitable sources for this pathway include Pseudomonas putida. A suitable gene is fdhA.

[0071] The above embodiments provide cells that are useful for generating reducing equivalents and energy for a cell in the form of NADH or NAD(P)H which can be used to increase biomass formation or biosynthesis of a valuable chemical that utilizes NADH or NAD(P)H. However, it is also useful to assimilate formaldehyde into central metabolic pathways for biomass production or biosynthesis of valuable chemicals as described in the following.

Formaldehyde assimilation

[0072] Provided are modified cells that catabolize formaldehyde into biomass and central metabolic pathway intermediates. Provided by invention are three metabolic pathways that assimilate formaldehyde into central metabolic and include the RuMP cycle, serine cycle, and DHAP cycle.

RuMP -cycle for formaldehyde catabolism.

[0073] Natural cells that catabolize formaldehyde into central metabolic pathways using the RuMP-cycle begin with the precursors formaldehyde and RuMP. A portion of this pathway is described above, where formaldehyde is assimilated into a six carbon intermediate that is oxidized and produces NADH, NAD(P)H and carbon dioxide. However, this pathway is also used for assimilation of formaldehyde. For assimilation, D-fructose 6-phosphate produced via the oxidation cycle is directed toward the pentose phosphate pathway that is common to many organisms. In this pathway, formaldehyde reacts with D-ribulose 5-phosphate (RuMP) to form hexulose 6-phosphate (H6P) with the enzyme hexulose 6-phosphate synthase. H6P is converted into D-fructose 6-phosphate (F6P) with the enzyme 6-phospho-3-hexuloisomerase. F6P is phosphorylated to fructose 1,6-bisphosphate and then cleaved into D- glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP) with standard central metabolic enzymes. DHAP is assimilated into central metabolism. Glyceraldehyde 3-phosphate and F6P are converted to D-erythrose 4-phosphate (E4P), releasing D-xylulose 5-phosphate by a transketolase enzyme. E4P and a third molecule of F6P are then converted into septulose 7-phosphate (S7P) and glyceraldehyde 3-phosphate (GAP) by a transaldolase. S7P and GAP are then converted into xylulose 5-phosphate and ribose 5-phosphate. Both xylulose 5- phosphate and ribose 5-phosphate are individually converted back to RuMP by a phosphate 3-epimerase and ribose 5-phosphate isomerase respectively. Suitable sources for this pathway include Bacillus subtilis, Brevibacillus brevis SI, Methylobacillus flagellatus, Methylomonas aminofaciens 77a, Mycobacterium gastri MN19. Suitable genes include hps, rmpA, rmpB. The invention here, provides recombinant host cells that assimilate formaldehyde into metabolic precursors via the RuMP-cycle pathway containing at a minimum the enzyme hexulose phosphate synthase (HPS), and hexuloisomerase (HI). Example 7 below provides Saccharomyces cerevisiae cells that have been modified to contain a HPS (AN WP_015473291.1) and HI (AN WP_003552753.1) from Lactobacillus brevis and are able to catabolize methanol and assimilate formaldehyde. Example 8 below provides P. kudriavzevii cells that have been modified to contain a HPS and HI from Lactobacillus brevis and are able to catabolize methanol and formaldehyde. However the invention provides a variety of host cells and enzymes as follows.

[0074] Suitable HPS enzymes include EKT32924.1, EKT38133.1,

EKT39703.1, CBG25728.1, AGK10387.1, WP_001683903.1, WP_008949169.1, WP_009004790.1, YP_007903878.1, NP_461681.1, YP_002046712.1, YP_005233750.1, YP_005238837.1, YP_005182631.1, YP_005248502.1, YP_005253248.1, YP_005243737.1, YP_005398127.1, BAG80018.1, BAI57622.1, CAQ65545.1, CAL59335.1, AE078189.1, CBH40934.1, AAT82636.1, ABG72382.1, ADK17704.1, ABR78363.1, ADM21913.1, ADQ90652.1, ADX18539.1, CCC17128.1, AGM22227.1, ΥΡ_001986403.1, YP_003515888.1, ΥΡ_003787554.1, ΥΡ_003856451.1, ΥΡ_005888551.1, ΥΡ_008025871.1, ΥΡ_001336593.1, ΥΡ_005243738.1, ΥΡ_003352072.1, AAL21641.1, ACY89742.1, AEF08615.1, ΝΡ_461682.1, ΥΡ_005238838.1, ΥΡ_005253249.1, AAL23206.1, ΑΑΧ68166.1 among others.

[0075] Suitable HI enzymes include WP_006215287.1, WP_009166436.1,

YP_416017.1, YP_709135.1, YP_006938326.1, YP_006938819.1, YP_006544334.1, YP_006544816.1, YP_006628530.1, YP_006709389.1, YP_007394192.1, YP_007491001.1, YP_007685385.1, YP_007867634.1, YP_008050389.1, EPC12662.1, EPC13144.1, EPC13509.1, EPC17633.1, EPC18642.1, EPC20316.1, EPC20344.1, EPC23618.1, EPC25770.1, EPC35016.1, EPC35045.1, EPC36952.1, EPC38837.1, WP_016383230.1, WP_016384253.1, CCW20899.1, WP_004607138.1, WP_009535380.1, AGP27571.1, YP_003852311.1, YP_005741297.1, YP_008167735.1, AGP67227.1, AGP71929.1, WP_020613988.1, EHP85623.1, AFJ02830.1, AFI85301.1, AEF96627.1, YP_004370221.1, ADK83279.1, ADN34938.1, AEB09040.1, YP_003805873.1, YP_003893376.1 among others.

Serine-cycle for formaldehyde catabolism

[0076] Natural cells that catabolize formaldehyde into central metabolic pathways using the serine-cycle begin with the precursors 5,10- methylenetetrahydrofolate and glycine, which are converted into serine via the enzyme serine hydroxymethylenetransferase. 5,10-methylenetetrahydrofolate is spontaneously formed from formaldehyde and tetrahydrofolate, a common metabolite. Serine is converted into hydroxypyruvate, producing glycerate and releasing glycine with the enzyme serine- glyoxylate aminotransferase. Hydroxypyruvate is reduced to glycerate with the cofactor NAD(P)H and the enzyme hydroxypyruvate reductase. Glycerate is converted into 2-phospho-D-glycerate with ATP and the enzyme glycerate 2-kinase. 2-phospho-D-glycerate is converted into either 3-phospho-D- glycerate, which is a central metabolite and used in biosynthetic pathways, by the enzyme phosphoglycerate mutase, or phosphoenolpyruvate (PEP) by an enolase. PEP is carboxylated with a PEP carboxylase to produce oxaloacetate. Oxaloacetate is reduced into S-malate with the enzyme malate dehydrogenase and releases NAD+. S- malate is converted into a CoA-derivative, S-malyl-CoA, with the enzyme malate thiokinase. S-malyl-CoA is then converted into acetyl-CoA, releaseing glyoxylate with the enzyme malyl-CoA lyase. Acetyl-CoA can then be converted back into glyoxylate to continue the cycle or be used as a central metabolic precursor for other biosynthetic pathways. Suitable sources for this pathway include Hyphomicrobium methylovorum GM2, Hyphomicrobium zavarzinii ZV580, Methylobacter whittenburyi, Methylobacterium extorquens AMI, Methylobacterium organophilum, Methylocystis echinoides, Methylocystis minimus, Methylocystis parvus, Methylocystis pyriformis, Methylosinus sporium, Methylosinus trichosporium. Suitable genes include glyA, sgaA, hprA, gckA, ppcA, mdh, mtkA, mtkB, mclA, and sgaA.

[0077] The present invention provides modified host cells that assimilate formaldehyde into metabolic precursors via the serine-cycle pathway containing at a minimum the enzyme serine hydroxymethyltransferase (SHMT) and serine glyoxylate aminotransferase (SGAT). Example 9 below provides Saccharomyces cerevisiae cells that have been modified to contain a SHMT (AN AAA64456.1) and SGAT (AN WP_003597639.1) from M. extorquens and is able to catabolize formaldehyde. However the invention provides a variety of host cells and enzymes as follows.

[0078] Suitable SHMT enzymes include NP 721474.1, NP_629503.1

YP_003859985.1, YP_003892463.1, YP_003912112.1, YP_003966576.1 YP_004004363.1, YP_004041630.1, YP .004049872.1, YP_004058129.1 YP_004060026.1, YP_004100792.1, YP_004162026.1, YP_004168653.1 YP_004176470.1, YP_004179261.1, YP_004194342.1, YP_004270502.1 YP_004411685.1, CAA20173.1, CAH58416.1, EHQ06730.1, EHQ06869.1 ADY57546.1, AEK23220.1, AFY34171.1, AFY39569.1, AFY42646.1, AFZ02160.1 AFZ11135.1, AFZ31432.1, AFZ55234.1, ELS33741.1, YP_007049796.1 YP_007067005.1, YP_007072403.1, YP_007140645.1, YP_007128592.1 YP_004740327.1, YP_004267547.1, YP_007138132.1, YP_007163278.1 CAN02604.1, CAL34552.1, WP_007920920.1, WP_008070862.1, WP_013565000.1 WP_013683034.1, YP_641292.1, and AAA33687.1.

[0079] Suitable SGAT enzymes include YP_002827132.1, YP_002582246.1

YP_003979610.1, YP_004358513.1, YP_004424802.1, YP_006902571.1 YP_008040311.1, CAQ71386.1, EDZ41023.1, EEB84781.1, WP_012355607.1 EOR24424.1, WP_011280657.1, WP_014149643.1, WP_014743098.1 YP_002007443.1, WP_010867962.1, NP_126522.1, EAU39726.1, ACG30854.1 EAP77421.1, EAQ24413.1, EHK64205.1, XP_002279236.1, EHK74228.1, EIE24183.1, EDZ44855.1, EDZ61119.1, EEE38769.1, EJO31580.1, EMD98318.1, WP_012251181.1, WP_014329402.1, BAB20811.1, CAE39480.1, XP_001702106.1, XP_001702107.1, ED097195.1, ED097196.1, NP_886332.1, ABV95058.1, YP_001534659.1, CCD91795.1, CCD86741.1, CCE00291.1, CCE12095.1, P84188.1, P84187.1, Q56YA5.2, NP_001148339.1 among others.

Dihydroxyacetone (DHAP) cycle for formaldehyde catabolism.

[0080] Natural cells that catabolize formaldehyde into central metabolic pathways using the DHAP cycle begin with the precursors formaldehyde and xylulose-5-phosphate, which are converted into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate with the enzyme dihydroxyacetone synthase. DHA is then phosphorylated to form dihydroxyacetone phosphate (DHAP) by a DHA kinase. DHAP and glyceraldehyde 3-phoshpate are converted into F16P. Most of the enzymes here are shared with the pentose phosphate pathway common to many organisms. Suitable sources for the dihydroxyacetone pathway include Candida boidinii, Candida methanolovescens, Candida methylica, Komagataella pastoris, Ogataea angusta, Pichia methanolica. Suitable genes include DAS1.

[0081] The present invention provides modified host cells that assimilate formaldehyde into metabolic precursors via the DHAP cycle pathway containing at a minimum the enzyme dihydroxyacetone synthase (DHAS). The invention provides Saccharomyces cerevisiae cells that have been modified to contain a DHAS (AN AAC83349.1) from Candida boidinii and are able to catabolize formaldehyde. However the invention provides a variety of host cells and enzymes as follows.

[0082] Suitable DHAS enzymes include 2QJH_Q, 2QJH_R, 2QJH_S,

2QJH_T, XP_001387002.2, EAZ62979.2, Q57843.1, CCF44904.1, ELA23390.1, ELA36946.1, ELA37692.1, EMT74378.1, ENH62895.1, EPE08051.1, EPE08463.1, ABV48736.1, XP_001217442.1, XP_001217703.1, XP_001941089.1, XP_003009479.1, EFW95760.1, XP_001400975.1, XP_001818857.1, XP_001820904.1, EAU30218.1, EAU30988.1, EDU43808.1, EEY15053.1, EGY14084.1, EGV61733.1, EJT78439.1, CCH43898.1, AFO55207.1, EMT65859.1, EMT74376.1, ENH62897.1, ENH75039.1, ENH86106.1, ENH88826.1, CBH48269.1, YP_004006953.1, XP_003720380.1, EHA48013.1, ELQ45089.1, ELQ70320.1, AAC83349.1, AAG12171.2, BAJ11494.1, P06834.3, 093884.3 among others. Modified cells for catabolizins methane and producing methanol

[0083] In various embodiments, the invention provides cells that are modified to catabolize methane, including prokaryotic and eukaryotic host cells. Suitable cells include cells from the following genera: Amycolatopsis, Bacillus, Brevibacillus, Burkholderia, Candida, Candidatus Methylomirabilis, Corynebacterium, Escherichia, Hansenula, Hyphomicrobium, Issatchenkia, Komagataella, Methanomonas, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Ogataea, Paracoccus, Pichia, Pseudomonas, Rhodobacter, Rhodococcus, Saccharomyces, Schizosaccharomyces, and Yarrowia. The invention provides methods for culturing these cells in a medium that comprises minerals, vitamins, a nitrogen source, methane, and optionally another carbon source.

[0084] This invention provides recombinant host cells and methods for producing chemicals using them. These recombinant host cells have been genetically engineered to utilize methane as a carbon source or to utilize it more efficiently than the wild type host cells from which they are derived. A wide variety of organisms are provided by and utilized in the methods of the invention; these include organisms that cannot naturally catabolize the single carbon substrate, methane, but have been modified to do so. The invention can be practiced with cells that are prokaryotic or eukaryotic. In some embodiments, cells have been genetically engineered to catabolize methane to methanol In some embodiments, cells provided by the invention naturally catabolize methane but do so via suboptimal pathways and have been modified to catabolize methane via more optimal pathways. In some embodiments the cells naturally catabolize methanol. In some embodiments the cells have been genetically engineered to catabolize methanol as described here. In various embodiments, the invention provides cells from the genera Amycolatopsis, Aspergillus, Bacillus, Brevibacillus, Burkholderia Candida, Candidatus Clostridium, Corynebacterium, Escherichia, Hansenula, Issatchenkia, Hyphomicrobium, Kluyveromyces, Komagataella, Lactobacillus, Manheimia, Methanomonas, Methylobacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylomirabilis, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Ogataea, Paracoccus, Pichia, Pseudomonas, Rhodobacter, Rhodococcus, Saccharomyces, Schizosaccharomyces, and Yarrowia. The cells provided by the invention are modified to possess one or more of the genes required for catabolizing methane. Provided by the invention are two classes of methane monooxygenase (MMO) enzymes that oxidize methane into methanol: 1) soluble MMOs and 2) insoluble MMOs. Host cells provided by the invention express at least one of the MMOs. In some embodiments the host cells express both MMOs. In all embodiments the cells contain or have been genetically engineered to contain some of the enzymes and metabolic pathways for catabolizing methanol into formaldehyde, central metabolic precursors / biomass, and carbon dioxide for energy. The cells provided by the invention are useful in producing valuable compounds from methane and/or methanol. While the methods of the invention can be used to produce any of a variety of useful compounds, such as organic acid, fatty acid, chemical, fuels, protein, and enzyme products, malonic acid is noteworthy as an exceptionally valuable product that can be produced in accordance with the invention. Also provided by the invention are methods for production of the products at commercial scale.

Soluble methane monooxygenases for methane oxidation to methanol

[0085] The present invention provides modified host cells that catabolize methane into methanol and methods for culturing such cells. Provided by the invention are host cells modified to contain a soluble methane monooxygenase enzyme. Suitable methane monooxygenases are encoded by 5 separate genes. Example 12 below provides S. cerevisiae that is modified to contain a soluble methane monooxygenase from M. trichosporium in a background strain that is engineered to catabolize methanol (Strain LSM001). This new strain containing the soluble MMO is able to catabolize methane. Example 13 below provides P. kudriavzevii that is modified to contain a soluble methane monooxygenase from M. trichosporium in a background strain that is engineered to catabolize methanol (Strain LPKM001). This new strain containing the soluble MMO is able to catabolize methane. The invention provides a wide variety of host cells and genes suitable for modifying a cell to catabolize methane in addition to those shown in the examples. Suitable host cells for modifying include those listed previously. Example 10 below demonstrates the use of the DNA expression construct for methane catabolism comprised of the promoter TEF1, a methane monooxygenase gene mmoX (AN CAA39068.2), a PGI terminator, a TEF2 promoter, mmoY (AN CAA39069.1), a TPIl terminator, a FBAl promoter mmoZ (AN CAA39070.1), a TDH3 terminator, a TDH3 promoter, mmoB (AN CAA39071.1), a FBAl terminator, an PDC1 promoter, mmoC (AN CAB45257.1), and finally a THD2 terminator to drive expression in S. cerevisiae. Example 13 below demonstrates the use of a similar construct, with promoter sequences modified to match promoter homologues of P. kudriavzevii and drive expression of methane monooxygenase in P. kudriavzevii. A variety of other promoters can be used in accordance with the invention to drive expression of genes in S. cerevisiae and P. kudriavzevii and include: TEF1, PGK1, TDH3, among others.

[0086] Suitable MmoX subunits of a methane monooxygenase include

AAC45289.1, CAJ26291.1, AAB62392.3, BAA84757.1, BAA84757.1, ABD46892.1, ABD46898.1, P27353.4, P22869.3, ABG56535.1, ABU89756.1, ABU89757.1, ABU89758.1, BAJ07233.1, AAU92736.1, BAM37167.1, YP_113659.1, BAA84751.1, BAA84757.1, CAD30344.1, CAA39068.2, CAJ26291.1, AAZ81974.1, AAV52905.1, AAV52906.1, AAC45289.1, AAZ81968.1, AAY83388.1, BAJ17645.1, BAE86875.1, AAB62392.3, AAZ06158.1, AAZ06159.1, AAZ06160.1, AAZ06161.1, AAZ06163.1, AAZ06164.1, AAZ06198.1, AAZ06199.1, AAZ06200.1, AAZ06201.1, AAF01268.1, and ABD13903.1.

[0087] Suitable MmoY subunits of a methane monooxygenase include

AAC45290.1, CAJ26292.1, AAB62393.2, BAA84758.1, BAA84758.1, Q53562.1, P22867.1, P27356.3, P18797.2, Q53563.1, P22868.2, P27354.3, P18798.4, AAU92727.1, YP_113660.1, BAA84752.1, BAA84758.1, CAA39069.1, CAJ26292.1, AAZ81975.1, AAC45290.1, AAZ81969.1, BAJ17646.1, BAE86876.1, AAB62393.2, AAF01269.1, and ABD46893.1.

[0088] Suitable MmoZ subunits of a methane monooxygenase include

AAC45291.1, CAJ26293.1, AAF04158.2, BAA84759.1, BAA84759.1, Q53562.1, P22867.1, P27356.3, P18797.2, Q53563.1, P22868.2, AAB21391.1, P27355.3, P11987.4, AAU92724.1, YP_113663.1, BAA84754.1, BAA84760.1, CAA39071.1, CAJ26294.1, AAZ81977.1, AAC45292.1, AAZ81971.1, BAJ17648.1, BAE86878.1, AAF04157.2, AAF01271.1, and ABD46895.1.

[0089] Suitable MmoB subunits of a methane monooxygenase include

AAC45292.1, CAJ26294.1, AAF04157.2, BAA84760.1, BAA84760.1, 1XMG_B, 1XMG_C, 1XMG_D, 1XMH_A, 1XMH_B, 1XMH_C, 1XMH_D, 1XMF_A, 1XMF_B, 1XMF_C, 1XMF_D, 2MOB_A, 1XMG_E, 1XMG_F, 1XMH_E, 1XMH_F, 1XMF_E, 1XMF_F, 4GAM_B, 4GAM_A, 4GAM_G, 4GAM_F, 4GAM_L, 4GAM_K, 4GAM_Q, 4GAM_P, 4GAM_C, 4GAM_H, 4GAM_M, 4GAM_R, 4GAM_D, 4GAM_I, 4GAM_N, 4GAM_S, P27356.3, PI 8797.2, AAU92726.1, YP_113661.1, CAA39070.1, CAJ26293.1, AAZ81976.1, AAC45291.1, AAZ81970.1, BAJ17647.1, AAF04158.2, BAE86877.1, BAA84753.1, BAA84759.1, AAFO 1270.1, and ABD46894.1.

[0090] Suitable MmoC subunits of a methane monooxygenase include

AAC45294.1, CAJ26296.1, AAB62391.2, BAA84762.1, BAA84762.1, AAB21393.1, Q53563.1, P22868.2, AEI77119.1, YP_004685600.1, BAA84756.1, AAC45294.1, AAZ81973.1, AAU92722.1, YP_113665.1, BAA84762.1, CAB45257.1, CAJ26296.1, AAZ81979.1, BAJ17650.1, BAE86880.1, AAB62391.2, CBI06592.1, CBI05225.1, AAF01273.1, and ABD46897.1.

[0091] In some embodiments and provided by the invention is co-expression of a methane monooxygenase chaperone. In one embodiment, S. cerevisiae host cells modified to catabolize methane are also modified to express a monooxygenase chaperone gene, mmoG (AN CAD61956.1). In another embodiment, P. kudriavezvii host cells modified to catabolize methane are also modified to express a monooxygenase chaperone gene, mmoG (AN CAD61956.1).

[0092] Suitable enzymes for the MMO chaperone include CAA39068.2,

CAJ26299.1, BAJ17652.1, Q7WZ32.1, BAE86883.1, CAD61956.1, and AAP80770.1.

[0093] Methods for detecting modified cells that catabolize methane are provided by the invention. Briefly, in Example 12 clonal isolates of the modified S. cerevisiae cells are isolated by and grown in accordance with the methods of the invention. After growth, cells are separated from fermentation broth. The fermentation broth is analyzed for catabolism of methane by high pressure liquid chromatography (HPLC), using an ion-exchange resin (e.g. Aminex HPX-87H, BioRAD, Hercules, CA), using a column temperature of 50C, a mobile phase of 5 mM sulfuric acid and flow rate of 0.7 ml/min. A standard of methanol (Sigma) is injected onto the HPLC and the corresponding peak is detected by RI detection at -17 min. Fermentation broth from modified S. cerevisiae cells containing the methane monooxygenase enzyme is analyzed and shown to catabolize methane and contain methanol, whereas wildtype S. cerevisiae cells catabolize no methane and no methanol is detected. When the suitable genes and enzymes for catabolism of methane and methanol are present in the host cell, the cell is grown with methods and media in accordance with the invention, and methane is catabolized. Further analysis of S. cerevisiae cells modified to catabolize methane by measuring biomass formation via OD 600nm demonstrates greater biomass production compared to the wildtype control cells. In some instances, methane catabolism may not be detectable via HPLC; in such instances, formation of biomass when methane is present in a fermentation as the sole-carbon source is a suitable method for detecting methane catabolism.

Particulate methane monooxygenase for methane oxidation to methanol

[0094] In another embodiment of the invention are host cells modified to contain a particulate methane monooxygenase enzyme. Provided by the invention are suitable particulate methane monooxygenases that are encoded by 3 genes. Example 12 below provides S. cerevisiae that is modified to contain a particulate methane monooxygenase from M. trichosporium in a background host that is previously engineered to catabolize methanol (Strain LSMOOl). This new strain containing a particulate MMO is able to grow on methane. Example 13 below provides P. kudriavzevii cells that are modified to contain a soluble methane monooxygenase from M. trichosporium and are able to catabolize methane. However, the invention provides a wide variety of host cells and genes suitable for modifying a cell to catabolize methane in addition to those shown in the examples. Suitable host cells for modifying include those listed previously. Example 12 below demonstrates the use of the construction of a DNA expression construct for methane catabolism comprised of the promoter TEF1, a particulate methane monooxygenase gene pmoCl (AN AAF37893.1), a PGI terminator, a TEF2 promoter, pmoA (AN AAA87220.2), a TPI1 terminator, a FBA1 promoter pmoB (AN AAF37894.1), and a TDH3 terminator to drive expression in S. cerevisiae. Example 13 below demonstrates the use of a similar construct, with promoter sequences modified to match homologues of P. kudriavzevii that drives expression of particulate methane monooxygenase in P. kudriavzevii. A variety of other promoters can be used in accordance with the invention to drive expression of genes in S. cerevisiae and P. kudriavzevii and include: TEF1, PGK1, TDH3, among others.

[0095] Suitable genes for the pmoA subunit of a methane monooxygenase include CAE48352.1, CAE47800.1, AAC45295.2, AAA87220.2, and AAB49821.1.

[0096] Suitable genes for the pmoB subunit of a methane monooxygenase include CAE48353.1, CAE47801.1, AAF37897.1, AAF37894.1, and AAB49822.1.

[0097] Suitable genes for the pmoC subunit of a methane monooxygenase include CAE48351.1, CAE47799.1, AAF37896.1, AAF37893.1, and AAB49820.1. [0098] Provided by the invention is a redox partner for the particulate methane monooxygenase. In some embodiments of the invention, an additional enzyme is expressed (AN YP_114352.1) and provides an optimized redox partner for the particulate methane monooxygenase. In other embodiments, a redox partner native to the host cell provides the redox partner.

[0099] Other suitable enzymes to those provided above can be identified by homology searching using any of the preceding enzymes. These homologous enzymes are provided and can be used in practice of the invention.

Chemicals / compounds produced from methanol or methane

[0100] Provided by the invention are a variety of processes for the production of compounds from cells that catabolize methanol or methanol. Some naturally occurring methanol or methane catabolizing strains produce valuable compounds; others are isolated and cultured under conditions that result in the desired production. In some embodiments, the invention provides recombinant host cells that have been modified to produce valuable compounds. Provided by the invention are host cells that produce a variety of valuable compounds including: organic acids, fatty acids, enzymes or proteins, chemicals, and fuels. These host cells have been modified to contain genes and enzymes for producing the compounds. Provided by the methods of the invention are methods for creating new host cells for use in the production methods of the invention, by transforming them with genes that produce the enzymes required for producing the valuable compounds.

Organic acid production

[0101] Provided by the invention are processes for producing organic acids using strains capable of catabolizing methanol or methane. Organic acids are useful in a variety of industries. Provided by the methods of the invention are processes for the production of the following organic acids: malonic acid, lactic acid, succinic acid, 3- hydroxypropionic acid, citric acid, fumaric acid, maleic acid, malic acid, pyruvic acid, among others.

Malonic acid production from methanol or methane catabolizing cells

[0102] The present invention provides modified host cells that produce malonic acid and methods for such production. Example 1 below demonstrates the production of malonic acid using the malonyl-CoA hydrolase enzyme F6AA82(3) in K. pastoris cells. Example 2 below demonstrates the production of malonic acid using the malonyl-CoA hydrolase enzyme F6AA82(3) in Methylococcus capsulatus cells. The invention provides a wide variety of host cells and genes suitable for producing malonic acid in addition to those shown in the examples. Suitable host cells for catabolizing methanol or methane and producing malonic acid as described above and new organisms that have been identified in accordance with the methods of the invention. Suitable genes and enzymes are described in PCT patent application US2013/029441 (PCT Pub No. WO 13/ 134424), incorporated herein by reference, including genes and expression vectors encoding enzymes that produce malonic acid, referred to as malonyl-CoA hydrolases. In various embodiments, the heterologous nucleic acid encodes a malonyl-CoA hydrolase selected from the group consisting of S. cerevisiae EHD3, EHD3 (E124S), EHD3 (E124A, E308V), EHD3 (E124H), EHD3 (E124K), EHD3 (E124R), EHD3 (E124Q), H. pneumoniae YciA, or F6AA82 (E95N/Q384A/F304R), referred to below as F6AA82(3). In various embodiments, the heterologous EHD3 also comprises mutations selected from the group F121I, F121L, F127I, F127L. In various embodiments, the heterologous EHD3 also comprises A or V mutations of amino acids selected from the group consisting of R3, K7, K14, K18, and R22. In various embodiments Various promoters can be used to express a malonyl-CoA hydrolase gene. Example 1 below demonstrates the use of the promoter AOX1, which is appended to the 5' end of the F6AA82(3) gene to drive expression in K. pastoris. Example 2 below demonstrates the use of the sigma 54 promoter, which is appended to the 5' end of the F6AA82(3) gene to drive expression in M. capsulatus. A variety of other promoters can be used in accordance with the invention to drive expression of F6AA82(3) in K. pastoris and include: MOX1, PMA1, GAP, TPS1, FMD, and TEFL A variety of other promoters can be used to drive expression of F6AA82(3) in M. capsulatus and include sigma70, among others.

[0103] Methods for transformation of a promoter - gene expression cassette are provided by the invention. In example 1 below, the AOX1 promoter - malonyl- CoA hydrolase gene expression cassette is transformed into K. pastoris and integrates into the genome. In example 2 below, the sigma54 promoter - malonyl-CoA hydrolase gene expression cassette is transformed into M. capsulatus and integrated into the genome.

[0104] Methods for detecting modified cells that produce malonic acid are provided by the invention. Clonal isolates of the modified K. pastoris cells are isolated by methods described above and grown in accordance with the methods of the invention. After growth, cells are separated from fermentation broth. The fermentation broth is analyzed for production of malonic acid by high pressure liquid chromatography (HPLC), using an ion-exchange resin (eg. Aminex HPX-87H, BioRAD, Hercules, CA), using a column temperature of 30C, a mobile phase of 5 mM sulfuric acid and flow rate of 0.8 ml/min. A standard of malonic acid (Sigma) is injected onto the HPLC and the corresponding peak is detected by UV detection at 210 nm at ~8 min. Fermentation broth from modified K. pastoris cells containing the F6AA82(3) enzyme is analyzed and shown to produce malonic acid, whereas wildtype K. pastoris cells produce no detectable levels of malonic acid. These cells are modified to contain the genes and enzymes for conversion of malonyl-CoA into malonic acid. When the suitable genes and enzymes for production of malonic acid are present in the methanol catabolizing host cell, the cell is grown with methods and media in accordance with the invention, and malonic acid is produced.

Lactic acid production from methanol or methane catabolizing cells

[0105] The present invention provides modified host cells that produce L or

D-lactic acid and methods for such production. Example 4 below demonstrates the production of L-lactic acid using a lactate dehydrogenase (LDH) enzyme from Lactobacillus helveticus, YP_001577351.1 (Accession Number) in modified Methylobacterium extorquens AMI. However the invention provides a variety of host cells as described and genes as follows.

[0106] Suitable LDH enzymes that convert pyruvate into lactic acid include

YP_007680079.1, AGI40061.1, YP_003999807.1, AEI42219.1, ADQ02883.1, ABS72728.1, CAC95475.1, CAD00737.1, AEW68427.1, ACC77762.1, ACC77751.1, ACX81196.1, ACX81178.1, ABL74518.1, ABL74516.1, ABL74510.1, AAB27184.1, AFC28011.1, Q27888.1, Q95028.1, P33571.3, P29038.1, Q27797.1, P93052.1, CBH40747.1, CAL59188.1, YP_005411613.1, YP_005388915.1, AFC68082.1, AFC66217.1, AAN58808.1, ABY60854.1, AAV68348.1, AAA67063.1, AAD15625.1, ABS18410.1, YP_005437454.1, YP_005435970.1, YP_005284196.1, YP_003515701.1, NP_001095933.1, AFA74830.1, BAL95955.1, BAL94471.1, ADK25713.1, ADV02470.1, ADK62519.1, AAA25172.1, AAN38977.1, AAN38976.1.

[0107] Suitable D-LDH enzymes that convert pyruvate into D-lactic acid include YP_003911139.1, YP_003941114.1, YP_004115070.1, YP_004115666.1, YP_004213794.1, YP .004272242.1, YP .004315016.1, YP_004365392.1, YP_004475667.1, YP .004481271.1, YP .004488769.1, YP_004499780.1, YP_004504732.1, YP_005277307.1, ΥΡ_005216326.1, ΥΡ_006024194.1, ΥΡ_005803320.1, EGJ71278.1, AEW02641.1, AEE53484.1, AFZ35623.1, ADY31655.1, ΥΡ_007132589.1, ΥΡ_004251835.1, ΥΡ_004450357.1, ΥΡ_005012044.1, AAA60530.1, P72357.1, ADV50239.1, ADV50298.1, EGN56622.1, AFZ03077.1, AFZ04725.1, ELS34055.1, AEM71879.1, ΥΡ_007113141.1, ΥΡ_004789301.1, ΥΡ_004165737.1, ΥΡ_004165796.1, ΥΡ_007139049.1, P30901.2, CBY96376.1, ΥΡ_006886585.1, P26298.1, ΝΡ_705690.2, ΝΡ_919417.1, Ρ26297.3, P06149.3, WP_007888018.1, and WP_007176620.1.

[0108] Suitable L-LDH enzymes that convert pyruvate into L-lactic acid include AEE95701.1, YP_001972658.1, YP_003825817.1, YP_004462523.1, EIC00523.1, ADL11844.1, AD077186.1, EH041556.1, AFY65878.1, AFZ47136.1, AFZ42738.1, YP_003826909.1, YP_005836346.1, YP_007108930.1, YP_007164785.1, YP_007166952.1, AFY38960.1, YP_007071794.1, AFZ13827.1, AEC02841.1, YP_007143337.1, YP_004412223.1, AFZ33285.1, ADV68710.1, ADY27109.1, YP_007130445.1, YP_004172375.1, YP_004256726.1, AEE17453.1, YP_004440584.1, AEB14220.1, AEE15700.1, AFZ38114.1, AFZ53918.1, AEB11000.1, YP_007151485.1, YP_004365517.1, YP_004367110.1, YP_004438831.1, YP_007161962.1, AFY70884.1, YP_007103312.1, CAA38914.1, WP_006040774.1, WP_009172973.1, YP_001037478.1, WP_002289717.1, WP_003665815.1, BAA14353.1, and CAA04010.1.

[0109] Other suitable enzymes to those provided above can be identified by homology searching using any of the preceding enzymes. These homologous enzymes are provided and can be used in practice of the invention.

[0110] Suitable genes for LDH enzymes that can carry out this reaction are available. Sources for lactate dehydrogenase (Idh) include Lactobacillus, Enterococcus, Corynebacterium, Mycoplasma, Camphylobacter, Clostridium, Bifidobacterium, Bacillus, Rhodopirellula, Streptococcus, Bordetella, Deinococcus, Staphylococcus, Listeria, Acidominococcus, Treponema, Rhodobacter, Azospirillum, Cyanothece, Stigmatella, Sorangium, Borrelia, among other sources. In some embodiments the Idh gene is isolated from Lactobacillus helveticus is synthesized for expression in Methylobacterium extroquens AMI.

[0111] Suitable promoters for expressing genes in accordance with the methods of the invention include any promoter proven to work in the host cell. In the example below, a suitable promoter is moxF. In addition if other promoters are desired they are identified. Identification of promoters for expression of heterologous genes via sequence alignments that determine DNA sequence for methanol or methane consumption genes, results in identification of the genome locations of the start codon. Analyzing and identifying the 400 nucleotides upstream of a gene's start codon position results in DNA sequence sufficient to achieve expression of heterologous genes when appended to the start codon of the heterologous gene. Expression is achieved by appending a suitable promoter element from Methylobacterium. Provided by this invention are such suitable promoters which have been identified by sequence analysis of DNA 400 nucleotides 5' to the start codon (ATG) of genes related to methane catabolism and include mxa, mxb, pqqABC/DE, pqqFG, mxc, mxaW, and others. In one embodiment, 400 base pairs of the promoter region 5' to the mxa gene (M. extorquens CM4 reference genome, nucleotide coordinates: 4451135 to 4451535 are synthesized and appended to the start codon of the Idh from Lactobacillus bulgaricus. An additional 200 nucleotides 3' to the mxa gene terminator region (M. extorquens CM4 reference genome, nucleotide coordinates: 4452647 to 4452847) are appended to the stop codon of the Idh gene. This promoter, Idh, terminator expression cassette is cloned into a plasmid containing a selectable kanamycin resistance cassette via electroporation and methods previously described (1991, Ueda et al. AEM).

[0112] Suitable methods for detecting modified cells that produce lactic acid are provided by the invention. Colonies are isolated and grown in liquid culture media with methane as a sole-carbon source as provided previously by methods of this invention. Cultures area analyzed by high-pressure liquid chromatography (HPLC). For HPLC analysis of lactic acid accumulation in the fermentation broth, a Shimadzu XR HPLC system equipped with a UV detector is employed. 5 μΐ of each sample is injected and separated with an Aminex HPX-87H fermentation-monitoring and guard column (Bio-Rad). The mobile phase is distilled water (pH 1.95 with sulfuric acid), the flow rate was 0.8 ml/min, the oven temperature was 30°C and the UV detector monitors 210 nm. Lactic acid elutes at -9.8 minutes post-injection under these conditions. A standard curve established with authentic lactic acid is used to determine lactic acid concentration in the fermentation broth. Analysis of cultures results in detection of lactic acid production compared to a negative control strain that lacks the Idh expression cassette, which produces no detectable or low-levels of lactic acid. Because M. extorquens is also capable of consuming methanol as a sole-carbon source, feeding of methanol to the cells modified for heterologous expression of an Idh also results in production of lactic acid compared to the negative control strain, which produces low-levels or undetectable levels lactic acid.

Succinate production from methanol or methane catabolizing host cells

[0113] The present invention provides modified host cells that produce succinate and methods for such production. Succinic acid is a natural intermediate of the tricarboxylic acid cycle and sometimes present at detectable levels in a variety of organisms. Succinic acid is produced via two biosynthetic pathways that rely upon the immediate precursor fumarate or isocitrate, but not detected at high levels in methanol or methane catabolizing host cells. Provided by the invention are cells that have been modified and produce succinic acid at higher levels than present in the naturally occurring cells. Example 5 below demonstrates higher production of levels of succinic acid in M. extorquens AMI that is modified to contain a pyruvate carboxylase (PYC) from Lactobacillus lactis and a citrate synthase from Bacillus subtilis compared to unmodified cells. However the invention provides a variety of host cells as described and genes as follows.

[0114] Provided by the methods of the invention are methods, genes and modifications made to the fumarate pathway in order to produce succinic acid in methanol or methane catabolizing cells. In the fumarate pathway, phosphoenolpyruvate is converted into oxaloacetate via the enzyme phosphoenolpyruvate carboxylase, oxaloacetate is converted into malate via the enzyme malate dehydrogenase, malate is converted into fumarate via the enzyme fumarase, and finally fumarate is converted into succinate via the enzyme fumarate reductase. Provided by the methods of the invention are methods, genes and modifications made to the isocitrate pathway for production of succinic acid from methanol or methane catabolizing cells. In the isocitrate pathway, PEP is converted into pyruvate via the enzyme pyruvate kinase, pyruvate is converted into acetyl-CoA via the enzyme pyruvate dehydrogenase, acetyl-CoA and oxaloacetate are converted into citrate via the enzyme citrate synthase, citrate is converted into isocitrate via the enzyme aconitase, and finally isocitrate is converted into succinic acid via the enzyme isocitrate lyase. Alternatively, acetyl-CoA and glyoxylate is converted into malate via the enzyme malate synthase and enter into the fumurate pathway for producing succinic acid. Provided by the invention are host cells that express recombinant pyruvate carboxylase, fumarate reductase, fumarate hydratase, and malate dehydrogenase such that the immediate central metabolic precursor, fumarate, is converted into succinic acid. In some embodiments a phosphoenolpyruvate (PEP) carboxylase is expressed or overexpressed. In one embodiment, these enzymes are native to the host cells but have been modified to result in increased production of succinic acid by metabolic engineering. In one embodiment, an isocitrate lyase is expressed or overexpressed to result in conversion of isocitrate into succinic acid. These chemical reactions are described by the enzyme class (EC) numbers 6.4.1.1, 1.3.1.6, 1.3.99.1, 1.3.5.4, 1.3.5.1, 1.1.1.37, 1.1.5.4, 1.1.99.16, 1.1.1.39, 1.1.1.40,

1.1.1.37, 1.1.1.82, 1.1.1.38, 1.1.1.299, 4.2.1.2, and 4.1.3.1, 4.1.1.31, 4.1.1.32,

4.1.1.38, and 4.1.1.49.

[0115] Suitable pyruvate carboxylase enzymes are employed and include

YP_004583696.1, YP_004588806.1, YP_004625808.1, YP_004805451.1, YP_004814968.1, CAB59603.1, CAL27650.1, AAG30411.1, AAA60033.1, ACV08954.1, ACV78266.1, WP_011406753.1, YP_003161257.1, NP_989677.1, CAD78963.1, AAB31500.1, EHP06100.1, ADH66593.1, AD077252.1, AD084262.1, ADV61203.1, AEH85752.1, ADU47818.1, ADR18408.1, EHQ03445.1, EIJ33384.1, ADR21001.1, YP_003679099.1, YP_005836412.1, YP_003968610.1, YP_004050571.1, YP_004053109.1, YP_004098545.1, YP_004177752.1, YP_004609846.1, AEV96968.1, ADV49631.1, ADY57205.1, ADY61171.1, AEA33111.1, AEE49459.1, ADY28393.1, YP_004165129.1, YP_004271193.1, YP_004261264.1, YP_004267206.1, YP_004339170.1, YP_004446332.1, YP_005006372.1, WP_014805828.1 among others.

[0116] Suitable fumarate reductase enzymes are employed and include

WP. _.012770922.1, WP. .012775406.1, WP. _.015722071.1, WP. _.015827337.1

WP. _.015854844.1, WP. .015854845.1, WP. _.015868004.1, WP. _.002772754.1

WP. .005032133.1, WP. .005032900.1, WP. .005033403.1, WP. .005463208.1

WP. _.006080372.1, WP. .006852705.1, WP. .006853915.1, WP. .007420066.1

WP. .007509949.1, WP. .007595990.1, WP. .007679151.1, WP. .007738831.1

WP. _.007828544.1, WP. _.007850711.1, WP. .007871888.1, WP. .008002743.1

WP. .008406793.1, WP. .009112246.1, WP. .009113520.1, WP. _.009113524.1

WP. .009114953.1, WP. .009548823.1, WP. .013119148.1, WP. _.013549271.1

WP. .013621358.1, WP. _.013702622.1, WP. _.013969821.1, WP. 015448449.1 WP_015765497.1, WP_002287050.1, WP_007413432.1, WP_012201302.1, WP_012385994.1, WP_015865668.1, WP_004339468.1, WP_005033600.1, WP_006743822.1, WP_007572896.1, WP_013546442.1, WP_013618092.1, WP_014809740.1, WP_008350680.1 among others.

[0117] Suitable fumarate hydratase enzymes are employed and include

EEQ01810.1, EEQ05100.1, EEQ11510.1, EEQ13383.1, EEQ19917.1, XP_002503527.1, CBI65711.1, CAL35476.1, XP_844042.1, AC064785.1, ACA12065.1, AEV15971.1, AEZ45128.1, AGH39439.1, WP_011859478.1, WP_011988872.1, AGI31428.1, AGI36463.1, WP_004236173.1, WP_013025710.1, WP_013027952.1, NP_669418.1, NP_885804.1, YP_003530916.1, YP_003728175.1, YP_005654045.1, YP_007549230.1, YP_007664936.1, YP_007669971.1, YP_310483.1, YP_407969.1, YP_001085015.1, YP_001393705.1, YP_001775695.1, YP_110392.1, YP_403429.1, YP_004115633.1, YP_004112836.1, YP_005216981.1, YP_005802923.1, WP_012698009.1, YP_386736.2, NP_001230517.1, YP_003657149.1, YP_003894296.1, YP_001972954.1, WP_013931726.1, WP_012253071.1, WP_003601902.1, WP_015722076.1 among others.

[0118] Suitable malate dehydrogenase enzymes are employed and include

WP_011444841.1, WP_011474984.1, WP. _.011644754.1, WP. .011878203.1,

WP_011952143.1, WP_011955047.1, WP. .011995831.1, WP. .011997820.1,

WP_012095736.1, WP_012256789.1, WP. .012288993.1, WP. _.012317922.1,

WP_012371076.1, WP_012526260.1, WP. .012660558.1, WP. .012726715.1,

WP_014097753.1, WP_015864778.1, WP. _.007690171.1, WP. .012871595.1,

WP_013524302.1, WP_015828083.1, WP. _.002659807.1, WP. _.002772524.1,

WP_003567261.1, WP_004443804.1, WP. .006743530.1, WP. .007505708.1,

WP_007673511.1, WP_007683090.1, WP. _.007709882.1, WP. .007720089.1,

WP_007725335.1, WP_007757753.1, WP. _.007786272.1, WP. .007796993.1,

WP_007822364.1, WP_007907923.1, WP. .008093736.1, WP. .008127205.1,

WP_009612077.1, WP_013545774.1, WP. .013705051.1, WP. .013892250.1,

WP_013899023.1, WP_014811607.1, WP. .015315009.1, WP. .015765856.1,

YP_770009.1, AAA39509.1 among others

[0119] Suitable isocitrate lyase enzymes are employed and include

YP_004156232.1, YP_004214829.1, YP_004228672.1, YP_004234858.1, YP_004388696.1, YP_004454339.1, YP_004487567.1, YP_004503002.1, YP_004507954.1, YP_004547792.1, YP_004541565.1, YP_004589107.1, YP_004599712.1, YP_005272604.1, ΥΡ_005279932.1, ΥΡ_006027416.1, CAC30940.1, CAC44332.1, ΥΡ_004022336.1, ADN76986.1, ΥΡ_003914060.1, EFH90336.1, AFK01787.1, ADH66861.1, ADH62916.1, AEH87023.1, ADU51871.1, EIJ36034.1, ADR23620.1, ADD27534.1, ADI14660.1, ΥΡ_003506554.1, ΥΡ_003679367.1, ΥΡ_003684424.1, ΥΡ_003705203.1, ΥΡ_004055728.1, ΥΡ_004102598.1, ΥΡ_004611117.1, ΥΡ_006871814.1, AEW03684.1, ADV66913.1, ADY26667.1, ΑΕΒ 11964.1, ΥΡ_004170578.1, ΥΡ_004256284.1, ΥΡ_004368074.1, ΥΡ_005255356.1, CAA84632.1, ΝΡ_188809.2, AEE76544.1 among others.

[0120] Suitable PEP carboxylase enzymes are employed and include

NP_866412.1, YP_003998059.1, YP_004045007.1, YP_004055360.1, YP_004165130.1, YP_004273452.1, YP_004373383.1, EFH90147.1, ADH69337.1, ADH63538.1, ADM28568.1, ADN50074.1, ADN74523.1, ADP77112.1, ADK68466.1, ADN35502.1, AEH60801.1, ADD27759.1, ADI15493.1, ADR36285.1, YP_003506779.1, YP_003681843.1, YP_003685046.1, YP_003706036.1, YP_003801346.1, YP_003860448.1, YP_003893940.1, YP_003901125.1, YP_003911597.1, YP_004003874.1, YP_004057458.1, YP_004616020.1, ADU51842.1, AEA47999.1, EJG06574.1, ADV67716.1, ADY25558.1, AEB11631.1, YP_004102569.1, YP_004171381.1, YP_004255175.1, YP_004342714.1, YP_004367741.1, WP .004636385.1, WP_012212910.1, WP_007363513.1, WP_007710188.1, WP_007738267.1, WP_008115278.1, BAA07723.1 among others.

[0121] Suitable citrate synthase enzymes are employed and include

AFY64559.1, AFY70761.1, AFY87747.1, AFZ03533.1, AFZ14343.1, AFZ29765.1, ELS34669.1, EFQ22922.1, EGJ70299.1, YP_933058.1, YP_003805022.1, YP_006441134.1, YP .007103189.1, YP_007065647.1, YP_007091616.1, YP_007107611.1, YP_007143853.1, YP_007126925.1, NP_867397.1, YP_004609930.1, YP_007139505.1, EFH80014.1, EHP10199.1, EHQ05301.1, AFM12222.1, ADN76694.1, ADW16919.1, ADQ18976.1, ADU47355.1, ADU49111.1, ADV46520.1, ADR34105.1, AEA44719.1, YP_003682305.1, YP_006439728.1, YP_003913768.1, YP_003999329.1, YP_004060305.1, YP_004098082.1, YP_004099838.1, YP_004168269.1, YP_004194210.1, YP_004345557.1, AAB35835.2, AAC73814.1, WP_005441312.1, WP_007670230.1, WP .007837365.1, NP_415248.1, CAA38996.1 [0122] Other suitable enzymes to those provided above can be identified by homology searching using any of the preceding enzymes. These homologous enzymes are provided and can be used in practice of the invention.

[0123] Provided by the invention are sources for the genes which include

Lactobacillus, Enterococcus, Corynebacterium, Mycoplasma, Camphylobacter, Clostridium, Bifidobacterium, Bacillus, Rhodopirellula, Streptococcus, Bordetella, Deinococcus, Staphylococcus, Listeria, Acidominococcus, Treponema, Rhodobacter, Azospirillum, Cyanothece, Stigmatella, Sorangium, Borrelia, among other sources. In some embodiments the pyc, mdh, gene is isolated from Bacillus subtilis is synthesized for expression in Methylobacterium extroquens AMI.

[0124] Suitable promoters for expressing genes in accordance with the methods of the invention include any promoter proven to work in the host cell. In the example below, a suitable promoter is moxF. In addition if other promoters are desired they are identified. Identification of promoters for expression of heterologous genes via sequence alignments that determine DNA sequence for methanol or methane consumption genes, results in identification of the genome locations of the start codon. Analyzing and identifying the 400 nucleotides upstream of a gene's start codon position results in DNA sequence sufficient to achieve expression of heterologous genes when appended to the start codon of the heterologous gene. Expression is achieved by appending a suitable promoter element from Methylobacterium. Provided by this invention are such suitable promoters which have been identified by sequence analysis of DNA 400 nucleotides 5' to the start codon (ATG) of genes related to methane catabolism and include mxa, mxb, pqqABC/DE, pqqFG, mxc, mxaW, and others. In one embodiment, 400 base pairs of the promoter region 5' to the mxa gene (M. extorquens CM4 reference genome, nucleotide coordinates: 4451135 to 4451535 are synthesized and appended to the start codon of the pyc gene from Lactobacillus lactis. A ribosome-binding site linker connecting the 3' termini of pyc gene to the 5' start codon of citZ. from B. subtilis results in an expression operon. An additional 200 nucleotides 3' to the mxa gene terminator region (M. extorquens CM4 reference genome, nucleotide coordinates: 4452647 to 4452847) are appended to the stop codon of the citZ gene. This promoter, pyc, ribosome binding site, citZ, terminator expression cassette is cloned into a plasmid containing a selectable kanamycin resistance cassette via electroporation and methods previously described (1991, Ueda et al. AEM). [0125] Suitable methods for detecting modified cells that produce succinic acid are provided by the invention. Colonies are isolated and grown in liquid culture media with methane as a sole-carbon source as provided previously by methods of this invention. Cultures area analyzed by high-pressure liquid chromatography (HPLC). For HPLC analysis of lactic acid accumulation in the fermentation broth, a Shimadzu XR HPLC system equipped with a UV detector is employed. 5 μΐ of each sample is injected and separated with an Aminex HPX-87H fermentation-monitoring and guard column (Bio-Rad). The mobile phase is distilled water (pH 1.95 with sulfuric acid), the flow rate was 0.8 ml/min, the oven temperature was 30°C and the UV detector monitors 210 nm. Succinic acid elutes at -9.48 minutes post-injection under these conditions. A standard curve established with a succinic acid standard is used to determine lactic acid concentration in the fermentation broth. Analysis of cultures results in detection of succinic acid production compared to a negative control strain that lacks the pyc and citZ expression cassette, which produces no detectable or low-levels of succinic acid. Because M. extorquens is also capable of consuming methanol as a sole-carbon source, feeding of methanol to the cells modified for heterologous expression of pyc and citZ also results in production of succinic acid compared to the negative control strain, which produces low-levels or undetectable levels succinic acid.

1,3 -propanediol production from methanol or methane catabolizing cells

[0126] The present invention provides modified host cells that produce 1,3- propanediol (PDO) and methods for such production. Example 6 below demonstrates the production of PDO using a glycerol phosphatase, GPP2, from S. cerevisiae, a glycerol dehydrogenase, DhaBl-3 from Klebsiella pneumoniae, and a PDO reductase enzyme, DhaT, from Klebsiella pneumoniae, in modified Methylobacterium extorquens AMI. However the invention provides a variety of host cells as described and genes as follows.

[0127] Suitable glycerol phosphatase enzymes that convert glycerol-3- phosphate into glycerol include ELQ16696.1, AGE26486.1, YP_007397982.1, YP_003349902.1, Q59544.1, Q9M8S8.1, 1U2Q_A, 4I9F_A, 4I9F_B, 4I9G_A, 4I9G_B, P65163.1, YP_002347523.1, WP_016557382.1, EPE95118.1, YP 767589.1, YP_001978018.1, EJT06061.1, EJZ21275.1, CAK44992.1, P13587.1, Q12163.1, CAK45289.1, P40106.1, P41277.3, EDN61395.1, CAY80401.1, CAY80456.1, GAA24014.1, GAA26776.1, DAA08446.1, NP_012159.1, EAL87462.1 EDN60500.1, EDN61441.1, XP 749500.1, EEU05032.1, CAY78665.1 CAY86759.1, GAA22392.1, EIW09908.1, DAA12002.1, DAA08494.1 DAA11234.1, NP_015123.1, NP_012211.2, NP_010446.3, EDN63036.1 EEU07776.1, CAY79234.1, GAA22894.1, GAA24061.1, EIW07337.1, EIW09761.1 EIW11365.1, EEU05940.1, EIW10851.1, DAA07721.1, NP_010984.3 NP_075020.2, NP_001158357.1, XP_752423.1, EAL90385.1, 2QLT_A, P32485.2 EDN60945.1, 3RF6_A, 3RF6_B, YP_002347523.1, YP_002347523.1 among others.

[0128] Suitable glycerol dehydrogenase enzymes that convert glycerol into 3- hydroxypropionaldehyde include EFE07756.1, EFU57594.1, YP_001004898.1 YP_005219156.1, ADY57065.1, AFZ30461.1, YP_007127621.1, YP_003919094.1 YP_004267066.1, AAN58240.1, AFY31496.1, AFY44343.1, AFY70790.1 AFZ01860.1, AFZ06653.1, AFZ15286.1, AFZ33907.1, AFZ60390.1 YP_007051493.1, YP_007103218.1, YP_007064330.1, YP_007144796.1 YP_007115069.1, YP_007130873.1, YP_007159300.1, NP_720934.1 YP_007137832.1, CBY98279.1, YP_006888481.1, AFY38496.1, AFZ55450.1 ELS30978.1, YP_007071330.1, YP_007163494.1, AFH03576.1, NP_001245902.1 AAF52304.1, AAN 10562.1, AAN 10563.1, AGB92647.1, AGB92648.1 AGB92649.1, NP .476565.1, NP_476566.1, NP_476567.1, NP_001260111.1 NP_001260112.1, NP_001260113.1, WP_015026272.1, WP_007910337.1 among others.

[0129] Suitable 1,3-propanediol oxidoreductase enzymes that convert 3- hydroxypropionaldehyde into PDO and NADH or NAD(P)H include YP_001573595.1, YP_001589870.1, YP_001723909.1, YP_002988900.1, YP_003005729.1, YP_003164647.1, YP_002936987.1, NP_461893.1, NP_696824.1, YP_151997.1, YP_002143487.1, YP_005239094.1, YP_005253460.1, Q59477.1, P45513.1, 2BI4_A, 2BI4_B, 2BL4_A, 2BL4_B, 3BFJ_A, 3BFJ_B, 3BFJ_C, 3BFJ_D, 3BFJ_E, 3BFJ_F, 3BFJ_G, 3BFJ_H, 3BFJ_I, 3BFJ_J, 3BFJ_K, 3BFJ_L, 3BFJ_M, 3BFJ_N, 3BFJ_0, 3BFJ_P, 3BFJ_Q, 3BFJ_R, 3BFJ_S, 3BFJ_T, CBG35825.1, CBJ02491.1, CAQ33123.1, YP_003000377.1, YP_006097293.1, YP_006116537.1, NP_667921.1, YP_004209572.1, YP_004221347.1, P0A9S2.2, P0A9S1.2 among others. [0130] Other suitable enzymes to those provided above can be identified by homology searching using any of the preceding enzymes. These homologous enzymes are provided and can be used in practice of the invention.

[0131] Provided by the invention are sources for the genes which include

Saccharomyces, Klebsiella, Lactobacillus, Enterococcus, Corynebacterium, Mycoplasma, Camphylobacter, Clostridium, Bifidobacterium, Bacillus, Rhodopirellula, Streptococcus, Bordetella, Deinococcus, Staphylococcus, Listeria, Acidominococcus, Treponema, Rhodobacter, Azospirillum, Cyanothece, Stigmatella, Sorangium, Borrelia, among other sources. In some embodiments the pyc, mdh, gene is isolated from Bacillus subtilis is synthesized for expression in Methylobacterium extroquens AMI.

[0132] Suitable promoters for expressing genes in accordance with the methods of the invention include any promoter proven to work in the host cell. In the example below, a suitable promoter is moxF. In addition if other promoters are desired they are identified. Identification of promoters for expression of heterologous genes via sequence alignments that determine DNA sequence for methanol or methane consumption genes, results in identification of the genome locations of the start codon. Analyzing and identifying the 400 nucleotides upstream of a gene's start codon position results in DNA sequence sufficient to achieve expression of heterologous genes when appended to the start codon of the heterologous gene. Expression is achieved by appending a suitable promoter element from Methylobacterium. Provided by this invention are such suitable promoters which have been identified by sequence analysis of DNA 400 nucleotides 5' to the start codon (ATG) of genes related to methane catabolism and include mxa, mxb, pqqABC/DE, pqqFG, mxc, mxaW, and others. In one embodiment, 400 base pairs of the promoter region 5' to the mxa gene (M. extorquens CM4 reference genome, nucleotide coordinates: 4451135 to 4451535 are synthesized and appended to the start codon of the GPP2 gene from Saccharomyces cerevisiae. A ribosome-binding site linker connecting the 3' termini of GPP2 to the 5' start codon of dhaBl, dhaBl, and dhaB3 operon from K. pneumoniae, followed by an ribosome binding site linker and the final dhaT gene results in an expression operon. An additional 200 nucleotides 3' to the mxa gene terminator region (M. extorquens CM4 reference genome, nucleotide coordinates: 4452647 to 4452847) are appended to the stop codon of the dhaT gene. This operon is cloned into a plasmid containing a selectable kanamycin resistance cassette via electrop oration and methods previously described (1991, Ueda et al. AEM).

[0133] Suitable methods for detecting modified cells that produce PDO are provided by the invention. Colonies are isolated and grown in liquid culture media with methane as a sole-carbon source as provided previously by methods of this invention. Cultures area analyzed by high-pressure liquid chromatography (HPLC). For HPLC analysis of PDO accumulation in the fermentation broth, a Shimadzu XR HPLC system equipped with a UV detector is employed. 5 μΐ of each sample is injected and separated with an Aminex HPX-87H fermentation-monitoring and guard column (Bio-Rad). The mobile phase is distilled water (pH 1.95 with sulfuric acid), the flow rate was 0.8 ml/min, the oven temperature was 30°C and the UV detector monitors 210 nm. A standard curve established with a PDO standard is used to determine PDO concentration in the fermentation broth. Analysis of cultures results in detection of PDO production compared to a negative control strain that lacks the expression cassette, which produces no detectable or low-levels of succinic acid. Because M. extorquens is also capable of consuming methanol as a sole-carbon source, feeding of methanol to the cells modified for production of PDO also results in production of PDO compared to the negative control strain, which produces low- levels or undetectable levels PDO.

[0134] The modified host cells provided by the invention produce a variety of products including: fatty acids, eicosapentanoic acid, docosahexanoic acid, hexanoic acid, ethanol, isobutanol, isopentanol, isoprene, farnesene, squalene, hexanol, heptanol, octanol, decanol, tetradecanoic acid, tetradecanedioic acid, 3- hydroxypropionic acid, citric acid, maleic acid, malic acid, fumaric acid, muconic acid, lysine, glutamic acid, serine, cysteine, proteins, enzymes, and insulin among others. In some embodiments the target chemical is 1,3-propanediol, 1,4-butanediol, succinate, malate, 1,4-butadiene, ethanol, isobutanol, isopentanol, isoprene, farnesene, squalene, 1 -hexanol, 1 -heptanol or 1 -octanol. In some cases the target compound is used as a fuel.

[0135] Provided by the methods of the invention are host cells that catabolize methanol or methane and have been modified to produce a valuable protein or enzyme. In one embodiment the target protein is insulin (Accession Number AAA59172.1). In one embodiment the target protein is erythropoeitin (Accession Number AAI43226.1). In one embodiment the target enzyme is used in laundry detergents and are classified as proteases and amylases. In one embodiment the enzyme is a phytase.

Methods for fermentation processes

[0136] Provided by the invention are methods of growing the organisms at a wide range of relevant scales, including <1L, 1L, 10L, 50L, 300L, 1000L, >10kL, >50kL, >100kL, >200kL, >250kL, >500kL, >1ML to produce a given compound from methanol or methane. One key parameter for growth and provided by the invention is oxygen transfer rate (OTR) and effective concentration. Depending upon what scale H. polymorpha is grown, there are different methods for controlling OTR. In general, increasing OTR increases growth rates and productivity of H. polymorpha. At smaller scales, generally less than 300L, OTR is increased via increased stirring rates. As fermentation volume increases, OTR is increased using a bubble column or direct oxygen infusion. There are a wide range of fermentation processes provided including batch, fed-batch and continuous. Provided by the invention is a fermentation process that overcomes typically decreased growth and productivity rates associated with feeding a gas substrate to a liquid fermentation broth due to limitations in gas-to-liquid phase transfer. In one embodiment, the gas is fed at a temperature and pressure where it exists as a liquid and increased growth rate or productivity results. In another embodiment, methane is fed in the fermentation process after biomass has grown upon a carbon source that occurs in the liquid phase (i.e. glucose, methanol). In one embodiment, the fermentation process takes place in at least two stages: the first stage includes a biomass formation stage that uses a liquid feedstock (glucose, methanol), which increases productivity and growth rates by relieving gas-to-liquid phase transfer limitations, the second stage includes a production phase that uses a liquid or gas feedstock (glucose, methanol, methane). In some embodiments production of a chemical also occurs during the biomass production phase.

Examples

[0137] Example 1. In this example, K. pastoris Y-1047 and Y-1603 modified host cells were cultured to produce malonic acid.

Construction of synthetic nucleic acid sequences conferring expression of a malonyl- CoA hydrolase and integration into methanol-consuming yeast strains [0138] An integrating plasmid was constructed that contained an expression cassette consisting of the AOXl promoter common to methanol catabolizing yeasts followed by a malonyl-CoA hydrolase gene. In this example and elsewhere we refer to the F6AA82(3) enzyme which is derived from the F6AA82 enzyme and contains three amino acid mutations (E95N/Q384A/F304R). The plasmid also contained the coding region for selection of geneticin resistant clones. This plasmid, pLCOOOl, was derived from the pJ901-15 vector (commercially available from DNA2.0, Menlo Park, CA, USA) The vector was assembled by amplifying the pJ901-15 vector and the F6AA82(3) by PCR. The primers for amplification were designed to contain 15 nucleotide base pair overlaps such that 15-30 base pairs of homology existed between the 3' end of the AOXl promoter and the 5' beginning of the F6AA82(3) gene. Homology was also designed between the 3' end of the F6AA82(3) gene and 5' beginning of the AOXl terminator. Primers for amplification of the F6AA82(3) gene are: F6AA82(3)-F: gaaagaattcaaaaagagaccaaaaaaaATGAATGTCACCTTTGAAGAAAGAG and F6AA82(3)-R: ctcttgagcccctgagaccactagtTTATGCCAAATCAGCTAAAGGGTG. Primers for amplification of the pJ901-15 vector are 15-R: tttttttggtctctttttgaattctttcaataattag and 15-F: actagtggtctcaggggctc. The two products were added in equimolar concentrations to a final PCR mix that contained no primers and subjected to 10 cycles of standard PCR thermocycling (98°C for 2min, then cycle 10 times: 55°C for 30s, 72°C for 5min). 10 uL of this reaction were transformed into E. coli chemically competent cells and selected for on LB agar plates containing 25 ug/ml kanamycin. Plasmids were isolated from cells, sequence verified and designated pLCOOOl.

Integration of synthetic nucleic acid sequences conferring expression of a malonyl- CoA hydrolase into the methanol consuming yeast strain genomes and production of malonate

[0139] 2 strains of K. pastoris (designated Y-1047 and Y-1603) were obtained from the USD A ARS Culture Collection (Peoria, IL, USA). Colonies were picked into 5 mL YPD and grown overnight at 29°C, 200 rpm. 1 mL of the overnight cultures were subcultured in 50mL of YPD for 6h. The cultures were pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4C ddH20, and re-pelleted. Finally cells were resuspended in lmL of 1M refrigerated at 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLCOOOl was prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLCOOOl was added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the USDA yeast strains (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains were recovered by growing in YPD for 21h at 25°C. 110 uL aliquots of the strains were then plated onto YPD agar plates containing 4 concentrations of G418 (0.25, 0.5, 1, and 4 mg/L). Colonies appeared within 8 days and were confirmed for genomic integration by colony PCR. All colonies screened were determined to contain the malonyl-CoA hydrolase gene.

[0140] Colonies were picked into 300 uL of minimal media containing IX

YNB, 1% glucose, and 12.5 mg/L G418 and grown 48h at 29°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains were grown in the same media, but lacking G418. 250 uL of minimal induction media was added and contained IX YNB and 1% (v/v) methanol. Samples of 30 uL were taken thereafter two times a day with replacement of 30 uL of fresh induction media containing IX YNB and 5% (v/v) methanol. The samples were clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. The final samples taken at 120 h was run on an HPLC and analyzed for production of malonic acid.

[0141] HPLC analysis, as described above, was used to measure malonate accumulation in the fermentation broth. A standard curve established with authentic malonic acid was used to determine malonate concentration in the fermentation broth.

[0142] In this example, 4 transformants of K. pas tons strain Y-1047 and 3 transformants of Y- 1603 were found to produce malonate in the media used. The wild type control strains did not produce detectable levels of malonate. To summarize the findings: 4 colonies from the strain Y-1047 produced variable levels of malonic acid ranging from 0.25mM to 7mM, compared to the negative control which produced no detectable malonic acid. 3 colonies from Y-1603 produced 0.25 mM to 1.1 mM malonic acid, compared to a negative control that produced no detectable levels of malonic acid. Variable production levels were observed due to different numbers of the hydrolase integrating.

[0143] Example 2. In this example, M. capsulatus modified host cells are cultured to produce malonic acid. Construction of synthetic nucleic acid sequences conferring expression of a malonyl- CoA hydrolase in the methane-consuming M. capsulatus cells

[0144] A plasmid is constructed that contains an expression cassette consisting of the M. capsulatus sigma 54 promoter followed by a malonyl-CoA hydrolase gene (F6AA82(3)). This plasmid, pLC0002 also contains the coding region for selection of kanamycin resistant clones.

Transformation of synthetic nucleic acid sequences conferring expression of a malonyl-CoA hydrolase into the methane consuming M. capsulatus cells and production of malonate

[0145] The M. capsulatus cells are prepared for transformation by electroporation. Colonies are picked into 5 mL media (as described above) and grown at 29°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of media until stationary growth as estimated by OD 600nm. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C 10% glycerol ddH20, and re- pelleted. Finally cells are resuspended in lmL of 10% 4°C glycerol and aliquoted into 50 uL volumes for electroporation. 50 ng of pLC0002 is added to 50uL of the cells, transferred to a 1mm cuvette and transformed by electroporation into the M capsulatus cells. Transformant strains are recovered by growing in media described above for 24h at 25°C with 0.5% methanol. Serially-diluted aliquots of the strains are then plated onto solid agar plates containing 25 ug/mL kanamycin. Colonies appear within 48h and are confirmed for plasmid transformation by colony PCR. Colonies are further screened and determined to contain the malonyl-CoA hydrolase gene.

[0146] Colonies are picked into 300 uL of minimal media as described above and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm in a dessicator that contains 1 : 1 methane : air. Wildtype control strains are grown in the same media, but lacking kanamycin. Samples of 30 uL were taken thereafter two times a day with replacement of the methane:air atmosphere. The samples are clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. Final samples are taken at 120 h was run on an HPLC and analyzed for production of malonic acid, as described previously.

[0147] In this example, M. capsulatus cells modified to express the

F6AAA82(3) malonyl-CoA hydrolase are determined to produce malonic. The wild type control strains do not produce detectable levels of malonate. [0148] Example 3. In this example a methanol consuming strain, K. pastoris, is cultured to produce lactic acid from methanol.

Construction of synthetic nucleic acid sequences conferring expression of a lactic acid dehydrogenase for integration into the methanol- consuming yeast cells, K. pastoris

[0149] An integrating plasmid is constructed that contains an expression cassette consisting of the AOX1 promoter common to methanol catabolizing yeasts followed by a Idh gene, (Prot Accession No YP_001577351.1). The plasmid also contains the coding region for selection of geneticin resistant clones. This plasmid, is designated pLC0003, was derived from the pJ901-15 vector obtained from DNA2.0 (Menlo Park, CA USA).

Integration of synthetic nucleic acid sequences conferring expression of a LDH into the methanol consuming yeast cell genomes and production of lactic acid

[0150] K. pastoris cells are prepared for transformation by electroporation.

Colonies are picked into 5 mL YPD and grown overnight at 29°C, 200 rpm. 1 mL of the overnight cultures were subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re- pelleted. Finally cells are resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0003 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0003 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the USDA yeast strains (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by growing in YPD for 21h at 25C. 110 uL aliquots of the strains are then plated onto YPD agar plates containing 4 concentrations of G418 (0.25, 0.5, 1, and 4 mg/L). Colonies appear within 8 days and are confirmed for genomic integration by colony PCR. Colonies screened are determined to contain the malonyl-CoA hydrolase gene.

[0151] Colonies are picked into 300 uL of minimal media containing IX

YNB, 1% glucose, and 12.5 mg/L G418 and grown 48h at 29°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains are grown in the same media, but lacking G418. 250 uL of minimal induction media is added and contains IX YNB and 1% (v/v) methanol. Samples of 30 uL are taken thereafter two times a day with replacement of 30 uL of fresh induction media containing IX YNB and 5% (v/v) methanol. The samples are clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. A final sample is taken at 120 h and run on an HPLC and analyzed for production of lactic acid as previously described.

[0152] In this example, transformants of K. pas tons strain Y-1047 are found to produce lactic acid. The wild type control strains do not produce detectable levels of lactic acid.

[0153] Example 4. In this example a methane consuming strain, M. extorquens, is cultured to produce lactic acid from methane.

Construction of synthetic nucleic acid sequences conferring expression of an LDH expression vector for transformation into the methane-consuming M. extorquens cells

[0154] A plasmid is constructed that contains an expression cassette consisting of the M. extorquens sigma 54 promoter followed by a lactic acid dehydrogenase (Prot Accession Number YP_001577351.1). This plasmid, pLC0004 also contains the coding region for selection of kanamycin resistant clones.

Transformation of synthetic nucleic acid sequences conferring expression of a LDH into the methane consuming M. extorquens cells and production of lactic acid

[0155] The M capsulatus cells are prepared for transformation by electroporation. Colonies are picked into 5 mL media (as described above) and grown at 29°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of media until stationary growth as estimated by OD 600nm. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C 10% glycerol ddH20, and re- pelleted. Finally cells are resuspended in lmL of 10% 4°C glycerol and aliquoted into 50 uL volumes for electroporation. 50 ng of pLC0004 is added to 50uL of the cells, transferred to a 1mm cuvette and transformed by electroporation into the M capsulatus cells. Transformant strains are recovered by growing in media described above for 24h at 25°C with 0.5% methanol. Serially-diluted aliquots of the strains are then plated onto solid agar plates containing 25 ug/mL kanamycin. Colonies appear within 48h and are confirmed for plasmid transformation by colony PCR. Colonies are further screened and determined to contain the Idh gene.

[0156] Colonies are picked into 300 uL of minimal media as described above and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm in a dessicator that contains 1 : 1 methane : air. Wildtype control strains are grown in the same media, but lacking kanamycin. Samples of 30 uL were taken thereafter two times a day with replacement of the methane:air atmosphere. The samples are clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. Final samples are taken at 120 h was run on an HPLC and analyzed for production of lactic acid, as described previously.

[0157] In this example, M. extorquens cells modified to express the

YP_001577351.1 LDH are determined to produce lactic acid. The wild type control strains do not produce detectable levels of lactic acid.

[0158] Example 5. In this example a methane consuming strain, M. extorquens, is cultured to produce succinic acid from methane.

[0159] DNA expression constructs are designed and constructed for transformation and expression of a pyruvate carboxylase and citrate synthase in M. extorquens AMI. A plasmid is constructed that contains an expression cassette consisting of the M. extorquens sigma 54 promoter followed by an operon that contains genes encoding a pyruvate carboxylase and citrate synthase (Prot Accession Numbers: AAF09095.1, NP_390792.1). This plasmid, pLC0005 also contains the coding region for selection of kanamycin resistant clones.

Transformation of synthetic nucleic acid sequences conferring expression of a pyruvate carboxylase and citrate synthase into the methane consuming M. extorquens cells and production of succinic acid

[0160] The M. extorquens cells are prepared for transformation by electroporation. Colonies are picked into 5 mL media (as described above) and grown at 29°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of media until stationary growth as estimated by OD 600nm. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C 10% glycerol ddH20, and re- pelleted. Finally cells are resuspended in lmL of 10% 4°C glycerol and aliquoted into 50 uL volumes for electroporation. 50 ng of pLC0005 is added to 50uL of the cells, transferred to a 1mm cuvette and transformed by electroporation into the M extorquens cells. Transformant strains are recovered by growing in media described above for 24h at 25°C with 0.5% methanol. Serially-diluted aliquots of the strains are then plated onto solid agar plates containing 25 ug/mL kanamycin. Colonies appear within 48h and are confirmed for plasmid transformation by colony PCR. Colonies are further screened and determined to contain the plasmid. [0161] Colonies are picked into 300 uL of minimal media as described above and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm in a dessicator that contains 1: 1 methane: air. Wildtype control strains are grown in the same media, but lacking kanamycin. Samples of 30 uL were taken thereafter two times a day with replacement of the methane: air atmosphere. The samples are clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. Final samples are taken at 120 h was run on an HPLC and analyzed for production of succinic acid, as described previously.

[0162] In this example, M. extorquens cells are modified to express a pyruvate carboxylase and citrate synthase are determined to produce higher levels of succinic acid than the wild type control strains. Those of skill in the art will appreciate from this disclosure that one need not introduce genes for all enzymes in a production pathway into a host cell to increase production levels of a desired compound. Instead, one can simply insert the one or more genes that result in the increased production of enzymes that are otherwise rate-limiting in the production of the desired product. Thus, consistent with the methods of the invention, succinic acid production can be achieved via recombinant expression of one or more enzymes in the metabolic pathway.

[0163] Example 6. In this example a methane consuming strain, M. extorquens, is cultured to produce 1,3-propanediol from methane.

[0164] DNA expression constructs are designed and constructed for transformation and expression of a glycerol phosphatase, GPP2, from S. cerevisiae, a glycerol dehydrogenase, DhaBl-3 from Klebsiella pneumoniae, and a PDO reductase enzyme, DhaT, from Klebsiella pneumoniae in M. extorquens AMI. A plasmid is constructed that contains an expression cassette consisting of the M. extorquens sigma 54 promoter followed by an operon that contains genes encoding a glycerol phosphatase, GPP2, from S. cerevisiae, a glycerol dehydrogenase, DhaBl-3 from Klebsiella pneumoniae, and a PDO reductase enzyme, DhaT, from Klebsiella pneumoniae. This plasmid, pLC0006 also contains the coding region for selection of kanamycin resistant clones.

Transformation of synthetic nucleic acid sequences conferring expression of a 1,3- PDO synthesis operon into the methane consuming M. extorquens cells and production of 1,3-PDO [0165] The M. extorquens cells are prepared for transformation by electroporation. Colonies are picked into 5 mL media (as described above) and grown at 29°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of media until stationary growth as estimated by OD 600nm. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C 10% glycerol ddH20, and re- pelleted. Finally cells are resuspended in ImL of 10% 4°C glycerol and aliquoted into 50 uL volumes for electroporation. 50 ng of pLC0006 is added to 50uL of the cells, transferred to a 1mm cuvette and transformed by electroporation into the M extorquens cells. Transformant strains are recovered by growing in media described above for 24h at 25°C with 0.5% methanol. Serially-diluted aliquots of the strains are then plated onto solid agar plates containing 25 ug/mL kanamycin. Colonies appear within 48h and are confirmed for plasmid transformation by colony PCR. Colonies are further screened and determined to contain the plasmid.

[0166] Colonies are picked into 300 uL of minimal media as described above and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm in a dessicator that contains 1 : 1 methane : air. Wildtype control strains are grown in the same media, but lacking kanamycin. Samples of 30 uL are taken thereafter two times a day with replacement of the methane:air atmosphere. The samples are clarified by centrifugation and filtered on a 0.45 μιη membrane prior to HPLC analysis. Final samples are taken at 120 h was run on an HPLC and analyzed for production of 1,3- PDO, as described previously.

[0167] In this example, M. extorquens cells are modified to express a 1,3-

PDO synthesis operon. Wild type control strains produce no detectable levels of 1,3- PDO. Those of skill in the art will appreciate from this disclosure that one need not introduce genes for all enzymes in a production pathway into a host cell to increase production levels of a desired compound. Instead, one can simply insert the one or more genes that result in the increased production of enzymes that are otherwise rate- limiting in the production of the desired product. Thus, consistent with the methods of the invention, 1,3-PDO production can be achieved via recombinant expression of one or more enzymes in the metabolic pathway.

[0168] Example 7. In this example, S. cerevisiae modified host cells are cultured to catabolize methanol. [0169] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1 promoter common to yeasts followed by methanol dehydrogenase (AN A42952), a PGI terminator, a TEF2 promoter, a formaldehyde dehydrogenase (BAA04743.1), a TPI1 terminator, a FBAl promoter, a formate dehydrogenase (AN EFW95288.1), a TDH3 terminator, a TDH3 promoter, an hexulose phosphate synthase (AN ABJ63600.1), a FBAl terminator, an PDC1 promoter, a hexuloisomerase (AN WP_003552753.1), and finally a THD2 terminator. The plasmid also contained the coding region for selection of uracil prototrophic clones and is designated pLC0201.

[0170] Integration of synthetic nucleic acid sequences conferring expression of methanol catabolizing genes into yeast cell genomes and catabolism of methanol. S. cerevisiae cells are prepared for transformation by electroporation. Colonies were picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in ImL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0201 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0201 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC-Ura agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette. This strain of yeast is designated LSM001.

[0171] The above example utilizes S. cerevisiae and illustrates an embodiment in which the host cell is modified to include recombinant versions of all 5 pathway genes. Not all S. cerevisiae (and not all other host cells) require all five genes for methanol catabolism. In many cases, the host cell will contain at least one endogenous enzyme that carries out the same function a desired pathway enzyme. Those of skill in the art will appreciate from this disclosure that one need not introduce genes for all enzymes in a production pathway into a host cell to increase production levels of a desired compound. Instead, one can simply insert the one or more genes that result in the increased production of enzymes that are otherwise rate-limiting or absent in the production of the desired product. Thus, consistent with the methods of the invention, methanol catabolism can be achieved via recombinant expression of one or more enzymes in the metabolic pathway.

[0172] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains are grown in the same media, but supplemented with uracil. Cells are pelleted, washed with ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 1% (v/v) methanol. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methanol catabolism at 24, 72, and 120h time points.

[0173] In this example, transformants of S.cerevisiae cells are determined to catabolize methanol and grow to higher cell densities compared to the wildtype control cells, which are unable to grow.

[0174] Example 8. In this example, P. kudriavzevii modified host cells are cultured to catabolize methanol.

[0175] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1 promoter common to yeasts followed by methanol dehydrogenase (AN A42952), a PGI terminator, a TEF2 promoter, an formaldehyde dehydrogenase (BAA04743.1), a TPI1 terminator, a FBA1 promoter, a formate dehydrogenase (AN EFW95288.1), a TDH3 terminator, a TDH3 promoter, an hexulose phosphate synthase (AN ABJ63600.1), a FBA1 terminator, an PDC1 promoter, a hexuloisomerase (AN WP_003552753.1), and finally a THD2 terminator. The plasmid also contains the coding region for selection of hygromycin resistant clones and is designated pLC0202.

[0176] Integration of synthetic nucleic acid sequences conferring expression of methanol catabolizing genes into yeast cell genomes and catabolism of methanol. P. kudriavzevii cells are prepared for transformation by electroporation. Colonies are picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0202 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0202 was added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC-Ura agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette. This strain is designated LPKM001.

[0177] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains were grown in the same media, but supplemented with uracil. Cells are pelleted, washed with ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 1% (v/v) methanol. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methanol catabolism at 24, 72, and 120h time points.

[0178] In this example, transformants of P. kudriavzevii cells are determined to catabolize methanol and grow to higher cell densities compared to the wildtype control cells, which are unable to grow.

[0179] Example 9. In this example, S. cerevisiae modified host cells are cultured to catabolize methanol.

[0180] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1 promoter common to yeasts followed by methanol dehydrogenase (AN A42952), a PGI terminator, a TEF2 promoter, an formaldehyde dehydrogenase (BAA04743.1), a TPI1 terminator, a FBA1 promoter, a formate dehydrogenase (AN EFW95288.1), a TDH3 terminator, a TDH3 promoter, SHMT (AN AAA64456.1), a FBA1 terminator, an PDC1 promoter, a SGAT (AN WP_003597639.1), and finally a THD2 terminator. The plasmid also contained the coding region for selection of uracil prototrophic clones.

[0181] Integration of synthetic nucleic acid sequences conferring expression of methanol catabolizing genes into yeast cell genomes and catabolism of methanol. S. cerevisiae cells are prepared for transformation by electroporation. Colonies were picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in ImL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0203 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0203 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC-Ura agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette.

[0182] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30 C in 96 well plates with shaking set to 350 rpm. Wildtype control strains were grown in the same media, but supplemented with uracil. Cells are pelleted, washed with ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 1% (v/v) methanol. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methanol catabolism at 24, 72, and 120h time points.

[0183] In this example, transformants of S.cerevisiae cells are determined to catabolize methanol and grow to higher cell densities compared to the wildtype control cells, which are unable to grow.

[0184] Example 10. In this example, the invention provides modified S. cerevisiae cells for the production of malonic acid from methanol.

[0185] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1 promoter common to yeasts followed by alcohol dehydrogenase (AN A42952), a PGI terminator, a TEF2 promoter, an aldehyde dehydrogenase (BAA04743.1), a TPI1 terminator, a FBAl promoter, a formate dehydrogenase (AN EFW95288.1), a TDH3 terminator, a TDH3 promoter, an hexulose phosphate synthase (AN ABJ63600.1), a FBAl terminator, an PDC1 promoter, a hexuloisomerase (AN WP_003552753.1), and finally a THD2 terminator. The plasmid also contained the coding region for selection of uracil prototrophic clones and designated pLC0201 previously in Example 7.

[0186] Integration of synthetic nucleic acid sequences conferring expression of methanol catabolizing genes into an S. cerevisiae strain designated LYM025, and catabolism of methanol. S. cerevisiae cells LYM025 were previously modified to contain a malonyl-CoA hydrolase and produce malonic acid. This strain was developed by constructing two integration cassettes that provide protorophy for histidine and leucine respectively. Each integration cassette was constructed to contain a TDH3 promoter followed by a malonyl-CoA hydrolase F6AA82(3) followed by a TPI terminator. In this example and elsewhere we refer to the F6AA82(3) enzyme which is derived from the F6AA82 enzyme and contains three amino acid mutations (E95N/Q384A/F304R). The two cassettes were transformed into B4741 background strain, integrated into the chromosome. The integration cassette containing the his3 and F6AA82(3) genes integrated into chromosome 16, coordinates 7776871-7776568. The integration cassette containing the leu2 gene integrated into chromosome 15, 969240-969471 (leu2). This strain that contains two copies of F6AA82(3) was designated LYM025. To enable these cells to catabolize methanol they are prepared for transformation by electroporation. Colonies are picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0201 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0201 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC-Ura agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette. This strain is designated as LSM002.

[0187] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains were grown in the same media, but supplemented with uracil. Cells are pelleted, washed once with 1 mL ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 1% (v/v) methanol. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methanol catabolism and malonic acid production at 24, 72, and 120h time points.

[0188] In this example, transformants of S.cerevisiae strain LYM025 are determined to catabolize methanol and grow to higher cell densities compared to the wildtype control cells, which are unable to grow. They are also determined to produce malonic acid.

[0189] Example 11. In this example, the invention provides modified P. kudriavzevii cells for the production of malonic acid from methanol.

[0190] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1 promoter common to yeasts followed by alcohol dehydrogenase (AN A42952), a PGI terminator, a TEF2 promoter, an aldehyde dehydrogenase (BAA04743.1), a TPI1 terminator, a FBA1 promoter, a formate dehydrogenase (AN EFW95288.1), a TDH3 terminator, a TDH3 promoter, an hexulose phosphate synthase (AN AN ABJ63600.1), a FBA1 terminator, an PDC1 promoter, a hexuloisomerase (AN WP_003552753.1), and finally a THD2 terminator. The plasmid also contains the coding region for selection of hygromycin resistant clones and was designated pLC0202 in Example 8 above.

[0191] Integration of synthetic nucleic acid sequences conferring expression of methanol catabolizing genes into P. kudriavzevii cell genome LPK2010, catabolism of methanol and production of malonic acid. P. kudriavzevii cells LPK2010 previously modified to contain a malonyl-CoA hydrolase (F6AA82(3)) and produce malonic acid are prepared for transformation by electroporation. Colonies are picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells were resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0202 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0202 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC-hygromycin agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette.

[0192] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains are grown in the same media, but without hygromycin. Cells are pelleted, washed once with 1 mL ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 1% (v/v) methanol. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methanol catabolism and malonic acid production at 24, 72, and 120h time points.

[0193] In this example, transformants of LPK2010 cells are determined to catabolize methanol and grow to higher cell densities compared to the wildtype control cells, which are unable to grow. They are also determined to produce malonic acid. [0194] Example 12. In this example, S. cerevisiae cells that catabolize methane and methods for their culturing are provided in accordance with the invention.

[0195] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1, a methane monooxygenase gene mmoX (AN CAA39068.2), a PGI terminator, a TEF2 promoter, mmoY (AN CAA39069.1), a TPI1 terminator, a FBA1 promoter mmoZ (AN CAA39070.1), a TDH3 terminator, a TDH3 promoter, mmoB (AN CAA39071.1), a FBA1 terminator, an PDC1 promoter, mmoC (AN CAB45257.1), and finally a THD2 terminator to drive expression in S. cerevisiae. The plasmid also contains the coding region for selection of hygromycin resistant clones and is designated pCL0301.

[0196] Integration of synthetic nucleic acid sequences conferring expression of methane catabolizing genes into S. cerevisiae strain LSMOOl and catabolism of methane. S. cerevisiae cells, designated as LSMOOl are modified to catabolize methanol as previously described (see Example 7), are prepared for transformation by electroporation. Colonies are picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0301 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0301 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC- Hyg agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette. This strain of yeast is designated LSM0301.

[0197] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains are grown in the same media. Cells are pelleted, washed with ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 50% methane:air atmosphere in a dessicator as described above. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methane catabolism at 24, 72, and 120h time points. The dessicator's atmosphere is replenished with fresh methane: air at every sampling point.

[0198] In this example, S.cerevisiae strain LSM0301 is determined to catabolize methane and grow to higher cell densities compared to the wildtype control cells, which are unable to grow.

[0199] Example 13. In this example, P. kudriavzevii cells that catabolize methane and methods for their culturing are provided in accordance with the invention.

[0200] A chromosomally-integrating DNA construct is built that contains tandem expression cassettes consisting of the TEF1, a methane monooxygenase gene mmoX (AN CAA39068.2), a PGI terminator, a TEF2 promoter, mmoY (AN CAA39069.1), a TPI1 terminator, a FBA1 promoter mmoZ (AN CAA39070.1), a TDH3 terminator, a TDH3 promoter, mmoB (AN CAA39071.1), a FBA1 terminator, an PDC1 promoter, mmoC (AN CAB45257.1), and finally a THD2 terminator to drive expression in P. kudriavzevii. The plasmid also contained the coding region for selection of hygromycin resistant clones and is designated pCL0302.

[0201] Integration of synthetic nucleic acid sequences conferring expression of methane catabolizing genes into P.kudriavzevii strain LPKMOOl and catabolism of methane. P. kudriavzevii cells, designated as LPKMOOl are modified to catabolize methanol as previously described (see Example 8), are prepared for transformation by electroporation. Colonies were picked into 5 mL YPD and grown overnight at 30°C, 200 rpm. 1 mL of the overnight cultures are subcultured in 50mL of YPD for 6h. The cultures are pelleted by centrifugation (2000 rpm, 5min, 4°C), washed with 4°C ddH20, and re-pelleted. Finally cells are resuspended in lmL of 1M 4°C sorbitol and aliquoted into 50 uL volumes for electroporation. The plasmid pLC0302 is prepared for transformation by digesting with Sacl and purifying on a silica column. 5uL of linearized pLC0302 is added to 50uL of the yeast cells, transferred to a 2mm cuvette and transformed by electroporation into the cells (1500V charging, 200ohm resistance, 25uF capacitance). Transformant strains are recovered by plating onto SC- Hyg agar plates. Colonies appear within 4 days and are confirmed for genomic integration by colony PCR. All colonies screened are determined to contain the 5 gene cassette. This strain of yeast is designated LPKM301.

[0202] Colonies are picked into 300 uL of minimal media containing IX

YNB, 2% glucose and grown 48h at 30°C in 96 well plates with shaking set to 350 rpm. Wildtype control strains are grown in the same media. Cells are pelleted, washed with ddH20 and resuspended in 500 uL of minimal media comprised of IX YNB and 50% methane:air atmosphere in a dessicator as described above. Culture densities are measured by spectrophotometry at OD 600nm and analyzed via HPLC for methane catabolism at 24, 72, and 120h time points. The dessicator's atmosphere is replenished with fresh methane: air at every sampling point.

[0203] In this example, P. kudriavzevii strain LPKM301 is determined to catabolize methane and grow to higher cell densities compared to the wildtype control cells, which are unable to grow.

Claims

Claims

1. A recombinant host cell that contains a methanol oxidation pathway and catabolizes methanol, wherein said host cell contains a heterologous enzyme, wherein said enzyme is either at least one enzyme of said methanol oxidation pathway or an enzyme in a pathway for making a product from methanol.

2. The host cell of claim 1 that further comprises a formaldehyde assimilation pathway.

3. The host cell of claim 2 that further comprises a formaldehyde oxidation pathway.

4. The host cell of claim 3 that further comprises a formate oxidation pathway.

5. The host cell of claim 1 that comprises a formaldehyde oxidation, and formate oxidation pathway.

6. The host cell of claim 1 that naturally catabolizes methane or has a methane oxidation pathway, at least one enzyme of which is heterologous to the host cell.

7. The recombinant host cell of claim 6 that comprises at least one heterologous enzyme of a methane oxidation pathway.

8. The host cell of any of the claims 1 to 7 wherein said methanol oxidation pathway includes a methanol dehydrogenase enzyme and said methanol dehydrogenase enzyme is heterologous to the host cell.

9. The host cell of any of the claims 1 to 7 wherein said methanol oxidation pathway includes a methanol oxidase enzyme and catalase enzyme and one or both of said enzymes is heterologous to the host cell.

10. The host cell of any of claims 2 to 4 wherein said formaldehyde assimilation pathway includes a hexulose 6-phosphate synthase enzyme and 6-phospho-3- hexuloisomerase enzyme and one or both of said enzymes is heterologous to the host cell.

11. The host cell of any of the claims 2 to 4 wherein said formaldehyde assimilation pathway includes a serine hydroxymethyltransferase enzyme and a serine glyoxylate aminotransferase enzyme and one or both of said enzymes is heterologous to the host cell.

12. The host cell of any of the claims 2 to 4 wherein said formaldehyde assimilation pathway is a dihydroxyacetone synthase enzyme that is heterologous to the host cell.

13. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway enzymes are hexulose 6-phosphate synthase and 6-phospho-3- hexuloisomerase and one or both of said enzymes are heterologous to the host cell.

14. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway is a thiol-independent formaldehyde dehydrogenase enzyme that is heterologous to the host cell.

15. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway is a glutathione-dependent formaldehyde dehydrogenase enzyme that is heterologous to the host cell.

16. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway is a THF-dependent formaldehyde dehydrogenase enzyme that is heterologous to the host cell.

17. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway is a H₄MPT-dependent formaldehyde dehydrogenase enzyme and said enzyme is heterologous to the host cell.

18. The host cell of any of the claims 3 to 5 wherein said formaldehyde oxidation pathway is a mycothiol-dependent formaldehyde dehydrogenase enzyme and said enzyme is heterologous to the host cell.

19. The host cell of claim 3 or 5 wherein said formate oxidation pathway is a formate dehydrogenase enzyme that is heterologous to the host cell.

20. The host cell of claim 8 wherein said methanol dehydrogenase enzyme is selected from the group consisting of: EIJ78800.1, EIJ83424.1, Q2NGI3.1, P42327.1, EDZ12794.1, AAL00968.1, EGW40324.1, ABM09061.1, ABK36659.1, EER41381.1, EKE58762.1, EMP12409.1, EMT62998.1, WP_011773405.1, WP_005325714.1, WP_009616336.1, WP_010634498.1, YP_856044.1, YP_946457.1, EEQ93101.1, AGM43456.1, YP_008042803.1, EGD44968.1, EKD15160.1, Q2NGV2.1, EEN88363.1, WP_003942095.1, AAM98772.1, EJO15902.1, P31005.3, Q2NEN0.1, P36234.2, Q07511.2, 013437.1, Q2NFL8.1, YP_448353.1, YP_447794.1, YP_447905.1, YP_448089.1, YP_448327.1, YP_448340.1, EIJ80893.1, ADJ47941.1, AF079652.1, WP_013227992.1, YP .006552597.1, YP_003768343.1, AEK44841.1, YP_005534298.1, A42952, WP_006360532.1, WP_006368341.1, WP_006368508.1, WP_006553557.1, WP_006865796.1, WP_007240717.1, WP_007315923.1, WP_008355702.1, WP_009154061.1, WP_009678089.1, WP_010229649.1, WP_010242003.1, WP_010591966.1, WP_013809089.1, WP_014361145.1, WP_014671061.1, WP_014805764.1, WP_015746107.1, WP_006932857.1, YP_118435.1, YP_705992.1, YP_006570807.1, YP_006668667.1, WP_005173993.1, WP_010842989.1, CCW10366.1, YP_890461.1, YP_004495541.1, CAM01686.1, AFM20522.1, AEV73570.1, EME62476.1, EHR50676.1, EON32465.1, YP_001104611.1, YP_006442847.1, YP_005000785.1, ABM16248.1, ACV77191.1, ACY21185.1, YP_956254.1, YP_003200180.1, YP_003273078.1, Q53062.2, EMP12409.1, EGD44968.1, ELQ86070.1, WP_003897664.1, EEN88363.1, WP_003942095.1, P14775.1, CAX26756.1, CCB63722.1, CCE25108.1, ACS42166.1, AAU92932.1, YP_113284.1, YP_002965443.1, YP_003070568.1, YP_004674298.1, YP_004918691.1, BAA23275.1, AAF43728.1, ABE77339.1, CBE67231.1, YP_003205076.1, ACB32199.1.

21. The host cell of claim 9 wherein said methanol oxidase enzyme is selected from the group consisting of: EIJ79022.1, EIJ79690.1, ELJ82004.1, ELJ82005.1, ELJ82370.1, EIJ82371.1, EIJ82924.1, EU84348.1, ACL41485.1, P81156.1, NP_566729.1, YP_002489574.1, AEE76762.1, EU78359.1, ELJ78393.1, EIJ79443.1, EIJ81633.1, EIJ78360.1, EIJ79444.1, EIJ81632.1, EU82043.1, EU77587.1, EIJ78292.1, EIJ78367.1, ELJ82955.1, 3Q9T_A, 3Q9T_B, 3Q9T_C, 4AAH_B, 4AAH_D, 4AAH_A, 4AAH_C, 2JBV_A, 2JBV_B, ELR66099.1, ELR66572.1, EJD40360.1, EJD41743.1, EJD50220.1, WP_007463330.1, WP_007464972.1, ABI14440.1, EJU01918.1, EOD81270.1, WP_002535908.1, AAA34321.1, AFO55203.1, Q00922.1, P04842.1, P04841.1.

22. The host cell of claim 9 wherein said catalase enzyme is selected from the group consisting of: WP_008574782.1, WP_009492216.1, WP_009550547.1, WP_013512707.1, WP_013964265.1, WP_014035677.1, WP_014813351.1, WP_015766802.1, WP_007917260.1, WP_007919761.1, WP_003599145.1, WP_006081102.1, WP_006085349.1, WP_011268380.1, WP_011336028.1, WP_011348926.1, WP_011548133.1, WP_011796211.1, WP_011982131.1, WP_012004841.1, WP_012313921.1, WP_012551601.1, WP_012768725.1, WP_015885989.1, WP_005989373.1, WP_005994599.1, WP_006086507.1, WP_006452102.1, WP_007681311.1, WP_007731213.1, WP_007805430.1, WP_007854113.1, WP_007895272.1, WP_007930032.1, WP_007937270.1, WP_007950986.1, WP_007963762.1, WP_008010502.1, WP_008069984.1, WP_008084630.1, WP_008109957.1, WP_008115964.1, WP_009128618.1, WP_013551748.1, WP_013594560.1, WP_007909771.1, WP_006079727.1, WP_008576465.1, CAB56850.1, and CAA38588.1.

23. The host cell of claim 10 wherein said hexulose-6-phosphate synthase is selected from the group consisting of: YP_115430.1, YP_176845.1, YP_185502.1, YP_301709.1, YP_301716.1, YP_302238.1, YP_544362.1, YP_809790.1, YP_001055461.1, YP_002429149.1, YP_002559440.1, YP_003485610.1, YP_003502426.1, YP_005707964.1, YP_005855367.1, YP_005858493.1, YP_006194711.1, YP_006470384.1, YP_006709388.1, YP_006700692.1, YP_006042078.1, YP_007969207.1, YP_187812.1, YP_003872970.1, YP_004479862.1, YP_004732614.1, YP_005398998.1, YP_005399644.1, EAR68750.1, EDJ89466.1, EAV47244.1, EIM05259.1, ABN07165.1, EKC82856.1, ELK44699.1, CCE75044.1, EON80240.1, EON82083.1, EON85721.1, WP_015489823.1, YP_007685384.1, NP_461682.1, YP_152642.1, YP_153252.1, ABN07618.1, AAM30911.1, NP_371094.1, YP_833183.1, NP_577949.1, YP_040023.1 among others.

24. The host cell of claim 10 wherein said 6-phospho-3-hexuloisomerase is selected from the group consisting of: WP_006215287.1, WP_009166436.1, YP_416017.1, YP_709135.1, YP_006938326.1, YP_006938819.1, YP_006544334.1, YP_006544816.1, YP_006628530.1, YP_006709389.1, YP_007394192.1, YP_007491001.1, YP_007685385.1, YP_007867634.1, YP_008050389.1, EPC12662.1, EPC13144.1, EPC13509.1, EPC17633.1, EPC18642.1, EPC20316.1, EPC20344.1, EPC23618.1, EPC25770.1, EPC35016.1, EPC35045.1, EPC36952.1, EPC38837.1, WP_016383230.1, WP_016384253.1, CCW20899.1, WP_004607138.1, WP_009535380.1, AGP27571.1, YP_003852311.1, YP_005741297.1, YP_008167735.1, AGP67227.1, AGP71929.1, WP_020613988.1, EHP85623.1, AFJ02830.1, AFI85301.1, AEF96627.1, YP_004370221.1, ADK83279.1, ADN34938.1, AEB09040.1, YP_003805873.1, and YP_003893376.1.

25. The host cell of claim 11 wherein said serine hydroxymethyltransferase is selected from the group consisting of: NP_721474.1, NP .629503.1, YP_003859985.1, YP .003892463.1, YP_003912112.1, YP_003966576.1, YP_004004363.1, YP_004041630.1, YP_004049872.1, YP_004058129.1, YP_004060026.1, YP_004100792.1, YP_004162026.1, YP_004168653.1, YP_004176470.1, YP_004179261.1, YP_004194342.1, YP_004270502.1, YP_004411685.1, CAA20173.1, CAH58416.1, EHQ06730.1, EHQ06869.1, ADY57546.1, AEK23220.1, AFY34171.1, AFY39569.1, AFY42646.1, AFZ02160.1, AFZ11135.1, AFZ31432.1, AFZ55234.1, ELS33741.1, YP_007049796.1, YP_007067005.1, YP_007072403.1, YP_007140645.1, YP_007128592.1, YP_004740327.1, YP_004267547.1, YP_007138132.1, YP_007163278.1, CAN02604.1, CAL34552.1, WP_007920920.1, WP_008070862.1, WP_013565000.1, WP_013683034.1, YP_641292.1, and AAA33687.1.

26. The host cell of claim 12 wherein said dihydroxyacetone synthase is selected from the group consisting of: YP_002827132.1, YP_002582246.1, YP_003979610.1, YP_004358513.1, YP_004424802.1, YP_006902571.1, YP_008040311.1, CAQ71386.1, EDZ41023.1, EEB84781.1, WP_012355607.1, EOR24424.1, WP_011280657.1, WP_014149643.1, WP_014743098.1, YP_002007443.1, WP_010867962.1, NP_126522.1, EAU39726.1, ACG30854.1, EAP77421.1, EAQ24413.1, EHK64205.1, XP_002279236.1, EHK74228.1, EIE24183.1, EDZ44855.1, EDZ61119.1, EEE38769.1, EJO31580.1, EMD98318.1, WP_012251181.1, WP_014329402.1, BAB20811.1, CAE39480.1, XP_001702106.1, XP_001702107.1, ED097195.1, ED097196.1, NP_886332.1, ABV95058.1, YP_001534659.1, CCD91795.1, CCD86741.1, CCE00291.1, CCE12095.1, P84188.1, P84187.1, Q56YA5.2, and NP_001148339.1.

27. The host cell of claim 14 wherein said thiol-independent formaldehyde dehydrogenase is selected from the group consisting of: YP_947616.1, YP_990317.1, YP_001061825.1, YP_001079157.1, YP_001074774.1, YP_001100315.1, YP_001108655.1, YP_001351520.1, YP_001853994.1, YP_002234932.1, YP_002443394.1, YP_002784270.1, YP_002875140.1, YP_004753733.1, YP_005978120.1, YP_005984383.1, YP_006264311.1, YP_006277264.1, YP_006326331.1, YP_006556791.1, YP_007598452.1, YP_004720850.1, NP_521614.1, YP_105240.1, YP_262781.1, ΝΡ_254108.1, ΝΡ_354566.2, ΥΡ_001024816.1, ΥΡ_003898523.1, ΥΡ_004278825.1, ΥΡ_004571417.1, ΥΡ_005189801.1, ΥΡ_005369175.1, AGK49647.1, ΥΡ_007921814.1, CAH38002.1, AFY22196.1, ΥΡ_110566.1, P46154.3, ΥΡ_770646.1, ΥΡ_007032001.1, AAN65959.1, AFR28906.1, AGL87404.1, ΥΡ_006661944.1, ΥΡ_008002908.1, NP 742495.1, BAN52208.1, ΥΡ_008111392.1, and AEV65401.1.

28. The host cell of claim 15 wherein said glutathione-dependent formaldehyde dehydrogenase is selected from the group consisting of: ELZ96420.1 EMA06490.1, EMA10473.1, EMA10775.1, EMA27812.1, EMA37918.1 EMA44468.1, EMA49083.1, EMA52447.1, EMA53947.1, AFY22196.1 YP_003736860.1, YP_007032001.1, YP_007227278.1, BAA04743.1 BAC16635.1, AEH35491.1, YP_004595370.1, EHB85873.1 YP_001265707.1, YP_003204070.1, YP_003394714.1, YP_005060061.1

YP_002487096.1 YP_003396210.1, YP_001583515.1, YP_004183484.1 YP_004216923.1 AAF54571.1, NP_524310.1, YP_001363686.1 YP_001666600.1 YP_001751724.1, YP_001859509.1, YP_001888795.1 YP_002489735.1 YP_003084575.1, YP_003389378.1, YP_003607370.1 YP_004333498.1 YP_004476269.1, NP_864907.1, AEW04442.1 YP 005256114.1 ACY58243.1, ACY62296.1, ADE64279.1, AEL74298.1 AAM86220.1, and CAC85637.1.

29. The host cell of claim 16 wherein said THF-dependent formaldehyde dehydrogenase is selected from the group consisting of FolD and MtdA.

30. The host cell of claim 17 wherein said H₄MPT-dependent formaldehyde dehydrogenase is selected from the group consisting of MtdB and MtdA.

31. The host cell of claim 18 wherein said mycothiol-dependent formaldehyde dehydrogenase is selected from the group consisting of: ACY98017.1 ADB31528.1, ADB75512.1, ADB77300.1, ADW06553.1, ADG96617.1 ADG98911.1, ADL46786.1, ADP80806.1, ADP81795.1, ADU10609.1 AEH09234.1, AEM85230.1, AEM88079.1, AEN09039.1, EGE45461.1 ADD41110.1, YP_002489517.1, YP_003101495.1, YP_003102324.1 YP .003200733.1, YP .003300055.1, YP .003380327.1, YP_003409883.1 YP_003411671.1, YP_003510203.1, YP .003657448.1, YP_003659742.1 YP_003836362.1, YP_003114870.1, YP_003271336.1, YP_004016676.1 YP_004017665.1, YP_004084760.1, ΥΡ_004583155.1, ΥΡ_004815510.1, ΥΡ_004818359.1, ΥΡ_004926070.1, EHB53720.1, EHI82325.1, ADB75524.1, ADG77049.1, AEA22904.1, AEM84190.1, ΥΡ_003409895.1, ΥΡ_003645388.1, ΥΡ_004330757.1, ΥΡ_004814470.1, ADH67817.1, and ΥΡ_003680323.1.

32. The host cell of claim 19 wherein said formate dehydrogenase is FdhA.

33. The host cell of any of any claims 1 to 32 wherein the cell is of a species of a genus selected from the group consisting of Amycolatopsis, Bacillus, Brevibacillus, Burkholderia, Candida, Candidatus Methylomirabilis, Corynebacterium, Hansenula, Hyphomicrobium, Komagataella, Methanomonas, Methyl obacillus, Methylobacter, Methylobacterium, Methylocaldum, Methylococcus, Methylocystis, Methylomicrobium, Methylophaga, Methylophilus, Methylosinus, Methylosphaera, Mycobacterium, Ogataea, Paracoccus, Pichia, Pseudomonas, Rhodobacter, Rhodococcus, Saccharomyces, Schizosaccharomyces, and Yarrowia.

34. The host cell of any of claims 1 to 32 wherein the species is a strain of a species selected from the group consisting of Amycolatopsis methanolica, Bacillus methanolicus, Bacillus subtilis, Brevibacillus brevis SI, Burkholderia xenovorans LB400, Candida boidinii, Candida methanolovescens, Candida methylica, Candidatus Methylomirabilis oxyfera, Corynebacterium glutamicum, Escherichia coli K-12 substr MG1655, Hansenula polymorpha, Hiphomicrobium methylovorum, Hiphomicrobium zavarzinii, Hyphomicrobium zavarzinii ZV580, Hyphomicrobium zavarzinii ZV580 , Issatchenkia orientalis, Komagataella methanolica, Komagataella pastoris, Methylobacillus flagellatus, Methylobacillus glycogenes, Methylobacter marinus, Methylobacter whittenburyi , Methylobacterium extorquens AMI, Methylobacterium organophilum, Methylobacterium rhodesianum, Methylococcus capsulatus Bath, Methylococcus thermophilus, Methylocystis echinoides, Methylocystis minimus, Methylocystis parvus, Methylocystis pyriformis, Methylomicrobium agile, Methylomicrobium album, Methylomicrobium pelagicum, Methylomonas aminofaciens 77a, Methylomonas aurantiaca, Methylomonas fodinarum, Methylomonas methanica, Methylomonas methanolica, Methylophilus methylotrophus, Methylosinus sporium, Methylosinus trichosporium OB3b, Methylosphaera japanese, Mycobacterium gastri MN19, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv, Ogataea angusta, Paracoccus denitrificans, Paracoccus versutus, Pichia kudriavzevii, Pichia methanolica, Pichia pastoris, Pseudomonas sp , Rhodobacter sphaeroides, and Rhodococcus erythropolis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowia lipolytica.

35. A method of producing an organic acid in a recombinant host cell of any claims 1 to 34, said method comprising culturing said host cell in a media containing methanol and under culture conditions wherein said organic acid is produced.

36. The method of claim 35, wherein said organic acid is malonic acid.

37. The method of claim 36, wherein said organism contains a heterologous gene that expresses a malonyl-CoA hydrolase.

38. The method of claim 37, wherein said malonyl-CoA hydrolase gene is selected from the group consisting of S. cerevisiae EHD3, EHD3 (E124S), EHD3 (E124A, E308V), EHD3 (E124H), EHD3 (E124K), EHD3 (E124R), EHD3 (E124Q), H. pneumoniae YciA, F6AA82 (E95N/V152A/E304R) and F6AA82 (E95N/Q384A/F304R).

39. The host cell of claim 1 that comprises both a methane oxidation and methanol oxidation pathway.

40. The host cell of claim 1 that comprises a soluble methane monooxygenase.

41. The host cell of claim 1 that comprises an insoluble methane monooxygenase.

42. The host cell of claim 1 that comprises a p450 monooxygenase that oxidizes methane to methanol.

43. The host cell of claim 40 wherein said soluble methane monooxygenase is an MmoX enzyme subunit selected from the following: AAC45289.1, CAJ26291.1, AAB62392.3, BAA84757.1, BAA84757.1, ABD46892.1, ABD46898.1, P27353.4, P22869.3, ABG56535.1, ABU89756.1, ABU89757.1, ABU89758.1, BAJ07233.1, AAU92736.1, BAM37167.1, YP_113659.1, BAA84751.1, BAA84757.1, CAD30344.1, CAA39068.2, CAJ26291.1, AAZ81974.1, AAV52905.1, AAV52906.1, AAC45289.1, AAZ81968.1, AAY83388.1, BAJ17645.1, BAE86875.1, AAB62392.3, AAZ06158.1, AAZ06159.1, AAZ06160.1, AAZ06161.1, AAZ06163.1, AAZ06164.1, AAZ06198.1, AAZ06199.1, AAZ06200.1, AAZ06201.1, AAF01268.1, and ABD13903.1.

44. The host cell of claim 40 wherein said soluble methane monooxygenase is an MmoY enzyme subunit selected from the following: AAC45290.1, CAJ26292.1, AAB62393.2, BAA84758.1, BAA84758.1, Q53562.1, P22867.1, P27356.3, P18797.2, Q53563.1, P22868.2, P27354.3, P18798.4, AAU92727.1, YP_113660.1, BAA84752.1, BAA84758.1, CAA39069.1, CAJ26292.1, AAZ81975.1, AAC45290.1, AAZ81969.1, BAJ17646.1, BAE86876.1, AAB62393.2, AAFO 1269.1, and ABD46893.1.

45. The host cell of claim 40 wherein said soluble methane monooxygenase is an MmoZ enzyme subunit selected from the following: AAC45291.1, CAJ26293.1, AAF04158.2, BAA84759.1, BAA84759.1, Q53562.1, P22867.1, P27356.3, P18797.2, Q53563.1, P22868.2, AAB21391.1, P27355.3, P11987.4, AAU92724.1, YP_113663.1, BAA84754.1, BAA84760.1, CAA39071.1, CAJ26294.1, AAZ81977.1, AAC45292.1, AAZ81971.1, BAJ17648.1, BAE86878.1, AAF04157.2, AAF01271.1, and ABD46895.1

46. The host cell of claim 40 wherein said soluble methane monooxygenase is an MmoB enzyme subunit selected from the following: AAC45292.1, CAJ26294.1, AAF04157.2, BAA84760.1, BAA84760.1, 1XMG_B, 1XMG_C, 1XMG_D, 1XMH_A, 1XMH_B, 1XMH_C, 1XMH_D, 1XMF_A, 1XMF_B, 1XMF_C, 1XMF_D, 2MOB_A, 1XMG_E, 1XMG_F, 1XMH_E, 1XMH_F, 1XMF_E, 1XMF_F, 4GAM_B, 4GAM_A, 4GAM_G, 4GAM_F, 4GAM_L, 4GAM_K, 4GAM_Q, 4GAM_P, 4GAM_C, 4GAM_H, 4GAM_M, 4GAM_R, 4GAM_D, 4GAM_I, 4GAM_N, 4GAM_S, P27356.3, P18797.2, AAU92726.1, YP_113661.1, CAA39070.1, CAJ26293.1, AAZ81976.1, AAC45291.1, AAZ81970.1, BAJ17647.1, AAF04158.2, BAE86877.1, BAA84753.1, BAA84759.1, AAF01270.1, and ABD46894.1.

47. The host cell of claim 40 wherein said soluble methane monooxygenase is an MmoC enzyme subunit selected from the following: AAC45294.1, CAJ26296.1, AAB62391.2, BAA84762.1, BAA84762.1, AAB21393.1, Q53563.1, P22868.2, AEI77119.1, YP_004685600.1, BAA84756.1, AAC45294.1, AAZ81973.1, AAU92722.1, YP_113665.1, BAA84762.1, CAB45257.1, CAJ26296.1, AAZ81979.1, BAJ17650.1, BAE86880.1, AAB62391.2, CBI06592.1, CBI05225.1, AAF01273.1, and ABD46897.1.

48. The host cell of claim 41 wherein said insoluble methane monooxygenase is a pMoA subunit is selected from the following: CAE48352.1, CAE47800.1, AAC45295.2, AAA87220.2, and AAB49821.1.

49. The host cell of claim 41 wherein said insoluble methane monooxygenase is a pMoB subunit selected from the following: CAE48353.1, CAE47801.1, AAF37897.1, AAF37894.1, and AAB49822.1.

50. The host cell of claim 41 wherein said insoluble methane monooxygenase is a pMoC subunit selected from the following: CAE48351.1, CAE47799.1, AAF37896.1, AAF37893.1, and AAB49820.1.

51. A method of producing an organic acid in a recombinant host cell of any claims 39 to 50, said method comprising culturing said host cell in a media containing methanol and under culture conditions wherein said organic acid is produced.

52. The method of claim 51, wherein said fermentation is carried out in a media that contains methanol at a concentration ranging from 0.5-2% (v/v).

53. The method of claim 51 or 52, wherein said fermentation is carried out in a media that contains methane.

54. The method of claim 51 or 52, wherein glucose is present in the media.

55. The method of claim 51 or 52, wherein the media is at pH less than 5.5.

56. The method of claim 51 or 52, wherein the media is at pH less than 2.

57. The method of any of claims 35-38 or 51-56, wherein both methane and methanol are both present in the media.