Development And Application Of Genome Continuous Integration System

Streptomyces,as an important industrial microorganism group,is the producer of antibiotics,immunosuppressants,anti-tumor drugs and other important compounds.Genes responsible for the synthesis of these important compounds in Streptomyces typically exist in clusters on the genome,ranging in length from tens to hundreds of kilobytes.Increasing the copy number of biosynthetic gene clusters is one of the main strategies for constructing industrial high-yield strains.However,the existing methods for realizing large fragment and multiple copies of gene clusters have many limitations such as long cycle and low efficiency,which limit the development process of industrial high-yield strains of Streptomyces.In this study,we developed a genome continuous integration vector system based on site-specific recombinases.Using this system,we can overcome the limitations of existing methods and efficiently construct multi-copy engineering strains of gene cluster.The specific research contents and results are as follows:(1)Design and screening of recombinant resistance genes containing integration locus(att B)within the reading framework: Apamycin resistance gene(acc(3)IV)and hygromycin resistance gene(aph)are two commonly used selective marker genes in Streptomyces.By analyzing the crystal structure of the protein encoded by aprthromycin resistance gene(acc(3)IV),the recombinant resistance gene was constructed by inserting the integration site att B10 into the 62,82,166,218 codon of aprthromycin resistance gene.By analyzing the crystal structure of the encoding protein of HYgromycin resistance gene(aph),the recombinant hygromycin resistance gene was constructed by inserting the integration site(att B)at the codon 123,135,168,215 of hygromycin resistance gene.By functional screening,the recombinant apr B10 obtained by inserting att B10 sequence into the 82 nd codon of aprmycin resistance gene still had the same aprmycin resistance function as the wild type gene.Hyg B was obtained by inserting att B into the 215 codon of hygromycin resistance gene,which had the same hygromycin resistance function as the wild type gene.(2)Construction of plasmid vectors for continuous integration: apr B10 and hyg B were used as selective marker genes to construct plasmid vectors p INT01 and p INT02 respectively.In order to realize the function of continuous integration,p INT01 also hasφBTI integrase coding gene and integrase recognition site(att P)and other functional elements;PINT01 also has φBTI integrase encoding gene and integrase recognition site(att P10)and other functional components.(3)Realization of genome continuous integration: Firstly,plasmid p INT01 was transformed into wild-type host,and the transformants were screened under apamycin condition.In the obtained transforters,the att P site on p INT01 and the att B site on the genome undergo site-specific recombination,and the plasmid is then integrated into the host genome and introduced into the att B10 site,thus achieving the first step of integration.Using the engineering strain obtained in the first step as the recipient,the transformation plasmid p INT02 was screened under hygromycin condition.In the obtained transforters,the att P10 site on p INT02 was recombined with the att B10 site introduced in the first step,and the plasmid was then integrated into the genome.As a result of integration,the apr B10 resistance gene was inactivated,and a new integration site att B was introduced to realize the second step integration.Similarly,the engineering strain obtained in the second step was used as the recipient,and the transformed plasmid p INT01 was screened under the condition of apamycin to realize the integration in the third step.Similarly,plasmid vectors p INT01 and p INT02 can be used alternately to achieve continuous genome integration.(4)Promotion and application of genome continuous integration system: We used the above genome continuous integration system to construct engineering strains containing different copy number biosynthetic gene clusters of indigo.To be specific,the recombinant plasmids p INT01::bps A and p INT02::bps A containing cytinoid gene clusters were firstly constructed,and the engineering strains containing single copy,double copy and triple copy cytinoid gene clusters were gradually obtained by alternate transformation in the host bacteria Streptomyces ceruvius M512.The results showed that with the increase of copy number of gene cluster,the yield of indigo increased significantly.In conclusion,we successfully constructed a genome continuous integration system based on the highly efficient site-specific recombination catalyzed by recombinant enzymeφBTI.This system overcomes the shortcomings of existing methods and can quickly realize the construction process of engineering strains containing arbitrary copy number target gene clusters,providing an effective method and means for industrial microbial breeding.