In this study,we employed a combination of genome mining and heteronuclear single quantum coherence(HSQC)-based small molecule accurate recognition technology(SMART)technology to search for fernane-type triterpenoids....In this study,we employed a combination of genome mining and heteronuclear single quantum coherence(HSQC)-based small molecule accurate recognition technology(SMART)technology to search for fernane-type triterpenoids.Initially,potential endophytic fungi were identified through genome mining.Subsequently,fine fractions containing various fernane-type triterpenoids were selected using HSQC data collection and SMART prediction.These triterpenoids were then obtained through targeted isolation and identification.Finally,their antifungal activity was evaluated.As a result,three fernane-type triterpenoids,including two novel compounds,along with two new sesquiterpenes and four known compounds were isolated from one potential strain,Diaporthe discoidispora.Their structures were elucidated through analysis of high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)and nuclear magnetic resonance(NMR)spectroscopic data.The absolute configurations were determined using single-crystal X-ray diffraction analysis and electron capture detector(ECD)analysis.Compound 3 exhibited moderate antifungal activity against Candida albicans CMCC 98001 and Aspergillus niger.展开更多
Genome mining for the search and discovery of two new globin-like enzymes,TriB from Fusarium poae and TutaA from Schizophyllum commne,are involved in the synthesis of two linear terpenes tricinonoic acid(1)and 2-buten...Genome mining for the search and discovery of two new globin-like enzymes,TriB from Fusarium poae and TutaA from Schizophyllum commne,are involved in the synthesis of two linear terpenes tricinonoic acid(1)and 2-butenedioic acid(3).Both in vivo heterologous biosynthesis and in vitro biochemical assays showed that these two enzymes catalyzed the C-C double bond cleavage of a cyclic sesquiterpene precursor(-)-germacrene D(7)and a linear diterpene backbone schizostain(2),respectively.Our work presents an unusual formation mechanism of linear terpenes from fungi and expands the functional skills of globin-like enzymes in the synthesis of terpene compounds.展开更多
Fungal alkylresorcinols are a class of polyketides,which are commonly synthesized by the hybridization of highly reducing polyketide synthase(hrPKS)with non-reducing polyketide synthase(nrPKS).In this study,we identif...Fungal alkylresorcinols are a class of polyketides,which are commonly synthesized by the hybridization of highly reducing polyketide synthase(hrPKS)with non-reducing polyketide synthase(nrPKS).In this study,we identified and demonstrated a new assembly model for synthesizing alkylresorcinol(scirpilin A,1),which was accomplished by collaboration of a hrPKS(FscA)and a typeⅢPKS(FscB).Furthermore,three post-tailoring enzymes(FscC,FscD,and FscE)act iteratively on 1 skeleton,including successive 14e-oxidation of inert carbons,di-halogenation,and di-methylation,to form highly oxidized and multisubstituted alkylresorcinols.Our work presents an unusual synthesis manner of alkylresorcinols,sheds light on the collaborative mechanism between hrPKS and typeⅢPKS and provides three valuable enzymatic catalysts for the tailoring of alkylresorcinol family natural products in future.展开更多
Nitrogen-nitrogen(N-N)bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism.Guided by a nitric oxide synthase(NOS)-based genome mining strategy,we report the discovery and charact...Nitrogen-nitrogen(N-N)bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism.Guided by a nitric oxide synthase(NOS)-based genome mining strategy,we report the discovery and characterization of a new heme-dependent enzyme system that catalyzes intermolecular N-N bond formation.Using both in vivo and in vitro reconstitution approaches,we demonstrated that a protein complex,comprising a heme enzyme and a 2[4Fe-4S]ferredoxin partner,mediates the coupling of theα-amine group of L-aspartate with inorganic nitrogen oxide species,such as nitrite or nitric oxide,to generate hydrazinosuccinic acid,a key biosynthetic precursor in several natural product pathways.Structural modeling and site-directed mutagenesis suggest a plausible catalytic mechanism involving the formation of a reactive nitrogen intermediate,potentially a heme-bound nitrene species.These findings reveal a new family of N-N bond-forming biocatalysts that leverage inorganic nitrogen sources,offering valuable tools for genome mining and the synthetic biology.展开更多
Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity.Recent advances in genomics and sy...Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity.Recent advances in genomics and synthetic biology,are facilitating us to discover a large number of new ribosomal natural products,including lanthipeptides,lasso peptides,sactipeptides,thiopeptides,microviridins,cyanobactins,linear thiazole/oxazole-containing peptides and so on.In this review,we summarize bioinformatic strategies that have been developed to identify and prioritize biosynthetic gene clusters(BGCs)encoding RiPPs,and the genome mining-guided discovery of novel RiPPs.We also prospectively provide a vision of what genomics-guided discovery of RiPPs may look like in the future,especially the discovery of RiPPs from dominant but uncultivated microbes,which will be promoted by the combinational use of synthetic biology and metagenome mining strategies.展开更多
Endophytic fungi are promising producers of bioactive small molecules.Bioinformatic analysis of the genome of an endophytic fungus Penicillium dangeardii revealed 43 biosynthetic gene clusters,exhibited its strong abi...Endophytic fungi are promising producers of bioactive small molecules.Bioinformatic analysis of the genome of an endophytic fungus Penicillium dangeardii revealed 43 biosynthetic gene clusters,exhibited its strong ability to produce numbers of secondary metabolites.However,this strain mainly produce rubratoxins alone with high yield in varied culture conditions,suggested most gene clusters are silent.Efforts for mining the cryptic gene clusters in P.dangeardii,including epigenetic regulation and one-strain-many-compounds(OSMAC)approach were failed probably due to the high yield of rubratoxins.A metabolic shunting strategy by deleting the key gene for rubratoxins biosynthesis combining with optimization of culture condition successfully activated multiple silent genes encoding for other polyketide synthases(PKSs),and led to the trace compounds detectable.As a result,a total of 23 new compounds including azaphilone monomers,dimers,turimers with unprecedented polycyclic bridged heterocycle and spiral structures,as well as siderophores were identified.Some compounds showed significant cytotoxicities,anti-inflammatory or antioxidant activities.The attractive dual PKS s gene clusters for azaphilones biosynthesis were mined by bioinformatic analysis and overexpression of a pathway specific transcription factor.Our work therefor provides an efficient approach to mine the chemical diversity of endophytic fungi.展开更多
Iron is essential for bacterial survival,and most bacteria capture iron by producing siderophores.Burkholde-riales bacteria produce various types of bioactive secondary metabolites,such as ornibactin and malleobactin ...Iron is essential for bacterial survival,and most bacteria capture iron by producing siderophores.Burkholde-riales bacteria produce various types of bioactive secondary metabolites,such as ornibactin and malleobactin siderophores.In this study,the genome analysis of Burkholderiales genomes showed a putative novel siderophore gene cluster crb,which is highly similar to the ornibactin and malleobactin gene clusters but does not have pvdF,a gene encoding a formyltransferase for N-δ-hydroxy-ornithine formylation.Establishing the bacteriophage recom-binase Redγ-Redδβ7029 mediated genome editing system in a non-model Burkholderiales strain Paraburkholderia caribensis CICC 10960 allowed the rapid identification of the products of crb gene cluster,caribactins A-F(1-6).Caribactins contain a special amino acid residue N-δ-hydroxy-N-δ-acetylornithine(haOrn),which differs from the counterpart N-δ-hydroxy-N-δ-formylornithine(hOrn)in ornibactin and malleobactin,owing to the absence of pvdF.Gene inactivation showed that the acetylation of hOrn is catalyzed by CrbK,whose homologs proba-bly not be involved in the biosynthesis of ornibactin and malleobactin,showing possible evolutionary clues of these siderophore biosynthetic pathways from different genera.Caribactins promote biofilm production and en-hance swarming and swimming abilities,suggesting that they may play crucial roles in biofilm formation.This study also revealed that recombineering has the capability to mine novel secondary metabolites from non-model Burkholderiales species.展开更多
Although sulfonation plays crucial roles in various biological processes and is frequently utilized in medicinal chemistry to improve water solubility and chemical diversity of drug leads,it is rare and underexplored ...Although sulfonation plays crucial roles in various biological processes and is frequently utilized in medicinal chemistry to improve water solubility and chemical diversity of drug leads,it is rare and underexplored in ribosomally synthesized and post-translationally modified peptides(RiPPs).Biosynthesis of RiPPs typically entails modification of hydrophilic residues,which substantially increases their chemical stability and bioactivity,albeit at the expense of reducing water solubility.To explore sulfonated RiPPs that may have improved solubility,we conducted co-occurrence analysis of RiPP class-defining enzymes and sulfotransferase(ST),and discovered two distinctive biosynthetic gene clusters(BGCs)encoding both lanthipeptide synthetase(LanM)and ST.Upon expressing these BGCs,we characterized the structures of novel sulfonated lanthipeptides and determined the catalytic details of LanM and ST.We demonstrate that SslST-catalyzed sulfonation is leader-independent but relies on the presence of A ring formed by LanM.Both LanM and ST are promiscuous towards residues in the A ring,but ST displays strict regioselectivity toward Tyr5.The recognition of cyclic peptide by ST was further discussed.Bioactivity evaluation underscores the significance of the ST-catalyzed sulfonation.This study sets up the starting point to engineering the novel lanthipeptide STs as biocatalysts for hydrophobic lanthipeptides improvement.展开更多
The biological signaling molecule nitric oxide(NO)has recently emerged as a metabolic precursor for the creation of microbial natural products with diversified structures and biological activities.Within the biosynthe...The biological signaling molecule nitric oxide(NO)has recently emerged as a metabolic precursor for the creation of microbial natural products with diversified structures and biological activities.Within the biosynthetic gene clusters(BGCs)of these compounds,genes associated with NO production pathways have been pinpointed.In this study,we employ a nitric oxide synthase(NOS)-guided genome mining strategy for the targeted discovery of NO-derived bacterial natural products and NO-utilizing biocatalysts.We show that a conserved NOS-containing BGC,distributed across several actinobacterial genomes,is responsible for the biosynthesis of lajollamycin,a unique nitro-tetraene-containing antibiotic whose biosynthetic mechanism remains elusive.Through a combination of in vivo and in vitro studies,we unveil the first cytochrome P450 enzyme capable of catalyzing olefin nitration in natural product biosynthesis.These results not only expand the current knowledge about biosynthetic nitration processes but also offer an efficient way for targeted identification of NO-utilizing metabolic pathways and novel nitrating biocatalysts.展开更多
Microbial natural products have been one of the most important sources for drug development.In the current postgenomic era,sequence-driven approaches for natural product discovery are becoming increasingly popular.Her...Microbial natural products have been one of the most important sources for drug development.In the current postgenomic era,sequence-driven approaches for natural product discovery are becoming increasingly popular.Here,we develop an effective genome mining strategy for the targeted discovery of microbial metabolites with antitumor activities.Our method employs uvrA-like genes as genetic markers,which have been identified in the biosynthetic gene clusters(BGCs)of several chemotherapeutic drugs of microbial origin and confer self-resistance to the corresponding producers.Through systematic genomic analysis of gifted actinobacteria genera,identification of uvrA-like gene-containing BGCs,and targeted isolation of products from a BGC prioritized for metabolic analysis,we identified a new tetracycline-type DNA intercalator timmycins.Our results thus provide a new genome mining strategy for the efficient discovery of antitumor agents acting through DNA intercalation.展开更多
Marine streptomycetes are rich sources of natural products with novel structures and interesting biological activities,and genome mining of marine streptomycetes facilitates rapid discovery of their useful products.In...Marine streptomycetes are rich sources of natural products with novel structures and interesting biological activities,and genome mining of marine streptomycetes facilitates rapid discovery of their useful products.In this study,a marine-derived Streptomyces sp.M10 was revealed to share a 99.02%16S rDNA sequence identity with that of Streptomyces marokkonensis Ap1T,and was thus named S.marokkonensis M10.To further evaluate its biosynthetic potential,the 7,207,169 bps of S.marokkonensis M10 genome was sequenced.Genomic sequence analysis for potential secondary metaboliteassociated gene clusters led to the identification of at least three polyketide synthases(PKSs),six non-ribosomal peptide synthases(NRPSs),one hybrid NRPS-PKS,two lantibiotic and five terpene biosynthetic gene clusters.One type I PKS gene cluster was revealed to share high nucleotide similarity with the candicidin/FR008 gene cluster,indicating the capacity of this microorganism to produce polyene macrolides.This assumption was further verified by isolation of two polyene family compounds PF1 and PF2,which have the characteristic UV adsorption at 269,278,290 nm(PF1)and 363,386 and 408 nm(PF2),respectively.S.marokkonensis M10 is therefore a new source of polyene metabolites.Further studies on S.marokkonensis M10 will provide more insights into natural product biosynthesis potential of related streptomycetes.This is also the first report to describe the genome sequence of S.marokkonensis-related strain.展开更多
Life may have begun in an RNA world,which is supported by increasing evidence of the vital role that RNAs perform in biological systems.In the human genome,most genes actually do not encode proteins;they are noncoding...Life may have begun in an RNA world,which is supported by increasing evidence of the vital role that RNAs perform in biological systems.In the human genome,most genes actually do not encode proteins;they are noncoding RNA genes.The largest class of noncoding genes is known as long noncoding RNAs(lncRNAs),which are transcripts greater in length than 200 nucleotides,but with no protein-coding capacity.While some lncRNAs have been demonstrated to be key regulators of gene expression and 3D genome organization,most lncRNAs are still uncharacterized.We thus propose several data mining and machine learning approaches for the functional annotation of human lncRNAs by leveraging the vast amount of data from genetic and genomic studies.Recent results from our studies and those of other groups indicate that genomic data mining can give insights into lncRNA functions and provide valuable information for experimental studies of candidate lncRNAs associated with human disease.展开更多
Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces s...Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.展开更多
Defining suitable enzymes for reaction steps in novel synthetic pathways is crucial for developing microbial cell factories for non-natural products.Here,we developed a computational workflow to identify C12 alcohol-a...Defining suitable enzymes for reaction steps in novel synthetic pathways is crucial for developing microbial cell factories for non-natural products.Here,we developed a computational workflow to identify C12 alcohol-active UDP-glycosyltransferases.The workflow involved three steps:(1)assembling initial candidates of putative UDP-glycosyltransferases,(2)refining selection by examining conserved regions,and(3)3D structure prediction and molecular docking.Genomic sequences from Candida,Pichia,Rhizopus,and Thermotoga,known for lauryl glucoside synthesis via whole-cell biocatalysis,were screened.Out of 240 predicted glycosyltransferases,8 candidates annotated as glycosyltransferases were selected after filtering out those with signal peptides and identifying conserved UDP-glycosyltransferase regions.These proteins underwent 3D structure prediction and molecular docking with 1-dodecanol.RO3G,a candidate from Rhizopus delemar RA 99-880 with a relatively high ChemPLP fitness score,was selected and expressed in Escherichia coli BL21(DE3).It was further characterized using a feeding experiment with 1-dodecanol.Results confirmed that the RO3G-expressing strain could convert 1-dodecanol to lauryl glucoside,as quantified by HPLC and identified by targeted LC-MS.Monitoring the growth and fermentation profiles of the engineered strain revealed that RO3G expression did not affect cell growth.Interestingly,acetate,a major fermentation product,was reduced in the RO3G-expressing strain compared to the GFP-expressing strain,suggesting a redirection of flux from acetate to other pathways.Overall,this work presents a successful workflow for discovering UDP-glycosyltransferase enzymes with confirmed activity toward 1-dodecanol for lauryl glucoside production.展开更多
Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analys...Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis.EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester[Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate],producing over 99%yield and 99%enantiomeric excess(e.e.)for(4S,5R)-monomethyl ester,a crucial chiral intermediate during the synthesis of d-biotin.Notably,the recombinant E.coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain.EstSIT01 displays a preference for short-chain p-NP esters.The optimal temperature and pH were 45°C and 10.0,with Km and kcat values of 0.147 mmol/L and 5.808 s−1,respectively.Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure.Collectively,EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.展开更多
Streptomyces sp.MJM3502 is a promising producer of rufomycins,which are a class of potent anti-tuberculosis lead compounds.Although the structure,activity,and mechanism of the main rufomycin 4/6 and its analogs have b...Streptomyces sp.MJM3502 is a promising producer of rufomycins,which are a class of potent anti-tuberculosis lead compounds.Although the structure,activity,and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied,a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of Streptomyces sp.MJM3502,and its metabolic engineering has not yet been reported.This study established the genetic manipulation platform for the strain.Using CRISPR/Cas9-based technology to in-frame insert the strong kasO^(*)p promoter upstream of the rufB and rufS genes of the rufomycin BGC,we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold,respectively.Furthermore,designing recombinant strains by inserting the kasO^(*)p promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives.These findings provide the basis for enhancing the production of valuable natural compounds in Streptomyces and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.展开更多
The increased number of annotated bacterial genomes provides a vast resource for genome mining.Several bacterial natural products with epoxide groups have been identified as pre-mRNA spliceosome inhibitors and antitum...The increased number of annotated bacterial genomes provides a vast resource for genome mining.Several bacterial natural products with epoxide groups have been identified as pre-mRNA spliceosome inhibitors and antitumor compounds through genome mining.These epoxide-containing natural products feature a common biosynthetic characteristic that cytochrome P450s(CYPs)and its patterns such as epoxidases are employed in the tailoring reactions.The tailoring enzyme patterns are essential to both biological activities and structural diversity of natural products,and can be used for enzyme pattern-based genome mining.Recent development of direct cloning,heterologous expression,manipulation of the biosynthetic pathways and the CRISPR-CAS9 system have provided molecular biology tools to turn on or pull out nascent biosynthetic gene clusters to generate a microbial natural product library.This review focuses on a library of epoxide-containing natural products and their associated CYPs,with the intention to provide strategies on diversifying the structures of CYP-catalyzed bioactive natural products.It is conceivable that a library of diversified bioactive natural products will be created by pattern-based genome mining,direct cloning and heterologous expression as well as the genomic manipulation.展开更多
Genome mining has revealed that Penicillium spp.possess numerous down-regulated or cryptic biosynthetic gene clusters(BGCs).This finding hinted that our investigation of fungal secondary metabolomes is limited.Herein,...Genome mining has revealed that Penicillium spp.possess numerous down-regulated or cryptic biosynthetic gene clusters(BGCs).This finding hinted that our investigation of fungal secondary metabolomes is limited.Herein,we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by Penicillium brasilianum CGMCC 3.4402.The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene berA responsible for berkeleyacetals biosynthesis from Neosartorya glabra.Chemical analysis of the extracts from the mutant strain Pb-OE:berA enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C(1),one daucane-type arpenicarotane C(4),along with four known sesquiterpenoids including arpenibisabolane A(2),eupenicisirenins A(3),arpenicarotane B(5)and aspterric acid(6).The assignments of their structures were elucidated from detailed analyses of spectroscopic data,electronic circular dichroism calculation,and biogenetic considerations.The bioassay of isolated compounds(1-6)exhibited no cytotoxic activities against three tumor cells including MCF-7,HepG2,and A549.Arpenibisabolane C(1)and A(2)showed weak inhibition bioactivities on aquatic pathogens Vibrio owensii and Vibrio algivorus.Moreover,phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed.Based on the chemical structures and biogenetic investigations,a hypothetic pathway of new compounds(1,4)was proposed.展开更多
The mainstream strategy of genome mining relies on the homologous activation and heterologous expression of target biosynthetic gene clusters(BGCs).However,the efficiency of the current techniques available for new co...The mainstream strategy of genome mining relies on the homologous activation and heterologous expression of target biosynthetic gene clusters(BGCs).However,the efficiency of the current techniques available for new compound discovery hardly complements these efforts.In a recent publication in Science,Xie et al.reported their breakthrough progress in expediting the discovery of untapped chemical diversity from bacteria by establishing the leveraged know-how of ACTIMOT(Advanced Cas9-mediaTed In vivo MObilization and mulTiplication of BGCs),offering a new avenue to access the unexploited,and even unpredictable,biosynthetic potential of bacteria.展开更多
基金supported by the Outstanding Youth Foundation of Heilongjiang Province(No.YQ2021H009).
文摘In this study,we employed a combination of genome mining and heteronuclear single quantum coherence(HSQC)-based small molecule accurate recognition technology(SMART)technology to search for fernane-type triterpenoids.Initially,potential endophytic fungi were identified through genome mining.Subsequently,fine fractions containing various fernane-type triterpenoids were selected using HSQC data collection and SMART prediction.These triterpenoids were then obtained through targeted isolation and identification.Finally,their antifungal activity was evaluated.As a result,three fernane-type triterpenoids,including two novel compounds,along with two new sesquiterpenes and four known compounds were isolated from one potential strain,Diaporthe discoidispora.Their structures were elucidated through analysis of high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)and nuclear magnetic resonance(NMR)spectroscopic data.The absolute configurations were determined using single-crystal X-ray diffraction analysis and electron capture detector(ECD)analysis.Compound 3 exhibited moderate antifungal activity against Candida albicans CMCC 98001 and Aspergillus niger.
基金supported by the National Natural Science Foundation of China(No.31870022)Chongqing Science Funds for Distinguished Young Scientists(No.cstc2020jcyjjqX0005)。
文摘Genome mining for the search and discovery of two new globin-like enzymes,TriB from Fusarium poae and TutaA from Schizophyllum commne,are involved in the synthesis of two linear terpenes tricinonoic acid(1)and 2-butenedioic acid(3).Both in vivo heterologous biosynthesis and in vitro biochemical assays showed that these two enzymes catalyzed the C-C double bond cleavage of a cyclic sesquiterpene precursor(-)-germacrene D(7)and a linear diterpene backbone schizostain(2),respectively.Our work presents an unusual formation mechanism of linear terpenes from fungi and expands the functional skills of globin-like enzymes in the synthesis of terpene compounds.
基金supported by the National Natural Science Foundation of China(No.22277100)the National Key R&D Program of China(No.2020YFA0907700)+2 种基金the Fundamental Research Funds for the Central Universities(Nos.SWU-KQ22034 and SWUXDPY22009)the Science and Technology Innovation Key R&D Program of Chongqing(No.CSTB2022TIAD-STX0015)the Chongqing Science Funds for Distinguished Young Scientists(No.cstc2020jcyj-jqX0005)。
文摘Fungal alkylresorcinols are a class of polyketides,which are commonly synthesized by the hybridization of highly reducing polyketide synthase(hrPKS)with non-reducing polyketide synthase(nrPKS).In this study,we identified and demonstrated a new assembly model for synthesizing alkylresorcinol(scirpilin A,1),which was accomplished by collaboration of a hrPKS(FscA)and a typeⅢPKS(FscB).Furthermore,three post-tailoring enzymes(FscC,FscD,and FscE)act iteratively on 1 skeleton,including successive 14e-oxidation of inert carbons,di-halogenation,and di-methylation,to form highly oxidized and multisubstituted alkylresorcinols.Our work presents an unusual synthesis manner of alkylresorcinols,sheds light on the collaborative mechanism between hrPKS and typeⅢPKS and provides three valuable enzymatic catalysts for the tailoring of alkylresorcinol family natural products in future.
基金supported by the National Key R&D Program of China(2025YFA0921000)the National Natural Science Foundation of China(32350051)to Y.-L.Du,+1 种基金China Postdoctoral Science Foun-dation(2024M752836,2024T170784)Postdoctoral Fellowship Program of CPSF(GZB20230658)to G.Zhao.
文摘Nitrogen-nitrogen(N-N)bond-forming enzymes are rare but play vital roles in both primary and secondary metabolism.Guided by a nitric oxide synthase(NOS)-based genome mining strategy,we report the discovery and characterization of a new heme-dependent enzyme system that catalyzes intermolecular N-N bond formation.Using both in vivo and in vitro reconstitution approaches,we demonstrated that a protein complex,comprising a heme enzyme and a 2[4Fe-4S]ferredoxin partner,mediates the coupling of theα-amine group of L-aspartate with inorganic nitrogen oxide species,such as nitrite or nitric oxide,to generate hydrazinosuccinic acid,a key biosynthetic precursor in several natural product pathways.Structural modeling and site-directed mutagenesis suggest a plausible catalytic mechanism involving the formation of a reactive nitrogen intermediate,potentially a heme-bound nitrene species.These findings reveal a new family of N-N bond-forming biocatalysts that leverage inorganic nitrogen sources,offering valuable tools for genome mining and the synthetic biology.
基金Z.Zhong,B.He,and Y-X Li acknowledge a partial financial support from the National Key R&D Program of China(2018YFA0903200)Southern Marine Science and Engineering Guangdong Laboratory(Guangzhou)(SMSEGL20SC01)J.Li acknowledges a partial financial support by a National Institutes of Health(NIH)grant P20GM103641 and a National Science Foundation EPSCoR Program OIA-1655740.
文摘Ribosomally synthesized and post-translationally modified peptides(RiPPs)are a class of cyclic or linear peptidic natural products with remarkable structural and functional diversity.Recent advances in genomics and synthetic biology,are facilitating us to discover a large number of new ribosomal natural products,including lanthipeptides,lasso peptides,sactipeptides,thiopeptides,microviridins,cyanobactins,linear thiazole/oxazole-containing peptides and so on.In this review,we summarize bioinformatic strategies that have been developed to identify and prioritize biosynthetic gene clusters(BGCs)encoding RiPPs,and the genome mining-guided discovery of novel RiPPs.We also prospectively provide a vision of what genomics-guided discovery of RiPPs may look like in the future,especially the discovery of RiPPs from dominant but uncultivated microbes,which will be promoted by the combinational use of synthetic biology and metagenome mining strategies.
基金supported financially by the National Key Research and Development Program of China(2018YFA0901900)the CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-010,2017-I2M-4-004)+1 种基金Fundamental Research Funds for the Central Universities(2017PT35001)supported by the Drug Innovation Major Project(2018ZX09711001-008-001)
文摘Endophytic fungi are promising producers of bioactive small molecules.Bioinformatic analysis of the genome of an endophytic fungus Penicillium dangeardii revealed 43 biosynthetic gene clusters,exhibited its strong ability to produce numbers of secondary metabolites.However,this strain mainly produce rubratoxins alone with high yield in varied culture conditions,suggested most gene clusters are silent.Efforts for mining the cryptic gene clusters in P.dangeardii,including epigenetic regulation and one-strain-many-compounds(OSMAC)approach were failed probably due to the high yield of rubratoxins.A metabolic shunting strategy by deleting the key gene for rubratoxins biosynthesis combining with optimization of culture condition successfully activated multiple silent genes encoding for other polyketide synthases(PKSs),and led to the trace compounds detectable.As a result,a total of 23 new compounds including azaphilone monomers,dimers,turimers with unprecedented polycyclic bridged heterocycle and spiral structures,as well as siderophores were identified.Some compounds showed significant cytotoxicities,anti-inflammatory or antioxidant activities.The attractive dual PKS s gene clusters for azaphilones biosynthesis were mined by bioinformatic analysis and overexpression of a pathway specific transcription factor.Our work therefor provides an efficient approach to mine the chemical diversity of endophytic fungi.
基金supported by the National Key R&D Program of China(2019YFA0905700)National Natural Science Foundation of China(32070060).
文摘Iron is essential for bacterial survival,and most bacteria capture iron by producing siderophores.Burkholde-riales bacteria produce various types of bioactive secondary metabolites,such as ornibactin and malleobactin siderophores.In this study,the genome analysis of Burkholderiales genomes showed a putative novel siderophore gene cluster crb,which is highly similar to the ornibactin and malleobactin gene clusters but does not have pvdF,a gene encoding a formyltransferase for N-δ-hydroxy-ornithine formylation.Establishing the bacteriophage recom-binase Redγ-Redδβ7029 mediated genome editing system in a non-model Burkholderiales strain Paraburkholderia caribensis CICC 10960 allowed the rapid identification of the products of crb gene cluster,caribactins A-F(1-6).Caribactins contain a special amino acid residue N-δ-hydroxy-N-δ-acetylornithine(haOrn),which differs from the counterpart N-δ-hydroxy-N-δ-formylornithine(hOrn)in ornibactin and malleobactin,owing to the absence of pvdF.Gene inactivation showed that the acetylation of hOrn is catalyzed by CrbK,whose homologs proba-bly not be involved in the biosynthesis of ornibactin and malleobactin,showing possible evolutionary clues of these siderophore biosynthetic pathways from different genera.Caribactins promote biofilm production and en-hance swarming and swimming abilities,suggesting that they may play crucial roles in biofilm formation.This study also revealed that recombineering has the capability to mine novel secondary metabolites from non-model Burkholderiales species.
基金supported by the National Natural Science Foundation of China(No.21907047,22077056,and 21907046)the Fundamental Research Funds for the Central Universities(No.lzujbky2019-ct03,lzujbky-2019-10,and lzujbky-2021-ct05,China).
文摘Although sulfonation plays crucial roles in various biological processes and is frequently utilized in medicinal chemistry to improve water solubility and chemical diversity of drug leads,it is rare and underexplored in ribosomally synthesized and post-translationally modified peptides(RiPPs).Biosynthesis of RiPPs typically entails modification of hydrophilic residues,which substantially increases their chemical stability and bioactivity,albeit at the expense of reducing water solubility.To explore sulfonated RiPPs that may have improved solubility,we conducted co-occurrence analysis of RiPP class-defining enzymes and sulfotransferase(ST),and discovered two distinctive biosynthetic gene clusters(BGCs)encoding both lanthipeptide synthetase(LanM)and ST.Upon expressing these BGCs,we characterized the structures of novel sulfonated lanthipeptides and determined the catalytic details of LanM and ST.We demonstrate that SslST-catalyzed sulfonation is leader-independent but relies on the presence of A ring formed by LanM.Both LanM and ST are promiscuous towards residues in the A ring,but ST displays strict regioselectivity toward Tyr5.The recognition of cyclic peptide by ST was further discussed.Bioactivity evaluation underscores the significance of the ST-catalyzed sulfonation.This study sets up the starting point to engineering the novel lanthipeptide STs as biocatalysts for hydrophobic lanthipeptides improvement.
基金supported by the National Natural Science Foundation of China(32122005 and 32370051)to Y.-L.D.
文摘The biological signaling molecule nitric oxide(NO)has recently emerged as a metabolic precursor for the creation of microbial natural products with diversified structures and biological activities.Within the biosynthetic gene clusters(BGCs)of these compounds,genes associated with NO production pathways have been pinpointed.In this study,we employ a nitric oxide synthase(NOS)-guided genome mining strategy for the targeted discovery of NO-derived bacterial natural products and NO-utilizing biocatalysts.We show that a conserved NOS-containing BGC,distributed across several actinobacterial genomes,is responsible for the biosynthesis of lajollamycin,a unique nitro-tetraene-containing antibiotic whose biosynthetic mechanism remains elusive.Through a combination of in vivo and in vitro studies,we unveil the first cytochrome P450 enzyme capable of catalyzing olefin nitration in natural product biosynthesis.These results not only expand the current knowledge about biosynthetic nitration processes but also offer an efficient way for targeted identification of NO-utilizing metabolic pathways and novel nitrating biocatalysts.
基金the National Key R&D Program of China(2019YFA0905400)the National Natural Science Foundation of China(32122005).
文摘Microbial natural products have been one of the most important sources for drug development.In the current postgenomic era,sequence-driven approaches for natural product discovery are becoming increasingly popular.Here,we develop an effective genome mining strategy for the targeted discovery of microbial metabolites with antitumor activities.Our method employs uvrA-like genes as genetic markers,which have been identified in the biosynthetic gene clusters(BGCs)of several chemotherapeutic drugs of microbial origin and confer self-resistance to the corresponding producers.Through systematic genomic analysis of gifted actinobacteria genera,identification of uvrA-like gene-containing BGCs,and targeted isolation of products from a BGC prioritized for metabolic analysis,we identified a new tetracycline-type DNA intercalator timmycins.Our results thus provide a new genome mining strategy for the efficient discovery of antitumor agents acting through DNA intercalation.
文摘Marine streptomycetes are rich sources of natural products with novel structures and interesting biological activities,and genome mining of marine streptomycetes facilitates rapid discovery of their useful products.In this study,a marine-derived Streptomyces sp.M10 was revealed to share a 99.02%16S rDNA sequence identity with that of Streptomyces marokkonensis Ap1T,and was thus named S.marokkonensis M10.To further evaluate its biosynthetic potential,the 7,207,169 bps of S.marokkonensis M10 genome was sequenced.Genomic sequence analysis for potential secondary metaboliteassociated gene clusters led to the identification of at least three polyketide synthases(PKSs),six non-ribosomal peptide synthases(NRPSs),one hybrid NRPS-PKS,two lantibiotic and five terpene biosynthetic gene clusters.One type I PKS gene cluster was revealed to share high nucleotide similarity with the candicidin/FR008 gene cluster,indicating the capacity of this microorganism to produce polyene macrolides.This assumption was further verified by isolation of two polyene family compounds PF1 and PF2,which have the characteristic UV adsorption at 269,278,290 nm(PF1)and 363,386 and 408 nm(PF2),respectively.S.marokkonensis M10 is therefore a new source of polyene metabolites.Further studies on S.marokkonensis M10 will provide more insights into natural product biosynthesis potential of related streptomycetes.This is also the first report to describe the genome sequence of S.marokkonensis-related strain.
基金supported by the Self Regional Healthcare Foundation,USA
文摘Life may have begun in an RNA world,which is supported by increasing evidence of the vital role that RNAs perform in biological systems.In the human genome,most genes actually do not encode proteins;they are noncoding RNA genes.The largest class of noncoding genes is known as long noncoding RNAs(lncRNAs),which are transcripts greater in length than 200 nucleotides,but with no protein-coding capacity.While some lncRNAs have been demonstrated to be key regulators of gene expression and 3D genome organization,most lncRNAs are still uncharacterized.We thus propose several data mining and machine learning approaches for the functional annotation of human lncRNAs by leveraging the vast amount of data from genetic and genomic studies.Recent results from our studies and those of other groups indicate that genomic data mining can give insights into lncRNA functions and provide valuable information for experimental studies of candidate lncRNAs associated with human disease.
基金supported in part by the National Natural Science Foundation of China (31170037)Ministry of Science and Technology of China (2013CB734003)the China Postdoctoral Science Foundation (2013M530755)
文摘Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.
基金This work(Grant No.RGNS 64-069)was financially supported by Office of the Permanent Secretary,Ministry of Higher Education,Science,Research and Innovationpartially supported by Chiang Mai University.
文摘Defining suitable enzymes for reaction steps in novel synthetic pathways is crucial for developing microbial cell factories for non-natural products.Here,we developed a computational workflow to identify C12 alcohol-active UDP-glycosyltransferases.The workflow involved three steps:(1)assembling initial candidates of putative UDP-glycosyltransferases,(2)refining selection by examining conserved regions,and(3)3D structure prediction and molecular docking.Genomic sequences from Candida,Pichia,Rhizopus,and Thermotoga,known for lauryl glucoside synthesis via whole-cell biocatalysis,were screened.Out of 240 predicted glycosyltransferases,8 candidates annotated as glycosyltransferases were selected after filtering out those with signal peptides and identifying conserved UDP-glycosyltransferase regions.These proteins underwent 3D structure prediction and molecular docking with 1-dodecanol.RO3G,a candidate from Rhizopus delemar RA 99-880 with a relatively high ChemPLP fitness score,was selected and expressed in Escherichia coli BL21(DE3).It was further characterized using a feeding experiment with 1-dodecanol.Results confirmed that the RO3G-expressing strain could convert 1-dodecanol to lauryl glucoside,as quantified by HPLC and identified by targeted LC-MS.Monitoring the growth and fermentation profiles of the engineered strain revealed that RO3G expression did not affect cell growth.Interestingly,acetate,a major fermentation product,was reduced in the RO3G-expressing strain compared to the GFP-expressing strain,suggesting a redirection of flux from acetate to other pathways.Overall,this work presents a successful workflow for discovering UDP-glycosyltransferase enzymes with confirmed activity toward 1-dodecanol for lauryl glucoside production.
基金supported by the Shanghai Committee of Science and Technology(No.13430503400)Project of Leading Talents in Shandong Taishan Industry(Grant No.LJNY202019)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry(No.ZX2012-05)Innovation Program of Shanghai Municipal Education Commission(No.11YZ227).
文摘Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis.EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester[Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate],producing over 99%yield and 99%enantiomeric excess(e.e.)for(4S,5R)-monomethyl ester,a crucial chiral intermediate during the synthesis of d-biotin.Notably,the recombinant E.coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain.EstSIT01 displays a preference for short-chain p-NP esters.The optimal temperature and pH were 45°C and 10.0,with Km and kcat values of 0.147 mmol/L and 5.808 s−1,respectively.Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure.Collectively,EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.
基金supported by grants from“Cooperative Research Program for Agriculture Science and Technology Development(Project No.PJ01319101)”Rural Development Administration,Republic of KoreaThe National Natural Science Foundation of China(Project No.31600038)+1 种基金the Key Research Development Program of the Natural Science Basic Research Plan in Shaanxi Province of China(Project No.2021NY-196)funding of their anti-TB cyclopetide research by grant U19AT142735.
文摘Streptomyces sp.MJM3502 is a promising producer of rufomycins,which are a class of potent anti-tuberculosis lead compounds.Although the structure,activity,and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied,a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of Streptomyces sp.MJM3502,and its metabolic engineering has not yet been reported.This study established the genetic manipulation platform for the strain.Using CRISPR/Cas9-based technology to in-frame insert the strong kasO^(*)p promoter upstream of the rufB and rufS genes of the rufomycin BGC,we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold,respectively.Furthermore,designing recombinant strains by inserting the kasO^(*)p promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives.These findings provide the basis for enhancing the production of valuable natural compounds in Streptomyces and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.
基金a US National Institute of Health/National Cancer Institute grant(R01 CA152212)awarded to Prof。
文摘The increased number of annotated bacterial genomes provides a vast resource for genome mining.Several bacterial natural products with epoxide groups have been identified as pre-mRNA spliceosome inhibitors and antitumor compounds through genome mining.These epoxide-containing natural products feature a common biosynthetic characteristic that cytochrome P450s(CYPs)and its patterns such as epoxidases are employed in the tailoring reactions.The tailoring enzyme patterns are essential to both biological activities and structural diversity of natural products,and can be used for enzyme pattern-based genome mining.Recent development of direct cloning,heterologous expression,manipulation of the biosynthetic pathways and the CRISPR-CAS9 system have provided molecular biology tools to turn on or pull out nascent biosynthetic gene clusters to generate a microbial natural product library.This review focuses on a library of epoxide-containing natural products and their associated CYPs,with the intention to provide strategies on diversifying the structures of CYP-catalyzed bioactive natural products.It is conceivable that a library of diversified bioactive natural products will be created by pattern-based genome mining,direct cloning and heterologous expression as well as the genomic manipulation.
基金supported by the National Natural Science Foundation of China(31872617)the CAMS Innovation Fund for Medical Sciences(CIFMS)(2021-I2M-1-055,2019-I2M-1-005)+1 种基金the National Microbial Resource Center(NMRC-2024-3)the central level,scientific research institutes for basic R&D fund business(3332018097).
文摘Genome mining has revealed that Penicillium spp.possess numerous down-regulated or cryptic biosynthetic gene clusters(BGCs).This finding hinted that our investigation of fungal secondary metabolomes is limited.Herein,we report a genetically-modified activation strategy to characterize the spectrum of sesquiterpenoids produced by Penicillium brasilianum CGMCC 3.4402.The cryptic or down-regulated pathways were stimulated by constitutive expression of pathway-specific regulator gene berA responsible for berkeleyacetals biosynthesis from Neosartorya glabra.Chemical analysis of the extracts from the mutant strain Pb-OE:berA enabled the isolation of two new compounds including one bisabolene-type arpenibisabolane C(1),one daucane-type arpenicarotane C(4),along with four known sesquiterpenoids including arpenibisabolane A(2),eupenicisirenins A(3),arpenicarotane B(5)and aspterric acid(6).The assignments of their structures were elucidated from detailed analyses of spectroscopic data,electronic circular dichroism calculation,and biogenetic considerations.The bioassay of isolated compounds(1-6)exhibited no cytotoxic activities against three tumor cells including MCF-7,HepG2,and A549.Arpenibisabolane C(1)and A(2)showed weak inhibition bioactivities on aquatic pathogens Vibrio owensii and Vibrio algivorus.Moreover,phylogenetic analysis and sequence alignments of crucial sesquiterpene synthases were performed.Based on the chemical structures and biogenetic investigations,a hypothetic pathway of new compounds(1,4)was proposed.
文摘The mainstream strategy of genome mining relies on the homologous activation and heterologous expression of target biosynthetic gene clusters(BGCs).However,the efficiency of the current techniques available for new compound discovery hardly complements these efforts.In a recent publication in Science,Xie et al.reported their breakthrough progress in expediting the discovery of untapped chemical diversity from bacteria by establishing the leveraged know-how of ACTIMOT(Advanced Cas9-mediaTed In vivo MObilization and mulTiplication of BGCs),offering a new avenue to access the unexploited,and even unpredictable,biosynthetic potential of bacteria.
