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
Many polyhydroxyalkanoates (PHAs) system genes, such as phaC, phaA, phaB, phaR, phaP and phaZ, are often found to be organised in the form of operon-like clusters. In this study, a genome survey was performed to identify such clustered PHA systems among 256 prokaryotic organisms. These data were then used to generate a comprehensive 16S rRNA species tree depicting the phylogenetic distribution of the observed clusters with diverse gene arrangements. In addition, the gene occurrences and physical linkages between PHA system genes were quantitatively estimated. From this, we identified a centrally connected hub gene, i.e. the phaC gene of PHA. Furthermore, a comparative investigation was performed between the clusters of PHA and glycogen, which decoded the role of the hub gene in the cluster organisation of both systems. Together, these findings suggest that the highly connected hub gene might contribute substantively towards the organisation and maintenance of the gene network connectivity in the clusters, particularly in the storage reserve systems.
Assuntos
Archaea/genética , Bactérias/genética , Família Multigênica , Filogenia , Poli-Hidroxialcanoatos/genética , Sequência de Aminoácidos , Sequência Conservada , Bases de Dados de Ácidos Nucleicos , Proteína Semelhante a ELAV 2/genética , Evolução Molecular , Ligação Genética , Glicogênio/genética , Óperon , RNA Ribossômico 16S/genéticaRESUMO
Polyphosphate accumulating organisms (PAOs) belong mostly to Proteobacteria and Actinobacteria and are quite divergent. Under aerobic conditions, they accumulate intracellular polyphosphate (polyP), while they typically synthesize polyhydroxyalkanoates (PHAs) under anaerobic conditions. Many ecological, physiological, and genomic analyses have been performed with proteobacterial PAOs, but few with actinobacterial PAOs. In this study, the whole genome sequence of an actinobacterial PAO, Microlunatus phosphovorus NM-1(T) (NBRC 101784(T)), was determined. The number of genes for polyP metabolism was greater in M. phosphovorus than in other actinobacteria; it possesses genes for four polyP kinases (ppks), two polyP-dependent glucokinases (ppgks), and three phosphate transporters (pits). In contrast, it harbours only a single ppx gene for exopolyphosphatase, although two copies of ppx are generally present in other actinobacteria. Furthermore, M. phosphovorus lacks the phaABC genes for PHA synthesis and the actP gene encoding an acetate/H(+) symporter, both of which play crucial roles in anaerobic PHA accumulation in proteobacterial PAOs. Thus, while the general features of M. phosphovorus regarding aerobic polyP accumulation are similar to those of proteobacterial PAOs, its anaerobic polyP use and PHA synthesis appear to be different.