Regulatory Mechanism Of Expression Of Lincomycin Gene Cluster

Lincomycin is a clinically improtant antimicrobial agent which was firstly discovered in 1962 and produced by Streptomyces lincolnensis.Lincomycin biosynthesis was speculated to include the PPL,MTL and condensation pathways,but the research on its biosynthetic mechanism lagged behind.Though lincomycin biosynthetic gene cluster has been initially cloned and analyzed in the early 1990s,the function of synthetic genes has only been identified in recent years attributed to the particularity and complexity of the intermediates.Moreover,the regulation of lincomycin biosynthesis has not been reported yet,since there are no typical regulators in the cluster.In this study,we identified the cluster-situated regulators(CSRs)in lincomycin biosynthetic gene cluster,and characterized the regulatory mechanisms of lincomycin biosynthesis.With these research,we intended to construct a corresponding gene regulatory network,and to provide a solid theoretical basis for the rational designs and modifications of the industrial strains.Firstly,we found three potential CSRs in the cluster based on bioinformatics analysis,LmbU,LmbIH and LmbQ.Gene knockout experiments showed that all the three regulators positively regulate lincomycin biosynthesis.RT-qPCR analysis showed that LmbU regulates transcription of multiple genes in the cluster,including lmbA,lmbC,lmbJ,and lmbW.However,LmbIH and LmbQ have little effect on transcription of these genes in the cluster,which suggested that they may regulate lincomycin production indirectly.Since LmbU has an important role in lincomycin biosynthesis and transcription of biosynthetic genes,the transcriptional regulatory function of LmbU was further confirmed by the neor reporter system in vivo.It was found that LmbU has dual functions of transcriptional activation and transcriptional repression.LmbU can positively regulate transcription of lmbA,lmbC,lmbJ,and lmbW,and negatively regulate transcription of lmbK and lmbU(selfregulating).Subsequently,a series of EMSA experiments showed that LmbU binds to the conserved palindrome 5'-CGCCGGCG-3'(binding site A)upstream of the lmbA and lmbW genes,but does not bind to the upstream regions of other target genes.In addition,LmbU can also bind to the palindrome 5'-TATTAATA-3'(binding site B),indicating that the transcriptional regulatory mechanism mediated by LmbU is much more complicated.Structural analysis showed that LmbU can be divided into multiple functional domains.Point mutations of cysteine and SDS-PAGE electrophoresis showed that C12 is a key site for LmbU dimerization.EMSA and xylTE reporter assays about LmbU mutants verified that a LmbU-DNA binding inhibitory region exists in the N-terminus of LmbU(1-57 aa),a DNAbinding domain(DBD)locates in the helix-turn-helix(HTH)motif(80-102 aa),and a potential functional region exists in the C-terminus of LmbU(162-225 aa).Interestingly,without the inhibitory region in the N-terminus,LmbU58-225 and LmbU58-161 mutants can bind to its own promoter lmbUp,which may be attributed to a non-fully conserved binding site 5'-AATTAATT3'(binding site B')within lmbUp.Due to the structural specificity and functional diversity of LmbU,we defines it as a novel family of regulatory proteins,the LmbU family.Homologous proteins of LmbU are present in a variety of actinomycetes,including Streptomyces and non-Streptomyces,and their coding genes are mainly located within the secondary metabolism gene clusters.Sequence alignments showed that HTH motifs of the homologous proteins were highly conserved(11 of 23 amino acids are identical,especially 10 of the 13 amino acids within the second helix are identical),indicating the similar regulatory function of these proteins.This was supported by the EMSA assays that LmbU homologue(LmbUSe)from Saccharopolyspora erythraea can also bind to the target probes lmbAp and PVw3 from S.lincolnensis.In addition,Western blotting and semi-quantitative reverse transcription and polymerase chain reaction(SqRT-PCR)assays showed that bldA controls translation of LmbU,subsequently indirectly regulates transcription of the target genes of LmbU.Thus,we demonstrated the regulatory cascades and network of lincomycin biosynthesis.