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

Abstract

BACKGROUND: 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. RESULTS: 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 (PhaC1EDQK), exhibiting broad substrate specificity to 3HA-CoAs of C4-C12, resulted in accumulation of only a small amount of PHA from soybean oil. This was caused by disappearance of PhaC1EDQK specifically in the soybean oil-grown cells, despite active transcription of the corresponding gene. Co-expression of pseudomonad phasins PhaIPs61-3 and PhaFPs61-3 tended to increase PHA production. During this investigation, we identified a spontaneously generated chimeric mutant of the two phasins (designated PhaIFchi), and found that PhaIFchi greatly increased P(3HB-co-mcl-3HA) accumulation on soybean oil. Further analysis revealed that both PhaC1EDQK 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, PhaIFchi, 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 PhaC1EDQK and PhaP1 on the granules, thereby promoting copolyester production. CONCLUSION: 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.

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