Synthetic methanotrophic and methylotrophic microorganisms

Abstract

Provided herein are non-naturally occurring microbial organisms comprising a methane-oxidizing metabolic pathway. The invention additionally comprises non-naturally occurring microbial organisms comprising pathways for the production of chemicals. The invention additionally provides methods for using said organisms for the production of chemicals.

1 . A synthetic microorganism, wherein said synthetic microorganism comprises a natural methanol-consuming microorganism and one or more genetic modifications that improve the production of a chemical. 
     
     
         2 . The synthetic microorganism of  claim 1 , wherein said chemical is selected from the group consisting of dicarboxylic acid, malic acid, fumaric acid, succinic acid, malic acid salt, fumaric acid salt, succinic acid salt, L-malic acid, D-malic acid, maleic acid, lactic acid, adipic acid, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, butadiene, fatty acid derivatives, fatty alcohols, fatty acids, fatty acid esters, fatty acid methyl esters, fatty acid ethyl esters, branched fatty acids, branched fatty acid derivatives, omega-3 fatty acids, isoprenoids, isoprene, farnesene, farnesane, squalene, squalane, carotenoids, amino acids, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, monosodium glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, ornithine, proline, selenocysteine, serine, tyrosine, ethanol, propanol, 1-butanol, 2-butanol, isobutanol (2-methylpropan-1-ol), alcohols, alkanes, alkenes, olefins, animal feed additives, mixtures of amino acids, and proteins. 
     
     
         3 . A synthetic microorganism comprising a natural non-methanol-consuming microorganism and one or more genetic modifications that allow said synthetic microorganism to oxidize methanol. 
     
     
         4 . The synthetic microorganism of  claim 3 , wherein the natural non-methanol-consuming microorganism is selected from the group consisting of  Escherichia coli, Bacillus subtilis, Pseudomonas putida, Saccharomyces cerevisiae, Salmonella enterica, Corynebacterium glutamicum, Kiebsiella oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, Rhizobium etli, Gluconobacter oxydans, Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus , and  Aspergillus niger.    
     
     
         5 . The synthetic microorganism of  claim 4 , wherein the natural non-methanol-consuming microorganism is not  Corynebacterium glutamicum  or  Escherichia coli.    
     
     
         6 . The synthetic microorganism of  claim 3 , further comprising exogenous polynucleotides, wherein said exogenous polynucleotides encode enzymes selected from the group consisting of methanol dehydrogenase (EC 1.1.1.224 or 1.1.99.37 or 1.1.2.7), alcohol dehydrogenase (EC 1.1.1.1), 3-hexulose-6-phosphate synthase (EC 4.1.2.43) and 6-phospho-3-hexuloisomerase (EC 5.3.1.27). 
     
     
         7 . The synthetic microorganism of  claim 3 , further comprising exogenous polynucleotides, wherein said exogenous polynucleotides encode enzymes selected from the group of alcohol oxidase (EC 1.1.3.13), methanol dehydrogenase (EC 1.1.1.224 or 1.1.99.37 or 1.1.2.7), alcohol dehydrogenase (EC 1.1.1.1), formaldehyde dehydrogenase (EC 1.2.1.46), formate dehydrogenase (EC 1.2.1.2), dihydroxyacetone synthase/formaldehyde transketolase (EC 2.2.1.3), and catalase (EC 1.11.1.6). 
     
     
         8 . A method for producing a chemical, comprising culturing the synthetic microorganism of  claim 3  under suitable culture conditions and for a sufficient period of time to produce said chemical. 
     
     
         9 . The method of  claim 8 , wherein the suitable culture conditions comprise a culture media containing methanol as a sole carbon source or as a major carbon source. 
     
     
         10 . The method of  claim 8 , wherein said chemical is selected from the group consisting of dicarboxylic acid, malic acid, fumaric acid, succinic acid, malic acid salt, fumaric acid salt, succinic acid salt, L-malic acid, D-malic acid, maleic acid, lactic acid, adipic acid, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, butadiene, fatty acid derivatives, fatty alcohols, fatty acids, fatty acid esters, fatty acid methyl esters, fatty acid ethyl esters, branched fatty acids, branched fatty acid derivatives, omega-3 fatty acids, isoprenoids, isoprene, farnesene, farnesane, squalene, squalane, carotenoids, amino acids, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, monosodium glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, ornithine, proline, selenocysteine, serine, tyrosine, ethanol, propanol, 1-butanol, 2-butanol, isobutanol (2-methylpropan-1-ol), alcohols, alkanes, alkenes, olefins, animal feed additives, mixtures of amino acids, and proteins. 
     
     
         11 . A synthetic microorganism, wherein said synthetic microorganism comprises a non-methanotrophic microorganism and one or more genetic modifications that allow said synthetic microorganism to oxidize methane. 
     
     
         12 . The synthetic microorganism of  claim 11 , wherein said non-methanotrophic organism is a naturally occurring microorganism selected from the group of  Hansenula, Pichia, Candida , and  Torulopsis.    
     
     
         13 . The synthetic microorganism of  claim 11 , wherein said non-methanotrophic microorganism is selected from the group of  Pichia pastoris  and  Pichia methanolica, Bacillus subtilis, Bacillus methanolicus, Pseudomonas putida, Escherichia coli  and  Corynebacterium  glutamicum. 
     
     
         14 . The synthetic microorganism of  claim 11 , wherein the non-methanotrophic microorganism is not  Corynebacterium glutamicum  or  Escherichia coli.    
     
     
         15 . The synthetic microorganism of  claim 11 , wherein said one or more genetic modifications comprise one or more exogenous polynucleotides encoding a methane monooxygenase enzyme selected from the group consisting of soluble methane monooxygenase (EC 1.14.13.25) and a particulate methane monooxygenase (EC 1.4.18.3) and a non-natural methane monooxygenase. 
     
     
         16 . The synthetic microorganism of  claim 11 , wherein said synthetic microorganism can grow using methane as a sole carbon source or as a major carbon source. 
     
     
         17 . The synthetic microorganism of  claim 16 , wherein said non-natural methane monooxygenase is the spmoB enzyme. 
     
     
         18 . The synthetic microorganism of  claim 11 , wherein said genetic modifications comprise one or more exogenous polynucleotides encoding accessory proteins, helper proteins, or protein-folding chaperones. 
     
     
         19 . A method for producing a chemical, comprising culturing the synthetic microorganism of  claim 11  under suitable culture conditions and for a sufficient period of time to produce said chemical. 
     
     
         20 . The method according to  claim 19 , wherein the suitable culture conditions comprises media containing methane. 
     
     
         21 . (canceled)