Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases
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Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 Pre , phaC2 Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 Pre and phaC2 Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.
Keywordsα/β-Hydrolase fold Hybrid gene Poly(hydroxyalkanoate) synthases
The authors thank Dr. DiRusso for the kind gift of E. coli LS1298. The assistance of Drs. Richard D. Ashby and Alberto Nuñez in acquiring the compositional data is acknowledged. The author also thanks Nicole Cross and Marshall Reed for technical assistance.
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