RESUMEN
Natamycin is a widely used antifungal antibiotic. For natamycin biosynthesis, the gene pimE encodes cholesterol oxidase, which acts as a signalling protein. To confirm the positive effect of the gene pimE on natamycin biosynthesis, an additional copy of the gene pimE was inserted into the genome of Streptomyces gilvosporeus 712 under the control of the ermE* promoter (permE*) using intergeneric conjugation. Overexpression of the target protein engendered 72% and 81% increases in the natamycin production and cell productivity, respectively, compared with the control strain. Further improvement in the antibiotic production was achieved in a 1 L fermenter to 7.0 g/l, which was a 153% improvement after 120 h cultivation. Exconjugants highly expressing pimE and pimM were constructed to investigate the effects of both genes on the increase of natamycin production. However, the co-effect of pimE and pimM did not enhance the antibiotic production obviously, compared with the exconjugants highly expressing pimE only. These results suggest not only a new application of cholesterol oxidase but also a useful strategy to genetically engineer natamycin production.
Asunto(s)
Antibacterianos/metabolismo , Colesterol Oxidasa/genética , Colesterol Oxidasa/metabolismo , Natamicina/biosíntesis , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/biosíntesis , Antibacterianos/aislamiento & purificación , Reactores Biológicos , Medios de Cultivo/química , ADN Intergénico , Escherichia coli/genética , Genes Bacterianos/genética , Ingeniería Genética , Genoma Bacteriano , Familia de Multigenes , Natamicina/aislamiento & purificación , Regiones Promotoras Genéticas , Streptomyces/crecimiento & desarrolloRESUMEN
A ß-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL(-1) with o-nitrophenyl-ß-D-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L(-1) in which the target protein (6.24 g L(-1)) occupied approximately 86 %. The recombinant ß-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the ß-galactosidase may have a good application potential in whey and milk industries.
Asunto(s)
Aspergillus oryzae/enzimología , Proteínas Fúngicas/química , Lactosa/química , Leche/química , beta-Galactosidasa/química , Animales , Aspergillus oryzae/química , Secuencia de Bases , Codón , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Expresión Génica , Ingeniería Genética , Glucosa/metabolismo , Glicerol/metabolismo , Hidrólisis , Cinética , Datos de Secuencia Molecular , Nitrofenilgalactósidos/química , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes , Especificidad por Sustrato , Temperatura , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. The activity of expressed VHb was confirmed by the observation of VHb-specific CO-difference spectrum with a maximal absorption at 419 nm for the recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.
Asunto(s)
Antifúngicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ingeniería Metabólica , Natamicina/metabolismo , Streptomyces/metabolismo , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/metabolismo , Western Blotting , Cromosomas Bacterianos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Streptomyces/genéticaRESUMEN
Response surface methodology (RSM) was employed to optimize medium composition for the production of a thermostable xylanase from T. lanuginosus SDYKY-1 using economical carbon and nitrogen sources (soybean meal and corncobs, respectively). Plackett-Burman (P-B) design was applied to evaluate the effects of nine variables (powdered corncobs, soybean meal, Tween-80, CaCl(2), MgSO(4)·7H(2)O, FeSO(4), KH(2)PO(4), initial pH and inoculum culture volume). Corncobs, soybean meal and FeSO(4) were found to significantly influence on the xylanase production. The concentrations of these three factors were therefore optimized using central composite design and RSM. Adjusting the concentration of corncobs to 38.7g/L, soybean meal to 17.5g/L and FeSO(4) to 0.26g/L favored maximum xylanase production. Xylanase activity of 3078U/mL was obtained after optimization, which was a 144% increase that obtained before optimization (1264U/mL).
Asunto(s)
Ascomicetos/enzimología , Biotecnología/métodos , Endo-1,4-beta Xilanasas/biosíntesis , Temperatura , Análisis de Varianza , Ascomicetos/efectos de los fármacos , Reactores Biológicos/microbiología , Modelos Biológicos , Nitrógeno/farmacología , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
In order to alleviate oxygen limitation and improve the yield of poly-gamma-glutamic acid (gamma-PGA) during fermentation, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosome of Bacillus subtilis and expressed during gamma-PGA production. The activity of the expressed Vitreoscilla hemoglobin (VHb) was confirmed by CO-difference spectrum. Expression of VHb enhanced cell growth under high viscosity fermentation conditions 1.26-fold and increased the yield of gamma-PGA 2.07-fold. These results indicate that the expression of VHb could be advantageous in high viscosity fermentation media.