A Construction Method Of Recombinant Streptomyces Viginiae IBL-14 Used For Penicillin Producation

Studies found that there is a repetitive structure in most of bacteria and archaea’s chromosomes, known as clustered regularly interspaced short palindromic repeat (CRISPR). CRISPR locus is a multi-segment R-S structures composed of a highly conserved direct repeats and spacers. Often there are a series of CRISPR-associated proteins (Cas proteins) near the CRISPR locus and a leader sequence upstream from the Cas site. Together, the CRISPR and Cas constitute an adaptive immune system (CRISPR-Cas) against invading of exogenous phages or viruses through three phases:adaptation, expression and interference. Currently, CRISPR-CAS systems are divided into three, type Ⅰ, type Ⅱ and type Ⅲ, among which type Ⅱ CRISPR-Cas technology has become a powerful tool for genetic editing.Penicillin is a antibiotic with high efficacy and low toxicity and hence has a broad application, especially for the treatment of infection caused by G+ bacteria. Currently, the acquirement of high-yield strains for penicillin production is mainly through mutation breeding, cell engineering and so on. Streptomyces Virginia IBL14, isolated and identified by our laboratory, is a kind of actinomyces that can biotransform and even degrade sterols. After whole genome sequencing of the strain IBL14 and related bioinformatic analysis, we found that there are a CRISPR-Cas I-SV14B system and a series of genes for penicillin biosynthesis and transfofmation in the strain’s chromosome. To construct a new strain for effective pencillin production, a new idea:the insertion, knockout, point mutation of these genes and further related genes in its chromosome by using CRISPR-Cas I-SV14B system itself becomes our tasks. The knockout of the three enzyme genes (penicillin-lactamase gene (sviPA), isopenicillin-N isomerase gene (sviIPI) and β- lactamase gene (sviLT)) for penicillin transformation and hydrolysis in its chromosome is the first step of our work.To knockout the three enzyme genes by using CRISPR-Cas I-SV14B system itself, we fisrt designed and syntheized corresponding editing templets and targeted constructs for each gene, then constructed corresponding knockout plasmids containing single guide RNA and gene editing template, transformed the knockout plasmids to the protoplasts of the strain IBL14, and finally screened and identified the recombinants. The results showed these genes have been successfully knocked out.Based on the successful knock-out of the three genes by using CRISPR-Cas I-SV14B system itself, in order to find the advantages and disadvantages of CRISPR-Cas I systems and CRISPR-Cas Π systems, we first selected the mutarotase (galM) used in E. coli blue-white screening for the gene knockout (white colonies of E. coli representing the success of the gene knockout) by using commercially applied CRISPR-Cas II systems. The results showed that the gene was successfully knocked out. These results discribed above show the technology of CRISPR-Cas systems for genetic editing in our laboratory has been on the right track, but is still in its infancy.