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.

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.

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.

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.

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.

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.

Fungal arginine-containing cyclodipeptide synthases are finally revealed

Biosynthetic pathways without any identifiable core enzymes may encode unknown(biosynthetic route)-unknown(molecular structure)natural products.However,bioinformatics-guided mining for such unknown-unknown metabolites is challenging.Recently,an unknown-unknown biosynthetic route has been deciphered in fungi.It was found that a class of enzymes previously annotated as hypothetical proteins catalyze the biosynthesis of arginine-containing cyclodipeptides(CDPs).This advances the understanding of the biosynthesis of CDPs and highlights the vast potential of unknown-unknown natural products encoded by microbial genomes.

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.

Epigenetic modification in histone deacetylase deletion strain of Calcarisporium arbuscula leads to diverse diterpenoids

Epigenetic modifications have been proved to be a powerful way to activate silent gene clusters and lead to diverse secondary metabolites in fungi. Previously, inactivation of a histone H3 deacetylase in Calcarisporium arbuscula had led to pleiotropic activation and overexpression of more than 75% of the biosynthetic genes and isolation of ten compounds. Further investigation of the crude extract of C. arbuscula Δhda A strain resulted in the isolation of twelve new diterpenoids including three cassanes(1-3), one cleistanthane(4), six pimaranes(5-10), and two isopimaranes(11 and 12) along with two know cleistanthane analogues. Their structures were elucidated by extensive NMR spectroscopic data analysis. Compounds 2 and 4 showed potent inhibitory effects on the expression of MMP1 and MMP2(matrix metalloproteinases family) in human breast cancer(MCF-7) cells.

Activation of microbial secondary metabolic pathways:Avenues and challenges

Microbial natural products are a tremendous source of new bioactive chemical entities for drug discovery.Next generation sequencing has revealed an unprecedented genomic potential for production of secondary metabolites by diverse micro-organisms found in the environment and in the microbiota.Genome mining has further led to the discovery of numerous uncharacterized‘cryptic’metabolic pathways in the classical producers of natural products such as Actinobacteria and fungi.These biosynthetic gene clusters may code for improved biologically active metabolites,but harnessing the full genetic potential has been hindered by the observation that many of the pathways are‘silent’under laboratory conditions.Here we provide an overview of the various biotechnological methodologies,which can be divided to pleiotropic,biosynthetic gene cluster specific,and targeted genome-wide approaches that have been developed for the awakening of microbial secondary metabolic pathways.

Puromycin A, B and C, cryptic nucleosides identified from Streptomyces alboniger NRRL B-1832 by PPtase-based activation

Natural product discovery is pivot for drug development,however,this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways.We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins.By applying this approach in the Streptomyces alboniger NRRL B-1832,we herein identified three cryptic nucleosides products,including one known puromycin A and two new derivatives(puromycin B and C).The biosynthesis of these products doesn't require the involvement of carrier protein,indicating the phosphopantetheinyl transferase(PPtase)indeed plays a fundamental regulatory role in metabolites biosynthesis.These results demonstrate that the PPtasebased approach have a much broader effective scope than the previously assumed carrier proteininvolving pathways,which will benefit future natural products discovery and biosynthetic studies.

Activation of an unconventional meroterpenoid gene cluster in Neosartorya glabra leads to the production of new berkeleyacetals

Fungal genomes carry many gene clusters seemingly capable of natural products biosynthesis,yet most clusters remain cryptic or down-regulated. Genome mining revealed an unconventional paraherquonin-like meroterpenoid biosynthetic gene cluster in the chromosome of Neosartorya glabra.The cryptic or down-regulated pathway was activated by constitutive expression of pathway-specific regulator gene ber A encoded within ber biosynthetic gene cluster. Chemical analysis of mutant Ng-OE:ber A extracts enabled the isolation of four berkeleyacetal congeners, in which two of them are new. On the basis of careful bioinformatic analysis of the coding enzymes in the ber gene cluster, the biosynthetic pathway of berkeleyacetals was proposed. These results indicate that this approach would be valuable for discovery of novel natural products and will accelerate the exploitation of prodigious natural products in filamentous fungi.

In silico methods for linking genes and secondary metabolites: The way forward

In silico methods for linking genomic space to chemical space have played a crucial role in genomics driven discovery of new natural products as well as biosynthesis of altered natural products by engineering of biosynthetic pathways.Here we give an overview of available computational tools and then briefly describe a novel computational framework,namely retro-biosynthetic enumeration of biosynthetic reactions,which can add to the repertoire of computational tools available for connecting natural products to their biosynthetic gene clusters.Most of the currently available bioinformatics tools for analysis of secondary metabolite biosynthetic gene clusters utilize the“Genes to Metabolites”approach.In contrast to the“Genes to Metabolites”approach,the“Metabolites to Genes”or retro-biosynthetic approach would involve enumerating the various biochemical transformations or enzymatic reactions which would generate the given chemical moiety starting from a set of precursor molecules and identifying enzymatic domains which can potentially catalyze the enumerated biochemical transformations.In this article,we first give a brief overview of the presently available in silico tools and approaches for analysis of secondary metabolite biosynthetic pathways.We also discuss our preliminary work on development of algorithms for retro-biosynthetic enumeration of biochemical transformations to formulate a novel computational method for identifying genes associated with biosynthesis of a given polyketide or nonribosomal peptide.

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.

Activation of three natural product biosynthetic gene clusters from Streptomyces lavendulae CGMCC 4.1386 by a reporter-guided strategy

Along with the fast developing of DNA sequencing technology,a great number of natural product biosynthetic gene clusters have been discovered by bioinformatic analysis,which demands novel high-throughput genome mining methods to obtain the diverse compounds dictated by those gene clusters.In this work,a method based on the reporter gene xylE was established to screen for the activation conditions of thirteen different gene clusters from Streptomyces lavendulae CGMCC 4.1386.In this reporter-guided method,the key structure gene was replaced by a xylE-kanaR cassette with the xylE gene being controlled by the transcription and translation machinery of the key structure gene.It not only facilitated the screening of activation conditions,but also provided the null mutants of specific natural product gene clusters as controls to link those clusters with their products conveniently.The potential activation conditions of eleven gene clusters from S.lavendulae CGMCC 4.1386 were obtained.In addition,activation of three of the eleven gene clusters was confirmed and their products were identified.

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.