RESUMEN
The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.
Asunto(s)
Acil-CoA Deshidrogenasa/genética , Cupriavidus necator/genética , Enoil-CoA Hidratasa/genética , Aceites de Plantas/metabolismo , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/genética , Acil-CoA Deshidrogenasa/metabolismo , Arabinosa/genética , Arabinosa/metabolismo , Caprilatos/metabolismo , Cupriavidus necator/metabolismo , Ácidos Decanoicos/metabolismo , Enoil-CoA Hidratasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Hidroxibutiratos/metabolismo , Plásmidos/genética , Polihidroxialcanoatos/metabolismo , Regiones Promotoras Genéticas/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transcripción Genética/genéticaRESUMEN
Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).