期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 79, 期 12, 页码 3813-3821出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.00564-13
关键词
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资金
- RIKEN Biomass Engineering Program
- USM APEX DE
- Universiti Sains Malaysia's Fellowship Scheme
- RIKEN's International Program Associate Fellowship
Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromo-bacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wildtype and mutant synthases for either SCL (C-4 and C-5) or MCL (C-6) substrates substantiates the fundamental classification of PHA synthases.
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