Biosynthesis of products from 1-carbon compounds
An engineered microbe that contains a designed platform for the conversion of one-carbon substrates to chemical products is described. The designed platform embodies a new metabolic architecture that consolidates carbon fixation, central metabolism, and product synthesis into a single pathway. This is made possible by the key finding that 2-hydroxyacyl-CoA lyase, an enzyme in the α-oxidation pathway, is capable of catalyzing the C—C bond formation between formyl-CoA and aldehydes of different chain lengths, allowing for the elongation of the carbon backbone of said aldehyde by one-carbon units. These novel microbes present an opportunity for the production of chemicals from single-carbon feedstocks such as carbon dioxide, carbon monoxide, formate, formaldehyde, methanol or methane.
1. A method for the production of a product from a single carbon substrate, the method comprising the steps of:
a) growing a genetically modified microorganism in a culture comprising the single carbon substrate and a growth media,
b) converting said single carbon substrate to a formyl-CoA,
c) condensing an initial C(n)-aldehyde with the formyl-CoA to produce 2-hydroxy-C(n+1)-acyl-CoA, wherein the 2-hydroxy-C(n+1)-acyl-CoA is one carbon longer than the initial C(n) aldehyde and wherein n is the number of carbons in the initial C(n)-aldehyde;
d) converting the 2-hydroxy-C(n+1)-acyl-CoA to a C(n+1)-aldehyde;
e) isolating a product from said microorganism, or said growth media, or both;
wherein the single carbon substrate is selected from the group consisting of methane, methanol, formaldehyde, formate, carbon monoxide, carbon dioxide, and combinations thereof; and
wherein the microorganism expresses:
i. a 2-hydroxyacyl-CoA lyase or an oxalyl-CoA decarboxylase that catalyzes the condensation of the initial C(n)-aldehyde with the formyl-CoA to produce the 2-hydroxy-C(n+1)-acyl-CoA;
ii. one or more polypeptides that catalyze the conversion of the single carbon substrate to the formyl-CoA; and
iii. one or more polypeptides that catalyze a conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the C(n+1)-aldehyde.
2. The method of claim 1 , wherein the microorganism expresses a 2-hydroxyacyl-CoA lyase.
3. The method of claim 1 , wherein the 2-hydroxyacyl-CoA lyase is encoded by a DNA molecule selected from Homo sapiens hacl1, Rattus norvegicus hacl1, Dictyostelium discoideum hacl1, and Mus musculus hacl1, and the oxalyl-CoA decarboxylase is encoded by a DNA molecule selected from Mycobacterium sp. MOTT36Y W7S_25140, Mycobacterium tuberculosis TKK-01-0051 K875_00487 , Polynucleobacter necessarius subsp. asymbioticus QLW-P1DMWA-1 Pnuc_0637, and E. coli oxc.
4. The method of claim 3 , wherein the 2-hydroxyacyl-CoA lyase is encoded by Homo sapiens hacl1.
5. The method of claim 1 , wherein the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the C(n+1)-aldehyde comprises the steps of:
i. conversion of the 2-hydroxy-C(n+1)-acyl-CoA to a 2-hydroxy-C(n+1)-aldehyde;
ii. conversion of the 2-hydroxy-C(n+1)-aldehyde to a 1,2-C(n+1)-diol; and
iii. conversion of the 1,2-C(n+1)-diol to the C(n+1)-aldehyde.
6. The method of claim 5 , wherein the one or more polypeptides that catalyzes the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the C(n+1)-aldehyde comprises:
an acyl-CoA reductase that catalyzes the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the 2-hydroxy-C(n+1)-aldehyde;
a polypeptide that catalyzes the conversion of the 2-hydroxy-C(n+1)-aldehyde to the 1,2-C(n+1)-diol, wherein the polypeptide is a 1,2-diol oxidoreductase or an alcohol dehydrogenase; and
a diol dehydratase that catalyzes the conversion of the 1,2-C(n+1)-diol to the C(n+1)-aldehyde.
7. The method of claim 6 , wherein:
a) the 2-hydroxyacyl-CoA lyase is encoded by a DNA molecule selected from Homo sapiens hacl1, Rattus norvegicus hacl1, Dictyostelium discoideum hacl1, and Mus musculus hacl1,
b) the acyl-CoA reductase is encoded by a DNA molecule selected from Acinetobacter calcoaceticus acr1, Acinetobacter sp Strain M-1 acrM, Clostridium beijerinckii ald, E. coli eutE, Salmonella enterica eutE, and E. coli mhpF;
c) the 1,2-diol oxidoreductase or alcohol dehydrogenase is encoded by a DNA molecule selected from E. coli betA, E. coli dkgA, E. coli eutG, E. coli fucO, E. coli ucpA, E. coli yahK, E. coli ybbO, E. coli ybdH, E. coli yiaY, and E. coli yjgB; and
d) the diol dehydratase is encoded by a DNA molecule selected from Klebsiella ocytoca pddABC, E. coli pduCDE, and S. enterica pduCDE.
8. The method of claim 1 , wherein the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the C(n+1)-aldehyde comprises the steps of:
i. conversion of the 2-hydroxy-C(n+1)-acyl-CoA to a trans-2-C(n+1)-enoyl-CoA;
ii. conversion the trans-2-C(n+1)-enoyl-CoA to a C(n+1)-acyl-CoA; and
iii. conversion the C(n+1)-acyl-CoA to the C(n+1)-aldehyde.
9. The method of claim 8 , wherein the one or more polypeptides that catalyzes the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to the C(n+1)-aldehyde comprises:
a 2-hydroxyacyl-CoA dehydratase that catalyzes the conversion of the 2-hydroxy-C(n+1)-acyl-CoA to a trans-2-C(n+1)-enoyl-CoA;
a trans-2-enoyl-CoA reductase that catalyzes the conversion the trans-2-C(n+1)-enoyl-CoA to a C(n+1)-acyl-CoA; and
an acyl-CoA reductase that catalyzes the conversion the C(n+1)-acyl-CoA to the C(n+1)-aldehyde.
10. The method of claim 9 , wherein:
a) the 2-hydroxyacyl-CoA lyase is encoded by a first heterologous DNA molecule encoding the selected from Homo sapiens hacl1, Rattus norvegicus hacl1, Dictyostelium discoideum hacl1, and Mus musculus hacl1, and wherein the oxalyl-CoA decarboxylase is encoded by a DNA molecule selected from Mycobacterium sp. MOTT36Y W7S_25140, Mycobacterium tuberculosis TKK-01-0051 K875_00487 , Polynucleobacter necessarius subsp. asymbioticus QLW-P1DMWA-1 Pnuc_0637, and E. coli oxc;
b) the 2-hydroxyacyl-CoA dehydratase is encoded by a DNA molecule selected from Clostridium propionicum lcdABC, and Clostridium difficile hadBCI; and
c) the trans-2-enoyl-CoA reductase is encoded by a DNA molecule selected from Rhodobacter sphaeroides AcuI, E. coli yhdH, E. coli fabI, Enterococcus faecalis fabK, Bacillus subtilis fabL, Euglena gracilis TER, Treponema denticola TER, and Vibrio cholera fabV; and
d) the acyl-CoA reductase is encoded by a DNA molecule selected from Acinetobacter calcoaceticus acr1, Acinetobacter sp Strain M-1 acrM, Clostridium beijerinckii ald, E. coli eutE, Salmonella enterica eutE, and E. coli mhpF.
11. The method of claim 1 , wherein the single carbon substrate is carbon dioxide, and wherein the conversion of the carbon dioxide to formyl-CoA comprises the steps of:
a) conversion of the carbon dioxide to formate;
b) conversion of the formate to formyl-phosphate; and
c) conversion of the formyl-phosphate to the formyl-CoA.
12. The method of claim 11 , wherein the one or more polypeptides that catalyze the conversion of the carbon dioxide to the formyl-CoA comprise:
a) a carbon dioxide reductase that catalyzes the conversion of the carbon dioxide to formate;
b) an acetate kinase or formate kinase that catalyzes the conversion of the formate to formyl-phosphate; and
c) a phosphate acetyl-transferase that catalyzes the conversion of the formyl-phosphate to the formyl-CoA.
13. The method of claim 6 , wherein the one or more polypeptides that catalyze the conversion of the carbon dioxide to the formyl-CoA comprise:
a) a carbon dioxide reductase that catalyzes the conversion of the carbon dioxide to formate;
b) an acetate kinase or formate kinase that catalyzes the conversion of the formate to formyl-phosphate; and
c) a phosphate acetyl-transferase that catalyzes the conversion of the formyl-phosphate to the formyl-CoA.
14. The method of claim 12 , wherein:
a) the carbon dioxide reductase is encoded by a DNA molecule selected from Acetobacterium woodii fdhF2, Acetobacterium woodii hycB2, Acetobacterium woodii hycB3, and Acetobacterium woodii hydA2;
b) the formate kinase is encoded by a DNA molecule selected from Salmonella typhimurium stAckA, and E. coli AckA and c) the phosphate acetyl-transferase is encoded by a DNA molecule selected from E. coli eutD, E. coli pta, and Salmonella typhimurium PduL.
15. The method of claim 1 , wherein the single carbon substrate is formate and wherein the one or more polypeptides that catalyze the conversion of the formate to formyl-CoA is acetyl-CoA synthetase.
16. The method of claim 15 , wherein the acetyl-CoA synthetase is encoded by Escherichia coli acs.
17. The method of claim 1 , wherein the single carbon substrate is methane or methanol, and wherein when the single carbon substrate is methane, the method comprises conversion of methane to methanol.
18. The method of claim 17 , wherein the conversion of methanol to formyl-CoA comprises:
a) conversion of methanol to formaldehyde; and
b) conversion of formaldehyde to formyl-CoA.
19. The method of claim 18 , wherein:
a) a methanol dehydrogenase catalyzes the conversion of the methanol to the formaldehyde; and
b) an acylating aldehyde dehydrogenase catalyzes the conversion of the formaldehyde to the formyl-CoA.
20. The method of claim 6 , wherein the single carbon substrate is methane or methanol, and wherein when the single carbon substrate is methane, the method comprises conversion of the methane to methanol, and wherein:
a) a methanol dehydrogenase catalyzes the conversion of the methanol to the formaldehyde; and
b) an acylating aldehyde dehydrogenase catalyzes the conversion of the formaldehyde to the formyl-CoA.
21. The method of claim 19 , wherein:
a) the methanol dehydrogenase is encoded by a DNA molecule selected from Bacillus methanolicus mdh, Mycobacterium sp. DSM 3803 mdo, and Methylobacterium extorquens moxI, moxF; and
b) the acylating aldehyde dehydrogenase is encoded by a DNA molecule selected from E. coli mhpF, and Pseudomonas sp. CF600 dmpF.
22. The method of claim 17 , wherein the single carbon substrate is methane and wherein a methane monooxygenase catalyzes the conversion of the methane to methanol.
23. The method of claim 1 , wherein the product is a carboxylic acid.
24. The method of claim 23 , wherein the carboxylic acid is selected from the group consisting of (3R)-β-hydroxy carboxylic acids, or (2R)-α-hydroxycarboxylic acids, and b-keto carboxylic acids, or α,β-unsaturated carboxylic acids.
25. The method of claim 23 , wherein the method comprises the conversion of an acyl-CoA to the carboxylic acid.
26. The method of claim 1 , wherein the product is selected from the group consisting of primary alcohols, or 1, (3R)-β diols, or 1, (2R)-α diols, or α-keto primary alcohols, or α,β-unsaturated primary alcohols.
27. The method of claim 26 , wherein the method comprises the conversion of an acyl-CoA to the alcohol.
28. The method of claim 1 , wherein the product is selected from the group consisting of linear alkanes, or linear alkan-3-ols, or linear alkan-2-ols, or linear methyl-ketones, or 1-alkenes.
29. The method of claim 1 , wherein the product is an amine selected from the group consisting of primary amines, or 3-hydroxy-amines, or 2-hydroxy-amines, or 3-keto-amines, or α,β-unsaturated primary amines.
30. The method of claim 29 , wherein the method comprises the conversion of an aldehyde to the amine.
31. The method of claim 1 , wherein the microorganism further expresses a polypeptide selected from the group consisting of a thioesterase, acyl-CoA:acetyl-CoA transferase, phosphotransacylase, carboxylate kinase, an alcohol-forming coenzyme-A thioester reductase, an aldehyde-forming coenzyme-A thioester reductase, an alcohol dehydrogenase, an aldehyde decarbonylase; and a transaminase.
32. The method of claim 1 , wherein the method further comprises regeneration of the C(n)-aldehyde and wherein the regeneration comprises:
i. conversion of a trans-2-enoyl-CoA to a 3-hydroxy-acyl-CoA;
ii. conversion of the 3-hydroxy-acyl-CoA to a 3-keto-acyl-CoA;
iii. conversion of the 3-keto-acyl-CoA to acetyl-CoA and an acyl-CoA shortened by two carbons; and
iv. conversion of the acetyl-CoA to the acetaldehyde.
33. The method of claim 32 , wherein:
a) an enoyl-CoA hydratase catalyzes the conversion of the trans-2-enoyl-CoA to the 3-hydroxy-acyl-CoA;
b) a 3-hydroxyacyl-CoA dehydrogenase catalyzes the conversion of the 3-hydroxy-acyl-CoA to the 3-keto-acyl-CoA;
c) a 3-ketoacyl-CoA thiolase catalyzes the conversion of the 3-keto-acyl-CoA to the acetyl-CoA and the acyl-CoA shortened by two carbons; and
d) an acetyl-CoA reductase catalyzes the conversion of the acetyl-CoA to the acetaldehyde.
34. The method of claim 33 , wherein:
a) the enoyl-CoA hydratase is encoded by a DNA molecule selected from E. coli fadB, E. coli fadJ, Aeromonas caviae phaJ, Pseudomonas aeruginosa phaJ1, Pseudomonas aeruginosa phaJ2, Pseudomonas aeruginosa phaJ3, and Pseudomonas aeruginosa phaJ4;
b) the 3-hydroxyacyl-CoA dehydrogenase is encoded by a DNA molecule selected from E. coli fadB, E. coli fadJ, Ralstonia eutropha phaB1, Ralstonia eutropha phaB2, and Ralstonia eutropha phaB3;
c) the 3-ketoacyl-CoA thiolase is encoded by a DNA molecule selected-from E. coli atoB, E. coli yqeF, E. coli fadA, E. coli fadI, Ralstonia eutropha bktB, Pseudomonas sp. Strain B13 catF, E coli paaJ, Rhodococcus opacus pcaF, Streptomyces sp. pcaF, Ralstonia eutropha phaA, Clostridium acetobutylicum thlA, and Clostridium acetobutylicum thlB;
d) the acetyl-CoA reductase is encoded by a DNA molecule selected from E. coli eutE, Salmonella enterica eutE, and E. coli mhpF.
35. The method of claim 32 , wherein the microorganism further expresses a polypeptide selected from the group consisting of a thioesterase, acyl-CoA:acetyl-CoA transferase, phosphotransacylase, carboxylate kinase, an alcohol-forming coenzyme-A thioester reductase, an aldehyde-forming coenzyme-A thioester reductase, an alcohol dehydrogenase, an aldehyde decarbonylase; and a transaminase.
36. The method of claim 1 , wherein the microorganism is selected from the group consisting of E. coli, S. cerevisiae, Methylococcus capsulatus, Pichia pastoris, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Laminaria japonica, Undaria pinnatifida, Pavlova lutheri , and Crypthecodinium cohni.