Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis.

BACKGROUND: The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities toward alternative substrates, particularly epoxidases, is a fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin. RESULTS: A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification. CONCLUSIONS: We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.     

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.     

Deacetylcephalosporin C production in Penicillium chrysogenum by expression of the isopenicillin N epimerization, ring expansion, and acetylation genes

Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics. We have constructed P. chrysogenum strains expressing cefD1, cefD2, cefEF, and cefG genes cloned from Acremonium chrysogenum. Northern analysis revealed that the four genes were expressed in P. chrysogenum. The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth.     

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR. Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and an array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids.     

By-products of the cider production: an alternative source of nutrients to produce bacterial cellulose

In the present work a culture process to produce bacterial cellulose (BC) using by-products of the cider production from the Basque Country was investigated. The apple pomace was mixed with sugar cane (AR/SC medium) and the mixture was found to be a potential carbon source for Gluconacetobacter medellinensis strain ID13488 since higher cellulose production was observed with respect to the commercial Hestrin and Shramm medium (H–S). The culture media were characterized in terms of pH, oxygen and sugars consumption. The expression level of the operon bcs (genes involved in BC biosynthesis) in apple residue containing medium respect to standard H–S medium was determined. It was found that in AR/SC medium the expression levels of bcsA gene, wich is the first gene of the bcs operon, was increased in 1.5-fold respect to the H–S media which correlates with the fact that BC production in AR/SC media is higher than in H–S media. The physico-chemical and mechanical properties, microstructure, crystallinity and water holding capacity of the biosynthesized BC membranes were analyzed and it was found that, in general, the BC obtained from AR/SC medium presented superior properties than that obtained from H–S medium. In this study an economic method for BC production is proposed with suitable properties for many applications.     

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C₁₀–C₁₀ in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.     

Screening of soil bacteria for production of biocleaner

Soil bacteria were studied for the production of biodegradable cleaning agents. Among 86 bacterial strains resistant to liquid paraffin, 58 showed hemolytic activity. These strains were cultured, and the supernatant of culture broths was evaluated for cleaning activity against a dirty porcelain tile. Potent activity was exhibited in 18 strains. The lowest value of surface tension was obtained from Bacillus sp. NKB03 suggesting the presence of a biosurfactant. Aeromonas sp. NKB26c and Bacillus cereus NKB46b exhibited enzymatic cleaning activity. A cleaning efficiency of 82% was achieved when using a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c in synthetic minimal media. The cleaning efficiency using this mixture was higher than that of sodium dodecyl sulfate. These results suggest that a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c has potential for commercial use as a biocleaner.     

Synthesis of hydrophospohoryl derivatives of the antifungal antibiotic pimaricin by the Kabachnik-Fields reaction

Reactions of the polyene macrolide antibiotic pimaricin with aromatic aldehydes and hypophosphorous acid are shown to afford pimaricin hydrophosphoryl derivatives. Physicochemical and biological properties of these compounds are explored. Biological studies indicated that the pimaricin hydrophosphoryl derivatives are low toxic agents possessing a high antifungal activity. The text was submitted by authors in English.     

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.     

Stereochemical determination and complex biosynthetic assembly of etnangien, a highly potent RNA polymerase inhibitor from the myxobacterium Sorangium cellulosum

A potent novel analogue of the natural macrolide antibiotic etnangien, a structurally unique RNA polymerase inhibitor from myxobacteria, is reported. It may be readily obtained from fermentation broths of Sorangium cellulosum and shows high antibiotic activity, comparable to that of etnangien. However, it is much more readily available than the notoriously labile authentic natural product itself. Importantly, it is stable under neutral conditions, allowing for elaborate NMR measurements for assignment of the 12 hydroxyl- and methyl-bearing stereogenic centers. The full absolute and relative stereochemistries of these complex polyketides were determined by a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and synthetic derivatization in combination with an innovative method based on biosynthetic studies of this polyketide which is also presented here. A first look into the solution conformation and 3D structure of these promising macrolide antibiotics is reported. Finally, the complete biosynthetic gene cluster was analyzed in detail, revealing a highly unusual and complex trans-AT type polyketide biosynthesis, which does not follow colinearity rules, most likely performs programmed iteration as well as module skipping, and exhibits HMG-CoA box-directed methylation.     

Hexaene derivatives of nystatin produced as a result of an induced rearrangement within the nysC polyketide synthase gene in S. noursei ATCC 11455

Genetic manipulation of the polyketide synthase (PKS) gene nysC involved in the biosynthesis of the tetraene antifungal antibiotic nystatin yielded a recombinant strain producing hexaene nystatin derivatives. Analysis of one such compound, S48HX, by LC-MS/MS suggested that it comprises a 36-membered macrolactone ring completely decorated by the post-PKS modification enzymes. Further characterization by bioassay has shown that S48HX exhibits antifungal activity. Genetic analysis of the hexaene-producing mutant revealed an in-frame deletion within the nysC gene via recombination between two homologous ketoreductase domain-encoding sequences. Apparently, this event resulted in the elimination of one complete module from NysC PKS, subsequently leading to the production of the nystatin derivative with a contracted macrolactone ring. These results represent the first example of manipulation of a PKS gene for the biosynthesis of a polyene antibiotic.     

利用代谢组学技术研究全氟辛酸的人肝脏毒性机制

采用超高效液相色谱-飞行时间质谱联用系统研究暴露于不同浓度全氟辛酸(PFOA)72 h的正常人L-02肝细胞内代谢谱的变化。将主成分分析法用于数据分析和生物标志物的初步筛选。在正离子和负离子扫描模式下,对照组和暴露组均可得到较好区分并呈现出明显的剂量-效应关系,筛选鉴定了18种与全氟辛酸毒性密切相关的潜在生物标志物,包括肉碱和酰肉碱、核苷及其同源物、氨基酸及其同源物等。在暴露组中,在脂肪酸代谢中起关键作用的肉碱类代谢物的含量变化显著,其中肉碱含量随剂量的升高呈现明显的下降趋势,而酰肉碱则呈现相反的变化趋势,表明全氟辛酸可通过诱导胆固醇等脂类物质代谢相关基因的异常表达从而扰乱胆碱类物质的正常合成和代谢。除了变化最显著的胆碱代谢通路之外,全氟辛酸的肝脏毒性还与三羧酸循环、嘌呤代谢、氨基酸代谢和核酸代谢等多个通路相关。这些结果表明,PFOA在体内经过长时间累积可通过干扰众多的代谢通路从而破坏人体的正常生理机能,造成潜在的健康危害。     

Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway

BACKGROUND: The polyene macrolide antibiotic nystatin produced by Streptomyces noursei ATCC 11455 is an important antifungal agent. The nystatin molecule contains a polyketide moiety represented by a 38-membered macrolactone ring to which the deoxysugar mycosamine is attached. Molecular cloning and characterization of the genes governing the nystatin biosynthesis is of considerable interest because this information can be used for the generation of new antifungal antibiotics. RESULTS: A DNA region of 123,580 base pairs from the S. noursei ATCC 11455 genome was isolated, sequenced and shown by gene disruption to be involved in nystatin biosynthesis. Analysis of the DNA sequence resulted in identification of six genes encoding a modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport and regulatory proteins. One of the PKS-encoding genes, nysC, was found to encode the largest (11,096 amino acids long) modular PKS described to date. Analysis of the deduced gene products allowed us to propose a model for the nystatin biosynthetic pathway in S. noursei. CONCLUSIONS: A complete set of genes responsible for the biosynthesis of the antifungal polyene antibiotic nystatin in S. noursei ATCC 11455 has been cloned and analyzed. This represents the first example of the complete DNA sequence analysis of a polyene antibiotic biosynthetic gene cluster. Manipulation of the genes identified within the cluster may potentially lead to the generation of novel polyketides and yield improvements in the production strains.     

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa

BACKGROUND: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.     

Biosynthesis of deoxyamphotericins and deoxyamphoteronolides by engineered strains of Streptomyces nodosus

Amphotericin B is an antifungal antibiotic produced by Streptomyces nodosus. During biosynthesis of amphotericin, the macrolactone core undergoes three modifications: oxidation of a methyl branch to a carboxyl group, mycosaminylation, and hydroxylation. Gene disruption was undertaken to block two of these modifications. Initial experiments targeted the amphDIII gene, which encodes a GDP-D-mannose 4,6-dehydratase involved in biosynthesis of mycosamine. Analysis of products by mass spectrometry and NMR indicated that the amphDIII mutant produced 8-deoxyamphoteronolides A and B. This suggests that glycosylation with mycosamine normally precedes C-8 hydroxylation and that formation of the exocyclic carboxyl group can occur prior to both these modifications. Inactivation of the amphL cytochrome P450 gene led to production of novel polyenes with masses appropriate for 8-deoxyamphotericins A and B. These compounds retained antifungal activity and may be useful new antibiotics.     

Steady-state detection of cholesterol contained in the plasma membrane of a single cell using lipid bilayer-modified microelectrodes incorporating cholesterol oxidase

Platinum microelectrodes modified with a lipid bilayer membrane incorporating cholesterol oxidase are used for detection of cholesterol contained in the plasma membrane of a single cell. Amperometric responses are consistent with enzymatic catalysis being rate limiting and cholesterol diffusing laterally in the plasma membrane to the electrode contact site. Importantly, electrode response appears to correlate with the cholesterol content of the cell plasma membrane. The electrodes should be useful for characterizing cellular cholesterol tracking pathways involved in pathogenesis of disease.     

Cloning and elucidation of the FR901464 gene cluster revealing a complex acyltransferase-less polyketide synthase using glycerate as starter units

FR901464, an antitumor natural product, represents a new class of potent anticancer small molecules targeting spliceosome and inhibiting both splicing and nuclear retention of pre-mRNA. Herein we describe the biosynthetic gene cluster of FR901464, identified by degenerate primer PCR amplification of a gene encoding the 3-hydroxy-3-methylglutaryl-CoA synthase (HCS) postulated to be involved in the biosynthesis of a β-branched polyketide from Pseudomonas sp. No. 2663. This cluster consists of twenty open reading frames (ORFs) and was localized to 93-kb DNA segment, and its involvement in FR901464 biosynthesis was confirmed by gene inactivation and complementation. FR901464 is biosynthesized by a hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS), HCS, and acyltransferases (AT)-less system. The PKS/NRPS modules feature unusual domain organization including multiple domain redundancy, inactivation, and tandem. Biochemical characterization of a glyceryl transferase and an acyl carrier protein (ACP) in the start module revealed that it incorporates D-1,3-bisphosphoglycerate, which is dephosphorylated and transferred to ACP as the starter unit. Furthermore, an oxidative Baeyer-Villiger reaction followed by chain release was postulated to form a pyran moiety. On the basis of in silico analysis and genetic and biochemical evidances, a biosynthetic pathway for FR901464 was proposed, which sets the stage to further investigate the complex PKS biochemically and engineer the biosynthetic machinery for the production of novel analogues.