Methods and Systems for Methylotrophic Production of Organic Compounds

Abstract

The present disclosure identifies pathways, mechanisms, systems and methods to confer production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and/or evolved methylotrophs such that these organisms efficiently convert C1 compounds, such as formate, formic acid, formaldehyde or methanol, to organic carbon-based products of interest, and in particular the use of organisms for the commercial production of various carbon-based products of interest.

1 . An engineered cell for producing a carbon-based product, comprising an at least partially engineered carbon product biosynthetic pathway introduced into a methylotrophic organism, wherein said engineered cell is capable of converting a C1 compound into a carbon-based product of interest. 
     
     
         2 . The engineered cell of  claim 1 , wherein the methylotrophic organism is capable of converting the C1 compound into a central metabolite. 
     
     
         3 . The engineered cell of  claim 1  or  2 , wherein the C1 compound is soluble in water, such as formate, formic acid, formaldehyde, methanol, or any combination thereof. 
     
     
         4 . The engineered cell of any one of  claims 1 - 3 , wherein the C1 compound is derived from electrolysis. 
     
     
         5 . The engineered cell of any one of  claims 1 - 4 , wherein said carbon-based product of interest is one or more of a sugar (for example, glucose, fructose, sucrose, xylose, lactose, maltose, pentose, rhamnose, galactose or arabinose), sugar phosphate (for example, glucose-6-phosphate or fructose-6-phosphate), sugar alcohol (for example, sorbitol), sugar derivative (for example, ascorbate), alcohol (for example, ethanol, propanol, isopropanol or butanol), fermentative product (for example, ethanol, butanol, lactic acid, lactose or acetate), ethylene, propylene, 1-butene, 1,3-butadiene, acrylic acid, fatty acid (for example, O-cyclic fatty acid), fatty acid intermediate or derivative (for example, fatty acid alcohol, fatty acid ester, alkane, olegin or halogenated fatty acid), amino acid or intermediate (for example, lysine, glutamate, aspartate, shikimate, chorismate, phenylalanine, tyrosine, tryptophan), phenylpropanoid, isoprenoid (for example, hemiterpene, monoterpene, sesquiterpene, triterpene, tetraterpene, polyterpene, isoprene, bisabolene, myrcene, amorpha-4,11-diene, farnesene, taxadiene, squalene, lanosterol, β-carotene, ζ-carotene, lycopene, phytoene, limonene, or polyisoprene), glycerol, 1,3-propanediol, 1,4-butanediol, 1,3-butadiene, polyhydroxyalkanoate, polyhydroxybutyrate, lysine, γ-valerolactone, and acrylate. 
     
     
         6 . The engineered cell of any one of  claims 1 - 5 , wherein
 when said carbon product biosynthetic pathway is for fatty acid biosynthesis, said carbon product biosynthetic pathway includes one or more of: fatty acid synthase, acetyl-CoA carboxylase, fatty-acyl-CoA reductase, aldehyde decarbonylase, lipase, thioesterase and acyl-CoA synthase peptides; or   when said carbon product biosynthetic pathway is for branched chain fatty acid biosynthesis, said carbon product biosynthetic pathway includes one or more of: branched chain amino acid aminotransferase, branched chain α-ketoacid dehydrogenase, dihydrolipoyl dehydrogenase, beta-ketoacyl-ACP synthase, crotonyl-CoA reductase, isobutyryl-CoA mutase, β-ketoacyl-ACP synthase I, trans-2,cis-3-decenoyl-ACP isomerase and trans-2-enoyl-ACP reductase II; or   when said carbon product biosynthetic pathway is for fatty alcohol biosynthesis, said carbon product biosynthetic pathway includes one or more of: fatty alcohol forming acyl-CoA reductase, fatty alcohol forming acyl-CoA reductase, alcohol dehydrogenase and alcohol reductase; or   when said carbon product biosynthetic pathway is for fatty ester biosynthesis, said carbon product biosynthetic pathway includes one or more of: alcohol O-acetyltransferase, wax synthase, fatty acid elongase, acyl-CoA reductase, acyltransferase, fatty acyl transferase, diacylglycerol acyltransferase, acyl-CoA was alcohol acyltransferase, bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase, and β-ketoacyl-ACP synthase I; or   when said carbon product biosynthetic pathway is for alkane biosynthesis, said carbon product biosynthetic pathway includes one or more of: decarbonylase and terminal alcohol oxidoreductase; or   when said carbon product biosynthetic pathway is for ω-cyclic fatty acid biosynthesis, said carbon product biosynthetic pathway includes one or more of: 1-cyclohexenylcarbonyl CoA reductase, 5-enopyruvylshikimate-3-phosphate synthase, acyl-CoA dehydrogenase, enoyl-(ACP) reductase, 2,4-dienoyl-CoA reductase, and acyl-CoA isomerase; or   when said carbon product biosynthetic pathway is for halogenated fatty acid biosynthesis, said carbon product biosynthetic pathway includes one or more of: fluorinase, nucleotide phosphorylase, fluorometabolite-specific aldolase, fluoroacetaldehyde dehydrogenase, and fluoroacetyl-CoA synthase; or   when said carbon product biosynthetic pathway is the deoxylylulose 5-phosphate (DXP) isoprenoid pathway, said carbon product biosynthetic pathway includes one or more of: 1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, 4-diphosphocytidyl-2C-methyl-D-erythritol kinase, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase, isopentyl/dimethylallyl diphosphate synthase and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; or   when said carbon product biosynthetic pathway is the mevalonate-dependent (MEV) isoprenoid pathway, said carbon product biosynthetic pathway includes one or more of: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase and isopentenyl pyrophosphate isomerase; or   when said carbon product biosynthetic pathway is the glycerol/1,3-propanediol biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: sn-glycerol-3-P dehydrogenase, sn-glycerol-3-phosphatase, glycerol dehydratase and 1,3-propanediol oxidoreductase; or   when said carbon product biosynthetic pathway is the 1,4-butanediol/1,3-butadiene biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: succinyl-CoA dehydrogenase, 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, 1,3-propanediol oxidoreductase and alcohol dehydratase; or   when said carbon product biosynthetic pathway is the polyhydroxybutyrate biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: acetyl-CoA:acetyl-CoA C-acetyltransferase, (R)-3-hydroxyacyl-CoA:NADP +  oxidoreductase and polyhydroxyalkanoate synthase; or   when said carbon product biosynthetic pathway is the lysine biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: aspartate aminotransferase, aspartate kinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, tetrahydrodipicolinate succinylase, N-succinyldiaminopimelate-aminotransferase, N-succinyl-L-diaminopimelate desuccinylase, diaminopimelate epimerase, diaminopimelate decarboxylase, L,L-diaminopimelate aminotransferase, homocitrate synthase, homoaconitase, homoisocitrate dehydrogenase, 2-aminoadipate transaminase, 2-aminoadipate reductase, aminoadipate semialdehyde-glutamate reductase and lysine-2-oxoglutarate reductase; or   when said carbon product biosynthetic pathway is the chorismate biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: 2-dehydro-3-deoxyphosphoheptonate aldolase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, NADPH-dependent shikimate dehydrogenase, NAD(P)H-dependent shikimate dehydrogenase, shikimate kinase, 3-phosphoshikimate-1-carboxyvinyltransferase and chorismate synthase; or   when said carbon product biosynthetic pathway is the phenylalanine biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: chorismate mutase, prephenate dehydratase and phenylalanine transaminase; or   when said carbon product biosynthetic pathway is the tyrosine biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: chorismate mutase, prephenate dehydrogeanse and tyrosine aminotransferase; or   when said carbon product biosynthetic pathway is the γ-valerolactone biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: propionyl-CoA synthase, beta-ketothiolase, acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ-isomerase, 4-hydroxybutyryl-CoA transferase and 1,4-lactonase; or   when said carbon product biosynthetic pathway is the butanol biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: beta-ketothiolase, acetoacetyl-CoA reductase, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase, butyryl-CoA dehydrogenase, trans-enoyl-coenzyme A reductase, butyrate CoA-transferase, aldehyde dehydrogenase, alcohol dehydrogenase, acetyl-CoA acetyltransferase, β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl CoA dehydrogenase, CoA-acylating aldehyde dehydrogenase and aldehyde-alcohol dehydrogenase; or   when said carbon product biosynthetic pathway is the acrylate biosynthesis pathway, said carbon product biosynthetic pathway includes one or more of: enoyl-CoA hydratase, propionyl-CoA synthase and acrylate CoA-transferase.   
     
     
         7 . The engineered cell of  claim 6 , wherein
 the 1-deoxy-D-xylulose-5-phosphate synthase is encoded by SEQ ID NO:1, or a homolog thereof having at least 80% sequence identity; or   the isopentenyl pyrophosphate isomerase is encoded by SEQ ID NO:2, or a homolog thereof having at least 80% sequence identity.   
     
     
         8 . The engineered cell of any one of  claims 1 - 7 , wherein when said carbon product biosynthetic pathway is the isoprene biosynthesis pathway, said carbon product biosynthetic pathway includes isoprene synthase. 
     
     
         9 . The engineered cell of  claim 8 , wherein the isoprene synthase is encoded by SEQ ID NO:3, or a homolog thereof having at least 80% sequence identity. 
     
     
         10 . The engineered cell of any one of  claims 1 - 7 , wherein when said carbon product biosynthetic pathway is the bisabolene biosynthesis pathway, said carbon product biosynthetic pathway includes E-alpha-bisabolene synthase. 
     
     
         11 . The engineered cell of  claim 10 , wherein the E-alpha-bisabolene synthase is encoded by SEQ ID NO:4, or a homolog thereof having at least 80% sequence identity. 
     
     
         12 . The engineered cell of any one of  claims 1 - 11 , wherein the methylotrophic organism is selected from the class Alphaproteobacterium. 
     
     
         13 . The engineered cell of any one of  claims 1 - 12 , wherein the methylotrophic organism is selected from the genus  Paracoccus.    
     
     
         14 . The engineered cell of any one of  claims 1 - 13 , wherein the methylotrophic organism is  Paracoccus denitrificans, Paracoccus versutus  or  Paracoccus  zeaxanthinifaciens. 
     
     
         15 . The engineered cell of any one of  claims 1 - 14 , further modified to have a less reduced growth rate on electrolytically generated C1 compound relative to non-evolved methylotrophic organism, or a substantially similar or enhanced growth rate on electrolytically generated C1 compound relative to non-electrolytically generated C1 compound. 
     
     
         16 . The engineered cell of any one of  claims 1 - 15 , further evolved to have a less reduced growth rate on electrolytically generated C1 compound relative to non-evolved methylotrophic organism, or a substantially similar or enhanced growth rate on electrolytically generated C1 compound relative to non-electrolytically generated C1 compound. 
     
     
         17 . An evolved methylotrophic organism, having a less reduced growth rate on electrolytically generated C1 compound relative to non-evolved methylotrophic organism, or having a substantially similar or enhanced growth rate on electrolytically generated C1 compound relative to non-electrolytically generated C1 compound. 
     
     
         18 . A method for selecting an evolved methylotrophic organism having improved growth on a C1 compound, comprising:
 incubating methylotrophic cells in a culture chamber with controlled temperature, cell concentration, and medium inflow and outflow rates, wherein a medium inflow includes a C1 compound;   continuously monitoring a concentration of biomass in the culture chamber; and   adjusting a flow rate of the C1 compound into the culture chamber so as to continually maintain an environment that selects for an improved growth rate.   
     
     
         19 . The method of  claim 18 , further comprising adjusting the medium inflow to be more permissive of growth or more suppressive of growth, so as to provide an adaptive environment to select for a fitness of the cells. 
     
     
         20 . The method of any one of  claims 18 - 19 , wherein the C1 compound is formate. 
     
     
         21 . The method of any one of  claims 18 - 20 , wherein the C1 compound is electrolytically generated. 
     
     
         22 . The method of any one of  claims 18 - 21 , wherein the C1 compound is soluble in water. 
     
     
         23 . A method of introducing a conjugative plasmid into methylotrophic host cells, comprising:
 incubating a mixture of predetermined ratios of a donor culture and a recipient culture, at temperatures between 4° C. and 37° C. for between 1 and 48 hours, wherein the donor culture comprises a conjugal donor containing a conjugative plasmid having a first selectable trait, and the recipient culture comprises methylotrophic host cell having a second selectable trait; and   subjecting the incubated mixture to a dually selective condition where only plasmid-containing transconjugants that have both the first selectable trait and the second selectable trait can grow,   wherein the method does not include centrifugation or filtration of the mixture or incubated mixture.   
     
     
         24 . The method of  claim 23 , wherein the conjugal donor is an  E. coli  strain such as  E. coli  S17-1, or an  E. coli  harboring plasmids such as pRK2013 or pRK2073, or any  E. coli  strain expressing a tra operon capable of mobilizing plasmids containing an RP4-derived sequence. 
     
     
         25 . The method of  claim 23  or  24 , wherein the conjugal donor is in a different species or genus of the host cell. 
     
     
         26 . The method of any one of  claims 23 - 25 , wherein the transconjugated plasmid contains an RP4 or similar mob element. 
     
     
         27 . The method of any one of  claims 23 - 26 , wherein the host cell is from the class Alphaproteobacterium. 
     
     
         28 . The method of any one of  claims 23 - 27 , wherein the host cell is from the genus  Paracoccus.    
     
     
         29 . The method of any one of  claims 23 - 28 , wherein the host cell is  Paracoccus denitrificans, Paracoccus versutus  or  Paracoccus zeaxanthinifaciens.    
     
     
         30 . A composition for bacterial culture, formulated to provide formate as the sole source of C1 compound and to enhance growth of methylotrophic bacteria. 
     
     
         31 . The composition of  claim 30 , comprising between 0 and 160 mM sodium bicarbonate, between 0 and 16 mM sodium chloride, between 0 and 100 mM sodium nitrate, between 0 and 30 mM sodium thiosulfate, and initially containing between 5 and 100 mM of a formate salt, such as sodium formate and/or ammonium formate. 
     
     
         32 . The composition of any one of  claims 30 - 31 , comprising 100 mM sodium bicarbonate, 6 mM sodium chloride, 6 mM sodium nitrate, 11 mM sodium thiosulfate, and 26 mM sodium formate or ammonium formate. 
     
     
         33 . The composition of any one of  claims 30 - 33 , further comprising a basal minimal medium. 
     
     
         34 . The composition of  claim 33 , wherein the basal minimal medium is MOPS minimal medium, M9 minimal medium, R medium or M63 medium, or a medium substantially similar thereto. 
     
     
         35 . A method for culturing methylotrophic bacteria, comprising incubating methylotrophic bacteria in the composition of any one of  claims 30 - 34 . 
     
     
         36 . A composition of bacterial culture, formulated to provide formate as the sole C1 compound and to enhance growth of methylotrophic bacteria in a fed-batch bioreactor. 
     
     
         37 . The composition of  claim 36 , comprising a medium initially charged in the fed-batch bioreactor which comprises R medium supplemented with between 1 and 100 micromolar sodium molybdate, between 10 and 1000 nanomolar sodium selenite, between 0.01 to 1 mg/L of thiamine, and between 0.001 to 1 mg/L of cobalamin. 
     
     
         38 . The composition of  claim 37 , wherein the medium comprises between 5 and 20 micromolar sodium molbydate, between 50 and 200 nanomolar sodium selenite, between 0.05 to 2 mg/L of thiamine, between 0.01 and 0.2 mg/L cobalamin. 
     
     
         39 . The composition of  claim 37  or  38 , further comprising a feed composition supplied to the fed-batch bioreactor comprising a formate salt at supramolar concentration. 
     
     
         40 . The composition of  claim 39 , wherein the formate salt is ammonium formate and/or sodium formate. 
     
     
         41 . The composition of  claim 39  or  40 , wherein feed composition further comprises a supramolar concentration of nitrate salt. 
     
     
         42 . The composition of  claim 41 , wherein the nitrate salt is sodium nitrate. 
     
     
         43 . The composition of any one of  claims 39 - 42 , wherein the nitrate salt and the formate salt are provided in a molar ratio of 3.0:8 or lower. 
     
     
         44 . A method for culturing methylotrophic bacteria, comprising incubating methylotrophic bacteria in the composition of any one of  claims 36 - 43  in a fed-batch bioreactor. 
     
     
         45 . The method of  claim 44 , wherein a volumetric rate of C1 feedstock consumption in the fed-batch reactor exceeds 1.5 g*L −1  hr −1 . 
     
     
         46 . The method of  claim 45 , wherein said incubating is conducted aerobically. 
     
     
         47 . The method of any one of  claims 44 - 46 , wherein said incubating is conducted in the presense of a nitrate salt as electron acceptor and formate salt as electron donor. 
     
     
         48 . The method of  claim 47 , wherein a molar ratio of the nitrate salt to the formate salt is kept below 3.2:8 in the fed-batch reactor. 
     
     
         49 . The method of  claim 47  or  48 , wherein the nitrate salt and the formate salt are provided to the fed-batch bioreactor in a feed composition in supramolar concentrations in a molar ratio of 3.0:8 or lower. 
     
     
         50 . The method of any one of  claims 47 - 49 , wherein the formate salt is ammonium formate and/or sodium formate. 
     
     
         51 . The method of any one of  claims 47 - 50 , wherein the nitrate salt is sodium nitrate.