Genome-wide characterization and metabolite profiling of Cyathus olla: insights into the biosynthesis of medicinal compounds.

Cyathus olla, belonging to the genus Cyathus within the order Agaricales, is renowned for its bird's nest-like fruiting bodies and has been utilized in folk medicine. However, its genome remains poorly understood. To investigate genomic diversity within the genus Cyathus and elucidate biosynthetic pathways for medicinal compounds, we generated a high-quality genome assembly of C. olla with fourteen chromosomes. The comparative genome analysis revealed variations in both genomes and specific functional genes within the genus Cyathus. Phylogenomic and gene family variation analyses provided insights into evolutionary divergence, as well as genome expansion and contraction in individual Cyathus species and 36 typical Basidiomycota. Furthermore, analysis of LTR-RT and Ka/Ks revealed apparent whole-genome duplication (WGD) events its genome. Through genome mining and metabolite profiling, we identified the biosynthetic gene cluster (BGC) for cyathane diterpenes from C. olla. Furthermore, we predicted 32 BGCs, containing 41 core genes, involved in other bioactive metabolites. These findings represent a valuable genomic resource that will enhance our understanding of Cyathus species genetic diversity. The genome analysis of C. olla provides insights into the biosynthesis of medicinal compounds and establishes a fundamental basis for future investigations into the genetic basis of chemodiversity in this significant medicinal fungus.

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.

The Complete Genomic Sequence of Microbial Transglutaminase Producer, Streptomyces mobaraensis DSM40587.

Actinomycetes are remarkable natural sources of active natural molecules and enzymes of considerable industrial value. Streptomyces mobaraensis is the first microorganism found to produce transglutaminase with broad industrial applications. Although transglutaminase in S. mobaraensis has been well studied over the past three decades, the genome of S. mobaraensis and its secondary metabolic potential were poorly reported. Here, we presented the complete genome of S. mobaraensis DSM40587 obtained from the German Collection of Microorganisms and Cell Cultures GmbH. It contains a linear chromosome of 7,633,041 bp and a circular plasmid of 23,857 bp. The chromosome with an average GC content of 73.49% was predicted to harbour 6683 protein-coding genes, seven rRNA and 69 tRNA genes. Comparative genomic analysis reveals its meaningful genomic characterisation. A comprehensive bioinformatics investigation identifies 35 putative BGCs (biosynthesis gene clusters) involved in synthesising various secondary metabolites. Of these, 13 clusters showed high similarity (> 55%) to known BGCs coding for polyketides, nonribosomal peptides, hopene, RiPP (Ribosomally synthesized and post-translationally modified peptides), and others. Furthermore, these BGCs with over 65% similarity to the known BGCs were analysed in detail. The complete genome of S. mobaraensis DSM40587 reveals its capacity to yield diverse bioactive natural products and provides additional insights into discovering novel secondary metabolites.

High-efficient production of mushroom polyketide compounds in a platform host Aspergillus oryzae.

BACKGROUND: Orsellinic acid (2,4-dihydroxy-6-methylbenzoic acid, OA) and its structural analog o-Orsellinaldehyde, have become widely used intermediates in clinical drugs synthesis. Although the research on the biosynthesis of such compounds has made significant progress, due to the lack of suitable hosts, there is still far from the industrial production of such compounds based on synthetic biology. RESULTS: With the help of genome mining, we found a polyketide synthase (PKS, HerA) in the genome of the Hericium erinaceus, which shares 60% amino acid sequence homology with ArmB from Armillaria mellea, an identified PKS capable of synthesizing OA. To characterize the function of HerA, we cloned herA and heterologously expressed it in Aspergillus oryzae, and successfully detected the production of OA. Subsequently, the introduction of an incomplete PKS (Pks5) from Ustilago maydis containing only three domains (AMP-ACP-R), which was into herA-containing A. oryzae, the resulted in the production of o-Orsellinaldehyde. Considering the economic value of OA and o-Orsellinaldehyde, we then optimized the yield of these compounds in A. oryzae. The screening showed that when maltose was used as carbon source, the yields of OA and o-Orsellinaldehyde were 57.68 mg/L and 15.71 mg/L respectively, while the yields were 340.41 mg/Kg and 84.79 mg/Kg respectively in rice medium for 10 days. CONCLUSIONS: Herein, we successfully expressed the genes of basidiomycetes using A. oryzae heterologous host. As a fungus of ascomycetes, which not only correctly splices genes of basidiomycetes containing multiple introns, but also efficiently produces their metabolites. This study highlights that A. oryzae is an excellent host for the heterologous production of fungal natural products, and has the potential to become an efficient chassis for the production of basidiomycete secondary metabolites in synthetic biology.

The First Five Mitochondrial Genomes for the Family Nidulariaceae Reveal Novel Gene Rearrangements, Intron Dynamics, and Phylogeny of Agaricales.

The family Nidulariaceae, consisting of five genera including Cyathus, is a unique group of mushrooms commonly referred to as bird's nest fungi due to their striking resemblance to bird's nests. These mushrooms are considered medicinal mushrooms in Chinese medicine and have received attention in recent years for their anti-neurodegenerative properties. However, despite the interest in these mushrooms, very little is known about their mitochondrial genomes (mitogenomes). This study is the first comprehensive investigation of the mitogenomes of five Nidulariaceae species with circular genome structures ranging in size from 114,236 bp to 129,263 bp. Comparative analyses based on gene content, gene length, tRNA, and codon usage indicate convergence within the family Nidulariaceae and heterogeneity within the order Agaricales. Phylogenetic analysis based on a combined mitochondrial conserved protein dataset provides a well-supported phylogenetic tree for the Basidiomycetes, which clearly demonstrates the evolutionary relationships between Nidulariaceae and other members of Agaricales. Furthermore, phylogenetic inferences based on four different gene sets reveal the stability and proximity of evolutionary relationships within Agaricales. These results reveal the uniqueness of the family Nidulariaceae and its similarity to other members of Agaricales; provide valuable insights into the origin, evolution, and genetics of Nidulariaceae species; and enrich the fungal mitogenome resource. This study will help to expand the knowledge and understanding of the mitogenomes in mushrooms.

Unprecedented Neoverrucosane and Cyathane Diterpenoids with Anti-Neuroinflammatory Activity from Cultures of the Culinary-Medicinal Mushroom Hericium erinaceus.

The culinary medicinal mushroom Hericium erinaceus holds significant global esteem and has garnered heightened interest within increasingly ageing societies due to its pronounced neuroprotective and anti-neuroinflammatory properties. Within this study, two novel diterpenes, 16-carboxy-13-epi-neoverrucosane (1) and Erinacine L (2); three known xylosyl cyathane diterpenoids, Erinacine A (3), Erinacine C (4), and Erinacine F (5); and four lanostane-type triterpenoids, and three cyclic dipeptides (10-12), in addition to orcinol (13), were isolated from the rice-based cultivation medium of H. erinaceus. Their structures were determined by NMR, HR-ESI-MS, ECD, and calculated NMR. Compound 1 marks a pioneering discovery as the first verrucosane diterpene originating from basidiomycetes, amplifying the scope of fungal natural product chemistry, and the intricate stereochemistry of Compound 5 has been comprehensively assessed for the first time. Compounds 2-5 not only showed encouraging neurotrophic activity in rat adrenal pheochromocytoma PC-12 cells, but also significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia cell cultures with IC50 values as low as 5.82 ± 0.18 µM. To elucidate the mechanistic underpinnings of these bioactivities, molecular docking simulation was used to analyze and support the interaction of 1 and 2 with inducible NO synthase (iNOS), respectively. In particular, compound 2, a cyathane-xyloside containing an unconventional hemiacetal moiety, is a compelling candidate for the prevention of neurodegenerative diseases. In summation, this investigation contributes substantively to the panorama of fungal diterpene structural diversity, concurrently furnishing additional empirical substantiation for the role of cyathane diterpenes in the amelioration of neurodegenerative afflictions.

Genome sequencing of Inonotus obliquus reveals insights into candidate genes involved in secondary metabolite biosynthesis.

BACKGROUND: Inonotus obliquus is an important edible and medicinal mushroom that was shown to have many pharmacological activities in preclinical trials, including anti-inflammatory, antitumor, immunomodulatory, and antioxidant effects. However, the biosynthesis of these pharmacological components has rarely been reported. The lack of genomic information has hindered further molecular characterization of this mushroom. RESULTS: In this study, we report the genome of I. obliquus using a combined high-throughput Illumina NovaSeq with Oxford Nanopore PromethION sequencing platform. The de novo assembled 38.18 Mb I. obliquus genome was determined to harbor 12,525 predicted protein-coding genes, with 81.83% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed the close evolutionary relationship of I. obliquus with Fomitiporia mediterranea and Sanghuangporus baumii in the Hymenochaetales clade. According to the distribution of reproduction-related genes, we predict that this mushroom possesses a tetrapolar heterothallic reproductive system. The I. obliquus genome was found to encode a repertoire of enzymes involved in carbohydrate metabolism, along with 135 cytochrome P450 proteins. The genome annotation revealed genes encoding key enzymes responsible for secondary metabolite biosynthesis, such as polysaccharides, polyketides, and terpenoids. Among them, we found four polyketide synthases and 20 sesquiterpenoid synthases belonging to four more types of cyclization mechanism, as well as 13 putative biosynthesis gene clusters involved in terpenoid synthesis in I. obliquus. CONCLUSIONS: To the best of our knowledge, this is the first reported genome of I. obliquus; we discussed its genome characteristics and functional annotations in detail and predicted secondary metabolic biosynthesis-related genes, which provides genomic information for future studies on its associated molecular mechanism.

Efficient production of a cyclic dipeptide (cyclo-TA) using heterologous expression system of filamentous fungus Aspergillus oryzae.

BACKGROUND: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. RESULTS: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. CONCLUSIONS: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.

Bioactive components of Laetiporus species and their pharmacological effects.

Laetiporus species are brown rot fungi belonging to the order Polyporales in the division Basidiomycota. These species produce a variety of metabolites and provide a great source of natural material for the screening of medicinally active natural products or their derivatives. This review summarizes the research progress on bioactive metabolites of Laetiporus species up to April 2022, including biological macromolecules, for instance, polysaccharides and lectins, as well as 80 reported small molecule chemical components (15 sterols, 29 triterpenes, 10 sesquiterpenes, 5 polyenes, 10 volatile compounds, and 11 other compounds). These metabolites exhibit antimicrobial, anticancer, antioxidant, hepatoprotective, anti-inflammatory, and antidiabetic activities. Genome mining predicted 23 terpene synthases, 7 polyketide synthases, and 9 non-ribosomal peptide synthases involved in bioactive metabolites biosynthesis, which were analyzed by antiSMASH in L. sulphureus genome. This review will provide a basis for the biosynthesis of active components in Laetiporus species and a reference for the research of medical precursors. KEY POINTS: • The mini-review summarized 80 secondary metabolites of Laetiporus spp. • The main pharmacological activities of Laetiporus spp. were summarized. • Biosynthetic genes of terpenoids, polyketides, and non-ribosomal peptides were also summarized.

Kanamycin-induced production of 2',3'-cyclic AMP in Escherichia coli.

In contrast to the well-characterized second messenger adenosine 3',5'-cyclic monophosphate (3',5'-cAMP), the biological roles of its isomer 2',3'-cAMP remain largely unknown, especially in bacteria. Recent work reported that RNase I-dependent elevation of 2',3'-cNMP levels in Escherichia coli correlated with reduced biofilm production, and separate studies demonstrated E. coli ribonuclease activation in response to aminoglycoside antibiotics. Here we report that E. coli produced 2',3'-cAMP in response to kanamycin at sub-inhibitory levels. Surprisingly, other aminoglycosides like streptomycin or gentamicin did not generate levels of 2',3'-cAMP detectable by 31P NMR. Interestingly, because 2',3'-cAMP is also produced in E. coli strains expressing a plasmid-encoded kanamycin resistance gene but not by other ribosome-targeting antibiotics, this kanamycin-specific production may not reflect disrupted protein synthesis. Overall, this finding provides a link between aminoglycoside-induced ribonuclease activity and 2',3'-cAMP production in E. coli.

Design, synthesis and interaction of BRC4 analogous peptides with RAD51(241-260).

Breast cancer susceptibility gene 2 (BRCA2) is an important tumor suppressor, which is participated in repair of damaged DNA by its highly conserved BRC repeat motifs regulating RAD51 protein homologous recombination and thereby preventing cell carcinogenesis. In this study, the BRCA2(1524-1548)-RAD51(241-260) complex structure was obtained based on PDB bank data 1N0W, which provided the basis for site-specific mutation of BRCA2(1524-1548). The BRC4 and BRC4 analogous peptides were synthesized, and the interaction between BRC peptide and RAD51(241-260) was studied by fluorescence spectroscopy, circular dichroism spectroscopy and microscale thermophoresis (MST). The results of circular dichroism showed that the changes in secondary structures of RAD51(241-260) occurred after adding BRC4 analogous peptides, and the α-helix content increased significantly. Fluorescence spectral data demonstrated that the model of BRC peptide binding to RAD51(241-260) was static quenching, and the binding constants of BRC4, P1, P2, P4 with RAD51(241-260) were 1.647 × 10-4 L mol-1, 2.532 × 10-4 L mol-1, 3.161 × 10-4 L mol-1, 1.705 × 10-4 L mol-1, respectively. The results of MST indicated that P2 and RAD51(241-260) have better affinity for dissociation constant 44.286 µM. The strongest affinity between P2 and RAD51(241-260) indicated that the mutation of amino acid residue constituting BRC α-helix affects the structure and interaction of BRC peptide and RAD51(241-260).

Design of BRC analogous peptides based on the complex BRC8-RAD51 and the preliminary study on the peptide structures.

BRCA2 is an important tumor suppressor gene that plays a critical role in preserving the stability of cellular genetic information, participating in DNA repair by engaging in binding interactions with RAD51 proteins. However, the lack of structural data on BRCA2 and RAD51 makes the study of their interaction mechanism still a great challenge. We characterize the structure of the BRC8-RAD51 complex using ZDOCK protein docking software and identify the potential non-conserved active site of BRC8 via virtual alanine scanning, utilizing the obtained results to synthesize BRC8, its six analogous peptides (BRC8-1 to BRC8-6), and critical peptide fragment of RAD51 (RAD51(231-260)) by Fmoc solid-phase synthesis. The analogous peptides are found to exhibit a secondary structure significantly different from that of BRC8 by circular dichroism spectroscopy, which indicates that mutation sites determined by computer-aided simulation correspond to key amino acid residues substantially affecting polypeptide structure. On the other hand, the secondary structure of RAD51(231-260) was also considerably influenced by its interaction with BRC8 and analogs, e.g., the fraction of the α-helical structure in RAD51(231-260) increased to 23.6, 15.1, and 13.5% upon interaction with BRC8-1, BRC8-3, and BRC8-6, respectively. The results show that the properties of C-terminal amino acid residues significantly influence peptide-peptide interactions, in agreement with the results of virtual alanine scanning. Therefore, computer-aided simulation was confirmed to be a technique that is useful for narrowing down the range of sites responsible for interactions between peptides or proteins, and provides new inspirations for the design of peptides with strong interactions.

Phaeosphaones: Tyrosinase Inhibitory Thiodiketopiperazines from an Endophytic Phaeosphaeria fuckelii.

Phaeosphaeria fuckelii, an endophytic fungus associated with the herbal medicine Phlomis umbrosa, produced four new thiodiketopiperazine alkaloids, phaeosphaones A-D (1-4), featuring an unusual ß-(oxy)thiotryptophan motif, along with four known analogues, phaeosphaone E (5), chetoseminudin B (6), polanrazine B (7), and leptosin D (8). Their structures were elucidated by extensive spectroscopic data analysis, and their absolute configurations were determined by single-crystal X-ray diffraction and ECD calculations. Compounds 4, 6, and 8 were found to display mushroom tyrosinase inhibitory activity with IC50 values of 33.2 ± 0.2, 31.7 ± 0.2, and 28.4 ± 0.2 µM, respectively, more potent than that of the positive control, kojic acid (IC50 = 40.4 ± 0.1 µM). A molecular-docking study disclosed the π-π stacking interaction between the indole moiety of 8 and the His243 residue of tyrosinase.

Discovery of 1,3-Disubstituted 2,5-Diketopiperazine Derivatives as Potent Class I HDACs Inhibitors.

Histone deacetylases (HDACs) as attractive targets in many diseases therapies has been studied extensively, and its application in cancer research is the most important. Here, we developed a series of derivatives containing natural 2,5-diketopiperazine (DKP) skeleton. Several compounds exhibited distinct HDAC1 inhibitory activities, in particular 2a (IC50 = 405 nM). The selectivity profile for representative 2a indicated that this series of compounds had a preference for HDAC1-3. Additionally, 2a showed the best growth inhibitory activities against K562 and HL-60 tumor cell line with IC50 values of 4.23 and 4.16 µM, respectively. This work may lay the foundation for developing DKP-based HDAC inhibitors as a potential anticancer agent.

Design, Synthesis and Biological Evaluation of Novel Benzoylimidazole Derivatives as Raf and Histone Deacetylases Dual Inhibitors.

In recent studies, combinations of histone deacetylases (HDACs) inhibitor with kinase inhibitor showed additive and synergistic effects. BRafV600E as an attractive target in many diseases treatments has been studied extensively. Herein, we present a novel design approach though incorporating the pharmacophores of BRafV600E inhibitor and HDACs inhibitor in one molecule. Several synthesized compounds exhibited distinct BRafV600E and HDAC1 inhibitory activities. The representative dual Raf/HDAC inhibitor, 7a, showed better antiproliferative activities against A549 and SK-Mel-2 in cellular assay than SAHA and sorafenib, with IC50 values of 9.11 µM and 5.40 µM, respectively. This work may lay the foundation for the further development of dual Raf/HDAC inhibitors as potential anticancer agents.

An ATP-Dependent Ligase with Substrate Flexibility Involved in Assembly of the Peptidyl Nucleoside Antibiotic Polyoxin.

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which the peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid [CPOAA]) and the nucleoside skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrate that PolG employs an ATP-dependent mechanism for amide bond formation and that the generation of the hybrid nucleoside antibiotic POL-N is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us to propose a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics.IMPORTANCE POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy.

Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

Characterization of the aurantimycin biosynthetic gene cluster and enhancing its production by manipulating two pathway-specific activators in Streptomyces aurantiacus JA 4570.

BACKGROUND: Aurantimycin (ATM), produced by Streptomyces aurantiacus JA 4570, is a potent antimicrobial and antitumor antibiotic. Although the chemical structure of ATM is highly distinctive and features a cyclohexadepsipeptide scaffold attached with a C14 acyl side chain, little is known about its biosynthetic pathway and regulatory mechanism. RESULTS: In this work, we report the identification and characterization of the ATM biosynthetic gene cluster from S. aurantiacus JA 4570. Targeted inactivation of artG, coding for a NRPS enzyme, completely abolished ATM production, thereof demonstrating the target gene cluster (art) is responsible for ATM biosynthesis. Moreover, four NRPS adenylation (A) domains including a freestanding enzyme ArtC have been characterized in vitro, whose substrate specificities are consistent with in silico analysis. Further genetic analysis of the two regulatory genes artB and artX unambiguously suggested both of them play positive roles in ATM biosynthesis, and ATM-A production was thus rationally enhanced to about 2.5 fold via tandem overexpression of artB and artX in S. aurantiacus JA 4570. CONCLUSIONS: These results will provide the basis for the understanding of precise mechanisms for ATM biosynthesis, and open the way for both rational construction of high-production ATM producer and orient-directed generation of designer ATM derivatives via synthetic biology strategies.

Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes.

Nucleoside antibiotics constitute an important family of microbial natural products bearing diverse bioactivities and unusual structural features. Their biosynthetic logics are unique with involvement of complex multi-enzymatic reactions leading to the intricate molecules from simple building blocks. Understanding how nature builds this family of antibiotics in post-genomic era sets the stage for rational enhancement of their production, and also paves the way for targeted persuasion of the cell factories to make artificial designer nucleoside drugs and leads via synthetic biology approaches. In this review, we discuss the recent progress and perspectives on the natural and engineered biosynthesis of nucleoside antibiotics.

Metabolic engineering of an industrial polyoxin producer for the targeted overproduction of designer nucleoside antibiotics.

Polyoxin and nikkomycin are naturally occurring peptidyl nucleoside antibiotics with potent antifungal bioactivity. Both exhibit similar structural features, having a nucleoside skeleton and one or two peptidyl moieties. Combining the refactoring of the polyoxin producer Streptomyces aureochromogenes with import of the hydroxypyridylhomothreonine pathway of nikkomycin allows the targeted production of three designer nucleoside antibiotics designated as nikkoxin E, F, and G. These structures were determined by NMR and/or high resolution mass spectrometry. Remarkably, the introduction of an extra copy of the nikS gene encoding an ATP-dependent ligase significantly enhanced the production of the designer antibiotics. Moreover, all three nikkoxins displayed improved bioactivity against several pathogenic fungi as compared with the naturally-occurring antibiotics. These data provide a feasible model for high efficiency generation of nucleoside antibiotics related to polyoxins and nikkomycins in a polyoxin cell factory via synthetic biology strategy.