Mining And Biosynthesis Of Natural Products From Three Strains Of Terrestrial Strepromyces

Natural products(NPs)of microorganisms are important sources of pharmaceutical lead compounds.Facing the challenges of increased resistance of pathogenic bacteria,frequent occurrence of infectious diseases,and incurable multiple diseases,many scientists still have been focusing on microbial natural products for screening and developing new drugs.Among many microorganisms,Streptomyces species have excellent potentials to produce active secondary metabolites,and made great contributions to medicine,agriculture,food,and other fields,with great commercial and medicinal values.Genome sequencing has revealed that Streptomyces species contain a rich variety of biosynthetic gene clusters(BGCs),which can provide strong support for the development of innovative drugs with diverse structures and activities,such as polyketides,peptides,lactams,terpenoids,and so on.Therefore,in this study,two strategies:"genome mining" and "activity-guided mining"were used to explore natural active products from different sources of Streptomyces.(1)Genome mining and biosynthesis of natural products in Streptomyces sp.AM-7161 and Streptomyces tanashiensis Kala DSM731Based on genome sequence analysis,more than 30 different types of biosynthetic gene clusters(BGCs)were found in each stain of AM-7161 and DSM731,indicating that they have great potentials to be exploited.Terpenoid BGCs were extremely abundant in two strains,among which,two gene clusters(BGC1.4 and BGC12.1)in AM-7161 and three gene clusters(BGC1.1,BGC1.2,and BGC1.29)in DSM731 were prioritized due to their low similarity with known terpenoids gene clusters,and the existence of putative terpene synthase-and modifying enzyme-encoding genes.Bioinformatics analysis using multiple sequence alignments and phylogenetic tree construction showed that:(ⅰ)The terpene synthases encoded by BGC1.4,BGC1.2,and BGC1.29 contained unique conserved sequences of class Ⅰ terpene synthases;(ⅱ)The terpene synthases encoded by BGC1.4,BGC1.29 and BGC1.2 are more closely related to sesquiterpene synthases.Therefore,it is speculated that three gene clusters(BGC1.4,BGC1.29,and BGC1.2)have the potential to produce sesquiterpenoids.These three gene clusters were directly cloned and engineered using Red/ET homologous recombination technology and expressed in the heterologous host of Streptomyces albus J1074(J1074).After that,it was found that:(ⅰ)BGC1.4 was successfully expressed in J1074,indicated by three differential peaks detected.Promoter engineering by replacing the original promoter region of the terpene synthase gene in BGC1.4 with a bidirectional efficient promoter increased the expression of the target products by 3-5 folds.α-Cadinol,a cadinane-type sesquiterpene.was isolated and identified structurally after large-scaled fermentation of J1074/P-BGC1.4.(ⅱ)BGC1.29 was found to be silent in J1074.A constitutive promoter was inserted into the upstream of the terpene synthase encoding gene,resulting in the successful expression of BGC1.29,evident by two differential peaks.After large-scaled fermentation of J1074/PBGC1.29 and subsequent chromatographic purification,isodauc-8-en-11-was isolated and identified structurally.(ⅲ)BGC1.2 was successfully cloned,and modified by promoter replacement and negative regulatory gene knockout,which activated the expression of BGC1.2 in J1074.Liquid chromatography-mass spectrometry showed that there were several different peaks in the expression of J1074/P-BGC1.2.Next,the predicted terpene synthases SaTS4 and SdTS29 encoded by BGC1.4 and BGC1.29,respectively,were introduced into a specific engineered Escherichia coli(which can provide precursors of terpenoids).By establishing the in vivo expression system of SaTS4 in E.coli,three peaks identical to the heterologous expression products by BGC1.4 in Streptomyces chassis were detected,indicating that SaTS4 is a promiscuous sesquiterpene synthase;The in vivo expression system of SaTS29 in E.coli produced sesquiterpene alcohol isodauc-8-en-11－ol,indicating that SaTS29 is isodauc-8-en-11-ol synthase.The co-expression of SdP450(P450 enzyme)and SdTS29 did not lead to the production of new compounds,indicating that this P450 enzyme is not functional in the subsequent oxidation of isodauc-8-en-11-ol in E.coli.(2)Activity-guided mining and biosynthesis of natural products in Streptomyces sp.CS-7"One strain many compounds"(OSMAC)strategy was used to further optimize the fermentation conditions of Streptomyces sp.CS-7,a strain exhibiting wonderful antibacterial activity in the previous work.After large-scaled fermentation and activity-guided mining,two angucyclines,mayamycin and mayamycin B,were isolated and identified.The biosynthetic pathway of mayamycin and mayamycin B have not yet been confirmed.Next steps were to explore its biosynthetic pathway through heterologous expression:By whole-genome sequencing and analysis,only one possible aromatic polyketide gene cluster(named mays7)was found in this strain and presumed to be related to the biosynthesis of mayamycins.Then,this gene cluster was directly cloned using Red/ET homologous recombination technology and expressed in the heterologous host S.albus J1074.Mayamycin and mayamycin B were successfully detected,indicating that the mays7 cluster is responsible for their biosynthesis.Two other peaks(m/z 466.11[M+H]+,m/z 480.13[M+H]+)were also detected in the crude extracts,suggesting that they may be mayamycin derivatives.Glycosyltransferases(GTs)play important roles in the structural diversities and biological activities of NPs.In the structure of mayamycin,a rare N-demethylangolosamine moiety is attached to the aglycone core structure at the C-5 position via a rare C-C glycosidic bond.A possible C-GT gene(mays7-ORF286)in mays7 cluster was inactivated to identify new GTs and explore their catalytic mechanisms.As expected,the inactivation of mays7-286 in CS-7 completely abolished the production of mayamycin and its derivatives.The functional complement of mays7-286 further indicated that this gene plays an important role in the Cglycosylation step.To sum up,in this study,two strategies "genome mining" and "activity-guided mining"were used to find natural products from three terrestrial Streptomyces,and characterized the functions of key enzymes in the products BGCs.Three terpene BGCs from AM-7161 and DSM731 were successfully expressed in Streplomyces heterologous hosts,and two sesquiterpene alcohols were isolated and identified.The functions of two sesquiterpene synthases were revealed by establishing the in vivo expression systems of SaTS4 and SaTS29 in E.coli cells;two angucyclines were isolated from CS-7,and their biosynthetic gene cluster was identified through heterologous expression.A new C-GT gene was identified by in-frame gene deletion and complementation.The above research further enriches the biosynthetic elements of relevant NPs,which can be used for combinatorial biosynthesis from different sources,and will help to promote the discovery of more novel natural products.