Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in metabolically recombinant Escherichia coli

In this study, a phaC_R gene encoding PHA synthase was identified in Rhodoligotrophos defluvii which was adjacent to β-ketothiolase encoded by phaA_R gene and acetoacetyl-CoA reductase encoded by phaB_R gene. Amino acid comparison of PhaC_R showed the highest homology of 65.98% with PhaC of R. appendicifer, while its homology with typical class I PHA synthase in Cupriavidus necator was only 42.54%. PHA synthesis genes were then transformed into E. coli harboring phaCAB_R and phaC_RAB_C which were cultured with 15 g/L glucose respectively, and 20.46 wt% and 16.95 wt% of CDW for poly(3-hydroxybutyrate) (PHB) were accumulated respectively. To further explore the effect of substrate specificity for PHA production, the ptsG gene was then deleted and 15 g/L glucose and 1.5 g/L propionate were co-employed as carbon sources, which enabled the synthesis of poly(3HB-co-3HV) copolymer. As a result, poly(3HB-co-3HV) was accumulated up to 24.74 wt% of CDW, and the highest content of 3-hydroxyvalerate (3HV) was 10.86 mol%. The T_d5 was 260 °C, which implied that it possessed good thermal stability, and the M_w of GPC in recombinant strains were between 22 and 26 × 10⁴ g/mol, and the highest PDI was 3.771. The structure of poly (3HB-co-3HV) copolymer was determined through ¹H NMR analysis.