Altered composition of Ralstonia eutropha poly(hydroxyalkanoate) through expression of PHA synthase from Allochromatium vinosum ATCC 35206.
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. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 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 poly(hydroxybutanoate) (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 PHB, albeit at a low level (4-5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.
Aneja
Kawalpreet K
KK
Fats, Oils and Animal Coproducts Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA, 19038, USA.
Ashby
Richard D
RD
Solaiman
Daniel K Y
DK
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002841:Chromatiaceae / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D023061:Gene Order; D008969:Molecular Sequence Data; D054813:Polyhydroxyalkanoates / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D054813:Polyhydroxyalkanoates; D011994:Recombinant Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase