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Patent US0210054427
Inventor

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Much More than Average Length Specification


1 Independent Claims

  • Claim 2) The method of claim 1, wherein: the conversion of said C(n)-acyl-CoA and acetyl-CoA into said C(n+2)-ketoacyl-CoA is catalyzed by a ketoacyl-CoA thiolase; or wherein the conversion of said C(n+2)-ketoacyl-CoA and acetyl-CoA into said C(n+4)-polyketoacyl-CoA is catalyzed by a polyketoacyl-CoA thiolase; or wherein the conversion of said polyketoacyl-CoA, 3-OH-polyketoacyl-CoA, polyketoenoyl-CoA, or. alpha.,.beta.-saturated-polyketoacyl-CoA and acetyl-CoA into said extended polyketoacyl-CoA, 3-OH-polyketoacyl-CoA, polyketoenoyl-CoA or .alpha.,.beta.-saturated-polyketoacyl-CoA is catalyzed by a polyketoacyl-CoA thiolase.
  • Claim 3) The method of claim 2, wherein said ketoacyl-CoA thiolase or polyketoacyl-CoA thiolase is encoded by a gene(s) selected from the group consisting of Rhodococcus opacus pcaF, Pseudomonas putida pcaF, Streptomyces sp. pcaF, P. putida fadAx, P. putida fadA, Acinetobacter sp. ADP1 dcaF, Ralstonia eutropha bktB, and homologs with the same catalytic activity.
  • Claim 4) The method of claim 1, wherein: the conversion of said C(n+4)-polyketoacyl-CoA into said 3-OH--C(n+4)-polyketoacyl-CoA is catalyzed by a 3-OH-polyketoacyl-CoA dehydrogenase; or wherein conversion of said 3-OH--C(n+4)-polyketoacyl-CoA into said C(n+4) polyketoenoyl-CoA is catalyzed by a 3-OH-polyketoacyl-CoA dehydratase; or wherein conversion of said C(n+4) polyketoenoyl-CoA into said C(n+4)-. alpha.,.beta.-saturated-polyketoacyl-CoA is catalyzed by a polyketoenoyl-CoA reductaseor wherein conversion of said polyketoacyl-CoA to a polyketide is catalyzed by a thioesterase or takes place spontaneously.
  • Claim 5) The method of claim 4, wherein said thioesterase is encoded by a gene(s) selected from the group consisting of E. coli tesA, E. coli tesB, E. coli yciA, E. coli fadM, E. coli ydiI, E. coli ybgC, E. coli paaI, Mus musculus acot8, Alcanivorax borkumensis tesB2, Fibrobacter succinogenes Fs2108, Prevotella ruminicola Pr655, Prevotella ruminicola Pr1687, Lycopersicon hirsutum glabratum mks2 and homologs with the same catalytic activity.
  • Claim 6) The method of claim 4, wherein said 3-OH-polyketoacyl-CoA dehydrogenase is encoded by a gene(s) selected from the group consisting of E. coli fabG, E. coli fadB, E. coli fadJ, E. coli paaH, P. putida fadB, P. putida fadB2x, Acinetobacter sp. ADP1 dcaH, Ralstonia eutrophus phaB, Clostridium acetobutylicum hbd and homologs with the same catalytic activity.
  • Claim 7) The method of claim 4, wherein said 3-OH-polyketoacyl-CoA dehydratase is encoded by a gene(s) selected from the group consisting of E. coli fabA, E. coli fabZ, E. coli fadB, E. coli fadJ, E. coli paaF, P. putida fadB, P. putida fadB1x, Acinetobacter sp. ADP1 dcaE, Clostridium acetobutylicum crt, Aeromonas caviae phaf and homologs with the same catalytic activity.
  • Claim 8) The method of claim 4, wherein said polyenoylacyl-CoA reductase is encoded by a gene(s) selected from the group consisting of E. coli fadE, E. coli ydiO, Euglena gracilis TER, Treponema denticola TER, Clostridium acetobutylicum TER, E. coli fabI, Enterococcus faecalis fabK, Bacillus subtilis fabL, Vibrio cholerea fabV and homologs with the same catalytic activity.
  • Claim 9) The method of claim 1, wherein said derivative of said polyketide is a prenylated aromatic, or a cannabinoid, or dehydroacetic acid, or olivetolic acid, or cannabigerolic acid, or orsellinic acid, or 6-methylsalicylic acid.
  • Claim 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 11) The method of claim 10, wherein said method is performed in vivo using a genetically engineered microorganism that expresses said polyketoacyl-CoA thiolase, or wherein said method is performed in vitro using a purified polyketoacyl-CoA thiolase.
  • Claim 12) The method of claim 10, wherein said acetyl-CoA is omega functionalized.
  • Claim 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. fwdarw.C(n+2)-ketoacyl-CoAb) C(n+2)-ketoacyl-CoA+acetyl-CoA.fwdarw.C(n+4)-polyketoacyl-CoAc) optionally, C(n+4)-polyketoacyl-CoA.fwdarw.3-OH-- C(n+4)-polyketoacyl-CoAd) optionally, 3-OH-- C(n+4)-polyketoacyl-CoA.fwdarw.C(n+4)-polyketoenoyl-CoAe) optionally, C(n+4)-polyketoenoyl-CoA.fwdarw.C(n+4)-.alpha.,.beta.-saturated-polyketoa- cyl-CoAf) 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 .alpha.,.beta.-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 .alpha.,.beta.-saturated-polyketoacyl-CoA chains by two carbons and add a beta-keto groupand, 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 14) The microorganism of claim 13, further comprising reduced activity of endogenous thioesterases.
  • Claim 15) The microorganism of claim 13, wherein said microorganism has at least 10 fold more polyketoacyl thiolase activity than polyketoacyl-CoA dehydrogenase activity.
  • Claim 16) A recombinant microorganism having expressed polyketoacyl-CoA thiolase(s) that catalyze(s) the following reaction: ##STR00010## and having reduced activity of an endogenous thioesterase activity.
  • Claim 17) The recombinant microorganism of claim 16, said recombinant microorganism having expressed genes encoding a type II thiolase (2. 3.1.9) and a polyketoacyl-CoA thiolase (2.3.1._).
  • Claim 18) The recombinant microorganism of claim 17, further comprising. DELTA.adhE, (.DELTA.pta or .DELTA.ackA or .DELTA.ack.DELTA.pta), .DELTA.poxB, .DELTA.ldhA, and .DELTA.frdA.
  • Claim 19) The microorganism of claim 18, further comprising reduced expression of fermentation enzymes leading to reduced production of lactate, acetate, ethanol and succinate.
  • Claim 20) A recombinant microorganism having expressed genes encoding at least two different thiolases, said thiolases including a ketoacyl thiolase and a polyketoacyl thiolase.


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