Chimeric pseudomonad phasin enhances biosynthesis of short-medium-chain-length polyhydroxyalkanoates (PHAs) by Cupriavidus necator employing class II PHA synthase

Abstract

Copolyesters of ( R )-3-hydroxybutyrate and medium-chain-length ( R )-3-hydroxyalkanoate) [P(3HB- co -mcl-3HA)] are a highly flexible kind of polyhydroxyalkanoates (PHAs), although the efficient biosynthesis still remains a challenge. Biosynthesis of P(3HB- co -mcl-3HA) copolyesters was attempted using a class II PHA synthase in Cupriavidus necator H16, a well-studied PHA producer. Expression of the mutant of PHA synthase 1 from Pseudomonas sp. 61-3 (PhaC1 EDQK ), exhibiting broad substrate specificity to 3HA-CoAs of C 4 –C 12 , resulted in accumulation of only a small amount of PHA from soybean oil. This was caused by disappearance of PhaC1 EDQK specifically in the soybean oil-grown cells, despite active transcription of the corresponding gene. Co-expression of pseudomonad phasins PhaI Ps 61-3 and PhaF Ps 61-3 tended to increase PHA production. During this investigation, we identified a spontaneously generated chimeric mutant of the two phasins (designated PhaIF chi ), and found that PhaIF chi greatly increased P(3HB- co -mcl-3HA) accumulation on soybean oil. Further analysis revealed that both PhaC1 EDQK and native phasin PhaP1 lacked binding ability to P(3HB- co -mcl-3HA) granules, which was likely associated with the low level of PHA production. In contrast, PhaIF chi, comprised of a fusion of PHA-binding domains and predicted to form a tetrameric structure, was able to not only bind to the granules but also assist in localization of PhaC1 EDQK and PhaP1 on the granules, thereby promoting copolyester production. This study provides new insights into a novel function of granule-associated proteins in PHA biosynthesis, and demonstrates that engineering of phasins is a promising strategy for establishing efficient biomanufacturing of practical bioplastics.