Construction And Evaluation Of Polyketide Chassis Derived From Streptomyces Chattanoogensis

Members of the genus Streptomyces are major producers of microbial drugs.They are rich in synthetic elements and precursors for various drugs and harbor a complete self-resistance system to avoid toxicity,and are excellent chassis for heterologous synthesis of drugs.They are also rich in secondary metabolic gene clusters and are reservoirs of natural products.Based on industrial Streptomyces to construct chassis cells and build an artificial synthetic system,we hope to innovate breeding technology,from a "one bacteria for one drug in multi-component" to a "one chassis for multi-drug in single component" new model.These chassis cells can serve for the prospecting of novel natural products,and the high quality and high yield of microbial drugs which is an important innovation in the field of microbial pharmaceuticals.Streptomyces chattanoogensis L10 is the industrial producer of natamycin(16g/L)and its genome is 9Mb.L10 harbors about 32 extra secondary metabolite biosynthesis gene clusters(BGCs)and most of them are polyketides.L10 is also rich in acyl donors,owns high-efficient regulatory network,genetic stability and mature genetic manipulation system.Therefore,it has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites and drug discovery.Based on comparative genomics,pan-genomics and functional genomics analyses,we developed the strategy to systematically decipher essential and dispensable genes in the genome of S.chattanoogensis L10.Firstly,by antiSMASH,IslandViewer 4 and ISsaga 2,the location of redundant gene elements like biosynthetic gene clusters(BGCs),genomic islands(GIs)and mobile genetic elements(MGEs)in the genome were determined.The results revealed that redundant gene elements mainly distributed in the end of chromosome(60%BGCs,73.5%GIs and 72%ISs).Then,multiple genome alignment and pan-genomic analysis were performed by Mauve and BPGA which suggested that the L10 genome can be definitely divided into two parts:highly conserved core gene region(6.0Mb)and two sub-telomeric regions(2.0Mb and 1.0Mb).Subsequently,multiple proteome alignment and functional genomic analysis were carried out by OrthoVenn and KEGG which indicated that there are about 2700 essential genes responsible for fundamental cell processes like cyto-architecture,primary metabolism(carbohydrate metabolism,TCA cycle,amino acid metabolism),DNA replication,transcription,translation,cell division et al and others mainly involved in secondary metabolism,pathogenic processes,transposon and bacteriophage infection et al.Finally,in overall view of functions and distributions of essential and dispensable genes,we determined two large non-essential gene regions(1.3Mb and 0.7Mb)accurately.Our strategy will provide a theoretical reference for rational construction of industrial Streptomyces chassis.In this study,we optimized the Cre/loxP site-specific recombination system.Based on pSET152 and pKC119,we constructed a series of suicide vectors or temperature-sensitive plasmids containing loxP or mutant loxP(lox66 or lox71)sites which can insert into genome by single or double crossover rapidly.In consideration of background expression and toxicity because of tipAp,we optimized the tsr-induced Cre/loxP recombination system by ε-caprolactam induced system.PnitA-NitR system has been proved a hyper-inducible expression system for Streptomyces which is tightly controlled by ε-caprolactam that is an inexpensive and non-toxic inducer which can be widely used in other Streptomyces.By above Cre/loxP system,two genome-streamlined mutants,designated S.chattanoogensis L320 and L321,were rationally constructed by depletion of 1.3Mb and 0.7Mb non-essential gene regions,respectively.We successfully developed large-scale deletion methods which will accelerate the process to rationally construct industrial Streptomyces chassis.This optimized Cre/loxP system could be widely applied in other Streptomyces to generate more miscellaneous and versatile chassis with minimized genome.Meanwhile,we improved the strategy to clone complete gene clusters and achieved assemble or direct cloning of three gene clusters which promoted the progress of enabling technologies for synthetic biology.Lastly,we systematically evaluated the performances of genome-reduced L321.Compared with wild type L10,genome reduction led to unanticipated emergent and excellent properties:enhanced intracellular energy(ATP)and reducing power(NADPH/NADP+),improved productivity of protein and secondary metabolite,more dispersed mycelia,increased transformation efficiency,simplified metabolite profiles,increased genetic stability.The changes of phenotype were beneficial for industrialization.Based on L321,we further blocked the biosynthesis pathway of azomycin,the main by-product,to obtained L325 with more simplified profile of secondary metabolites.Our results suggested that the L321 and L325 can serve as platform cell for heterologous biosynthesis of secondary metabolites,especially polyketides and has high application value and enormous development potential in industries.