Present address: Dyax, Corp., 300 Technology Square, 8th Floor, Cambridge, MA 02139, USA
A novel thiolase–reductase gene fusion promotes the production of polyhydroxybutyrate in Arabidopsis
Article first published online: 24 JUN 2005
Plant Biotechnology Journal
Volume 3, Issue 4, pages 435–447, July 2005
How to Cite
Kourtz, L., Dillon, K., Daughtry, S., Madison, L. L., Peoples, O. and Snell, K. D. (2005), A novel thiolase–reductase gene fusion promotes the production of polyhydroxybutyrate in Arabidopsis. Plant Biotechnology Journal, 3: 435–447. doi: 10.1111/j.1467-7652.2005.00136.x
- Issue published online: 24 JUN 2005
- Article first published online: 24 JUN 2005
- Received 13 September 2004; revised 23 December 2004; accepted 19 January 2005.
- gene fusion;
- hybrid enzyme;
The 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.