Heterologous Expression Of RiPP Biosynthetic Gene Cluster BGC2 Of Marine Streptomyces Sp.HNS054 And Testing Of Chassis

Marine actinomycetes have great potential to produce new and active natural products due to the specificity of the growth environment.However,this potential of most marine actinomycetes is silent in the normal culture conditions,which is not conducive to the development and utilization of marine active natural resources.According to the genomic characteristics of actinomycetes,to activate the production capacity of a certain natural product,it is necessary to activate the secondary metabolic gene clusters that synthesize this natural product.Therefore,how to activate the target gene clusters is an important scientific problem in the field of exploitation and utilization of marine active natural products.Heterologous expression of silenced gene clusters by actinomycetes chassis cells is one of the effective methods to activate silenced gene clusters.In this paper,we used the RiPP(ribosomally synthesized and post-translationally modified peptides)gene clusters of marine actinomycetes as markers to explore the problem of efficient heterogene expression.Firstly,we investigated the effect of gene cluster copy number on gene yield.The research process is described as follows:two RiPP gene clusters BGC1 and BGC2 of marine Streptomyces sp.HNS054 were obtained by using the long term PCR method,and episomal plasmids and integrated plasmids were constructed(chapter 2);the episomal plasmid was transferred to S.coelicolor M512,and the integrated plasmid was integrated into a series chassis of S.coelicolor M512,M1 146,M1246,M1346,M1446 and M1546 to obtain the strains with different copy numbers of heterologous gene clusters;the relative concentration of RiPP products was detected by LC-MS(chapter 3).The results showed that compared with the episomal expression,the integrated expression was more favorable to the efficient expression of heterologous gene clusters.In the integrated expression,when the number of copies of the BGC2 gene cluster increased to 2,the relative concentration of BGC2 products increased by about 22 times,but there was no significant effect on the production improvement of BGC2 when the number of copies of gene clusters continued to increase,indicating that there was a limit to the improvement of BGC2 products when the number of copies of gene clusters increased without changing other factors.Secondly,we studied the effect of overexpression of regulatory genes on product yield(chapter 4).Four different promoters were used to overexpress the positive regulatory gene bgc2R2 of BGC2 in the form of integration or multiple copies.The lcms results showed that the overexpression of bgc2R2 regulated by promoter P5 could improve the synthesis of BGC2 to a certain extent,which indicated that increasing the expression of regulatory gene was also an effective method to increase the gene cluster yield.Finally,we used CRISPR/Cas 9 gene editing technology to simplify the genome of Streptomyces coelicolor(chapter 5).In this study,we successfully knocked out 6 secondary metabolic gene clusters,laying a foundation for the construction of actinomycetes chassis with clean metabolic background.In addition,we also tried to knock out multiple gene clusters of the heterogenously expressed BGC2 strain M/pS2.The deletion of L02 gene cluster increased the relative concentration of BGC2 by more than 2 times.The establishment of this method provided a feasible idea for the activation of other marine actinomycetes silent gene clusters and the increase of the yield of secondary metabolites.To sum up,this paper made a series of exploration on the heterologous expression of marine actinomycetes RiPP gene clusters by studying the increase copy number of gene cluster,the optimization of gene expression and the simplification of genome,and a series of actinomycetes chassis cells were tested.This study established a whole set of experimental programs from the cloning and the construction of heterologous expression system of gene cluster to the detection of expression products,which laid a solid foundation for the future research on natural compounds of marine actinomycetes.