Discovery of New Antimicrobial Metabolites in the Coculture of Medicinal Mushrooms.

Bioactivity screening revealed that the antifungal activities of EtOAc extracts from coculture broths of Trametes versicolor SY630 with either Vanderbylia robiniophila SY341 or Ganoderma gibbosum SY1001 were significantly improved compared to that of monocultures. Activity-guided isolation led to the discovery of five aromatic compounds (1-5) from the coculture broth of T. versicolor SY630 and V. robiniophila SY341 and two sphingolipids (6 and 7) from the coculture broth of T. versicolor SY630 and G. gibbosum SY1001. Tramevandins A-C (1-3) and 17-ene-1-deoxyPS (6) are new compounds, while 1-deoxyPS (7) is a new natural product. Notably, compound 2 represents a novel scaffold, wherein the highly modified p-terphenyl bears a benzyl substituent. The absolute configurations of those new compounds were elucidated by X-ray diffraction, ECD calculations, and analysis of physicochemical constants. Compounds 1, 2, and 5-7 exhibited different degrees of antimicrobial activity, and the antifungal activities of compounds 6 and 7 against Candida albicans and Cryptococcus neoformans are comparable to those of fluconazole, nystatin, and sphingosine, respectively. Transcriptome analysis, propidium iodide staining, ergosterol quantification, and feeding assays showed that the isolated sphingolipids can extensively downregulate the late biosynthetic pathway of ergosterol in C. albicans, representing a promising mechanism to combat antibiotic-resistant fungi.

Characterization of C30 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 C30 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-ß-glucopyranosyl]-4,4'-diapolycopenedioc acid (1), a glycosylated C30 carotenoid. Furthermore, the genes related to the C30 carotenoid synthesis were investigated. Squalene, the precursor of the C30 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.

Discovery of Novel Terpenoids from the Basidiomycete Pleurotus ostreatus through Genome Mining and Coculture Optimization.

In our previous work, postredienes A-C, three unusual linear sesterterpenes with high antifungal activities, were isolated from Pleurotus ostreatus SY10 when cocultured with Trametes robiniophila SY636. However, their titers were low, and exploration of newly biosynthesized trace analogues is required. Herein, genome mining analysis predicted that 17 gene clusters are involved in terpenoid biosynthesis in P. ostreatus. Thus, coculture conditions for strains SY10 and SY636 were optimized using a single-factor test and Box-Behnken design. As a result, the titers of postredienes A-C were increased by over 2.5-fold, reaching 1.28 to 8.40 mg/L. Moreover, five new terpenoids, named postredienes D-H (1-5), were successfully isolated. Compound 1 exhibited activities against the human pathogenic fungi Candida albicans and Cryptococcus neoformans comparable to those of amphotericin B. Compound 2 represents a novel sesterterpene with a five-membered ring at C-7. The absolute configurations of 1-5 were elucidated by making the methoxyphenylacetic acid esters and acetonide derivatives, combined with ECD and NMR calculation. Two potential gene clusters and relevant biosynthetic pathways for 1-5 were subsequently proposed based on real-time reverse transcription-quantitative PCR (RT-qPCR) analysis. The current study provides new insights into the research of terpenoid biosynthesis genes in P. ostreatus and other basidiomycetes.

Biosynthetic strategies for tetramic acid formation.

Covering: up to the end of 2020Natural products bearing tetramic acid units as part of complex molecular architectures exhibit a broad range of potent biological activities. These compounds thus attract significant interest from both the biosynthetic and synthetic communities. Biosynthetically, most of the tetramic acids are derived from hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machineries. To date, over 30 biosynthetic gene clusters (BGCs) involved in tetramate formation have been identified, from which different biosynthetic strategies evolved in Nature to assemble this intriguing structural unit were characterized. In this Highlight we focus on the biosynthetic concepts of tetramic acid formation and discuss the molecular mechanism towards selected representatives in detail, providing a systematic overview for the development of strategies for targeted tetramate genome mining and future applications of tetramate-forming biocatalysts for chemo-enzymatic synthesis.

Metabolomic analysis improves bioconversion of methanol to isobutanol in Methylorubrum extorquens AM1.

BACKGROUND: Methylorubrum extorquens AM1 can be engineered to convert methanol to value-added chemicals. Most of these chemicals derive from acetyl-CoA involved in the serine cycle. However, recent studies on methylotrophic metabolism have suggested that C3 pyruvate is a good potential precursor for broadening the types of synthesized products. METHODS AND RESULTS: In the present study, we found that isobutanol was a model chemical that could be generated from pyruvate through a 2-keto acid pathway. Initially, the engineered M. extorquens AM1 could only produce a trace amount of isobutanol at 0.62 mgL-1 after introducing the heterologous 2-ketoisovalerate decarboxylase and alcohol dehydrogenase. Furthermore, the metabolomic analysis revealed that insufficient carbon fluxes through 2-ketoisovalerate and pyruvate were the key limitation steps for efficient biosynthesis of isobutanol. Based on this analysis, the titer of isobutanol was improved by over 20-fold after overexpressing alsS gene encoding acetolactate synthase and deleting ldhA gene for lactate dehydrogenase. Moreover, substituting the cell chassis with the isobutanol-tolerant strain isolated from adaptive evolution of M. extorquens AM1 further increased the production of isobutanol by 1.7-fold, resulting in the final titer of 19 mgL-1 in flask cultivation. CONCLUSION: Our current findings provided promising insights into engineering methylotrophic cell factories capable of converting methanol to isobutanol or value-added chemicals using pyruvate as the precursor.

Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens.

Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO2 as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.

Complete Genome of Nocamycin-Producing Strain Saccharothrix syringae NRRL B-16468 Reveals the Biosynthetic Potential for Secondary Metabolites.

The bacterium Saccharothrix syringae NRRL B-16468 is the producer of nocamycin I and nocamycin II which feature tetramic acid and bicyclic ketal groups. In this study, we presented the complete genome of S. syringae NRRL B-16468 obtained from ARS Culture Collection. It contains a circular chromosome of 10,929,570 bp with an average GC content of 73.49%, 9316 genes, 12 rRNAs and 54 tRNAs. Bioinformatics analyses of the genome has demonstrated that it harbors 55 putative biosynthetic gene clusters (BGCs) involved in synthesizing diverse secondary metabolites. The backbones of the natural products synthesized by these BGCs encoding for type I polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and hybrid type I PKS-NRPS were analyzed, furthermore, the natural products synthesized by these BGCs with > 40% similarity to known BGCs were described in detail. The complete genome of S. syringae reveals its capacity in producing diverse bioactive natural products, and it will also shed lights on mining novel secondary metabolites from S. syringae through rational strategies.

Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis.

The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of Streptococcus pyogenes-derived deactivated cas9 (dcas9). When the dcas9 and sgRNA were respectively controlled by medium PR/tetO and strong PmxaF-g promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene glyA was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene mCherry over 50% and to reduce the expression of phytoene desaturase gene crtI by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene META1_3670 from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene shc involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of M. extorquens as well as in biotechnology for producing high-valued chemicals from methanol. KEY POINTS: Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene Improvement of carotenoid production by repressing a competitive pathway.

Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis.

Nocamycins I and II, featured with a tetramic acid scaffold, were isolated from the broth of Saccharothrix syringae NRRL B-16468. The biosynthesis of nocamycin I require an intermediate bearing a hydroxyl group at the C-10 position. A short chain dehydrogenase/reductase NcmD was proposed to catalyze the conversion of the hydroxyl group to ketone at the C-10 position. By using the λ-RED recombination technology, we generated the NcmD deletion mutant strain S. syringae MoS-1005, which produced a new intermediate nocamycin F with a hydroxyl group at C-10 position. We then overexpressed NcmD in Escherichia coli BL21 (DE3), purified the His6-tagged protein NcmD to homogeneity and conducted in vitro enzymatic assays. NcmD showed preference to the cofactor NAD+, and it effectively catalyzed the conversion from nocamyin F to nocamycin G, harboring a ketone group at C-10 position. However, NcmD showed no catalytic activity toward nocamyin II. NcmD achieved maximum catalytic activity at 45°C and pH 8.5. The kinetics of NcmD toward nocamycin F was investigated at 45°C, pH 8.5 in the presence of 2 mM NAD+. The K m and k cat values were 131 ± 13 µM and 65 ± 5 min-1, respectively. In this study, we have characterized NcmD as a dehydrogenase, which is involved in forming the ketone group at the C-10 position of nocamycin F. The results provide new insights to the nocamycin biosynthetic pathway.

CytA, a reductase in the cytorhodin biosynthesis pathway, inactivates anthracycline drugs in Streptomyces.

Antibiotic-producing microorganism can develop strategies to deal with self-toxicity. Cytorhodins X and Y, cosmomycins A and B, and iremycin, are produced as final products from a marine-derived Streptomyces sp. SCSIO 1666. These C-7 reduced metabolites show reduced antimicrobial and comparable cytotoxic activities relative to their C-7 glycosylated counterparts. However, the biosynthetic mechanisms and relevant enzymes that drive C-7 reduction in cytorhodin biosynthesis have not yet been characterized. Here we report the discovery and characterization of a reductase, CytA, that mediates C-7 reduction of this anthracycline scaffold; CytA endows the producer Streptomyces sp. SCSIO 1666 with a means of protecting itself from the effects of its anthracycline products. Additionally, we identified cosmomycins C and D as two intermediates involved in cytorhodin biosynthesis and we also broadened the substrate specificity of CytA to clinically used anthracycline drugs.

Bioconversion of Methanol into Value-added Chemicals in Native and Synthetic Methylotrophs.

Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of native methylotrophic bacteria serving as whole cell catalysts for production of chemicals and materials using methanol as a feedstock is highly attractive. In recent years, the available omics data for methylotrophic bacteria, especially for Methylobacterium extorquens, the most well-characterized model methylotroph, have provided a solid platform for rational engineering of methylotrophic bacteria for industrial production. In addition, there is a strong interest in converting the more traditional heterotrophic production platforms toward the use of single carbon substrates, including methanol, through metabolic engineering. In this chapter, we review the recent progress toward achieving the desired growth and production yields from methanol, by genetically engineered native methylotrophic strains and by the engineered synthetic methylotrophs.

Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr.

Candida albicans and Cryptococcus neoformans, human-pathogenic fungi found worldwide, are receiving increasing attention due to high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by coculturing 16 wood-decaying basidiomycetes, among which coculture of Trametes robiniophila Murr and Pleurotus ostreatus was found to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis revealed that 44 features were either newly synthesized or produced at high levels in this coculture system and that 6 of the features that belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with MICs of 1 to 32 µg/ml against pathogenic fungi. Furthermore, dynamic 13C-labeling analysis revealed an association between induced features and the corresponding fungi. Unusual sesterterpenes were 13C labeled only in P. ostreatus in a time course after stimulation by the coculture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr. Sesterterpene compounds 1 to 3 were renamed postrediene A to C. Real-time reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that transcriptional levels of three genes encoding terpene synthase, farnesyl-diphosphate farnesyltransferase, and oxidase were found to be 8.2-fold, 88.7-fold, and 21.6-fold higher, respectively, in the coculture than in the monoculture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A to postrediene C was then proposed based on structures of sesterterpenes and molecular network analysis.IMPORTANCE A number of gene clusters involved in biosynthesis of secondary metabolites are presumably silent or expressed at low levels under conditions of standard laboratory cultivation, resulting in a large gap between the pool of discovered metabolites and genome capability. This work mimicked naturally occurring competition by construction of an artificial coculture of basidiomycete fungi for the identification of secondary metabolites with novel scaffolds and excellent bioactivity. Unusual linear sesterterpenes of postrediene A to C synthesized by P. ostreatus not only were promising lead drugs against human-pathogenic fungi but also highlighted a distinct pathway for sesterterpene biosynthesis in basidiomycetes. The current work provides an important basis for uncovering novel gene functions involved in sesterterpene synthesis and for gaining insights into the mechanism of silent gene activation in fungal defense.

Cytochrome P450 oxidase SlgO1 catalyzes the biotransformation of tirandamycin C to a new tirandamycin derivative.

In the present study, an Escherichia coli whole cell system with overexpression of a cytochrome P450 oxidase SlgO1 involved in streptolydigin biosynthetic pathway, an E. coli flavodoxin NADP+ oxidoreductase (EcFLDR), and an E. coli flavodoxin A (EcFLDA) were constructed. Biotransformation experiments revealed that SlgO1 can convert tirandamycin C to tirandamycin F, indicating that it can introduce a hydroxyl group into the C-10 position of tirandamycin C. Subsequently, slgO1 was cloned into pSET152AKE vector under the downstream of ermE* promoter, which was, respectively, introduced into Streptomyces sp. SCSIO1666 (tirandamycin B producer), Streptomyces sp. Ju1008 (tirandamycin C producer), and Streptomyces sp. Ju1009 (tirandamycin E producer). A novel tirandamycin derivative tirandamycin L accumulated in the engineered strain Streptomyces sp. Ju1008::slgO1 was isolated and its structure was determined on the basis of nuclear magnetic resonance (NMR) and mass spectrometry. Unlike most of the identified tirandamycins, tirandamycin L possessed a rare C-11-C-12 saturated bond as well as a C-10 ketone moiety. In addition, tirandamycin L showed weaker antibacterial activity. Based on the structure of tirandamycin L, SlgO1 was proposed to be responsible for multiple modifications toward tirandamycin C, including the formation of C-10 hydroxyl and C-11-C-12 saturated bond.

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor.

BACKGROUND: Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis. RESULTS: In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The Km and kcat of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s-1, 1.28 mM and 153.14 s-1, and 2.34 mM and 1.15 s-1 towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THKM82V) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THKM82V under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 µg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase. CONCLUSIONS: These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.

Cytotoxic Anthracycline Metabolites from a Recombinant Streptomyces.

The C7 (C9 or C10)- O-l-rhodosamine-bearing anthracycline antibiotic cytorhodins and their biosynthetic intermediates were recently isolated from Streptomyces sp. SCSIO 1666. Cosmid p17C4 from the Streptomyces lydicus genomic library, which harbors both the biosynthetic genes for l-rhodinose (or 2-deoxy-l-fucose) and its glycosyltransferase (encoded by slgG), was introduced into SCSIO 1666 to yield the recombinant strain Streptomyces sp. SCSIO 1666/17C4. Chemical investigations of this strain's secondary metabolic potential revealed the production of different anthracyclines featuring C7- O-l-rhodinose (or 2-deoxy-l-fucose) instead of the typically observed l-rhodosamine. Purification of the fermentation broth yielded 12 new anthracycline antibiotics including three new ε-rhodomycinone derivatives, 1, 4, and 8, nine new ß-rhodomycinone derivatives, 2, 3, 5-7, and 9-12, and three known compounds, l-rhodinose-l-rhodinose-l-rhodinoserhodomycinone (13), ε-rhodomycinone (14), and γ-rhodomycinone (15). All compounds were characterized on the basis of detailed spectroscopic analyses and comparisons with previously reported data. These compounds exhibited cytotoxicity against a panel of human cancer cell lines. Significantly, compounds 4 and 13 displayed pronounced activity against HCT-116 as characterized by IC50 values of 0.3 and 0.2 µM, respectively; these IC50 values are comparable to that of the positive control epirubicin.

Elucidating the Sugar Tailoring Steps in the Cytorhodin Biosynthetic Pathway.

Anthracycline antitumor cytorhodins X and Y feature a rare 9α-glycoside and 7-dexoy-aglycone. Characterization of the cytorhodin gene cluster from Streptomyces sp. SCSIO 1666 through gene inactivations and metabolite analyses reveals three glycosyltransferases (GTs) involved in the sugar tailoring steps. The duo of CytG1 and CytL effects C-7 glycosylation with l-rhodosamine whereas the iterative GT CytG3 and CytW similarly modifies both C-9 and C-10 positions. CytG2 also acts iteratively by incorporating the second and third sugar moiety into the trisaccharide chains at the C-7 or C-10 position.

Elucidation of a carboxylate O-methyltransferase NcmP in nocamycin biosynthetic pathway.

Nocamycins belong to the tetramic acid family natural products and show potent antimicrobial activity. Recently, the biosynthetic gene cluster of nocamycin was identified from the rare actinomycete Saccharothrix syringae and an S-adenosylmethionine (SAM) dependent methyltransferase gene NcmP was found to be located within the gene cluster. In this report, the methyltransferase gene NcmP was disrupted and a new nocamycin intermediate nocamycin E was isolated from the mutant strain. Meanwhile, NcmP was heterologously expressed in Escherichia coli BL21 (DE3) and biochemically characterized as a carboxylate O-methyltransferase in nocamycin biosynthetic pathway. Compared to nocamycin I, nocamycin E showed inferior antibacterial activity, indicating the methyl group is essential to antibacterial activity.

Identification of nocamycin biosynthetic gene cluster from Saccharothrix syringae NRRL B-16468 and generation of new nocamycin derivatives by manipulating gene cluster.

BACKGROUND: Nocamycins I and II, produced by the rare actinomycete Saccharothrix syringae, belong to the tetramic acid family natural products. Nocamycins show potent antimicrobial activity and they hold great potential for antibacterial agent design. However, up to now, little is known about the exact biosynthetic mechanism of nocamycin. RESULTS: In this report, we identified the gene cluster responsible for nocamycin biosynthesis from S. syringae and generated new nocamycin derivatives by manipulating its gene cluster. The biosynthetic gene cluster for nocamycin contains a 61 kb DNA locus, consisting of 21 open reading frames (ORFs). Five type I polyketide synthases (NcmAI, NcmAII, NcmAIII, NcmAIV, NcmAV) and a non-ribosomal peptide synthetase (NcmB) are proposed to be involved in synthesis of the backbone structure, a Dieckmann cyclase NcmC catalyze the releasing of linear chain and the formation of tetramic acid moiety, five enzymes (NcmEDGOP) are related to post-tailoring steps, and five enzymes (NcmNJKIM) function as regulators. Targeted inactivation of ncmB led to nocamycin production being completely abolished, which demonstrates that this gene cluster is involved in nocamycin biosynthesis. To generate new nocamycin derivatives, the gene ncmG, encoding for a cytochrome P450 oxidase, was inactivated. Two new nocamycin derivatives nocamycin III and nocamycin IV were isolated from the ncmG deletion mutant strain and their structures were elucidated by spectroscopic data analyses. Based on bioinformatics analysis and new derivatives isolated from gene inactivation mutant strains, a biosynthetic pathway of nocamycins was proposed. CONCLUSION: These findings provide the basis for further understanding of nocamycin biosynthetic mechanism, and set the stage to rationally engineer new nocamycin derivatives via combinatorial biosynthesis strategy.

Euzebyella marina sp. nov., isolated from seawater.

A Gram-stain-negative, aerobic, yellow-pigmented, non-flagellated, non-gliding, oxidase- and catalase-positive bacterium, designated CY01T, was isolated from seawater of the Yellow Sea. CY01T grew at 15-37 °C (optimum, 30 °C), pH 5-8 (optimum, 6.5-7.5) and with 0.5-12 % (w/v) NaCl (optimum, 0.5-3.5 %). It could not produce flexirubin-type pigment or reduce nitrate to nitrite. CY01T showed the highest 16S rRNA gene sequence similarity to the type strain of Euzebyella saccharophila (97.0 %) and clustered tightly with the species of the genus Euzebyella in the phylogenetic trees based on the 16S rRNA gene sequences. The major cellular fatty acids of CY01T were iso-C15 : 0, iso-C15 : 1G and iso-C17 : 0 3-OH and the major respiratory quinone was menaquinone MK-6. Polar lipids included phosphatidylethanolamine (PE), four unidentified lipids and one unidentified aminolipid. The genomic DNA G+C content was 38.2 mol%. Based on the results of the polyphasic characterization of CY01T, it represents a novel species of the genus Euzebyella, for which the name Euzebyella marina sp. nov. is proposed. The type strain is CY01T (=CCTCC AB 2014348T=KCTC 42440T).

Discovery of a new family of Dieckmann cyclases essential to tetramic acid and pyridone-based natural products biosynthesis.

Bioinformatic analyses indicate that TrdC, SlgL, LipX2, KirHI, and FacHI belong to a group of highly homologous proteins involved in biosynthesis of actinomycete-derived tirandamycin B, streptolydigin, α-lipomycin, kirromycin, and factumycin, respectively. However, assignment of their biosynthetic roles has remained elusive. Gene inactivation and complementation, in vitro biochemical assays with synthetic analogues, point mutations, and phylogenetic tree analyses reveal that these proteins represent a new family of Dieckmann cyclases that drive tetramic acid and pyridone scaffold biosynthesis.