The Function Study Of LmbIH And LmbQ Genes Of S.lincolnensis

Lincomycin is one of the important members of lincosamide antibiotics.As a class of antibiotics widely used clinically,lincomycin affects the growth of bacteria by inhibiting the synthesis of bacterial proteins and is therefore used to treat various infectious diseases.Although the production of lincomycin can be improved through the improvement of fermentation process and the selection of strains,they all have certain limitations,and genetic engineering is an effective strategy to achieve a leap in lincomycin production capacity.The biosynthetic pathway of lincomycin has been preliminarily analyzed,and its regulatory mechanism has gradually become clear,but some gene functions in the gene cluster(lmb)responsible for lincomycin biosynthesis are still unknown.Among them,the genes lmbIH and lmbQ bioinformatics predict that the encoded product belongs to the PmbA-TldD family,but the mechanism of action is unknown,therefore,exploring the functions of lmbIH and lmbQ is expected to improve the biosynthesis and regulation process of lincomycin,and provide an operation target for the genetic modification of high-lincomycin-producing strainsIn order to explore the functions of the genes lmbIH and lmbQ in the lincomycin cluster,this paper uses the CRISPR/Cas-mediated streptomyces genome editing technology,using the wild strain NRRL2936 of Streptomyces lincolnensis as the starting strain,constructed lmbIH and lmbQ single gene knockout strain and double gene combined knockout strain.The fermentation results showed that the loss of lmbIH and/or lmbQ significantly reduced the synthesis of lincomycin,but did not affect the formation of spores.At the same time,in order to detect whether the encoded products of lmbIH and lmbQ existed the PmbA-TldD family peptide cleavage activity,in vitro activity and in vivo activity analysis were carried out.In the in vitro bioassay,the recombinant expression of lmbIH and lmbQ was first achieved in E.coli,and the recombinant LmbIH and LmbQ proteins obtained by renaturation purification of inclusion bodies;the analysis of short peptide cleavage activity based on mass spectrometry analysis showed that the positive compared with the control,the recombinant protein had no in vitro short peptide cleavage activity under the selected conditions.In vivo activity analysis,for the potential substrates of LmbIH and LmbQ,LmbU,LmbN and LmbA,the Flag tag was introduced in situ at the C-terminus of the coding gene.Based on Western-Blotting analysis,it was found that the target protein Flag marker did not show obvious positive bands in the wild and deleted strains of LmbIH and LmbQ.It is possible that the expression level of the target protein in Streptomyces is low.In the future,it is necessary to further increase the expression of the target protein for activity analysisIn summary,the genes lmbIH and lmbQ are regulating the biosynthesis of lincomycin,but their specific mechanism of action needs further analysis.