Abstract
Polyhydroxyalkanoate (PHA) synthase from the bacterium Bacillus cereus YB-4 (PhaRCYB4), a Class IV PHA synthase, shows both PHA polymerization and alcoholysis activities. In PhaRCYB4-expressing Escherichia coli in the presence of ethanol, biosynthesized PHA is subjected to an ethyl-esterification at the carboxy terminus as a result of alcoholytic cleavage of the PHA chain by PhaRCYB4. In this study, we investigated the range of alcohol species available for alcoholysis by PhaRCYB4 using E. coli JM109 as an assay host by monitoring the molecular weight of PHA during the second step of a two-step cultivation. PhaRCYB4 utilized various alcohols other than ethanol for alcoholysis. Through the use of bifunctional compounds for alcoholysis, the PHA carboxy terminus could be modified with thiol, alkynyl, hydroxy, and benzyl groups, resulting in the functionalization of the PHA carboxy terminus. Furthermore, one-step production of carboxy-terminal functionalized PHA was developed using E. coli XL1-Blue as a production host.
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