Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.

SummaryThe production of polyhydroxybutyrate (PHB) involves a multigene pathway consisting of thiolase, reductase and synthase genes. In order to simplify this pathway for plant-based expression, a library of thiolase and reductase gene fusions was generated by randomly ligating a short core linker DNA sequence to create in-frame fusions between the thiolase and reductase genes. The resulting fusion constructs were screened for PHB formation in Escherichia coli . This screen identified a polymer-producing candidate in which the thiolase and reductase genes were fused via a 26-amino-acid linker. This gene fusion, designated phaA-phaB , represents an active gene fusion of two homotetrameric enzymes. Expression of phaA-phaB in E. coli and Arabidopsis yielded a fusion protein observed to be the expected size by Western blotting techniques. The fusion protein exhibited thiolase and reductase enzyme activities in crude extracts of recombinant E. coli that were three-fold and nine-fold less than those of the individually expressed thiolase and reductase enzymes, respectively. When targeted to the plastid, and coexpressed with a plastid-targeted polyhydroxyalkanoate (PHA) synthase, the fusion protein enabled PHB formation in Arabidopsis , yielding roughly half the PHB formed in plants expressing individual thiolase, reductase and synthase enzymes. This work represents a first step towards simplifying the expression of the PHB biosynthetic pathway in plants.

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