RESUMEN
Three strains of the genus Diaporthe were isolated from different plant hosts in south-western China. Phylogenetic analyses of the combined ITS, ß-tubulin, tef1 and calmoudulin dataset indicated that these strains represented three independent lineages in Diaporthe. Diaporthe millettiae sp. nov. clustered with D. hongkongensis and D. arecae, Diaporthe osmanthi sp. nov. grouped with D. arengae, D. pseudomangiferae and D. perseae and Diaporthe strain GUCC9146, isolated from Camellia sinensis, was grouped in the D. eres species complex with a close relationship to D. longicicola. These species are reported with taxonomic descriptions and illustrations.
RESUMEN
The alcohol dehydrogenase promoter PadhE and mixed acid fermentation pathway deficient mutants of Escherichia coli were employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Escherichia coli/genética , Hidroxibutiratos/metabolismo , Ingeniería Metabólica , Poliésteres/metabolismo , Aerobiosis , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fermentación , Glucosa/metabolismo , Plásmidos/genéticaRESUMEN
A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.
Asunto(s)
Butileno Glicoles/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Oxígeno/metabolismo , Butileno Glicoles/química , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , IsomerismoRESUMEN
A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kgammaori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/genética , Marcación de Gen/métodos , Genoma Bacteriano , Bacterias/genética , Cromosomas Bacterianos/genética , Plásmidos/genética , Recombinación GenéticaRESUMEN
Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (PadhE) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring PadhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Deltapta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of PadhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.
Asunto(s)
Proteínas Bacterianas/genética , Cupriavidus necator/genética , Escherichia coli/metabolismo , Ingeniería Genética , Polímeros/metabolismo , Regiones Promotoras Genéticas , Aerobiosis , Anaerobiosis , Proteínas Bacterianas/metabolismo , Clonación Molecular , Escherichia coli/genética , Fermentación , Regulación Bacteriana de la Expresión Génica , Peso Molecular , Polímeros/químicaRESUMEN
Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Coenzima A Ligasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Polihidroxialcanoatos/biosíntesis , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Caproatos/metabolismo , Caprilatos/metabolismo , Hidrolasas de Éster Carboxílico/genética , Coenzima A Ligasas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Ácidos Grasos/genética , Expresión Génica , Pseudomonas stutzeri/enzimología , Pseudomonas stutzeri/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
Dissolved oxygen (DO) plays an important role in cell growth, especially in industry-scale microbial production. To alleviate the defects of hypoxic conditions, Vitreoscilla hemoglobin (VHb) has been used to enhance respiration and energy metabolism by promoting oxygen delivery. Heterologous expression of VHb in a variety of hosts has been shown to improve cell growth, protein synthesis, metabolite productivity, and bioremediation under oxygen-restricted conditions. In this chapter, many well-studied areas are presented to illustrate the potential of VHb application in microbial metabolic engineering industry. Also, applications of the vgb promoter have been discussed.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fermentación/genética , Genes Bacterianos , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/metabolismo , Vitreoscilla/genética , Vitreoscilla/metabolismo , Antibacterianos/biosíntesis , Biodegradación Ambiental , Biotecnología , Escherichia coli/genética , Escherichia coli/metabolismo , Oxígeno/metabolismo , Polihidroxialcanoatos/biosíntesis , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more L -glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more L -glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA' was able to produce L: -glutamine effectively. Co-expression of vgb and glnA' genes in C. glutamicum produced 17 g/l L -glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA' gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l L -glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, L -glutamate, and L -glutamine production by recombinant C. glutamicum.