Abstract

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), P(3HB-co-3HHx), is one of the most practical PHAs as it exhibits higher flexibility and lower melting temperature and crystallinity compared to P(3HB) homopolymer. While P(3HB-co-3HHx) is generally produced from plant oils and fatty acids by several wild and recombinant bacteria, biosynthesis of this copolyester from unrelated sugars is expected to be another way for the low-cost production, as well as an intriguing challenge in the view point of metabolic engineering. Our group previously reported an artificial pathway for P(3HB-co-3HHx) biosynthesis from fructose by which C₆-acyl-CoA intermediates were generated via condensation of acetyl-CoA with butyryl-CoA formed from crotonyl-CoA by NADPH-dependent crotonyl-CoA reductase (Ccr). When the pathway was installed into Ralstonia eutropha strain PHB^-4 using ccr from Streptomyces cinnamonensis and phaC from Aeromonas caviae, the recombinant strain actually accumulated P(3HB-co-3HHx) from fructose although the 3HHx composition was lower than 1.6 mol% (Biomacromolecules, 2002, 3:618). The present study aimed to improve the pathway in R. eutropha for production of the copolyester with high 3HHx composition. It was observed that Ccr from Methylobacterium extorquens gave stronger impacts on PHA biosynthesis by R. eutropha than that from S. cinnamonensis. The results further clarified effective modifications in the Ccr-driven pathway for simultaneous supply of (R)-3HB-CoA and (R)-3HHx-CoA from acetyl-CoA molecules. The resulting engineered strain of R. eutropha equipped with the advanced artificial pathway could produce P(3HB-co-3HHx) composed of 22 mol% 3HHx fraction with high cellular content from fructose.