Optimal Design Of Thiostrepton And Its Antibacterial Activities Against Oral Pathogens

OBJECTIVE: Thiopeptide antibiotics have been a research hotspot over the last 10 years for their complex chemical structures and remarkable biological activities.We chose thiostrepton(TSR),a star molecule of the thiopeptide antibiotic family,as the research object.Based on the interruption of the TSR precursor peptide,we knocked out tsr T gene which is responsible for the quinoline acid(QA)synthesis pathway.Then by complementation of the precursor peptide of siomycin(SIO)which exhibits better biological activities than TSR to acquire the potential of producing SIO.The chemically modified QA analogs were fed during the fermentation of the S.laurentii mutant strain to obtain novel SIO analogs with targeted chemical site modifications,and investigated their antibacterial activities against oral pathogens.MATERIALS AND METHODS: Adopting prevailing CRISPR/Cas9 gene editing technology,the plasmid p WHU1002 containing all elements of tsr T all-frame deletion was constructed.Then the plasmid was introduced into TSR mutation strain SL2051 by E.coli-Streptomyces conjunction.After screening for the successful knockout strain SL3051,the precursor peptide of SIO(sio H)was complemented into the above tsr T-deletion stain(SL3051),The chemically modified QA analog(5'-F-QA)was fed while fermentating SL3052 to obtain a novel SIO analog(5'-F-SIO).The chemical structure of 5'-F-SIO was analyzed by nuclear magnetic data after isolation and purification.5'-F-SIO,together with TSR,5'-F-TSR and SIO,were employeed to operate water soluability tests.The antibacterial activities against oral pathogens(Streptococcus mutans,Actinomyces viscosus,Lactobacillus acidophilus,Porphyromonas gingivalis,Fusobacterium nucleatum)were conducted by MIC tests with above 4 compounds.RESULTS: 1.A recombinant plasmid p WHU1002 for tsr T knocking out was successfully constructed in vitro via CRISPR/Cas 9 gene editing technology,and TSR mutation strain SL3052 was obtained after two E.coli-Streptomyces conjunctions.2.By chemically synthesizing the modified QA analog(5'-F-QA),the yield of 5'-FSIO was achieved by fermentating SL3052 strain with 5'-F-QA fed,and the structure of 5'-F-SIO was identified after isolating and purifying.3.The water-solubility tests showed that 5'-F-SIO has greater preponderance over TSR,5'-F-TSR and SIO.MIC experiments exhibited that 5'-F-SIO has better resistance against oral pathogens with the aforementioned compounds.CONCLUSIONS: From the perspective of biosynthesis,TSR,a thiopeptide molecule with excellent biological activities,was selected for our experiment.Under the guidance of mutation synthesis therory,tsr T gene knockout in the TSR mutant strain SL2051 was completed via CRISPR/Cas9 technology.Fermentation of 5'-F-QA after replenishment of sio H revealed a novel TSR analog,5'-F-SIO,which has not been reported previously.Subsequent water-soluble tests and quantitative bioassays against oral pathogens showed that the 5'-F-SIO has better property enhancement than the parent SIO and other compounds.The TSR family of natural product families has been introduced into the field of oral microbes for the first time,laying the foundation for the application of Ri PPs antibiotics like TSR in the field of stomatology.