Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

Cloning and identification of saprolmycin biosynthetic gene cluster from Streptomyces sp. TK08046

Saprolmycins A–E are anti-Saprolegnia parasitica antibiotics. To identify the gene cluster for saprolmycin biosynthesis in Streptomyces sp. TK08046, polymerase chain reaction using aromatase and cyclase gene-specific primers was performed; the spr gene cluster, which codes for angucycline biosynthesis, was obtained from the strain. The cluster consists of 36 open reading frames, including minimal polyketide synthase, ketoreductase, aromatase, cyclase, oxygenase, and deoxy sugar biosynthetic genes, as defined by homology to the corresponding genes of the urdamycin, Sch-47554, and grincamycin biosynthetic gene clusters in Streptomyces fradiae, Streptomyces sp. SCC-2136, and Streptomyces lusitanus, respectively. To establish the function of the gene cluster, an expression cosmid vector containing all 36 open reading frames was introduced into Streptomyces lividans TK23. The transformant was confirmed to express the biosynthetic genes and produce saprolmycins by liquid chromatography–mass spectrometry analysis of the extract.     

Biosynthesis of new lipopentapeptides by an engineered strain of Streptomyces sp.

Arylomycins are type I signal peptidase inhibitors and have a potential as a new type of antibiotics. They were identified from the broth of Streptomyces sp. HCCB10043. The arylomycin biosynthetic gene cluster in this strain was identical to that in S. roseosporus. Within the gene cluster, aryC, encoding a P450 enzyme, was deduced to be responsible for biaryl bond formation in, the arylomycins. Inactivation of aryC abolished arylomycin production and led to the generation of two novel linear lipopentapeptides lacking the aryl–aryl linkage. These derivatives had lost their antibacterial activities against Staphylococcus epidermidis which is sensitive to arylomycins A2 and A4.     

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.     

Valinomycin Biosynthetic Gene Cluster in Streptomyces: Conservation,Ecology and Evolution

Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values≥95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.     

Biochemical detection of a metallo-β-lactamase in carbapenem resistant strain ofStreptomyces sp. CN229 isolated from soil

Streptomyces sp. CN229 was isolated from Tunisia soil. This strain displayed antimicrobial activity against Gram positive and Gram negative bacteria. In addition it is resistant to most β-lactam antibiotics including imipenem and meropenem (MIC imipenem >70 μg/ml). Metallo-β-lactamase (MβL) production was confirmed by either imipenem MIC decrease in the presence of ethylene diamine tetraactic acid (EDTA) or the inhibition zone enhancement around EDTA-impregnated imipenem, or meropenem discs. Isolectric focusing analysis demonstrated the production of β-lactamase with pI of 5.8 that is inhibited by EDTA.Streptomyces sp. CN229 was screened for the imipenem resistance genes,bla _VIM andbla _IMP previously identified inPseudomonas aeruginosa. The presence of these genes was not confirmed by specific PCR analysis. We concluded that carbapenem resistance inStreptomyces sp. CN229 strain is mainly due to production of a novel carbapenemase. Our data show for the first time that MβL is produced byStreptomyces sp. MβL-mediated imipenem and meropenem resistance inStreptomyces is a cause for concern in the study of resistance evolution and antibiotic cluster biosynthetic genes.     

Identification and characterization of the biosynthetic gene cluster of divergolides from Streptomyces sp. W112

Divergolides are a group of structurally unprecedented ansamacrolactam antibiotics with antibacterial and antitumor activities. A biosynthetic gene cluster predicted to encode the biosynthesis of divergolides was cloned and sequenced from endophytic Streptomyces sp. W112. The gene cluster of divergolides (div) spans a DNA region of 61-kb and consists of 20 open reading frames (ORFs) that encode polyketide synthases (PKSs), enzymes for the synthesis of AHBA and PKS extender units, and post-PKS modifications, proposed regulators, and putative transporters. Disruption of the AHBA synthase gene (divK) completely abolished the production of divergolides proved its involvement in the biosynthesis of divergolides. Bioinformatics analysis suggested that the regulatory gene div8 in div gene cluster might encode a positive regulator for the biosynthesis of divergolides. Constitutive overexpression of div8 improved the production of divergolides E, implying that div gene cluster maybe responsible for the biosynthesis of divergolides. These findings set the stage for fully investigating the biosynthesis of divergolides and rational engineering of new divergolide analogs by genetic modifications, and pave the way to further improve the production of divergolides.     

Strain-Level Diversity of Secondary Metabolism in Streptomyces albus

Streptomyces spp. are robust producers of medicinally-, industrially- and agriculturally-important small molecules. Increased resistance to antibacterial agents and the lack of new antibiotics in the pipeline have led to a renaissance in natural product discovery. This endeavor has benefited from inexpensive high quality DNA sequencing technology, which has generated more than 140 genome sequences for taxonomic type strains and environmental Streptomyces spp. isolates. Many of the sequenced streptomycetes belong to the same species. For instance, Streptomyces albus has been isolated from diverse environmental niches and seven strains have been sequenced, consequently this species has been sequenced more than any other streptomycete, allowing valuable analyses of strain-level diversity in secondary metabolism. Bioinformatics analyses identified a total of 48 unique biosynthetic gene clusters harboured by Streptomyces albus strains. Eighteen of these gene clusters specify the core secondary metabolome of the species. Fourteen of the gene clusters are contained by one or more strain and are considered auxiliary, while 16 of the gene clusters encode the production of putative strain-specific secondary metabolites. Analysis of Streptomyces albus strains suggests that each strain of a Streptomyces species likely harbours at least one strain-specific biosynthetic gene cluster. Importantly, this implies that deep sequencing of a species will not exhaust gene cluster diversity and will continue to yield novelty.     

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.     

Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn–Arg–hOrn–hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.     

Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128

Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.     

Identification and catalytic characterization of a nonribosomal peptide synthetase-like (NRPS-like) enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35

Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.     

Mutational biosynthesis of tacrolimus analogues by fkbO deletion mutant of Streptomyces sp. KCTC 11604BP

Tacrolimus (FK506) is an important macrocyclic polyketide showing antifungal and immunosuppressive activities, as well as neuroregenerative properties. Tacrolimus biosynthetic machinery should incorporate the shikimate-derived 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) as a biosynthetic starter unit into the biosynthetic line of tacrolimus. fkbO is a homologue of rapK encoding chorismatase related to the biosynthesis of starter unit DHCHC from chorismate in the rapamycin biosynthetic gene cluster. FkbO and RapK are good targets for mutational biosynthesis to produce novel analogues of tacrolimus, ascomycin, and rapamycin, which could be important drugs for clinical application in the treatment of cancer and immune and neurodegenerative diseases. To make novel tacrolimus analogues, we prepared an fkbO in-frame deletion mutant, Streptomyces sp. GT110507, from a tacrolimus high producer. We scrutinized the cyclic carboxylic acids that were possibly incorporated instead of DHCHC by precursor-directed mutasynthesis using Streptomyces sp. GT110507 to lead tacrolimus analogues. Among them, trans-4-hydroxycyclohexanecarboxylic acid and 3-hydroxybenzoic acid were successfully incorporated into the tacrolimus backbone, which led to the production of 31-desmethoxytacrolimus and TC-225, respectively. Especially, adding of trans-4-hydroxycyclohexanecarboxylic acid produced a high amount (55 mg/L) of 31-desmethoxytacrolimus. Interestingly, in the rapK mutant, it has been reported that the incorporation of cyclohexanecarboxylic acid (CHC) led to 39-desmethoxy rapamycin. However, in Streptomyces sp. GT110507, CHC is not successfully incorporated. This discrepancy should reflect the differences in the DHCHC biosynthesis mechanism and/or substrate specificity of starter unit loading machineries (FkbP and RapP) of tacrolimus and rapamycin.     

Nature’s combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in <Emphasis Type="Italic">Streptomyces</Emphasis>

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.     

Identification and characterization of <Emphasis Type="Italic">gerPI</Emphasis> and <Emphasis Type="Italic">gerPII</Emphasis> involved in epoxidation and hydroxylation of dihydrochalcolactone in <Emphasis Type="Italic">Streptomyces</Emphasis> species KCTC 0041BP

The macrolide antibiotics are biosynthesized by initial assembly of a macrolactone ring, followed by a series of post-polyketide (PKS) modifications. In general, the additional hydroxyl or epoxy groups are installed by cytochrome P450 enzymes, improving the bioactivity profile through structural diversification of natural products. The biosynthetic gene cluster for the 16-membered macrolide antibiotic dihydrochalcomycin (DHC) has been cloned from Streptomyces sp. KCTC 0041BP. Three cytochrome P450 genes are found in the DHC biosynthetic gene (ger) cluster. Two P450 enzymes were characterized from this cluster. Disruption of gerPI accumulated predominantly 12,13-de-epoxydihydrochalcomycin while disruption of gerPII accumulated 8-dehydroxy-12,13-de-epoxydihydrochalcomycin; DHC production was abolished in both cases. The results suggest that GerPII P450 catalyzes hydroxylation at the C₈ position followed by an epoxidation reaction catalyzed by GerPI P450 at the C₁₂–C₁₃ position.     

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42

Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent.

Methodology/Principal Findings

The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens.

Conclusions/Significance

The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.