This disclosure generally relates to the use of microorganisms to make various functionalized polyketides through polyketoacyl-CoA thiolase-catalyzed non-decarboxylative condensation reactions instead of decarboxylative reactions catalyzed by polyketide synthases. Native or engineered polyketoacyl-CoA thiolases catalyze the non-decarboxylative Claisen condensation in an iterative manner (i.e. multiple rounds) between two either unsubstituted or functionalized ketoacyl-CoAs (and polyketoacyl-CoAs) serving as the primers and acyl-CoAs serving as the extender unit to generate (and elongate) polyketoacyl-CoAs. Before the next round of polyketoacyl-CoA thiolase reaction, the β-keto group of the polyketide chain of polyketoacyl-CoA can be reduced and modified step-wise by 3-OH-polyketoacyl-CoA dehydrogenase or polyketoenoyl-CoA hydratase or polyketoenoyl-CoA reductase. Dehydrogenase converts the β-keto group to β-hydroxy group. Hydratase converts the β-hydroxy group to α-β-double-bond. Reductase converts the α-β-double-bond to single bond. Spontaneous or thioesterase catalyzed termination reaction terminates the elongation of polyketide chain of polyketoacyl-CoA at any point through CoA removal and spontaneous reactions rearrange the structure, generating the final functional polyketide products.
Claim CLM-00001. 1) A method of making a polyketide, comprising growing a genetically engineered microorganism in a nutrient broth for a time sufficient to produce a polyketide and isolating said polyketide or a spontaneously rearranged form of said polyketide or a derivative of said polyketide, wherein said microorganism has a polyketide-producing pathway comprising the following substrate(s) to product(s) conversions:
a) C(n)-acyl-CoA+acetyl-CoA→C(n+2)-ketoacyl-CoA; b) C(n+2)-ketoacyl-CoA+acetyl-CoA→C(n+4)-polyketoacyl-CoA; c) optionally, C(n+4)-polyketoacyl-CoA→3-OH— C(n+4)-polyketoacyl-CoA; d) optionally, 3-OH— C(n+4)-polyketoacyl-CoA→C(n+4)-polyketoenoyl-CoA; and e) optionally, C(n+4)-polyketoenoyl-CoA→C(n+4)-α,β-saturated-polyketoacyl-CoA; f) iterations of at least one of the reactions in steps b), c), d), and e) wherein said iterations are achieved by utilizing the polyketoacyl-CoAs, 3-OH-polyketoacyl-CoAs, polyketoenoyl-CoA and α,β-saturated-polyketoacyl-CoA generated in reactions steps b), c), d), and e), respectively, as substrates for condensation with acetyl-CoA to elongate said polyketoacyl-CoAs, 3-OH-polyketoacyl-CoAs, polyketoenoyl-CoA, and α,β-saturated-polyketoacyl-CoA chains by two carbons and add a beta-keto group; and g) conversion of said polyketoacyl-CoA in steps b), c), d), e), or f) to a polyketide or a spontaneously rearranged form of said polyketide or a derivative of said polyketide; wherein n>0 and <30.
Claim CLM-00010. 10) A method, comprising:
a) combining a polyketoacyl-CoA thiolase with acetyl-CoA and an acetoacetyl-CoA primer, or a ketoacyl-CoA primer or a polyketoacyl-CoA primer under conditions sufficient to allow said polyketoacyl-CoA thiolase to perform non-decarboxylative Claisen condensations with said acetyl-CoA and said primer to form a polyketoacyl-CoA; b) hydrolyzing said polyketoacyl-CoA to form polyketide or a spontaneous rearrangement form of said polyketide and free Co-A; and c) isolating said polyketide or said spontaneous rearrangement form of said polyketide or a derivative of said polyketide.
Claim CLM-00013. 13) A genetically engineered microorganism, wherein said microorganism has a polyketide-producing pathway comprising the following substrate(s) to product(s) conversions:
a) C(n)-acyl-CoA+acetyl-CoA→C(n+2)-ketoacyl-CoA; b) C(n+2)-ketoacyl-CoA+acetyl-CoA→C(n+4)-polyketoacyl-CoA; c) optionally, C(n+4)-polyketoacyl-CoA→3-OH— C(n+4)-polyketoacyl-CoA; d) optionally, 3-OH— C(n+4)-polyketoacyl-CoA→C(n+4)-polyketoenoyl-CoA; e) optionally, C(n+4)-polyketoenoyl-CoA→C(n+4)-α,β-saturated-polyketoacyl-CoA; f) iterations of at least one of the reactions in steps b), c), d), and e) wherein said iterations are achieved by utilizing the polyketoacyl-CoAs, 3-OH-polyketoacyl-CoAs, polyketoenoyl-CoA and α,β-saturated-polyketoacyl-CoA generated in reactions steps b), c), d), and e) as substrates for condensation with acetyl-CoA to elongate said polyketoacyl-CoAs, 3-OH-polyketoacyl-CoAs, polyketoenoyl-CoA, and α,β-saturated-polyketoacyl-CoA chains by two carbons and add a beta-keto group; and, g) conversion of said polyketoacyl-CoA in steps b), c), d), e), or f) to a polyketide or a spontaneously rearranged form of said polyketide.
Claim CLM-00016. 16) A recombinant microorganism having expressed polyketoacyl-CoA thiolase(s) that catalyze(s) the following reaction: and having reduced activity of an endogenous thioesterase activity.
Claim CLM-00020. 20) A recombinant microorganism having expressed genes encoding at least two different thiolases, said thiolases including a ketoacyl thiolase and a polyketoacyl thiolase.
