Genome mining of sulfonated lanthipeptides reveals unique cyclic peptide sulfotransferases

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.

Nitric oxide synthase-guided genome mining identifies a cytochrome P450 enzyme for olefin nitration in bacterial specialized metabolism

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.

Genome mining of multi-substituted alkylresorcinols from a hybrid highly reducing-and typeⅢ-polyketide pathway

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.

Genomic data mining for functional annotation of human long noncoding RNAs

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.

Targeted genome mining for microbial antitumor agents acting through DNA intercalation

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.

Recombineering enables genome mining of novel siderophores in a non-model Burkholderiales strain

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.

Genus-specific secondary metabolome in Allokutzneria and Kibdelosporangium

Rare actinomycete genera are highly recognized as a promising source of structurally diverse and bioactive natural products.Among these genera,Allokutzneria and Kibdelosporangium are two phylogenetically closely related and have been reported to encode some valuable biosynthetic enzymes and secondary metabolites.However,there is currently no relevant systematic research available to outline the linkage of genomic and metabolomics for specific secondary metabolites in these two promising genera.In this study,we first investi-gated the genus-specific secondary metabolic potential in Allokutzneria and Kibdelosporangium by comparing the diversity and novelty of their secondary metabolite biosynthetic gene clusters(BGCs).The specific secondary metabolites produced by two representative strains of these genera were comprehensively investigated using untargeted metabolomics techniques.The findings unveiled that the majority(95.4%)of the gene cluster families(GCFs)encoded by Allokutzneria and Kibdelosporangium were genus-specific,including NRPS GCFs encoding siderophores.The untargeted metabolomics analysis revealed that the metabolic profiles of two representative strains exhibit extensive specificity,with the culture medium having a big impact on the metabolic profiles.Besides,an MS-cluster featuring a series of hydroxamate-type siderophores was identified from Allokutzneria albata JCM 9917,with two of them,including a novel one(N-deoxy arthrobactin A),being experimentally validated.The present study offers valuable insights for the targeted discovery of genus-specific natural products from microorganisms.

Discovery of angucyclinone polyketides from marine actinomycetes with a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthase

Angucyclinones are aromatic polyketides produced by type Ⅱ polyketide synthases（PKS） and are mainly found in terrestrial actinomycetes. To discover more angucyclinones from marine actinomycetes, a genomic DNA-based PCR assay targeting type Ⅱ polyketide synthases was performed. Among the 167 marine actinomycetes strains screened, twelve strains were identified as the ＂positive＂ strains possessing type Ⅱ PKS-encoding genes based on the sequencing of PCR products. One of the 12 ＂positive＂ strains, Streptomyces sp. PKU-MA00218 was selected for the large-scale fermentation based on the HPLC and TLC analysis. Four angucyclinones, 6-deoxy-8-O-methylrabelomycin（1）, 8-O-methylrabelomycin（2）, 8-O-methyltetrangulol（3）, C-ring cleavage product of angucyclinone C（4）, were isolated and their structures were elucidated based on spectroscopic analyses. The isolation of angucyclinones 1–4 highlights the power of genome mining technologies based on biosynthetic knowledge in natural products discovery.

Discovery of thiazostatin siderphores from Streptomyces sp. HMU0027 with a genomic DNA-based PCR assay targeting nonribosomal peptide synthetase

Nonribosomal peptides （NRPs） represent a large family of natural products with great diversities of chemical structures and biological activities. The peptide backbones of NRPs are synthesized by nonribosomal peptide synthetases （NRPSs） that minimally consist of one adenylation （A） domain, one peptidyl carrier protein （PCP） and one condensation （C） domain. In this study, we carded out a PCR screening and identified 21 ＂positive＂ strains from 100 actinomycete strains with the degenerate primers designed from the conserved sequences of A domains of NRPSs. One of the 21 ＂positive＂ strains, Streptomyces sp. HMU0027, was selected for large-scale fermentation （9 L） based on HPLC analysis, and subsequent isolation and structural elucidation afforded seven NRPS-synthesized thiazostatin siderophore analogues （1-7）. Compound 1 was a new compound containing an unusual linkage between a phenolate siderophore and a sugar moiety. These results laid the foundation for further biosynthetic research of these thiazostatin analogues and highlighted the power of genome mining technologies based on biosynthetic knowledge in NRP discovery.

Genome mining combined metabolic shunting and OSMAC strategy of an endophytic fungus leads to the production of diverse natural products

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.

Mining of a streptothricin gene cluster from Streptomyces sp.TP-A0356 genome via heterologous expression

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.

Challenges and advances in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs)

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.

Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns

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.

Development of fungal cell factories for the production of secondary metabolites:Linking genomics and metabolism

The genomic era has revolutionized research on secondary metabolites and bioinformatics methods have in recent years revived the antibiotic discovery process after decades with only few new active molecules being identified.New computational tools are driven by genomics and metabolomics analysis,and enables rapid identification of novel secondary metabolites.To translate this increased discovery rate into industrial exploitation,it is necessary to integrate secondary metabolite pathways in the metabolic engineering process.In this review,we will describe the novel advances in discovery of secondary metabolites produced by filamentous fungi,highlight the utilization of genome-scale metabolic models(GEMs)in the design of fungal cell factories for the production of secondary metabolites and review strategies for optimizing secondary metabolite production through the construction of high yielding platform cell factories.

Genomics-guided discovery of a new and significantly better source of anticancer natural drug FK228

FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.

Genome mining of fungal globin-like enzymes for catalyzing the synthesis of linear terpenes

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.

Genome mining reveals the biosynthetic potential of the marinederived strain Streptomyces marokkonensis M10

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.

Recent advances in the direct cloning of large natural product biosynthetic gene clusters

Large-scale genome-mining analyses have revealed that microbes potentially harbor a huge reservoir of unchar-acterized natural product(NP)biosynthetic gene clusters(BGCs),and this has spurred a renaissance of novel drug discovery.However,the majority of these BGCs are often poorly or not at all expressed in their native hosts under laboratory conditions,and thus are regarded as silent/orphan BGCs.Currently,connecting silent BGCs to their corresponding NPs quickly and on a large scale is particularly challenging because of the lack of universal strategies and enabling technologies.Generally,the heterologous host-based genome mining strategy is believed to be a suitable alternative to the native host-based approach for prioritization of the vast and ever-increasing number of uncharacterized BGCs.In the last ten years,a variety of methods have been reported for the direct cloning of BGCs of interest,which is the first and rate-limiting step in the heterologous expression strategy.Es-sentially,each method requires that the following three issues be resolved:1)how to prepare genomic DNA;2)how to digest the bilateral boundaries for release of the target BGC;and 3)how to assemble the BGC and the capture vector.Here,we summarize recent reports regarding how to directly capture a BGC of interest and briefly discuss the advantages and disadvantages of each method,with an emphasis on the notion that direct cloning is very beneficial for accelerating genome mining research and large-scale drug discovery.

Identification and heterologous expression of the globomycin biosynthetic gene cluster

Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens.Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II(LspA),which is absent in eukaryotes and considered an attractive target for the development of new antibiotics.Despite its interesting biological properties,the gene cluster encoding its biosynthesis has not yet been identified.In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp.CA-278952 to identify a candidate gene cluster responsible for its biosynthesis.A null mutant was constructed using CRISPR base editing where production was abolished,strongly suggesting its involvement in the biosynthesis.The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146,therefore unambigu-ously linking globomycin and its biosynthetic gene cluster.Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties.

Nobachelins,new siderophores from Nocardiopsis baichengensis protecting Caenorhabditis elegans from Pseudomonas aeruginosa infection

The biosynthetic potential of actinobacteria to produce novel natural products is still regarded as immense.In this paper,we correlated a cryptic biosynthetic gene cluster to chemical molecules by genome mining and chemical analyses,leading to the discovery of a new group of catecholate-hydroxamate siderophores,nobachelins,from Nocardiopsis baichengensis DSM 44845.Nobachelin biosynthesis genes are conserved in several bacteria from the family Nocardiopsidaceae.Structurally,nobachelins feature fatty-acylated hydroxy-ornithine and a rare chlorinated catecholate group.Intriguingly,nobachelins rescued Caenorhabditis elegans from Pseudomonas aeruginosa-mediated killing.