|Ashby, Richard - Rick|
Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2009
Publication Date: 6/26/2009
Citation: Aneja, K.K., Ashby, R.D., Solaiman, D. 2009. Altered Composition of Ralstonia eutropha Poly(hydroxyalkanoate) through Expression of PHA Synthase from Allochromatium vinosum ATCC 35206. Biotechnology Letters. 31:1601-1612.
Interpretive Summary: Poly(hydroxyalkanoates) (PHAs) are bioplastics synthesized and stored inside the cell by many bacteria. PHAs possess properties that make them useful as environmentally friendly alternatives to the fossil fuel-based polymers in various applications such as biomedical devices, consumer products, packaging materials, and adhesives. We have previously demonstrated that agricultural co/products can be used as feedstocks to produce PHA by many natural and genetically engineered bacteria, thus creating a new outlet for agricultural products. One approach to get new PHA is by manipulating the genes responsible for PHA. PHA production in a strain of photosynthetic bacterium called Allochromatium vinosum ATCC 35206. We found that genetically engineered bacterium could produce an unique PHA. Further research to achieve high-yield production of this new biopolymer should make available new materials useful for casting biodegradable articles such as containers and films to benefit the consumer.
Technical Abstract: The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. We employed a PCR technique coupled with a chromosomal gene-walking method to clone and subsequently sequence the contiguous phaC (1068 bps) and phaE (1065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved alpha/beta-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a PHB-negative mutant of Ralstonia eutropha PHB-4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHA, albeit at a low level (1%-cell dry weight). However, in contrast to the wild-type R. eutropha which produces poly(hydroxybutyrate), the recombinant strain synthesized either a blend or a copolymer of PHA that also contains a high content of beta-hydroxyoctanoate and beta-hydroxydecanoate as its repeat-unit monomers. (Supported by USDA/CSREES NRI grant 2003-35504-13751.)