Studies On The Screen And Function Of Related Regulatory Genes Involved In Clavulanic Acid Biosynthesis In Streptomyces Clavuligerus

BACKGROUND: The long-term widespread use of β-lactam antibiotics has led to emergence a large number of drug-resistant strains,and the main resistance mechanism of these resistant strains is to product β-lactamases,which inactivate the β-lactam antibiotics.As a broad-spectrum β-lactamase inhibitor,clavulanic acid has been widely used in the clinical treatment of β-lactam antibiotic resistant bacteria infection.The main production strain of clavulanic acid is Streptomyces clavuligerus.At present,the regulation mechanism between primary metabolism and secondary metabolism and clavulanic acid synthesis in Streptomyces clavuligerus is not yet clear.OBJECTIVES:(1)To find out the regulatory genes regulating the synthesis of clavulanic acid in Streptomyces clavuligerus;(2)To elucidate the regulatory functions and regulatory pathways of newly discovered regulatory genes.METHODS: Knockout mutants were constructed by homologous recombination and double exchange.At the same time,the production of clavulanic acid in mutant strains was detected by the biological activity assay of solid fermentation and liquid fermentation HPLC,and the regulatory genes regulating clavulanic acid synthesis were found.Subsequently,using liquid fermentation,transcriptome comparison,and RT-qPCR to analyze the regulatory genes’ functions,and ChIP-seq and EMSA techniques were used to analyze target genes for regulatory genes.RESULTS:(1)Knockout of the two-component signaling system orf1717/1718 could significantly affect the biosynthesis of clavulanic acid,and the effect was different under different fermentation conditions.(2)The transcriptome and RT-qPCR analysis showed that the expression levels of various primary metabolic pathways such as fatty acid degradation,arginine synthesis and oxidative phosphorylation,and secondary metabolic pathways such as clavulanic acid synthesis were significantly different in △1717/1718 strains.(3)ChIP-seq shows that ORF1717 can bind to the transcriptional regulatory regions for SCN_RS01115,SCN_RS21190,SCN_RS24630(gene of fatty acid degradation pathway),cyp450 and claR(gene of clavulanic acid cluster),SCN_RS06875(glutamic acid synthesis pathway gene),SCN_RS05510(Glycolytic pathway genes)and SCN_RS02260(γ-butyrolactone synthesis gene).CONCLUSIONS:(1)orf1717/1718 is involved in the regulation of the clavulanic acid biosynthesis in Streptomyces clavuligerus.(2)ORF1717 can indirectly regulate clavulanic acid synthesis through the γ-butyrolactone pathway.(3)ORF1717 inhibited arginine synthesis,inhibited carbon metabolism(including fatty acid degradation,pyruvate metabolism,oxidative phosphorylation,etc.)activity and negatively regulated the expression of claR gene under high dissolved oxygen conditions.