Genome-Wide Metabolic Reconstruction of the Synthesis of Polyhydroxyalkanoates from Sugars and Fatty Acids by Burkholderia Sensu Lato Species

Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner–Doudoroff (ED), pentose-phosphate (PP), and lower Embden–Meyerhoff–Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via β-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)

Polyhydroxybutyrate is a component of bioplastics that is synthesized under the control of enzymes encoded by pha multigenes. The genes are naturally present in Ralstonia eutropha. However, the production of bioplastics in bacteria is inefficient because the bacterial biomass is relatively small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objectives of this study were to transform the tissue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The genetic transformation of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon tissue of J. curcas in a suspension of the bacterium for 30 min, followed by cocultivation for 3 d on Murashige and Skoog (MS) medium. The tissue was then placed on a selection medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addition of 20 mg/L kanamycin. The results showed that 12.35% of the tissue survived and regenerated into a shoot after 1–2 months. Molecular analysis of the transformed tissue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respectively.

The genetics of feeding in Caenorhabditis elegans.

The pharynx of Caenorhabditis elegans is a nearly self-contained neuromuscular organ responsible for feeding. To identify genes involved in the development or function of the excitable cells of the pharynx, I screened for worms with visible defects in pharyngeal feeding behavior. Fifty-two mutations identified 35 genes, at least 22 previously unknown. The genes broke down into three broad classes: 2 pha genes, mutations in which caused defects in the shape of the pharynx, 7 phm genes, mutations in which caused defects in the contractile structures of the pharyngeal muscle, and 26 eat genes, mutants in which had abnormal pharyngeal muscle motions, but had normally shaped and normally birefringent pharynxes capable of vigorous contraction. Although the Eat phenotypes were diverse, most resembled those caused by defects in the pharyngeal nervous system. For some of the eat genes there is direct evidence from previous genetic mosaic and pharmacological studies that they do in fact affect nervous system. In eat-5 mutants the motions of the different parts of the pharynx were poorly synchronized. eat-6 and eat-12 mutants failed to relax their pharyngeal muscles properly. These pharyngeal motion defects are most easily explained as resulting from abnormal electrical excitability of the pharyngeal muscle membrane.