A High-yield Strain Of Antibiotic-resistant Bacterial Antibiotic-Puramycin I Was Constructed Using A Combination Of Metabolic Engineering Strategies

Pristinamycin,produced by Streptomyces pristinaespiralis,is a streptogramin-like antibiotic,which consists of two compounds with different chemical structure: pristinamycin I(PI)(30%,non-ribosomal peptide antibiotic)and pristinamycin II(PII)(70%,hybrid PK/NRP antibiotic).The synergistic effect of PI and PII can produce an antibacterial activity with 100-fold higher than the single component.Pristinamycin has antibacterial activity against G+ bacteria,especially drug-resistant bacteria,such as methicillin-resistant Staphylococcus aureus(MRSA),vancomycin-resistant Staphylococcus aureus and Enterococcus faecium(VREF),and it makes bacteria tolerant to the drug difficultly.Pristinamycin and its chemically modified water-soluble derivatives,like Quinupristin and Dalfopristin,have been on the market in Europe and the United States for treatment of pneumonia,bacteremia and skin infections caused by drug-resistant bacteria.However,domestic enterprises are still lack of high-yielding excellent strains and the cost of production is high,which prevents the process of industrialization of this important antibiotic internally.Based on literature researches,the main reason which effects the yield of pristinamycin is: due to the strong synergistic effect of PI and PII,they have serious toxic effects on S.pristinaespiralis when they exist in fermentation medium simultaneously,which results in restrained growth of bacteria,declined biomass and finally low fermentation levels of pristinamycin.Hence,generating PI-and PII-single component producing strains,respectively,can effectively solve these problems.In our lab's previous work,we have successfully utilized metabolic engineering strategies to get a PII high-yielding engineered strain with a maximum PII titer of approximately 2.2 g/L,which basically meet the requirement of industrialization.On this basis,the research work is mainly focused on the construction of PI high-yielding engineered strain and the parental strains are Streptomyces pristinaespiralis HCCB10218(producing both PI and PII)and HCCB10187(only producing PI)obtained from an enterprise.Firstly,using HCCB10218 as the starting strain,we used CRISPR/Cas9 gene editing technology to construct a PI single component engineered strain ?PII by deleting two PII biosynthetic genes snaE1 and snaE2 encoding two polyketide synthases.Then,we used two kinds of metabolic engineering strategies,including deletion of the pathway-specific repressor gene papR3 and chromosomal integration of the PI biosynthetic gene cluster(BGC)based on ?C31 integrase site-specific recombination technique(adding one extra cope),to construct the PI high-yielding strain.The final engineered strain ?PII?papR3/PI can produce a maximum PI titer of 132 mg/L,with an approximately 2.4-fold higher than that of the parental strain Streptomyces pristinaespiralis HCCB10218.These results indicate that metabolic engineering strategies used in this study are very effective and provide molecular breeding of Streptomyces with convenient technological means.In addition,using Streptomyces pristinaespiralis HCCB10187,a high PI-producing strain,as the starting strain,an engineered strain named as HCCB10189 with a maximum PI level of 520 mg/L was developed using the above two metabolic engineering approaches.The production level of PI by HCCB10189 showed approximately 94% higher than that of the parental strain HCCB10187.However,the fermentation levels of PI high-yielding strain are still low,and we will combine other metabolic engineering technologies in the future work,such as increasing the supply of precursors/cofactors,deleting competitive pathways and improving fermentation medium and technology,to further improve PI production,which would accelerate the process of the industrialization of pristinamycin.