A novel thiolase–reductase gene fusion promotes the production of polyhydroxybutyrate in Arabidopsis

Abstract

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.

Diversion of Flux toward Sesquiterpene Production in Saccharomyces cerevisiae by Fusion of Host and Heterologous Enzymes

“…The process likely benefits from a proximity effect of the two active sites to reduce loss of intermediates to other competing pathways. Previous attempts to use this concept in vivo have generally been unsuccessful (1,18,36) despite the fact that fusion enzymes have proven superior to free enzymes in several in vitro studies (6,7,22,29). At least two parameters should be considered in order to obtain successful exploitation of fusion proteins in vivo.…”

“…However, the most simple and most studied approach is to simply fuse two open reading frames (ORFs) which encode different enzymatic activities. This allows expression of chimeric proteins with two enzymes fused end-to-end into a single polypeptide (1,18,36). A number of observations suggest that this strategy can be used to improve the flux through a metabolic pathway.…”

Plastics Completely Synthesized by Bacteria: Polyhydroxyalkanoates

“…PHB synthesis using genetic engineering approaches was reported in some plants, including switchgrass (Somleva et al 2008), sugarcane (Purnell et al 2007), sugar beet (Menzel et al 2003), tobacco (Lossl et al 2005), flax (Wrobel et al 2004), Arabidopsis thaliana (Kourtz et al 2005), rape, and corn (Poirier 2002).…”

Metabolic Engineering of Plants for the Synthesis of Polyhydroxyalkanaotes

“…In this respect, a novel fusion protein composed of the 3-ketothiolase and acetoacetyl-CoA reductase from R. eutropha was created (Kourtz et al 2005). This was a challenging project since the native thiolase and reductase enzymes act as homotetramers in bacteria.…”