Engineering of stable recombinant bacteria for production of chiral medium-chain-length poly-3-hydroxyalkanoates.
In order to scale up medium-chain-length polyhydroxyalkanoate (mcl-PHA) production in recombinant microorganisms, we generated and investigated different recombinant bacteria containing a stable regulated expression system for phaC1, which encodes one of the mcl-PHA polymerases of Pseudomonas oleovorans. We used the mini-Tn5 system as a tool to construct Escherichia coli 193MC1 and P. oleovorans POMC1, which had stable antibiotic resistance and PHA production phenotypes when they were cultured in a bioreactor in the absence of antibiotic selection. The molecular weight and the polydispersity index of the polymer varied, depending on the inducer level. E. coli 193MC1 produced considerably shorter polyesters than P. oleovorans produced; the weight average molecular weight ranged from 67,000 to 70,000, and the polydispersity index was 2.7. Lower amounts of inducer added to the media shifted the molecular weight to a higher value and resulted in a broader molecular mass distribution. In addition, we found that E. coli 193MC1 incorporated exclusively the R configuration of the 3-hydroxyoctanoate monomer into the polymer, which corroborated the enantioselectivity of the PhaC1 polymerase enzyme.
Prieto
M A
MA
Institute of Biotechnology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Kellerhals
M B
MB
Bozzato
G B
GB
Radnovic
D
D
Witholt
B
B
Kessler
B
B
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D005818:Genetic Engineering; D008970:Molecular Weight; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D013237:Stereoisomerism
D001426:Bacterial Proteins; D017931:DNA Primers; D011091:Polyesters; D000217:Acyltransferases; C067032:poly(3-hydroxyalkenoate)polymerase