Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases.
Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes ( phaC1(Pre), phaC2(Pre)) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved alpha/beta-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the alpha/beta-hydrolase-fold coding portions of phaC1(Pre) and phaC2(Pre) at the 3' terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73-75% beta-hydroxydecanoate (beta-HD) and 25-27% beta-hydroxyoctanoate (beta-HO). Deletion mutants, Delta pha7 and Delta pha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55-60 mol% beta-HD and 40-45 mol% beta-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.
Solaiman
Daniel K Y
DK
Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA. dsolaiman@arserrc.gov
D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003652:Decanoic Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000254:growth & development; D030781:Organisms, Genetically Modified / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D017384:Sequence Deletion / Q000235:genetics*
D003652:Decanoic Acids; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase