Study On The Regulatory Mechanism Of CseB-C_SR In Streptomyces Rapamycinious And Development Of SPACE Base Editor

Streptomyces rapamycinious,also known as Streptomyces hygroscopicus,could produce a variety of biologically active secondary metabolites such as rapamycin,elaiophylin,and actinoplanic acids,which have a relatively important role in agriculture,industry,and medicine.Prokaryotes often include the two-component system（TCS）,which can affect physiological processes such as morphological differentiation,growth and development,primary and secondary metabolism,toxicity,and osmotic pressure.The Base Editor is an editing tool based on the CRISPR/Cas system that is suited for genome editing of strains with poor homologous recombination capacity since it is not dependent on double-strand breaks.The fermentation of rapamycin and elaiophylin produced by S.rapamycinious has low yield and high production cost,and the ability of S.rapamycinious to recombine is poor.Therefore,investigating the molecular mechanism of the two-component system regulation of antibiotic biosynthesis and developing a base editor in S.rapamycinious,will help to address the metabolic regulatory network of S.rapamycinious and provide suggestions for the genetic modification of Streptomyces.（1）Three pairs of two-component systems,M271＿22640/M271＿22645(CseB-C_SR),M271＿28325/M271＿28330 and M271＿14685/M271＿14690,were knocked out in Streptomyces rapamycinious NRRL 5491.Compared with the original strain NRRL 5491,knockout of M271＿28325/M271＿28330 and M271＿14685/M271＿14690 increased rapamycin yield by 54.71%and 55.36%,respectively,while knockout of cseB-C_SR reduced rapamycin yield by 91.18%.Interestingly,the yield of elaiophylin inΔcseB-C_SR was increased by 52.45%.The diphenylamine colorimetric method was used to analyze the growth curves,and it was revealed that knockout cseB-C_SR had no obvious impact on growth.Gene complementation experiments onΔcseB-C_SR resulted in restoration of both rapamycin and elaiophylin yield.（2）The gene transcript levels of the original strain NRRL 5491 and the mutant strainΔcseB-C_SR were analyzed by semi-quantitative RT-PCR in this study to further clarify the molecular mechanism by which CseB-C_SR regulates the production of rapamycin and elaiophylin in S.rapamycinious.The results showed that the transcripts of sigE_SR,cse A_SR,rapA,rapP,rapG,rapH,and elaI were down-regulated;rapS/R,rapY,elaA,elaB,ela3,and M271＿22625 were up-regulated;while rapT and M271＿22615 were not substantially altered.Overexpression of sigE_SR in theΔcseB-C_SR strain could restore rapamycin and elaiophylin yield.Electrophoretic mobility shift assay（EMSA）demonstrated that the response regulator CseB_SR binds directly to the sigE_SR promoter region,suggesting that CseBSR is involved in the regulation of rapamycin and elaiophylin biosynthesis by regulating sigE_SR transcription.（3）Due to the low homologous recombination capacity of S.rapamycinious,a SPACE base editor,allowing simultaneous conversion of cytosine（C）to thymine（T）and adenine（A）to guanine（G）,was developed to facilitate its genetic modification.The base editing plasmid p KC-ABE-CBE was obtained by introducing TadA-TadA*into the cytosine base editor d Cas9-CDA-UL_str.Then five positions were examined at rapS,rapS2,rapH,rapX,and rapY,all of which enabled C to T and A to G base editing.The SPACE base editor has an edit window of-14 to-16 bp for A to G and-16 to-20 bp for C to T.In summary,the two-component system CseB-C_SR was identified as a positive regulator of rapamycin and a negative regulator of elaiophylin biosynthesis in S.rapamycinious,and identification of sigE_SR as a target gene of CseB_SR by transcriptional analysis combined with EMSA results.In addition,a base editor capable of simultaneous cytosine to thymine and adenine to guanine conversions has been developed for the first time in Streptomyces.These will facilitate the promotion of S.rapamycinious functional gene screening,and metabolic engineering to improve rapamycin fermentation level.