MICROBIAL PRODUCTION OF CHEMICAL PRODUCTS AND RELATED COMPOSITIONS, METHODS AND SYSTEMS
Metabolically engineered microorganism strains are disclosed, such as bacterial strains, in which there is an increased utilization of malonyl-CoA for production of a chemical product. Such chemical products include polyketides, 3-hydroxypropionic acid, and various other chemical products described herein. Methods of production also may be applied to further downstream products, such as consumer products. In various embodiments, modifications to a microorganism and/or culture system divert, at least transiently, usage of malonyl-coA from the fatty acid biosynthesis pathway and thereby provides for usage of the malonyl-coA for a chemical product other than a fatty acid. In various embodiments, the fatty acid biosynthesis pathway is modulated to produce specific fatty acids or combinations of fatty acids.
1 .- 45 . (canceled)
46 . A method for producing a C4-C18 fatty acid or fatty acid derivative comprising:
combining a carbon source, a microorganism, and a cell culture to produce the C4-C18 fatty acid, wherein
a) said cell culture comprises an inhibitor of fatty acid synthase and/or the microorganism is genetically modified for reduced enzymatic activity in at least one of the microorganism's native fatty acid synthase pathway enzymes, providing for reduced conversion of malonyl-CoA to fatty acyl-ACPs; and
b) the microorganism additionally has one or more genetic modifications increasing fatty acid production.
47 . A method for producing a fatty acid or fatty acid derivative comprising:
combining a carbon source, a microorganism, and a cell culture to produce the fatty acid, wherein
a) said cell culture comprises an inhibitor of fatty acid synthase and/or the microorganism is genetically modified for reduced enzymatic activity in at least one of the microorganism's native fatty acid synthase pathway enzymes, providing for reduced conversion of malonyl-CoA to fatty acyl-ACPs; and
b) the microorganism additionally has one or more genetic modifications providing for increased conversion of malonyl-CoA to fatty acyl-CoAs, thereby increasing fatty acid production through a non-native fatty acid production pathway.
48 . The method of claim 46 or 47 , wherein the at least one fatty acid synthase pathway enzymes with reduced enzymatic activity is selected from the group consisting of: a beta-ketoacyl-ACP synthase, enoyl-ACP reductase, malonyl-coA-ACP transacylase, β-ketoacyl-ACP reductase, and β-hydroxyacyl-ACP dehydratase.
49 . The method of claim 46 or 47 , wherein the native fatty acid synthase pathway enzyme is selected from the group consisting of: fabI, a polypeptide of 80% or more homology to SEQ ID NO: 14, fabB, a polypeptide of 80% or more homology to SEQ ID NO: 9, fabF, a polypeptide of 80% or more homology to SEQ ID NO: 8, fabD, and a polypeptide of 80% or more homology to SEQ ID NO: 7.
50 . The method of claim 48 , wherein the enoyl-ACP reductase is fabI or a polypeptide of 80% or more homology to SEQ ID NO: 14, the beta-ketoacyl-ACP synthase is selected from the group consisting of fabB or a polypeptide of 80% homology or more to SEQ ID NO: 9, and fabF or a polypeptide of 80% homology or more to SEQ ID NO: 8, and the malonyl-coA-ACP transacylase is fabD or a polypeptide of 80% or more homology to SEQ ID NO: 7.
51 . The method of claim 49 or 50 wherein fabI, fabB, and fabD are temperature-sensitive mutants.
52 . The method of claim 51 , wherein the native fatty acid synthase pathway enzyme is a mutant temperature-sensitive fabI of E. coli having 80% homology or more to SEQ ID NO: 28 or SEQ ID NO: 29.
53 . The method of claim 46 , wherein the C4-C18 fatty acid is selected from the group consisting of C4, C6, C8, C10, C12, C14, C16, and C18 fatty acids.
54 . The method of claim 47 , wherein the genetic modifications providing for increased conversion of malonyl-CoA to fatty acyl-CoAs comprises genetic modifications to increase β-ketoacyl-CoA reductase activity, increase β-hydroxyacyl-CoA dehydratase activity, and increase enoyl-acyl-CoA reductase activity
55 . The method of claim 54 wherein the microorganism further comprises increased thioesterase activity.
56 . The method of claim 55 wherein the thioesterase is selected from the group consisting of tesA, ‘tesA, and tesB.
57 . The method of claim 46 or 47 , wherein the microorganism is genetically modified for increased enzymatic activity of an enzyme selected from the group consisting of: hbd, fadJ, crt, ech, and ter.
58 . The method of any of claims 1 - 57 , wherein the microorganism is E. coli.
59 . The method of any one of claims 1 - 58 , wherein the microorganism is further genetically modified to have modified malonyl-CoA dependent acetoacetyl-CoA synthase activity.
60 . The method of claim 59 , wherein the modified malonyl-CoA dependent acetoacetyl CoA synthase is nphT7 or a polypeptide of 80% or more homology to nphT7.
61 . The method of any one of claims 1 - 60 , wherein the microorganism is further genetically modified to have altered elongase activity.
62 . The method of claim 61 , wherein the microorganism has one or more modifications in the group consisting of: elo1, elo2, and elo3, or a polypeptide of 80% or more homology to a polypeptide from SEQ ID NOs: 199-201.
63 . The method of any one of claims 1 - 62 , wherein acetoacetyl-CoA is converted to a linear acyl-CoA with a chain length from 4-18 carbons.
64 . The method of any one of claims 1 - 62 , wherein the fatty acid derivative is selected from the group consisting of a fatty aldehyde, a fatty alcohol, and a fatty acid ester.
65 . The method of claim 64 , wherein the chemical is produced at a concentration higher than a concentration of said chemical produced by a wild type microorganism.
66 . A genetically modified microorganism for use according to any one of claims 1 - 65 .
67 . The genetically modified microorganism of claim 66 , wherein said microorganism comprises a sequence selected from group consisting of: SEQ ID NO: 1-215 or a sequence of 80% or more homology to SEQ ID NO: 1-215.
68 . The genetically modified microorganism of claim 66 , wherein said microorganism comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more sequences selected from group consisting of SEQ ID NO: 1-215 or sequences of 80% or more homology to SEQ ID NO: 1-215.
69 . The method of any one of claims 1 - 65 , wherein the microorganism is further genetically modified to induce enzyme expression from the yibD gene promoter with a decreased environmental phosphate concentration.
70 . A consumer product produced from the fatty acid or fatty acid derivative of claim 46 or 47 .
71 . A consumer product of claim 70 , wherein the product is selected from the group consisting of: a detergent, soap, resin, emulsifier, lubricant, grease, and wax.