Research On The Regulation Of Streptomyces Secondary Metabolites Biosynthesis By The Aqueous Extracts Of Traditional Chinese Medicine

In recent years,the emergence of multi-drug resistant "superbugs" due to the abuse of antibiotics has posed a serious threat to the health of all humanity,and there is an urgent demand for new antibiotics with novel structures and the unique mechanisms of action.Chinese herbs such as Rheum palmatum,Polygonum cuspidatum,Eucommia ulmoides,Lonicera japonica Thunb,and Isatis tinctoria Linnaeus not only treat infectious diseases by inhibiting the reproduction of pathogens in the human body,but also promote the growth of gut probiotics in the human body.Studies had shown that certain small-molecule active compounds in herbal medicines can be used as elicitors to activate biosynthetic pathway of microbial secondary metabolites.Microbial secondary metabolites provide a rich source of antibiotic compounds,in which ribosomal synthesis and post-translation modification peptides(RiPPs)show powerful antibiotic activity.Lanthipeptides,as important members of the RiPPs family,have attracted much attention because of their unique structures and antibacterial mechanism.The secondary metabolites of Streptomyces have long been considered as a molecular treasure trove of natural products of microbial drugs and an important source of drug lead compounds,genome mining shows that Streptomyces has great potential to produce lanthipeptides,but a large number of gene clusters are "silent" or low expression under conventional culture conditions.Therefore,it is necessary to study how to activate the lanthipeptide gene cluster in Streptomyces.Traditional Chinese medicines and their active ingredients can regulate the production and activity of microbial secondary metabolites,but its mechanism of action is not clear.Therefore,studying the regulatory mechanism of traditional Chinese medicine on microbial secondary metabolites will not only conduce to develop more novel active compounds,but also provide theoretical basis for studying the mechanism of activating silent gene clusters by low-molecule weight compounds.The research in this thesis is divided into three major parts.Firstly,we analyzed the genomes with probiotic potential from bacterial genome database,and used various bioinformatic methods to mine and illustrate the secondary metabolites biosynthetic gene clusters(BGCs)of secondary metabolites,classify and map the BGCs profiles for RiPPs.Secondly,the aqueous extracts of Eucommia ulmoides Olive,Rheum palmatum and Polygonum cuspidatum were selected to test their ability to activate the "silent" BGCs for RiPPs from four different Streptomyces strains.Finally,we focused on a class I lanthipeptide biosynthetic gene cluster from Streptomyces sp.CB02414 and aimed at activating this silent BGC by various approaches,including heterologous expression,bi-directional promoter replacement,and gene knockout,to study the mechanism of aqueous extracts of traditional Chinese medicine on activating the gene cluster.The specific research contents and results are as follows.1.Genome mining and biosynthetic analysis of lanthipeptideIn this thesis,the genomes of 195 actinobacterial strains were analyzed for secondary metabolites using genome mining tools such as antiSMASH 5.0,BAGEL 3,and RiPPMiner.The types of RiPPs and their BGCs were categorized and analyzed,with emphasis on the multiple sequence alignment of precursor peptide and analysis of biosynthetic genes of the lanthipeptide BGCs.The results showed that the next-generation probiotics(NGPs)included seven genera: Bacteroides,Faecalibacterium,Anaerobutyricum,Prevotella,Christensenella,Clostridium,and Akkermansia.The types of RiPPs compounds excavated in the genomes of161 strains of these seven genera were rich,but few in number.Only five class I lanthipeptide BGCs and one class II lanthipeptide BGC were found in six NGP strains.However,in 34 Streptomyces strains,lanthipeptide is not only rich in types but also abundant in a number of gene clusters.Comparing the number and types predicted by antiSMASH 5.0 and BAGEL 3genome mining tools,the BGCs of 132 lanthipeptides were predicted by using antiSMASH 5.0in 34 Streptomyces strains,among which class III lanthipeptide accounted for the most and class V accounted for the least.The BGCs of a class I lanthipeptide from Streptomyces sp.CB02414 was specifically analyzed,predicting its complete biosynthetic pathway and possible post-translational modification structure.Therefore,Streptomyces were selected for traditional Chinese medicine intervention.2.The influence of traditional Chinese medicine aqueous extracts on the production of Streptomyces secondary metabolitesWe selected the Eucommia ulmoides Olive aqueous extract,Rheum palmatum aqueous extract,and Polygonum cuspidatum aqueous extract as the herbs to test the influence of traditional Chinese medicine aqueous extracts on the fermentation of Streptomyces species(Streptomyces sp.CB02414;Streptomyces sp.CB03234;Streptomyces sp.CB02130 and Streptomyces sp.NRRL S-1813).The fermentation products were extracted by adding the aqueous extract of traditional Chinese medicine to the liquid medium with Streptomyces at different concentration ratios for seven days,and the secondary metabolites of the four Streptomyces strains were analyzed differently by high-performance liquid chromatography and liquid chromatography-mass spectrometry,and the antibacterial activity of crude substance in the fermentation broth was determined by drug-sensitive tablet diffusion method.The results show that the metabolites of the four Streptomyces strains had no inhibitory effect on Escherichia coli under the intervention of the aqueous extract of traditional Chinese medicine,but had obvious inhibitory activity on Staphylococcus aureus and Micrococcus luteus.Compared with the control groups,there was no significant difference between R.palmatum aqueous extract and P.cuspidatum aqueous extract on the secondary metabolites of Streptomyces strains,but the co-cultured fermentation products had weak antibacterial activity.The aqueous extract of E.ulmoides Olive can significantly promote the secondary metabolism of Streptomyces to produce new compounds and promote the antibacterial activity of its metabolites.3.Study on the regulatory mechanism of traditional Chinese medicine aqueous extracts on the production of Planosporicin-like compound from Streptomyces sp.CB02414In the first chapter,a potential precursor peptide structural gene of class I lanthipeptide(Planosporicin-like)was identified in Streptomyces sp.CB02414 by genome mining,but it was found that the fermentation broth of Streptomyces sp.CB02414 had weak antibacterial activity against Micrococcus luteus.Although E.ulmoides Olive aqueous extract can promote the production of metabolites,the antibacterial activity is weak.In this chapter,strategies such as heterologous expression of E.coli BL21 for detecting the transporter enzyme LanT,the core peptide bidirectional promoter substitution using CRISPR-Cas9 system in the original host Streptomyces sp.CB02414,and homologous recombination for knockout the negative regulatory gene lanW,to identify the gene targets that activate the gene cluster and enhance its high production or antibacterial activity,and then the antibacterial activity was detected after the intervention of E.ulmoides Olive aqueous extract.The key role of the target in promoting Planosporicin-like production by Streptomyces sp.CB02414 by E.ulmoides Olive aqueous extract were clarified.The results showed that the transporter LanT could not be efficiently heterologously expressed in E.coli BL21,bidirectional promoter substitution of core peptide had no significant effect on the production and activity of Planosporicin-like.The mutant strainΔlanW,which knocked out of the negative regulatory gene lanW in the gene cluster,showed significantly enhanced its antibacterial activity against M.luteus.Therefore,lanW is the key regulatory gene of Planosporicin-like production by Streptomyces sp.CB02414.Compared with the wild type strain,the Eucommia ulmoides Olive aqueous extract could promote the antibacterial activity of the mutant ΔlanW.Therefore,Lan W is a negative regulator of E.ulmoides Olive aqueous extract to promote Planosporicin-like production by Streptomyces sp.CB02414.In summary,this study revealed that NGPs and Streptomyces have the potential to produce RiPPs compounds through genome mining.Streptomyces has a higher potential to produce lanthipeptides.Using traditional Chinese medicine aqueous extracts and Streptomyces coculture fermentation method to confirm that E.ulmoides Olive aqueous extract has the ability to improve the antibacterial activity of Streptomyces strains.The BGC of a class I lanthipeptide in Streptomyces sp.CB02414 by genetic manipulation,and the mutant strain has obvious antibacterial activity.This study lays a molecular foundation for the study of the secondary metabolites of Streptomyces and NGPs,and provides a reference for the research on the regulatory mechanism of traditional Chinese medicine on Streptomyces secondary metabolites.