Discovery Of Juanlimycin-like Compounds And Genome Mining Of Two Rare Actinomycetes

Microorganisms,especially actinomycetes,are the main source of natural product drugs,more than 10,000 biologically active substances have been isolated from actinomycetes up to date.However,after the 1980s,more and more actinomycetes were discovered and isolated repeatedly,leading to lots of repeating compounds been discovered from actinomycetes.Researchers once thought that microbial natural product resources have been exhausted.However,genome sequencing show that microorganisms contain a large number of secondary metabolic gene clusters to produce abundant new compounds.due to the limitations of traditional mining methods before microorganisms can not be fully explored,based on this background,various genomic mining methods have emerged and played an increasingly important role in the field of natural products’discovery.In this paper,we attempt to make genome mining for three AHBA synthase positive strains including Streptomycete sp.LC6,Verrucosispora sp.NS0172,Micromonospora sp.FXY A29 and discover natural products with new structures or good activities.Junalimycin A and B are novel dimerized pentaketo ansamycin isolated from,Streptomyces sp.LC6 by laboratory in the early stage.In order to obtain more compounds with similar skeleton to juanlimycins and deduce their biosynthesis pathway,we attempted to increase the yield and type of juanlimycin-like compounds by knocking out the competitive metabolite metacycloprodigiosin’s gene cluster red and overexpressing pathway-specific positive regulatory genes.This thesis ferment mutant strain LC6-deRED-SARP in SFM medium for 95 L,four compounds of A.F.H,I were isolated and identified through HPLC guided separation,in which A and I were new compounds of juanlimvcin type.We also tried to activate silent gene cluster and identified their secondary metabolites by overexpressing regulatory genes and promoter engineering for Verrucosisipora sp.NSO0172 and Micromonspora sp.FXY A29.Based on the previous genetic manipulation methods,we further improved the efficiency of conjugal transfer by exploring the spore germination conditions and spore heat shock conditions,which laid a good foundation for the follow-up work.In Verrucosispora sp.NS0172,we partially "activated" the type Ⅱ PKS-NRPS hybrid gene cluster NS0172-GC2 by two rounds of promoter replacement,and isolated two compounds numbered A1,A2 from the mutant strain NS01172-GC2-1.wherein A1 was identified by NMR as O--4-carboxybutenyl phenylpropionic acid,A2 was a O--5-hydroxy-6-carboxyhexenyl phenylpropionic acid.A1 and A2 are presumed to be synthesized by the type Ⅱ polyketide sub-gene cluster orf50-59,but further gene knockout experiments are needed for confirmation or falsification.In addition,by overexpressing the SARP family regulatory gene orf39,we obtained the mutant strain 0172-GC5-SARP,which fermentation extract showed a dark green color,and there were multiple differential peaks compared to the wild type.The fermentation medium of 0172-GC5-SARP had no color change,but a large amount of black oily spores were produced.We speculated that the dark green component in the extract was certain"spore pigment".We used YMG medium to carry out 5 L fermentation and tried to isolate target compounds,but the dark green component could not be purified and identified due to poor solubility in common organic reagents.In addition,the yeild of several differential compounds at 275 nm is very low,so their structure also cannot be identified.It is necessary to further expand the fermentation scale or optimize the fermentation conditions for next fermentation.In Micromono.spora sp.FXY A29.we attempted to activate GC1(PKS-NRPS-Lantibiotic hybrid gene cluster).GC9(Type Ⅱ PKS gene cluster)and GC15(trans-AT PKS-NRPS hybrid gene cluster).From the two-way promoter replacement mutant strain A29-GC1.we isolated compounds B1 and B2.Surprisingly,the structure of B1 is the same with A1 that obtained in 0172-GC2-1.Although there are two type Ⅱpolyketide pathways in the geno,me of A29.there is a large difference from the type Ⅱpolyketide subgene cluster of 0172-GC2.Since we have ruled out the posibility of fermentation contamination.we speculate that the possible reasons are:1)Some sequences are lost due to incomplete genomic sequencing.2)A29 uses different routes to synthesize such compounds.Using the same promoter replacement strategy as A29-GC1,we play two rounds promoter replacement of GC15 to obtain A29-GC15-1 and A29-GC15-2 mutants,respectively.The A29-GC15-2 mutant had two distinct differential peaks compared with the wild type by HPLC detection,numbered B3.B4.Owing to transformation or degradation during the isolation process,the two compounds were not identified,we need to ferment again and modified the isolation method,attempting to separate at low temperature or dark conditions.In summary,based on the optimization of the conjugal transfer conditions of two rare actinomycetes,we used a variety of strategies to mine multiple secondary metabolic pathway’s in the above three actinomycetes and identified at least 4 new compounds in 2 types.Mining work has a large workload and low success rate.Therefore,for rare actinomycetes or other microorganisms with difficult genetic manipulation and slow growth,the two common mining strategies of promoter replacement and regulatory gene overexpression may not be very good choices.Methods that can bypassing the genetic manipulation of the original bacteria such as pathway assembly in vitro based on synthetic biology strategy and heterologous expression in suitable actinomycetes,or only need simple genetic manipulation such as medium optimization and double reporter gene selection may be more suitable for the genome mining of new natural products of (rare) actinomycetes.