Identification of anrF gene, a homology of admM of andrimid biosynthetic gene cluster related to the antagonistic activity of Enterobacter cloacae B8

AIM： To identify the gene （s） related to the antagonistic activity of Enterobacter cloacae B8 and to elucidate its antagonistic mechanism. METHODS： Transposon-mediated mutagenesis and tagging method and cassette PCR-based chromosomal walking method were adopted to isolate the mutant strain （s） of B8 that lost the antagonistic activity and to clone DNA fragments around Tn5 insertion site. Sequence compiling and open reading frame （ORF） finding were done with DNAStar program and homologous sequence and conserved domain searches were performed with BlastN or BlastP programs at www.ncbi.nlm.nih.gov. To verify the gene involved in the antagonistic activity, complementation of a full-length clone of the anrFgene to the mutant B8F strain was used. RESULTS： A 3 321 bp contig around the Tn5 insertion site was obtained and an ORF of 2 634 bp in length designated as anrFgene encoding for a 877 aa polyketide synthase-like protein was identified. It had a homology of 83% at the nucleotide level and 79% ID/87% SIM at the protein level, to the admM gene of Pantoea agglomerans andrimid biosynthetic gene cluster （AY192157）. The Tn5 was inserted at 2 420 bp of the gene corresponding to the COG3319 （the thioesterase domain of type I polyketide synthase） coding region on BSF. The antagonistic activity against Xanthomonas oryzae pv. oryzae was resumed with complementation of the full-length anrFgene to the mutant B8F. CONCLUSION： The anrFgene obtained is related to the antagonistic activity of BS, and the antagonistic substances produced by B8 are andrimid and/or its analogs.

Rational Engineering of Secondary Metabolic Pathways in a Heterologous Host to Enable the Biosynthesis of Hibarimicin Derivatives with Enhanced Anti-Melanomic Activity

A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Genome-resolved metagenomics of Venice Lagoon surface sediment bacteria reveals high biosynthetic potential and metabolic plasticity as successful strategies in an impacted environment

Bacteria living in sediments play essential roles in marine ecosystems and deeper insights into the ecology and biogeochemistry of these largely unexplored organisms can be obtained from‘omics’approaches.Here,we characterized metagenome-assembled-genomes(MAGs)from the surface sediment microbes of the Venice Lagoon(northern Adriatic Sea)in distinct sub-basins exposed to various natural and anthropogenic pressures.MAGs were explored for biodiversity,major marine metabolic processes,anthropogenic activity-related functions,adaptations at the microscale,and biosynthetic gene clusters.Starting from 126 MAGs,a non-redundant dataset of 58 was compiled,the majority of which(35)belonged to(Alpha-and Gamma-)Proteobacteria.Within the broad microbial metabolic repertoire(including C,N,and S metabolisms)the potential to live without oxygen emerged as one of the most important features.Mixotrophy was also found as a successful lifestyle.Cluster analysis showed that different MAGs encoded the same metabolic patterns(e.g.,C fixation,sulfate oxidation)thus suggesting metabolic redundancy.Antibiotic and toxic compounds resistance genes were coupled,a condition that could promote the spreading of these genetic traits.MAGs showed a high biosynthetic potential related to antimicrobial and biotechnological classes and to organism defense and interactions as well as adaptive strategies for micronutrient uptake and cellular detoxification.Our results highlighted that bacteria living in an impacted environment,such as the surface sediments of the Venice Lagoon,may benefit from metabolic plasticity as well as from the synthesis of a wide array of secondary metabolites,promoting ecosystem resilience and stability toward environmental pressures.

Characterization of C_(30) carotenoid and identification of its biosynthetic gene cluster in Methylobacterium extorquens AM1

Methylobacterium species,the representative bacteria distributed in phyllosphere region of plants,often synthesize carotenoids to resist harmful UV radiations.Methylobacterium extorquens is known to produce a carotenoid pigment and recent research revealed that this carotenoid has a C_(30) backbone.However,its exact structure remains unknown.In the present study,the carotenoid produced by M.extorquens AM1 was isolated and its structure was determined as 4-[2-O-11Z-octadecenoyl-β-glucopyranosyl]-4,4′-diapolycopenedioc acid(1),a glycosylated C_(30) carotenoid.Furthermore,the genes related to the C_(30)carotenoid synthesis were investigated.Squalene,the precursor of the C_(30) carotenoid,is synthesized by the co-occurrence of META1p1815,META1p1816 and META1p1817.Further overexpression of the genes related to squalene synthesis improved the titer of carotenoid 1.By using gene deletion and gene complementation experiments,the glycosyltransferase META1p3663 and acyltransferase META1p3664 were firstly confirmed to catalyze the tailoring steps from 4,4′-diapolycopene-4,4′-dioic acid to carotenoid 1.In conclusion,the structure and biosynthetic genes of carotenoid 1 produced by M.extorquens AM1 were firstly characterized in this work,which shed lights on engineering M.extorquens AM1 for producing carotenoid 1 in high yield.

Horizontal transfer and evolution of the biosynthetic gene cluster for benzoxazinoids in plants

Benzoxazinoids are a class of protective and allelopathic plant secondary metabolites that have been identified in multiple grass species and are encoded by the Bx biosynthetic gene cluster(BGC)in maize.Data mining of 41 high-quality grass genomes identified complete Bx clusters(containing genes Bx1–Bx5 and Bx8)in three genera(Zea,Echinochloa,and Dichanthelium)of Panicoideae and partial clusters in Triticeae.The Bx cluster probably originated from gene duplication and chromosomal translocation of native homologs of Bx genes.An ancient Bx cluster that included additional Bx genes(e.g.,Bx6)is presumed to have been present in ancestral Panicoideae.The ancient Bx cluster was putatively gained by the Triticeae ancestor via horizontal transfer(HT)from the ancestral Panicoideae and later separated into multiple segments on different chromosomes.Bx6 appears to have been under less constrained selection compared with the Bx cluster during the evolution of Panicoideae,as evidenced by the fact that it was translocated away from the Bx cluster in Zea mays,moved to other chromosomes in Echinochloa,and even lost in Dichanthelium.Further investigations indicate that purifying selection and polyploidization have shaped the evolutionary trajectory of Bx clusters in the grass family.This study provides the first candidate case of HT of a BGC between plants and sheds new light on the evolution of BGCs.

A chromosome-level genome assembly reveals that a bipartite gene cluster formed via an inverted duplication controls monoterpenoid biosynthesis in Schizonepeta tenuifolia

Biosynthetic gene clusters(BGCs)are regions of a genome where genes involved in a biosynthetic pathway are in proximity.The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear.In this study,we have assembled a chromosome-scale genome of Japanese catnip(Schizonepeta tenuifolia)and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids.This BGC has an unprecedented bipartite structure,with mirrored biosynthetic regions separated by 260 kilobases.This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme,a type of flavin-dependent reductase.In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase.This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp.,indicating convergent evolution of this pathway step.Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S.tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases.The cluster gained its bipartite structure via an inverted duplication.The discovered bipartite BGC for p-menthane biosynthesis in S.tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome,providing an example of the convergent evolution of gene order.This work expands our understanding of plant BGCs with respect to both form and evolution,and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.

Landscape of global urban environmental resistome and its association with local socioeconomic and medical status

Antimicrobial resistance(AMR)poses a critical threat to global health and development,with environmental factors—particularly in urban areas—contributing significantly to the spread of antibiotic resistance genes(ARGs).However,most research to date has been conducted at a local level,leaving significant gaps in our understanding of the global status of antibiotic resistance in urban environments.To address this issue,we thoroughly analyzed a total of 86,213 ARGs detected within 4,728 metagenome samples,which were collected by the Meta SUB International Consortium involving diverse urban environments in 60 cities of 27 countries,utilizing a deep-learning based methodology.Our findings demonstrated the strong geographical specificity of urban environmental resistome,and their correlation with various local socioeconomic and medical conditions.We also identified distinctive evolutionary patterns of ARG-related biosynthetic gene clusters(BGCs)across different countries,and discovered that the urban environment represents a rich source of novel antibiotics.Our study provides a comprehensive overview of the global urban environmental resistome,and fills a significant gap in our knowledge of large-scale urban antibiotic resistome analysis.

Heterologous expression facilitates the discovery and characterization of marine microbial natural products

Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals.Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals.However,discovering compounds with new skeletons from marine microbes remains challenging.In recent decades,multiple approaches have been de-veloped to discover novel marine microbial natural products,among which heterologous expression has proven to be an effective method.Facilitated by large DNA cloning and comparative metabolomic technologies,a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters(BGCs)in heterologous hosts.Heterologous expression is advantageous for character-izing gene functions and elucidating the biosynthetic mechanisms of natural products.This review provides an overview of recent progress in heterologous expression-guided discovery,biosynthetic mechanism elucidation,and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.

Comparative analysis of rapamycin biosynthesis clusters between Actinoplanes sp. N902-109 and Streptomyces hygroscopicus ATCC29253

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology(RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases(PKS) genes: Rap A, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ(encoding a methyltransferase) and rap G in N902-109 were found in ATCC29253, however, an extra rap M gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.

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.

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.

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.

Oryzalexin S biosynthesis:a cross-stitched disappearing pathway

Rice produces many diterpenoid phytoalexins and,reflecting the importance of these natural products in this important cereal crop plant,its genome contains three biosynthetic gene clusters(BGCs)for such metabolism.The chromosome 4 BGC(c4BGC)is largely associated with momilactone production,in part due to the presence of the initiating syn-copalyl diphosphate(CPP)synthase gene(OsCPS4).Oryzalexin S is also derived from syn-CPP.However,the relevant subsequently acting syn-stemarene synthase gene(OsKSL8)is not located in the c4BGC.Production of oryzalexin S further requires hydroxylation at carbons 2 and 19(C2 and C19),presumably catalyzed by cytochrome P450(CYP)monooxygenases.Here it is reported the closely related CYP99A2 and CYP99A3,whose genes are also found in the c4BGC catalyze the necessary C19-hydroxylation,while the closely related CYP71Z21 and CYP71Z22,whose genes are found in the recently reported chromosome 7 BGC(c7BGC),catalyze subsequent hydroxylation at C2α.Thus,oryzalexin S biosynthesis utilizes two distinct BGCs,in a pathway cross-stitched together by OsKSL8.Notably,in contrast to the widely conserved c4BGC,the c7BGC is subspecies(ssp.)specific,being prevalent in ssp.japonica and only rarely found in the other major ssp.indica.Moreover,while the closely related syn-stemodene synthase OsKSL11 was originally considered to be distinct from OsKSL8,it has now been reported to be a ssp.indica derived allele at the same genetic loci.Intriguingly,more detailed analysis indicates that OsKSL8(j)is being replaced by OsKSL11(OsKSL8i),suggesting introgression from ssp.indica to(sub)tropical japonica,with concurrent disappearance of oryzalexin S production.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces hosts for natural product discovery

With the advent of rapid automated in silico identification of biosynthetic gene clusters(BGCs),genomics pre-sents vast opportunities to accelerate natural product(NP)discovery.However,prolific NP producers,Strepto-myces,are exceptionally GC-rich(>80%)and highly repetitive within BGCs.These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing.Here,we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes.Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions.We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb with a median N50 of 8.2 Mb.Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri.Further comprehensive characterization of their biosynthetic,pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase(PKS)BGCs reflected their potential as alternative NP hosts.Thus,the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.

Unlocking plant metabolic diversity: A (pan)- genomic view

Plants produce a remarkable diversity of structurally and functionally diverse natural chemicals that serve as adaptive compounds throughout their life cycles.However,unlocking this metabolic diversity is significantly impeded by the size,complexity,and abundant repetitive elements of typical plant genomes.As genome sequencing becomes routine,we anticipate that links between metabolic diversity and genetic variation will be strengthened.In addition,an ever-increasing number of plant genomes have revealed that biosynthetic gene clusters are not only a hallmark of microbes and fungi;gene clusters for various classes of compounds have also been found in plants,and many are associated with important agronomic traits.We present recent examples of plant metabolic diversification that have been discovered through the exploration and exploitation of various genomic and pan-genomic data.We also draw attention to the fundamental genomic and pan-genomic basis of plant chemodiversity and discuss challenges and future perspectives for investigating metabolic diversity in the coming pan-genomics era.

Coordinated regulation for nature products discovery and overproduction in Streptomyces

Streptomyces is an important treasure trove for natural products discovery.In recent years,many scientists focused on the genetic modification and metabolic regulation of Streptomyces to obtain diverse bioactive compounds with high yields.This review summarized the commonly used regulatory strategies for natural products discovery and overproduction in Streptomyces from three main aspects,including regulator-related strategies,promoter engineering,as well as other strategies employing transposons,signal factors,or feedback regulations.It is expected that the metabolic regulation network of Streptomyces will be elucidated more comprehensively to shed light on natural products research in the future.

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.

Metabolic engineering of Streptomyces to enhance the synthesis of valuable natural products

The mycelial bacterium Streptomyces is a workhorse for producing natural products,serving as a key source of drugs and other valuable chemicals.However,its complicated life cycle,silent biosynthetic gene clusters(BGCs),and poorly characterized metabolic mechanisms limit efficient production of natural products.There-fore,a metabolic engineering strategy,including traditional and emerging tools from different disciplines,was developed to further enhance natural product synthesis by Streptomyces.Here,current trends in systems metabolic engineering,including tools and strategies,are reviewed.Particularly,this review focuses on recent developments in the selection of methods for regulating the Streptomyces life cycle,strategies for the activation of silent gene clusters,and the exploration of regulatory mechanisms governing antibiotic production.Finally,future challenges and prospects are discussed.

Improvement of pristinamycin I(PI)production in Streptomyces pristinaespiralis by metabolic engineering approaches

Pristinamycin,produced by Streptomyces pristinaespiralis,which is a streptogramin-like antibiotic consisting of two chemically unrelated components:pristinamycin I(PI)and pristinamycin II(PII),shows potent activity against many antibiotic-resistant pathogens.However,so far pristinamycin production titers are still quite low,particularly those of PI.In this study,we constructed a PI single component producing strain by deleting the PII biosynthetic genes(snaE1 and snaE2).Then,two metabolic engineering approaches,including deletion of the repressor gene papR3 and chromosomal integration of an extra copy of the PI biosynthetic gene cluster(BGC),were employed to improve PI production.The final engineered strain DPIIDpapR3/PI produced a maximum PI level of 132 mg/L,with an approximately 2.4-fold higher than that of the parental strain S.pristinaespiralis HCCB10218.Considering that the PI biosynthetic genes are clustered in two main regions in the 210 kb“supercluster”containing the PI and PII biosynthetic genes as well as a cryptic polyketide BGC,these two regions were cloned separately and then were successfully assembled into the PI BGC by the transformation-associated recombination(TAR)system.Collectively,the metabolic engineering approaches employed is very efficient for strain improvement in order to enhance PI titer.

Fusaricidins in Paenibacillus polymyxa A21 and their antagonistic activity against Botrytis cinerea on tomato

Four kinds of antifungal compounds from an extract of Paenibacillus polymyxa A21 with molecular masses of 883.56, 897.59, 947.55, and 961.58 Da were characterized as the members of fusaricidin-type of antibiotics according to LC-MS analysis. Fusaricidins isolated from culture filtrate displayed high antagonistic activity against several plant fungal pathogens, especially Botrytis cinerea, the causal agent of gray mold. The fusaricidins biosynthetic gene cluster(BGC) from A21 was cloned by PCR and comparative cluster analysis revealed that gene fus TE, the 3′ boundary of the fusaricidin BGC in strain PKB1, was not present in fusaricidin BGC of A21, indicating that fus TE is not necessary for fusaricidin synthesis. Fusaricidin extract from A21 significantly reduced gray mold disease incidence and severity on tomato. The m RNA levels for three pathogenesis-related proteins(PRs) revealed that treatment of tomato leaves with fusaricidin extract induced the expression of PR genes to different levels, suggesting that one reason for the reduction of gray mold infection by fusaricidin is induction of PR proteins, which lead to increased resistance to pathogens. This is the first report of the application of fusaricidins to control tomato gray mold and the comparative cluster analysis provides important molecular basis for research on fusaricidin biosynthesis.