RESUMO
An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE" commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in (3-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
RESUMO
An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in β-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.
Assuntos
Conjugação Genética , Glucuronidase/genética , Regiões Promotoras Genéticas , Streptomyces rimosus/genéticaRESUMO
ABSTRACTOxytetracycline (OTC) production byStreptomyces rimosus was studied in batch and fed-batch cultures in shake flask and bioreactor levels using semi-defined medium. First, the effect of glucose concentration on OTC production and growth kinetics was studied intensively. The optimal glucose concentration in the medium was 15 g/L. Higher glucose concentrations supported higher biomass production by less volumetric and specific antibiotic production. Based on these data, cultivations were carried out at semi-industrial scale 15 L bioreactor in batch culture. At bioreactor level, cell growth and OTC production were higher compared to the shake flask culture by about 18 and 38%, respectively. During the bioreactor cultivation, glucose was totally consumed after only 48 h. Thus, the fed-batch experiment was designed for mono-glucose feeding and complete medium feeding to increase the OTC production by overcoming carbon limitations. The results showed that the fed-batch culture using constant glucose feeding strategy with rate of 0.33 g/L/h produced 1072 mg/L. On the other hand, feeding with complete medium resulted in 45% higher biomass but less OTC production by about 26% compared to mono-glucose fed culture. A further improvement in this process was achieved in by keeping the dissolved oxygen (DO) value at 60% saturation by cascading the glucose feeding pump with the DO controller. The later feeding strategy resulted in higher antibiotic production, reaching 1414 mg/L after 108 h.
RESUMO
The antioxidant activity of lycopene is highest among the current carotenoids.Recently the researches on lycopene are focused on ingredient of functional foods in the world.The fermentation production of lycopene by Streptomyces rimosus was reported first time in China.The determination methods,such as spectrophotometric method and HPLC were constructed.Using a stain Fc of Streptomyces rimosus as a original strain,a high-yield lycopene producing mutant strain Fc' was selected after UV mutation.The lycopene yield of the strain is 2.5 times higher than the original strain Fc.The optimal fermentation conditions were determined by flask experiments,and the lycopene yield of strain Fc' reached 230mg/L in flask under the optimal fermentation condition,meanwhile the Streptomyces rimosus strain could produce purer lycopene without adding any blocking agents in its fermentation process.This results lay a good foundation for lycopene commercial production by fermentation using Streptomyces rimosus.