Propionyl-CoA dependent biosynthesis of 2-hydroxybutyrate containing polyhydroxyalkanoates in metabolically engineered Escherichia coli

• Escherichia coli strain was metabolically engineered to produce 2-hydroxybutyrate (2HB)-containing polyhydroxyalkanoate (PHA).
• Propionyl-CoA was used as precursor for 2HB monomer for PHA synthesis.
• PHA with modified monomer compositions could be synthesized by engineering precursor synthesis pathway for PHA monomers.

We have previously reported in vivo biosynthesis of 2-hydroxyacid containing polyesters including polylactic acid (PLA), poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], and poly(3-hydroxybutyrate-co-2-hydroxybutyrate-co-lactate) [P(3HB-co-2HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6–19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6–19). In this study, we further engineered in vivo PLA biosynthesis system in E. coli to synthesize 2HB-containing PHA, in which propionyl-CoA was used as precursor for 2-ketobutyrate that was converted into 2HB-CoA by the sequential actions of Lactococcus lactis (d)-2-hydroxybutyrate dehydrogenase (PanE) and PctCp and then 2HB-CoA was polymerized by PhaC1Ps6–19. The recombinant E. coli XL1-blue expressing the phaC1437 gene, the pct540 gene, and the Ralstonia eutropha prpE gene together with the panE gene could be grown to 0.66 g/L and successfully produced P(70 mol%3HB-co-18 mol%2HB-co-12 mol%LA) up to the PHA content of 66 wt% from 20 g/L of glucose, 2 g/L of 3HB and 1 g/L of sodium propionate. Removal of the prpC gene in the chromosome of E. coli XL1-blue could increase the mole fraction of 2HB in copolymer, but the PHA content was decreased.The metabolic engineering strategy reported here suggests that propionyl-CoA can be successfully used as the precursor to provide PHA synthase with 2HB-CoA for the production of PHAs containing 2HB monomer.