Metabolic engineering studies for the production of polyhydroxyalkanoate (PHA) in Escherichia coli have been carried out. For these tasks, metabolic pathways of E. coli including glycolytic pathway and fatty acid β-oxidation pathway have been engineered to support PHA production.
At first, poly(3-hydroxybutyrate) [P(3HB)], a member of short-chain-length (SCL) PHA was efficiently produced by the amplification of triosephosphate isomerase (TpiA) or fructose-bisphosphate aldolase (FbaA) enzyme, which were previously identified by proteome analysis of E. coli XL1-Blue producing P(3HB).
Secondly, β-oxidation pathway was metabolically engineered to supply medium-chain-length (MCL) PHA precursors. MCL PHA enriched in the specific monomers could be produced by the amplification of FadD and FadE enzymes in the recombinant E. coli strains deficient in the FadA and/or FadB. Also, amplification of FabG enzymes in fadA mutant E. coli strain supported the production of MCL PHA enriched in the specific monomer. Enhanced production of MCL-PHA from fatty acid was achieved by the amplification of FadB homologous enzymes. These enzymes include YfcX, PaaF, PaaG, and YdbU. A new enoyl-CoA hydratase MaoC involved in the production of MCL PHA in fadB mutant E. coli strain was identified and characterized. Enzymatic and genetic analyses revealed that MaoC enzyme constructs the metabolic link between beta-oxidation pathway and PHA biosynthetic pathway when the FadB is removed.
Thirdly, PHA consisting of SCL and MCL monomers could be produced. Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] was produced from fatty acid by the expression of Aeromonas PHA synthase and enoyl-CoA hydratase genes. Also, terpolymer PHA consisting of 3HB, 3-hydroxyvalerate (3HV), and 3HHx could be produced by supplying odd-carbon-number fatty acid and lauric acid concomitantly. Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] was produced from fatty acid by the expression of Pseu...