Study On Mutagenesis Screening Of High-yield Salinomycin Mutant And The Global Regulation Mechanism Of Two-component System RspA1/A2 In Streptomyces Albus

Salinomycin,being one of the useful antibiotic biosynthesized by Streptomyces albus,has been widely used for the treatment of coccidiosis in agriculture field due to its ability to inhibit the growth of an array of gram-positive bacteria.In addition,it has also been proved effective to use as a potential anti-tumor drug.In this study,we aimed to carry out the research on the mutation breeding screening technology of Streptowyces albus and the global regulation of two-component system RspAl/A2 on primary metabolism and secondary metabolism such as the central carbon source metabolism and salinomycin biosynthesis.Firstly,a high-efficiency mutagenesis breeding screening platform was established to obtain the high-yield salinomycin mutants through high-throughput screening with the industrial strain of S.albus S12 as the parent strain.Meanwhile,transcriptome data were used to analyze the mechanism of intensive salinomycin biosynthesis capacity by mutagenesis breeding.Secondly,the global regulation mechanism of two-component system RspAl/A2 on the central metabolism related to cell growth(glucose metabolism,nitrogen metabolism and phosphate metabolism)and salinomycin biosynthesis in Streptomyces albus were investigated,in order to understand the relationship between the primary metabolism(cell growth)and secondary metabolism(salinomycin biosynthesis).The main results of this thesis work are as follows:1.Study on mutagenesis screening of industrial strains and mechanism of mutagenesis breeding to improve the salinomycin production capacity of high-yielding mutantA mutagenesis screening method for obtaining high-yield salinomycin mutants has been developed by our research group,but it still needs to cope with the disadvantages of long breeding cycles,laborious works and low efficiency.To overcome these drawbacks,an agar block diffusion method was utilized to replace the original 24-well plate fermentation method to characterize the titer of salinomycin based on the established mutagenesis screening method.The obtained results show that the diameter of the inhibition zone produced by Streptomyces albus with bacillus pumilus as an indicator bacteria is positively correlated with the titer of salinomycin(correlation coefficient R2 is 0.955).Finally,the screening cycle was shortened by 18 days,and the efficiency was significantly improved.Subsequently,atmospheric and room temperature plasma(ARTP)was applied to generate mutants library,and then resistance screening was conducted on plates containing single antibiotic or multiple antibiotic to select positive mutants.As a result,a positive mutant Tet30Chl25 and a negative mutant TK.were obtained by high-throughput screening from 106 to 108 mutants after preliminary screening and secondary screening.Compared with the initial strain S12,mutant strain Tet30Chl25 showed a 2-fold increase in production of salinomycin and a 20%increase in biomass accumulation.The differences among mutant strain Tet30Chl25,(a positive mutant with high yield of salinomycin),TK(a negative mutant with low yield of salinomycin)and the parental industrial strain S12 transcriptomes were analyzed at day 5 of fermentation.The results are as follows:(1)A total of 1603 genes showed differences in expression among mutant strain TK,Tet30Chl25,and the initial strain S12,these differentially expressed genes(DEGs)covered most of the metabolic pathways of Streptomyces albus,including glycolysis pathways,TCA cycle in primary metabolism and antibiotic biosynthesis in secondary metabolism.(2)The relevant differential pathways affecting salinomycin biosynthesis were mainly related to butanoate metabolism,starch and sucrose metabolism,and glyoxylate metabolism.In short,these metabolic pathways were more active in high-yield salinomycin production mutant.(3)A tentative regulatory model of high-yield salinomycin mutant Tet30Chl25 after ARTP treatment and resistance screening is proposed.This model involved in three aspects of the signal transduction process.Firstly,stringent response was activated along with accumulation of(p)ppGpp and mutation of ribosomal proteins.Secondly,accumulation of(p)ppGpp and mutational ribosomal protein may further activate the signal transduction system,including activation of sigma factors and transcriptional factors.Thirdly,the activated transcriptional regulators and ECF sigma factor further affected the expression of genes in salinomycin biosynthesis gene cluster(slnA1-A9,slnR,orf3,orf15),salinomycin precursors synthesis pathway(accA1,accA2,pcc,mcm,crr)and antibiotic synthesis-related pathways like butanoate metabolism,starch and sucrose metabolism,and glyoxylate metabolism.Furthermore,these factors are consequently resulted in an increase in the ability of salinomycin biosynthesis.2.Two-component system RspA1/A2 regulates the saliomycin biosynthesisA two-component system(named RspA1/A2)was found in genome of S.albus DSM41398,which is a homologous protein with the two-component system AfsQl/Q2 and SmrA/B based on protein sequence blast.In order to study the regulatory function of RspA1/A2 on salinomycin biosynthesis,a two-component system RspA1/A2 gene rspAl deletion mutant ArspAl and complemented mutant?rspAla were constructed by homologous double exchange on the basis of industrial strain Streptomyces albus A30.Then the fermentation results showed that the two-component system RspA1/A2 positively regulates the saliomycin biosynthesis.It was further found that the response protein RspA1 could affect the biosynthesis of salinomycin in two ways:On one hand,RspA1 regulated salinomycin biosynthesis by directly controlling the transcript level of pathway-specific regulator gene slnR.On the other hand,The gene sigW encodes a SigW-like sigma factor.RT-qPCR results found that SigW negatively regulated the transcription of the gene slnR,thus inhibiting the synthesis of salinomycin.Meanwhile,the fermentation results showed that SigW promoted the growth of Streptomyces albus.The phosphorylated response protein RspAl could bind to promoter region of the downstream gene sigW and promote the expression of sigW,thereby indirectly affecting the biosynthesis of salinomycin.3.The regulation mechanism of two-component system RspA1/A2 on the primary metabolism in Streptomyces albusIt was found that the gene rspAl deletion mutant ?rspAl had a lower glucose consumption rate and less biomass accumulation as compared to the starting strain A30 when cultured in a fermentation medium containing 75 mM glutamate,indicating that the two-component system RspA1/A2 response protein RspAl could enhance glucose catabolism and promote cell growth under specific conditions.Further research found that the two-component system RspA1/A2 plays a regulatory role in the metabolism of carbon,nitrogen and phosphorus sources,thus affecting glucose metabolism and cell growth of Streptomyces albus.For carbon source metabolism,protein RspAl promotes the glucose consumption rate Rglu of the starting strain A30 to be higher than that of the rspA1-deficient mutant ?rspA1 by enhancing the pyruvate-driven glucose decomposition pathway and weakening the glutamate-driven glycogenic pathway.The experimental results show that protein RspAl directly regulates the transcript levels of genes gap,pdh,and cit,and thus enhancing the flux of glucose catabolism pathways including glycolysis and TCA cycle.At the same time,the gluconeogenesis pathway of the original strain A30 with pyruvate and glutamate as substrates was significantly weakened.Transcriptome data analysis revealed that transcript levels of three key genes in the gluconeogenesis pathway(pyc,pck and glpX)and genes(sdh,gdhA,sucA,fum)in the flux of glutamate converting to oxaloacetate through the TCA cycle are strikingly down-regulated.For nitrogen source metabolism,Two-component system RspA1/A2 promotes glutamate intake and facilitates it into the urea cycle.The results showed that two-component system RspA1/A2 enhanced the transcription level of the glutamate ABC transporter genes gluA-D,thereby promoting the intake of glutamate by the starting strain A30.Meanwhile,compared with the gene rspA 1-deletion mutant ?rspAl,the transcript level of arg gene located in flux of glutamate conversion to the urea cycle were obviously increased,thereby enhancing the abundant glutamate entry into the urea cycle decomposition in the starting strain A30.As for phosphate metabolism,protein RspA1 can promote the intake of phosphate in Streptomyces alhus.EMSAs results showed that the response protein RspA1 can directly bind to promoter region of gene pstS and promote its expression.Gene pstS encodes an ABC transporter,cooperating with genes pstA,pstB,and pstC to form a protein complex which is responsible for the intake of phosphate sources.Therefore,protein RspA1 facilitates the uptake of phosphate source in Streptomyces albus by promoting the transcription levels of genes pstS,pstA,pstB,pstC.In short,the two-component system RspA1/A2 promotes the intake of glutamate and phosphate sources and prompt glutamate entering into the urea cycle.This global regulation on carbon,nitrogen and phosphate sources can maintain C/N/P metabolism at a specific balance,thus promoting cell growth and the biomass accumulation in starting bacteria A30.