otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and ot...otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coeficolor M145 by intergenedc conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M14-~OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin （Act） production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction （RT-PCR） analysis revealed that the transcrip- tional level of pathway-specific regulatory gene acUl-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.展开更多
The genus Streptomyces exhibits a complex life cycle of morphological differentiation and an extraordinary ca-pacity to produce numerous bioactive secondary metabolites.In submerged cultures,Streptomyces species usual...The genus Streptomyces exhibits a complex life cycle of morphological differentiation and an extraordinary ca-pacity to produce numerous bioactive secondary metabolites.In submerged cultures,Streptomyces species usually grow in the form of mycelial networks and aggregate into large pellets or clumps,which is generally unfavorable for industrial production.This study aimed to construct efficient microbial cell factories by manipulating morphology-related genes.We herein employed a morphology engineering approach to generate eight engi-neered derivatives(MECS01~MECS08)of Streptomyces coelicolor M1146,a versatile chassis widely used for the heterologous production of various secondary metabolites.We found that genetic manipulation of morphology-related genes exerted a substantial influence on the growth and mycelial characteristics of the engineered strains.Once the native actinorhodin gene cluster was introduced into these strains,antibiotic production increased in all engineered strains compared to the parental strain.Notably,a significant elevation of actinorhodin production was observed in three of the engineered strains,MECS01,MECS03 and MECS05.Similar scenarios occurred when expressing the staurosporine gene cluster and the carotenoid gene cluster in these three engineered derivatives,respectively.Our study demonstrates that morphology engineering represents an effective strategy for alleviating mycelial aggregation.It has also expanded the toolkit of Streptomyces chassis available for the heterologous expression of gene clusters encoding a variety of secondary metabolites.展开更多
基金Project supported by the National Natural Science Foundation of China(No.31772213)the Excellent Youth Fund of Zhejiang Province,China(No.LR17C140002)
文摘otrA resembles elongation factor G （EF-G） and is considered to be an oxytetracycline （OTC）-resistance determinant in Streptomyces rimosus. In order to determine whether otrA also conferred resistance to OTC and other aminoglycosides to Streptomyces coelicolor, the otrA gene from S. rimosus M527 was cloned under the control of the strong ermE promoter. The resulting plasmid, pIB139-otrA, was introduced into S. coeficolor M145 by intergenedc conjugation, yielding the recombinant strain S. coelicolor M145-OA. As expected S. coelicolor M145-OA exhibited higher resistance levels specifically to OTC and aminoglycosides gentamycin, hygromycin, streptomycin, and spectinomycin. However, unexpectedly, S. coelicolor M14-~OA on solid medium showed an accelerated aerial mycelia formation, a precocious sporulation, and an enhanced actinorhodin （Act） production. Upon growth in 5-L fermentor, the amount of intra- and extracellular Act production was 6-fold and 2-fold higher, respectively, than that of the original strain. Consistently, reverse transcription polymerase chain reaction （RT-PCR） analysis revealed that the transcrip- tional level of pathway-specific regulatory gene acUl-orf4 was significantly enhanced in S. coelicolor M145-OA compared with in S. coelicolor M145.
基金supported in part by a grant from the National Key Research and Development Program of China(No.2023YFD1700700)grants from the National Natural Science Foundation of China(No.32370077,U22A20582,32100051)the Shaanxi Provincial Science and Technology Projects(2024RSCXTD-67 and QCYRCXM-2023-040).
文摘The genus Streptomyces exhibits a complex life cycle of morphological differentiation and an extraordinary ca-pacity to produce numerous bioactive secondary metabolites.In submerged cultures,Streptomyces species usually grow in the form of mycelial networks and aggregate into large pellets or clumps,which is generally unfavorable for industrial production.This study aimed to construct efficient microbial cell factories by manipulating morphology-related genes.We herein employed a morphology engineering approach to generate eight engi-neered derivatives(MECS01~MECS08)of Streptomyces coelicolor M1146,a versatile chassis widely used for the heterologous production of various secondary metabolites.We found that genetic manipulation of morphology-related genes exerted a substantial influence on the growth and mycelial characteristics of the engineered strains.Once the native actinorhodin gene cluster was introduced into these strains,antibiotic production increased in all engineered strains compared to the parental strain.Notably,a significant elevation of actinorhodin production was observed in three of the engineered strains,MECS01,MECS03 and MECS05.Similar scenarios occurred when expressing the staurosporine gene cluster and the carotenoid gene cluster in these three engineered derivatives,respectively.Our study demonstrates that morphology engineering represents an effective strategy for alleviating mycelial aggregation.It has also expanded the toolkit of Streptomyces chassis available for the heterologous expression of gene clusters encoding a variety of secondary metabolites.
