Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme.

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Genome Mining and Metabolic Profiling Reveal Cytotoxic Cyclodipeptides in Streptomyces hygrospinosus var. Beijingensis.

Two new cyclodipeptide (CDP) derivatives (1-2) and another seven known cyclodipeptides (3-9) were isolated from Streptomyces 26D9-414 by the genome mining approach combined with genetic dereplication and the "one strain many compounds" (OSMAC) strategy. The structures of the new CDPs were established on the basis of 1D- and 2D-NMR and comparative electronic circular dichroism (ECD) spectra analysis. The biosynthetic gene clusters (BGCs) for these CDPs were identified through antiSMASH analysis. The relevance between this cdp cluster and the identified nine CDPs was established by genetic interruption manipulation. The newly discovered natural compound 2 displayed comparable cytotoxicity against MDA-MB-231 and SW480 with that of cisplatin, a widely used chemotherapeutic agent for the treatment of various cancers.

Chemistry and biosynthesis of bacterial polycyclic xanthone natural products.

Covering: up to the end of 2021Bacterial polycyclic xanthone natural products (BPXNPs) are a growing family of natural xanthones featuring a pentangular architecture with various modifications to the tricyclic xanthone chromophore. Their structural diversities and various activities have fueled biosynthetic and chemical synthetic studies. Moreover, their more potent activities than the clinically used drugs make them potential candidates for the treatment of diseases. Future unraveling of structure activity relationships (SARs) will provide new options for the (bio)-synthesis of drug analogues with higher activities. This review summarizes the isolation, structural elucidation and biological activities and more importantly, the recent strategies for the microbial biosynthesis and chemical synthesis of BPXNPs. Regarding their biosynthesis, we discuss the recent progress in enzymes that synthesize tricyclic xanthone, the protein candidates for structural moieties (methylene dioxygen bridge and nitrogen heterocycle), tailoring enzymes for methylation and halogenation. The chemical synthesis part summarizes the recent methodology for the division synthesis and coupling construction of achiral molecular skeletons. Ultimately, perspectives on the biosynthetic study of BPXNPs are discussed.

Acyltransferase AniI, a Tailoring Enzyme with Broad Substrate Tolerance for High-Level Production of Anisomycin.

Anisomycin (compound 1), a pyrrolidine antibiotic, exhibits diverse biological and pharmacologic activities. The biosynthetic gene cluster of compound 1 has been identified previously, and the multistep assembly of the core benzylpyrrolidine scaffold was characterized. However, enzymatic modifications, such as acylation, involved in compound 1 biosynthesis are unknown. In this study, the genetic manipulation of aniI proved that it encoded an indispensable acetyltransferase for compound 1 biosynthesis. Bioinformatics analysis suggested AniI as a member of maltose (MAT) and galactoside O-acetyltransferases (GAT) with C-terminal left-handed parallel beta-helix (LbH) subdomain, which were referred to as LbH-MAT-GAT sugar O-acetyltransferases. However, the biochemical assay identified that its target site was the hydroxyl group of the pyrrolidine ring. AniI was found to be tolerant of acyl donors with different chain lengths for the biosynthesis of compound 1 and derivatives 12 and 13 with butyryl and isovaleryl groups, respectively. Meanwhile, it showed comparable activity toward biosynthetic intermediates and synthesized analogues, suggesting promiscuity to the pyrrolidine ring structure of compound 1. These data may inspire new viable synthetic routes for the construction of more complex pyrrolidine ring scaffolds in compound 1. Finally, the overexpression of aniI under the control of strong promoters contributed to the higher productivities of compound 1 and its analogues. These findings reported here not only improve the understanding of anisomycin biosynthesis but also expand the substrate scope of O-acetyltransferase working on the pyrrolidine ring and pave the way for future metabolic engineering construction of high-yield strains. IMPORTANCE Acylation is an important tailoring reaction during natural product biosynthesis. Acylation could increase the structural diversity and affect the chemical stability, volatility, biological activity, and even the cellular localization of specialized compounds. Many acetyltransferases have been reported in natural product biosynthesis. The typical example of the LbH-MAT-GAT sugar O-acetyltransferase subfamily was reported to catalyze the coenzyme A (CoA)-dependent acetylation of the 6-hydroxyl group of sugars. However, no protein of this family has been characterized to acetylate a nonsugar secondary metabolic product. Here, AniI was found to catalyze the acylation of the hydroxyl group of the pyrrolidine ring and be tolerant of diverse acyl donors and acceptors, which made the biosynthesis more efficient and exclusive for biosynthesis of compound 1 and its derivatives. Moreover, the overexpression of aniI serves as a successful example of genetic manipulation of a modification gene for the high production of final products and might set the stage for future metabolic engineering.

Development of Methods Derived from Iodine-Induced Specific Cleavage for Identification and Quantitation of DNA Phosphorothioate Modifications.

DNA phosphorothioate (PT) modification is a novel modification that occurs on the DNA backbone, which refers to a non-bridging phosphate oxygen replaced by sulfur. This exclusive DNA modification widely distributes in bacteria but has not been found in eukaryotes to date. PT modification renders DNA nuclease tolerance and serves as a constitute element of bacterial restriction-modification (R-M) defensive system and more biological functions are awaiting exploration. Identification and quantification of the bacterial PT modifications are thus critical to better understanding their biological functions. This work describes three detailed methods derived from iodine-induced specific cleavage-an iodine-induced cleavage assay (ICA), a deep sequencing of iodine-induced cleavage at PT site (ICDS) and an iodine-induced cleavage PT sequencing (PT-IC-Seq)-for the investigation of PT modifications. Using these approaches, we have identified the presence of PT modifications and quantized the frequency of PT modifications in bacteria. These characterizations contributed to the high-resolution genomic mapping of PT modifications, in which the distribution of PT modification sites on the genome was marked accurately and the frequency of the specific modified sites was reliably obtained. Here, we provide time-saving and less labor-consuming methods for both of qualitative and quantitative analysis of genomic PT modifications. The application of these methodologies will offer great potential for better understanding the biology of the PT modifications and open the door to future further systematical study.

DNA Phosphorothioate Modifications Are Widely Distributed in the Human Microbiome.

The DNA phosphorothioate (PT) modification existing in many prokaryotes, including bacterial pathogens and commensals, confers multiple characteristics, including restricting gene transfer, influencing the global transcriptional response, and reducing fitness during exposure to chemical mediators of inflammation. While PT-containing bacteria have been investigated in a variety of environments, they have not been studied in the human microbiome. Here, we investigated the distribution of PT-harboring strains and verified their existence in the human microbiome. We found over 2000 PT gene-containing strains distributed in different body sites, especially in the gastrointestinal tract. PT-modifying genes are preferentially distributed within several genera, including Pseudomonas, Clostridioides, and Escherichia, with phylogenic diversities. We also assessed the PT modification patterns and found six new PT-linked dinucleotides (CpsG, CpsT, ApsG, TpsG, GpsC, ApsT) in human fecal DNA. To further investigate the PT in the human gut microbiome, we analyzed the abundance of PT-modifying genes and quantified the PT-linked dinucleotides in the fecal DNA. These results confirmed that human microbiome is a rich reservoir for PT-containing microbes and contains a wide variety of PT modification patterns.

Metabolic engineering of a methyltransferase for production of drug precursors demecycline and demeclocycline in Streptomyces aureofaciens.

Demecycline (DMTC) and demeclocycline (DMCTC) are C6-demethylated derivatives of tetracycline (TC) and chlortetracycline (CTC), respectively. They are precursors of minocycline and tigecycline, which showed remarkable bioactivity against TC-resistant bacteria and have been used clinically for decades. In order to biosynthesize drug precursors DMTC and DMCTC, the function of a possible C-methyltransferase encoding gene ctcK was studied systematically in the CTC high-yielding industrial strain Streptomyces aureofaciens F3. The ΔctcK mutant accumulated two new products, which were turned out to be DMTC and DMCTC. Meanwhile, time-course analysis of the fermentation products detected the epimers of DMTC and DMCTC transformed spontaneously. Finally, an engineering strain with higher productivity of DMCTC was constructed by deleting ctcK and overexpressing ctcP of three extra copies simultaneously. Construction of these two engineering strains not only served as a successful example of synthesizing required products through metabolic engineering, but also provided original strains for following elaborate engineering to synthesize more effective tetracycline derivatives.

Flavin Adenine Dinucleotide-Dependent Halogenase XanH and Engineering of Multifunctional Fusion Halogenases.

Xantholipin (compound 1), a polycyclic xanthone antibiotic, exhibited strong antibacterial activities and showed potent cytotoxicity. The biosynthetic gene cluster of compound 1 has been identified in our previous work, and the construction of xanthone nucleus has been well demonstrated. However, limited information of the halogenation involved in compound 1 biosynthesis is available. In this study, based on the genetic manipulation and biochemical assay, we characterized XanH as an indispensable flavin adenine dinucleotide (FAD)-dependent halogenase (FDH) for the biosynthesis of compound 1. XanH was found to be a bifunctional protein capable of flavin reduction and chlorination and exclusively used the NADH. However, the reduced flavin could not be fully and effectively utilized, and the presence of an extra flavin reductase (FDR) and chemical-reducing agent could promote the halogenation. XanH accepted its natural free-standing substrate with angular fused polycyclic aromatic systems. Meanwhile, it exhibited moderate halogenation activity and possessed high substrate specificity. The requirement of extra FDR for higher halogenation activity is tedious for future engineering. To facilitate efforts in engineering XanH derivative proteins, we constructed the self-sufficient FDR-XanH fusion proteins. The fusion protein E1 with comparable activities to that of XanH could be used as a good alternative for future protein engineering. Taken together, these findings reported here not only improve the understanding of polycyclic xanthones biosynthesis but also expand the substrate scope of FDH and pave the way for future engineering of biocatalysts for new active substance synthesis.IMPORTANCE Halogenation is important in medicinal chemistry and plays an essential role in the biosynthesis of active secondary metabolites. Halogenases have evolved to catalyze reactions with high efficiency and selectivity, and engineering efforts have been made to engage the selective reactivity in natural product biosynthesis. The enzymatic halogenations are an environmentally friendly approach with high regio- and stereoselectivity, which make it a potential complement to organic synthesis. FDHs constitute one of the most extensively elucidated class of halogenases; however, the inventory awaits to be expanded for biotechnology applications and for the generation of halogenated natural product analogues. In this study, XanH was found to reduce flavin and halogenated the freely diffusing natural substrate with an angular fused hexacyclic scaffold, findings which were different from those for the exclusively studied FDHs. Moreover, the FDR-XanH fusion protein E1 with comparable reactivity to that of XanH serves as a successful example of genetic fusions and sets an important stage for future protein engineering.

Three Recently Diverging Duplicated Methyltransferases Exhibit Substrate-Dependent Regioselectivity Essential for Xantholipin Biosynthesis.

Polycyclic xanthones are characterized by highly oxygenated, angular hexacyclic frameworks and exhibit diverse biological activities. Although many of them have been isolated and chemically synthesized, the detailed biosynthetic machinery awaits discovery. Recently, xanthone construction in the xantholipin (1) pathway was shown to involve cryptic demethoxylation. This suggested a rationale for the existence of three O-methyltransferase (OMT) genes in the gene cluster, although there are only two O-methyl groups in the structure of 1. Here, in vivo and in vitro analysis have been used to show that the three paralogous OMTs, XanM1-M3, introduce individual methyl groups at specific points in the biosynthetic pathway. Each OMT can to some extent take over the role of the other OMTs, although they exhibit highly substrate-dependent regiospecificity. In addition, phylogenetic analysis suggests their evolution from a common ancestor. Four putative ancestral proteins were constructed, and one of them performed all the functions of XanM1-M3, while the others possessed more limited catalytic functions. The results suggest that a promiscuous common ancestor may have been able to catalyze all three reactions prior to gene duplication and functional divergence. The characterization of XanM1-M3 expands the enzyme inventory for polycyclic xanthone biosynthesis and suggests novel directed evolution approaches to diversifying natural product pathways.

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage.

DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3'-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3'-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

CtcS, a MarR family regulator, regulates chlortetracycline biosynthesis.

BACKGROUND: Chlortetracycline (CTC) is one of the commercially important tetracyclines (TCs) family product and is mainly produced by Streptomyces. CTC is still in a great demand due to its broad-spectrum activity against pathogens. Engineering transcriptional control allows the cell to allocate its valuable resources towards protein production and provides an important method for the build-up of desired metabolites. Despite extensive efforts concerning transcriptional regulation for increasing the productivities of TCs, the regulatory mechanisms of the CTC biosynthesis remain poorly understood. RESULTS: In this study, the possible regulatory function of CtcS, a potential member of MarR (multiple antibiotic resistance regulator) family of transcriptional regulators in S. aureofaciens F3, was demonstrated. Knockdown of ctcS altered the transcription of several biosynthesis-related genes and reduced the production of tetracycline (TC) and CTC, without obvious effect on morphological differentiation and cell growth. Especially, CtcS directly repressed the transcription of the adjacent divergent gene ctcR (which encodes a putative TC resistance efflux protein). A CtcS-binding site was identified within the promoter region of ctcR by DNase I footprinting and an inverted repeat (5'-CTTGTC-3') composed of two 6-nt half sites in the protected region was found. Moreover, both CTC and TC could attenuate the binding activity of CtcS with target DNA. CONCLUSION: ctcS regulated the production of TC and CTC in S. aureofaciens F3 and the overexpression of it could be used as a simple approach for the construction of engineering strain with higher productivity. Meanwhile, CtcS was characterized as a TC- and CTC-responsive MarR family regulator. This study provides a previously unrecognized function of CtcS and will benefit the research on the regulatory machinery of the MarR family regulators.

Novel Iodine-induced Cleavage Real-time PCR Assay for Accurate Quantification of Phosphorothioate Modified Sites in Bacterial DNA.

DNA Phosphorothioate (PT), replacing a non-bridging phosphate oxygen atom with a sulfur atom, is one kind of common DNA modification in bacteria. Whole genome scale description of the location and frequency of PT modification is the key to understand its biological function. Herein we developed a novel method, named with iodine-induced cleavage quantitative real-time PCR (IC-qPCR), to evaluate the frequency of PT modification at a given site in bacterial DNA. The efficiency, dynamic range, sensitivity, reproducibility and accuracy of IC-qPCR were well tested and verified employing an E. coli B7A strain as example. The amplification efficiency of IC-qPCR assay ranged from 91% to 99% with a high correlation coefficient ≥0.99. The limit of quantification was determined as low as 10 copies per reaction for the 607710 and 1818096 sites, and 5 copies for the 302695 and 4120753 sites. Based on the developed IC-qPCR method, the modification frequency of four PTs in E. coli B7A was determined with high accuracy, and the results showed that the PT modification was partial and that the modification frequency varied among investigated PT sites. All these results showed that IC-qPCR was suitable for evaluating the PT modification, which would be helpful to further understand the biological function of PT modification.

Quantitative mapping of DNA phosphorothioatome reveals phosphorothioate heterogeneity of low modification frequency.

Phosphorothioate (PT) modifications of the DNA backbone, widespread in prokaryotes, are first identified in bacterial enteropathogens Escherichia coli B7A more than a decade ago. However, methods for high resolution mapping of PT modification level are still lacking. Here, we developed the PT-IC-seq technique, based on iodine-induced selective cleavage at PT sites and high-throughput next generation sequencing, as a mean to quantitatively characterizing the genomic landscape of PT modifications. Using PT-IC-seq we foud that most PT sites are partially modified at a lower PT frequency (< 5%) in E. coli B7A and Salmonella enterica serovar Cerro 87, and both show a heterogeneity pattern of PT modification similar to those of the typical methylation modification. Combining the iodine-induced cleavage and absolute quantification by droplet digital PCR, we developed the PT-IC-ddPCR technique to further measure the PT modification level. Consistent with the PT-IC-seq measurements, PT-IC-ddPCR analysis confirmed the lower PT frequency in E. coli B7A. Our study has demonstrated the heterogeneity of PT modification in the bacterial population and we also established general tools for rigorous mapping and characterization of PT modification events at whole genome level. We describe to our knowledge the first genome-wide quantitative characterization of PT landscape and provides appropriate strategies for further functional studies of PT modification.

A LuxR family transcriptional regulator AniF promotes the production of anisomycin and its derivatives in Streptomyces hygrospinosus var. beijingensis.

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi. It is one of the important effective components in Agricultural Antibiotic120, which has been widely used as naturally-originated agents for treatment of crop decay in China. The chemical synthesis of anisomycin has recently been reported, but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts. The biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis has been identified in our previous work, while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S. hygrospinosus var. beijingensis. In this study here, we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis. The genetic manipulations of aniF and the real-time quantitative PCR (RT-qPCR) revealed that it positively regulated the transcription of the anisomycin BGC. Moreover, the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives. Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis. Especially, one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence (5'-GGGC-3') composed of two 4-nt half sites in the protected region was found. Taken together, our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives.

Characterization of the positive SARP family regulator PieR for improving piericidin A1 production in Streptomyces piomogeues var. Hangzhouwanensis.

Piericidin A1, a member of ɑ-pyridone antibiotic, exhibits various biological activities such as antimicrobial, antifungal, and antitumor properties and possesses potent respiration-inhibitory activity against insects due to its competitive binding capacity to mitochondrial complex I. The biosynthetic pathway of piericidin A1 has been reported in Streptomyces piomogeues var. Hangzhouwanensis, while the regulatory mechanism remains poorly understood. In this study, a Streptomyces antibiotic regulatory protein (SARP) family transcriptional regulator PieR was characterized. Genetic disruption and complementation manipulations revealed that PieR positively regulated the production of piericidin A1. Moreover, the overexpression of pieR contributed to the improvement of piericidin A1 productivity. The real-time quantitative PCR (RT-qPCR) was carried out and the data showed that pieR stimulated the transcription of all the biosynthesis-related genes for piericidin A1. In order to explore the regulatory mechanism, electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments have been conducted. A protected region covering 50 nucleotides within the upstream region of pieR was identified and two 5-nt direct repeat sequences (5'-CCGGA-3') in the protected region were found. These findings, taken together, set stage for transcriptional control engineering in the view of optimizing piericidin A1 production and thus provide a viable potent route for the construction of strains with high productivity.

Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability.

Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h-1 in unstressed Escherichia coli and Salmonella enterica. Whereas PT levels were unaffected by exposure to hydrogen peroxide (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8-10% h-1 after HOCl treatment and was unchanged by H2O2, consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.

Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps.

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.

[Genetic manipulation system and genomic library of Streptomyces luteosporeus NRRL 2401].

OBJECTIVE: To clone the biosynthetic gene clusters for secondary metabolites, we developed the genetic modification system and constructed a genomic library of Streptomyces luteosporeus NRRL 2401. METHODS: The genetic modification system was developed by using conjugal transfer vectors pSET152, pPM927 and pJTU1278 which were transferred from Escherichia coli ET12567/pUZ8002 to S. luteosporeus. The genomic library of S. luteosporeus NRRL 2401 was constructed by the fosmid vector pCClFOS, with E. coli EP1300 -T1 as the host strain. A PCR-based method was then developed for screening the biosynthetic gene clusters of secondary metabolites in the constructed genomic library. RESULTS: Vectors pSET152, pPM927 and pJTU1278 were successfully transferred into S. luteosporeus for genetic modification, with pSET152 presenting the highest transformation efficiency. The constructed genomic library of S. luteosporeus NRRL 2401 contained 2880 clones with an average -35 kb inserted DNA fragment in each clone, indicating the 99.99% coverage of the genome in the library. In this genomic library, we detected 9 clones containing possible indolmycin biosynthesis genes by the PCR-based screening method. CONCLUSION: A stable, efficient genetic modification system and high-quality genomic library could be used for discovery of the biosynthetic gene clusters for secondary metabolites in S. luteosporeus NRRL 2401.

A Multifunctional Monooxygenase XanO4 Catalyzes Xanthone Formation in Xantholipin Biosynthesis via a Cryptic Demethoxylation.

Xantholipin and several related polycyclic xanthone antibiotics feature a unique xanthone ring nucleus within a highly oxygenated, angular, fused hexacyclic system. In this study, we demonstrated that a flavin-dependent monooxygenase (FMO) XanO4 catalyzes the oxidative transformation of an anthraquinone to a xanthone system during the biosynthesis of xantholipin. In vitro isotopic labeling experiments showed that the reaction involves sequential insertion of two oxygen atoms, accompanied by an unexpected cryptic demethoxylation reaction. Moreover, characterizations of homologous FMOs of XanO4 suggested the generality of the XanO4-like-mediated reaction for the assembly of a xanthone ring in the biosynthesis of polycyclic xanthone antibiotics. These findings not only expand the repertoire of FMO activities but also reveal a novel mechanism for xanthone ring formation.

[Function of Streptomyces antibiotic regulatory proteins family transcriptional regulator ctcB in the biosynthetic cluster of chlortetracycline].

Objective: In order to understand the regulatory mechanisms of chlortetracycline biosynthesis in an industrial strain, function of an Streptomyces antibiotic regulatory proteins (SARP) family transcriptional regulator ctcB in the biosynthetic gene cluster of chlortetracycline was studied. Methods: By double crossover recombination, we constructed Streptomyces aureofaciens F3 with disrupted SARP family transcriptional regulator ctcB gene. The amplicons of RT-PCR were designed to cover the adjacent genes for verification of the operons in chlortetracycline biosynthetic cluster. Transcriptional level was analyzed in the chlortetracycline biosynthetic gene cluster in the wild type strain and the ctcB gene disrupted mutant LJIA02 by quantitative real-time RT-PCR. Results: The disruption mutant LJIA02 abolished tetracycline and chlortetracycline production. In RT-PCR six operons were confirmed in chlortetracycline biosynthetic cluster. Quantitative real-time RT-PCR indicated that ctcB directly activated five promoters from ctcG-D, ctcH-K, ctcN-P, ctcW-T and ctcQ. Conclusion: CtcB is an essential activator as an SARP family transcriptional regulator in the chlortetracycline biosynthetic gene cluster.