Mining Of Bioactive Natural Products From Two Habitat-Special Bacteria

In recent years,with the abuse of antibiotics,the emergence of multidrug-resistant bacteria seriously threatening human health.It is urgent to develop new antibacterial drugs to cope with the prevalence of drug-resistant bacteria.However,in recent decades the development of antibiotic drugs has been slow,and only few of the newly approved antibiotics possess new chemical skeletons.Microbial natural products are important sources of clinical drugs,especially antibiotics,and are widely used in medicine,agriculture,food processing and personal care.For a long time,the mining of microbial natural products mainly based on the activity screening of microbial fermentation products,which often encounters the repeated discovery of known compounds.Microorganisms in special habitats harbor unique metabolic pathways during long-term evolution,which have the potential to produce secondary metabolites with novel structures and a wide range of bioactivities.With the development of DNA sequencing and bioinformatics technologies,a large number of unknown secondary metabolite biosyntheic gene clusters(BGCs)have been found in the genomes of microorganisms from special habitats.Exploitation of these BGCs is of great potential for the discovery of novel compounds and development of new drugs.This research focuses on the mining of natural products from an anaerobic bacterium Clostridium cellulovorans 743B and a psychrophilic bacterium Pedobacter cryoconitis A37.BGC1.4 and BGC1.16 in C.cellulovorans 743B were successfully cloned using Gibson assembly,and the native promoters of both gene clusters were replaced with constitutive promoters by Red/ET recombination.Both gene clusters were introduced into a heterologous host for expression.LC-MS analysis of the crude extracts from fermentation broth uncovered a set of novel compounds,indicating successful activation of the BGC1.16.The BGC1.13 in the strain 743B contains two NRPS gene clusters,which is difficult to be cloned due to the large number of repetitive sequences.The predicted peptide products of this gene cluster attached with SNAC-tagwere chemically synthesized,and the T-TE didomains were expressed and purified.In vitro biochemical reactions were performed with chemically synthesized linear peptides and T-TE didomains.LC-MS analysis showed that the linear peptides were released from the SNAC group,but not cyclic peptide was detected.Activity tests of the fermentation products of the strain P.cryoconitis A37 revealed good siderophore activity.Guided by the activity,the siderophore compounds were separated by Sephadex gel and HPLC.HPLC-MS analysis and NMR structural elucidation identified the compounds as flavolipids.To verify the biosynthesis pathway of flavolipids,two siderophore biosynthetic gene clusters BGC2.1 and BGC10.1 in P.cryoconitis A37 were successfully cloned by using Gibson assembly and expressed in heterologous host.However,production of the corresponding flavolipids could not be detected.Meanwhile,we tried to identify the biosynthetic pathway of flavolipids by in situ gene knockout.A CRISPR-Cas gene knockout plasmid was constructed and transferred into the strain S37,indicating stable replication in the strain.In this study,secondary metabolites in two bacterial strains from specific habitat were mined by activity screening,gene cluster cloning,modification and heterologous expression.The BGC1.16 in C.cellulovorans 743B was successfully activated,and an approach for mining complex novel cyclic peptides by a combination of chemical and enzymatic catalysis was carried out.A known class of iron carrier flavolipids was obtained from P.cryoconitis A37 and its biosynthetic pathway was investigated.This study established the foundation for further expoitation of active secondary metabolites from the strains 743B and A37,and also provided technical support for the discovery of natural products from other microbes in special habitats.