Discovery And Biosynthetic Investigations Of Antibacterial Substances From Three Anaerobic Bacteria

Microorganisms produce a large number of natural products with significant biological activities and complex chemical structures.Some of these compounds are widely used in various fields such as clinical therapy,agriculture,livestock,and manufacturing.With the current epidemic of multi-drug resistant bacteria,the development of novel antibiotics is imminent.Genomic DNA sequencing and bioinformatic analysis have shown that anaerobic microbial genomes contain a large number of natural product biosynthetic gene clusters with the potential to synthesize novel natural products.Discovery of new chemical entities from anaerobic bacteria is of great significance for the development of novel drugs and chemical products.This research focused on discovery of bioactive natural products from three anaerobic microorganisms,Clostridium sardiniense LY3-1,Pelosinus fermentans DSM17108 and Clostridium saccharoperbutylacetonicum DSM 2152,by combining metabonomic analysis and genome mining.Our work laid the foundation for the construction of mining system for the natural products from anaerobic microbes.Four bioactive compounds were isolated from fementation broth of Clostridium sardiniense LY3-1 and Pelosinus fermentans DSM 17108 in an activity-oriented route.Firstly,crude extracts from culture broth of LY3-1 and DSM 17108 were found harboring good inhibiting activity against both Gram-positive and Gram-negative bacteria.Two known active compounds,3-indole-propionic acid(IPA)and p-hydroxyphenylpropionic acid(HPPA),were then isolated from both strains.In addition,two small molecule compounds,anisole and aniline,were isolated from fementation broth of the strain DSM 17108.Two alkenyl reductases that can hydrogenate the substrate p-coumaric acid to produce HPPA were identified in the genome of the strain LY3-1.The expression vectors of 2-enoate-reductases were then successfully constructed and transferred into E.coli,but no soluble protein was obtained.We then performed codon optimization and synthesized the encoding genes of 2-enoate-reductases and expressed the proteins in E.coli.Although soluble expressions of 2-enoate-reductases were achieved after codon optimization,the synthesis of HPPA was not detected in E.coli.Furthermore,the biosynthetic gene cluster of HPPA was cloned by Gibson assembly and transferred into E.coli for heterologous expression,but no production of HPPA was detected.A NRPS gene cluster BGC2.3 in LY3-1 and a NRPS-type I PKS hybrid gene cluster BGC6 in DSM 2152 were successfully cloned and transferred into heterologous hosts for expression.Genomic analysis of LY3-1 and DSM 2152 revealed that both NRPS BGC2.3 in LY3-1 and NRPS-type I PKS heterozygous gene cluster BGC6 in DSM 2152 are expected to synthesize compounds containing thiazole rings.We attempted to activate these two gene clusters by heterologous expression to obtain the corresponding natural products.BGC2.3 and BGC6 were cloned through enzymatic ligation,Red/ET recombination technique and Gibson assembly.Dual expression plasmids containing gene cluster parts and different replicons were successfully constructed and introduced into the heterologous host E.coli GB05-MtaA.However,no new compound production was detected by LC-MS analysis.The original promoter of the gene cluster was subsequently replaced with the strong promoter Plaps and transferred into Bacillus subtilis 1A751 for expression,but no new compound production was detected.In summary,this study focused on the mining of bioactive natural products from anaerobic bacteria.Bioactivity-guided mining led to identification of four known compounds which harboring potential industrial value.Two new 2-enoate-reductases were identified in the genome of the strain LY3-1 and solubly expressed in in E.coli after codon optimization.The cloning of HPPA biosynthetic gene cluster laid the foundation for de novo synthesis of HPPA in E.coli.In addition,two silent natural product biosynthetic gene clusters BGC2.3 and BGC6 were cloned.Although activation of the gene clusters was not achieved in E.coli and Bacillus subtilis,it provided feasible methods for the excavation of natural products from anaerobic bacteria.