RESUMO
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.
Assuntos
Acil-CoA Desidrogenase/genética , Cupriavidus necator/genética , Enoil-CoA Hidratase/genética , Óleos de Plantas/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Poli-Hidroxialcanoatos/genética , Acil-CoA Desidrogenase/metabolismo , Arabinose/genética , Arabinose/metabolismo , Caprilatos/metabolismo , Cupriavidus necator/metabolismo , Ácidos Decanoicos/metabolismo , Enoil-CoA Hidratase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Hidroxibutiratos/metabolismo , Plasmídeos/genética , Poli-Hidroxialcanoatos/metabolismo , Regiões Promotoras Genéticas/genética , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transcrição Gênica/genéticaRESUMO
The aim of this study was to increase the density of wild type Cupriavidus necator H16 biomass grown on fructose in order to produce sufficient copolymer of short-chain-length (scl) and medium-chain-length (mcl) polyhydroxyalkanoate (PHA) from canola oil for mechanical testing of the PHA. Initial batch cultivation on fructose was followed by exponential feeding of fructose at a predetermined µ to achieve 44.4 g biomass/l containing only 20 % w/w of polyhydroxybutyrate (PHB) with a Y(x/fructose) of 0.44 g/g. In a third stage, canola oil was added under N-limited conditions to produce 92 g/l of biomass with 48 % w/w scl-mcl PHA. Using known standards, the PHA composition was confirmed by GC-MS analysis as 99.81 % 3-hydroxybutyrate, 0.06 % 3-hydroxyvalerate, 0.09 % 3-hydroxyhexanoate and 0.04 % 3-hydroxyoctanoate. The melting temperature (179 °C), crystallinity (54 %), tensile stress (25.1 Mpa) and Young's modulus (698 Mpa) for a PHB standard decreased to 176 °C, 52 %, 19.1 and 443 Mpa respectively for C. necator PHA produced in the 3-stage process.