Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus

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.

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus

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.