Site-directed Mutation Of Streptomyces RpsL Gene Based On CRISPR-Cas9 System

Streptomyces is an important source of active products,which can be used as antifungal drugs,antiviral drugs,antitumor drugs,antihypertensive drugs,immunosuppressants and antibiotics.However,the probability of discovering new compounds from Streptomyces continues to decline.The recent studies showed that the genome of Streptomyces contains 20 or more potential secondary metabolite gene clusters,of which only a part is expressed in fermentation,suggesting that most of secondary metabolite gene clusters in Streptomyces are cryptic.Thus there still has a great potential to explore new compounds and discovery new drugs from Streptomyces.In this thesis,we expected that K88E and P91S mutations were introduced into Streptomyces rpsL gene via CRISPR-Cas9 technology to inspire its synthesis potential.In other words,ribosome engineering can wake up cryptic biosynthetic pathways of Streptomyces.The K88E and P91S mutations in the rpsL gene were the marked enhancement of antibiotic production observed types in the model strain S.coelicolor 1147.CRISPR-Cas9 used in Streptomyces is a novel subject.The earliest literature was published at the end of 2014.The establishment of an efficient and universally applicable genome editing system is of great importance for subsequent study,like activation of cryptic gene clusters and optimization of strains.CRISPR-Cas9 system was established for Streptomycetes genome editing.Plasmid pCas9-1 was synthesized artificially and 11 strains of sponge-associated Streptomyces were genetically manipulated.The pSG5-derived temperature-sensitive replicon that cannot replicate at high temperature,the apramycin resistance gene(acc)and the thiostrepton resistance gene(trs)contained in the plasmid were tested respectively,and the results showed that all of them could work properly.And we optimized conditions for culture,selection,and plasmid removal.But no positive result was obtained for the site-directed mutation of the rpsL gene in Streptomyces.Another two plasmids used in this study named pCas9-2 and pCas9-3 were derived from plasmid pCas9-1.These three plasmids contain the same sgRNA(used to target the same site)and the same HR template.The sgRNA and Cas9 of different plasmids with different promoters.There was still no positive result for the re-operation of the sponge-associated Steptomyces.Subsequently,we successfully performed genome editing in the terrestrial S.coelicolor AS4.242 using plasmid pCas9-2,which with a positive rate of more than 50%.And the repeated experiments showed that the system could work stably.Since these three plasmids failed to achieve site-directed mutagenesis of sponge-associated Streptomyces,we suspect that the CRISPR-Cas9 system may not work properly in host,especially sgRNA and Cas9.Even in S.coelicolor AS4.242,only pCas9-2 was successfully edited.The expression levels of sgRNA and Cas9 in S.coelicolor AS4.242 transformed with three plasmids were compared by semiquantitative RT-PCR.For pCas9-2,the expression levels of sgRNA and Cas9 between pCas9-1 and pCas9-3,and the level of sgRNA higher than Cas9.Therefore,we speculated that the expression level of sgRNA and Cas9 should be carefully adjusted in the genome editing of sponge-associated Streptomyces.The expression of Cas9 should not be too high,otherwise it may damage the host,and the appropriate expression ratio of sgRNA and Cas9 is approximately 13:1.We need to introduce more accurate quantification methods such as Real-time RT-PCR and to carry out more experiments on a variety of hosts to verify our speculation.At the end of the thesis,the activity of S.coelicolor AS4.242 with rpsL K88E and P91S mutations was preliminary detected.The result shows that the mutated strains have higher resistant level to streptomycin:from<2 ?g/mL(wild-type)to 70-80 ?g/mL(mutated strains),indicating that ribosome engineering of S.coelicolor AS4.242 was successful.But the fermentation product has no antibacterial activity.Because of that fermentation and extraction steps are not optimized,more sophisticated experiments should be designed to determine whether a secondary metabolic gene cluster is activated.