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Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus 被引量:11
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作者 Shouliang Yin Xuefeng Wang +10 位作者 Mingxin Shi Fang Yuan Huizhuan Wang Xiaole Jia Fang Yuan Jinliang Sun Tiejun Liu Keqian Yang Yuxiu Zhang Keqiang Fan Zilong Li 《Science China(Life Sciences)》 SCIE CAS CSCD 2017年第9期992-999,共8页
Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics.To increase the oxytetracycline(OTC) production in Streptomyces rimosus,we investigated the coope... Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics.To increase the oxytetracycline(OTC) production in Streptomyces rimosus,we investigated the cooperative effect of three co-overexpressing OTC resistance genes:one gene encodes a ribosomal protection protein(otrA) and the other two express efflux proteins(otrB and otrC).Results indicated that combinational overexpression of otrA,otrB,and otrC(MKABC) exerted a synergetic effect.OTC production increased by 179%in the recombinant strain compared with that of the wild-type strain M4018.The resistance level to OTC was increased by approximately two-fold relative to the parental strain,thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production.Furthermore,the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC;such strain can produce OTC of approximately7.49 g L^((-1)),which represents an increase of 19%in comparison with that of the OtcR-overexpressing strain alone.Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces. 展开更多
关键词 resistance genes OXYTETRACYCLINE combinatorial overexpression cluster-situated activator streptomyces rimosus
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Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527 被引量:5
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作者 Zhang-qing SONG Zhi-jun LIAO +3 位作者 Ye-feng HU Zheng MA Andreas BECHTHOLD Xiao-ping YU 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2019年第11期891-900,共10页
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... 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 recipie nt was obtai ned.Subseque ntly,based on the above developed and optimized tran sformati on 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 p-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. 展开更多
关键词 streptomyces rimosus M527 Intergeneric conjugation PROMOTER β-Glucuronidase(GUS)
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Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus 被引量:1
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作者 Yiying YANG Qingqing SUN +16 位作者 Yang LIU Hanzhi YIN Wenping YANG Yang WANG Ying LIU Yuxian LI Shen PANG Wenxi LIU Qian ZHANG Fang YUAN Shiwen QIU Jiong LI XuefengWANG Keqiang FAN Weishan WANG Zilong LI Shouliang YIN 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2021年第5期383-396,共14页
Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces i... Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species. 展开更多
关键词 Counterselectable system pyrF 5-Fluoroorotic acid(5-FOA) Gene deletion streptomyces rimosus
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