Submitted to: Biophysical Society
Publication Type: Abstract Only
Publication Acceptance Date: 11/22/2006
Publication Date: 3/3/2007
Citation: Aneja, K., Solaiman, D., Zhu, J., Zu, H., Wang, B. 2007. Cloning and Expression of Poly(hydroxyalkanoate) Synthase Genes from Photosynthetic bacterium Allochromatium vinosum ATCC 35206 [abstract]. 51st Biophysical Society Annual Meeting. p. 212a.
Technical Abstract: Poly(hydroxyalkanoate) (PHA) synthases catalyze the polymerization of beta-hydroxy fatty acids to form PHA biopolyesters. These enzymes are grouped into four classes (classes I to IV) based on their subunit composition and substrate specificity. Since PHA biopolymers are naturally synthesized by bacteria and are biodegradable, they are of intense interest to researchers in the packaging industry, food industry, agriculture and medicine. There is a continued need to improve the material properties of PHA via compositional modification to expand its applications and market potential. Protein engineering and gene-shuffling of PHA synthase (PHAS) genes (pha) can help achieve this goal, and the availability of new pha genes and the 3-dimensional structure of PHAS are in turn important for these undertakings. In this presentation, we report the cloning, sequencing and expression of class III PHAS genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206. Class III PHAS’s are of interest because they are uniquely composed of 2 hetero-subunits and are responsible for the synthesis of short-chain-length, thermoplastic PHA. We employed a PCR technique coupled with chromosomal gene-walking method to clone and subsequently sequence the contiguous phaC (1068 bps) and phaE (1065 bps) genes of A. vinosum ATCC 35206. A BLASTP search of protein databases showed that the gene-products of phaC and phaE are different from the previously reported pha genes of A. vinosum DSM180. Domain analysis also revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. The individual phaC and phaE genes, the contiguous phaE-C operon and the alpha/beta-hydrolase domain of phaC were expressed in E. coli using a His6-tagged expression vector (pProEX-HTc). High-level expression of these 4 sequences was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The isolated gene products were subjected to a high-throughput crystallization screening to identify conditions suitable for crystal growth. The genes obtained in this study add to the genetic variety of phaC genes for gene-shuffling investigation, and the high expression of the gene-products afforded protein samples for crystallographic study. (Supported by USDA/CSREES NRI grant 2003-35504-13751.)