Abstract:来源于微生物的次生代谢产物是新药发现和发展的重要源泉，也是行之有效、研究生物学问题的探针工具。微生物产生次生代谢产物的目的并非为人类所用，而是以之为工具或媒介，调控其内在的生物化学过程并响应各种外部环境的变化。另一方面，微生物也通过其产物的结构改变、优化和最终选择，适应各种动态、可变的生物学过程。化学结构与生物功能的共进化，体现了自然中小分子基于普适性与特异性的演变规律。围绕微生物次生代谢产物的生物合成机制，《微生物学通报》本期推出的《微生物天然产物发现及生物合成主题刊》包括16篇论文，内容涵盖了天然产物相关的化学结构、生物学功能、合成代谢途径、酶促反应机理、生物信息学分析等多个方面。期望该主题刊的出版有助于加强我国相关领域专家之间的交流与合作，促进微生物生物化学方向的学科发展。

Abstract:[Background] Marine microorganisms are one of the important biological resources in the complex marine ecological environment. The natural products produced by marine microorganisms are extremely abundant, which are the rich source of drug discovery. [Objective] Exploration of the optimal growth conditions of Streptomyces sp. S52-B, a sponge-derived Streptomyces, and excavation of its secondary metabolites in order to isolate new bioactive natural products. [Methods] According to the "One Strain Many Compounds" strategy, we optimized the cultural conditions of sponge-derived Streptomyces sp. S52-B which are suitable for mycelium growth as well as the production of secondary metabolites. Combining mass spectrometry and characteristic UV absorption spectra, one medium is selected for large scale fermentation. After purification by column chromatography and preparative high performance liquid chromatography (HPLC), the structure of the compounds were elucidated by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance spectroscopy. [Results] The optimal medium of marine Streptomyces sp. S52-B was determined. Based on UV absorption spectroscopy and mass spectrometry analysis, three compounds with pyrrolo[4,3,2-de]quinolone were isolated and identified from a large scale fermentation using medium A. The chlorinated compounds with the pyrrolo[4,3,2-de]quinolone core structure belongs to the natural product of aminoamides, among which ammosalic acid is a new structure compound. [Conclusion] The aminosamide family compounds are known to have excellent anti-cancer activity. Three aminoamide compounds have been isolated and identified from the sponge-derived Streptomyces sp. S52-B, which not only enriches the structural types of such compounds, but also provides opportunity to combining culture conditions and genomic information to mine new structural active natural products from this strain.

Abstract:[Background] As an essential element for bacterial growth, iron in its ferric form is almost insoluble in aqueous environments. Bacteria have evolved to produce various siderophores to facilitate iron uptake. For Streptomyces, the characteristic siderophores are desferrioxamines, while they can also produce other structurally different siderophores, such as ceolichelin, albomycin, enterobactin, and griseobactin. [Objective] We aimed to reveal the distribution and characteristic of siderophore biosynthetic gene clusters (BGCs), and to explore their product structures in streptomycetes. [Methods] We systematically investigated the distribution and conservation of siderophore BGCs in 308 annotated Streptomyces genomes using bioinformatics tools. Chromatographic and spectroscopic methods were utilized to isolate and characterize the enterobactin-related natural products. [Results] This enabled us to identify an orphan enterobactin BGC, which lacked genes encoding enzymes for the biosynthesis of 2,3-dihydroxybenzoic acid (2,3-DHB), together with a griseobactin BGC in Streptomyces albofaciens JCM 4342 and other strains. Four enterobactin-derived natural products, including linear trimer and dimer of 2,3-dihydroxybenzoyl-L-serine (2,3-DHBS), and their dehydrated products, were identified from S. albofaciens JCM 4342. [Conclusion] These results suggested an interesting synergistic biosynthetic mechanism executed by the two BGCs. The orphan enterobactin BGC encoding enzymes hijacked the 2,3-DHB, which was biosynthesized by the griseobactin BGC, to complete biosynthesis of the four aforementioned enterobactin-related natural products.

Abstract:[Background] Lechevalieria rhizosphaerae NEAU-A2 was identified as a novel rare actinobacteria, and genomic analysis shows that it contains a variety of biosynthetic gene clusters with the potential to generate multiple natural products. [Objective] Study on the secondary metabolites in L. rhizosphaerae NEAU-A2 to discover compounds with novel structures or unique biological activities, and further establish the genetic system of the strain. [Methods] The secondary metabolites were isolated and purified by using silica gel column chromatography, high performance liquid chromatography and other methods. The structures of compounds were elucidated by analysis of NMR and MS data. The mutant cosmid with the disruption of the gene 1609 (a PKS-NRPS hybrid gene) was constructed using PCR-targeting method. Intergeneric conjugation was conducted for plasmid the transformation into wild type strain. [Results] Two new dimeric indole alkaloids (1-2), along with five known compounds, N-acetyltryptamine (3), 4-((2-(1H-indol-3-yl)ethyl)amino)-4-oxobutanoic acid (4), brevianamide F (5), 4S,7R-germacra-1(10)E,5E-diene-11-ol (6), 1H-pyrrole-2-carboxylic acid (7), were isolated and identified from the fermentation broth of L. rhizosphaerae NEAU-A2. The double crossover recombinant strains were obtained after culturing two generations, and further verified by polymerase chain reaction (PCR). [Conclusion] Seven compounds were isolated and identified from strain NEAU-A2, and the genetic system of this novel rare actinobacteria L. rhizosphaerae NEAU-A2 was successfully established.

Abstract:[Background] Secondary metabolites from mangrove actinomycetes are an important source for the discovery of small molecule drugs. [Objective] To isolate and identify the antibacterial secondary metabolites from mangrove actinomycetes. [Methods] The mangrove actinomycete strains were separated and purified using dilution plate method, and antibacterial activity of the strains were assayed by agar block and filter disk methods. The 16S rRNA gene sequence of an actinomycete strain was used for species analysis and construction of the phylogenetic tree. The fermentation broth of the actinomycete strain was analyzed by high performance liquid chromatography (HPLC), and the antibacterial compounds were purified by silica gel column chromatography and HPLC combining with activity-tracking method. The structures of the antibacterial compounds were elucidated by high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) and nuclear magnetic resonance (NMR). [Results] An actinomycetes strain ZFSM1-146 with strong antibacterial activity was obtained from mangrove soil. Species and phylogenetic tree analysis showed that the strain belongs to Streptomyces antibioticus. Strain ZFSM1-146 could produce antibacterial compounds 1-3, which were identified as actinomycin X_Oβ, X₂ and D, respectively. After preliminary medium optimization, the yield of actinomycin X₂, which showed the strongest antibacterial activity among the 3 compounds, was almost twice as the original yield. [Conclusion] S. antibioticus ZFSM1-146 was obtained from mangrove soil and could produce antibacterial compound actinomycins. This study provided a valuable strain for improving the yield of actinomycins as well as producing new analogs through genetic manipulation.

Abstract:[Background] Tetramycin and tetrin are tetraene macrolides with broad-spectrum antifungal activity. Streptomyces sp. CB02959 was initially screened as a potential producer of leinamycin-like compounds. We noticed the existence of a putative natamycin biosynthetic gene cluster in CB02959 using antiSMASH analysis. [Objective] In this study, we aim to characterize the secondary metabolites from Streptomyces sp. CB02959, determine whether CB02959 is a tetraene macrolide-producing strain. We also aim to characterize the major products from CB02959 and improve their titers by media optimization.[Methods] Guided by bioinformatic analysis and high-resolution mass spectrum data, we predicted the structures of the compounds to be isolated. Streptomyces sp. CB02959 was then cultivated in different media to determine the right medium for large-scale fermentation. The tetrA gene, which encodes the first polyketide synthase of the polyketide assembly line, was disrupted to correlate the target gene cluster with the production of tetraene macrolides. The structures of major metabolites from CB02959 were elucidated based on extensive spectra analysis. The concentration of glucose, malt extract and tryptone were adjusted to improve the titers of the produced tetraenes. [Results] Based on the analysis of the predicted natamycin biosynthetic gene cluster in CB02959 and phylogenetic analysis of the 16S rRNA gene, CB02959 was proposed as a new tetramycin and tetrin-producing strain. After the large-scale fermentation in YEME medium, we isolated four metabolites from CB02959, which were determined as tetramycin A (1), tetramycin B (2), tetrin A (3), and tetrin B (4). We increased the titers of compounds 1-4 to 208.1, 100.0, 1 315.6, 109.9 mg/L via preliminary optimization of the fermentation medium. [Conclusion] In this study, we identified Streptomyces sp. CB02959 as a new producer for tetramycins and tetrins using the genome mining strategy, the titers of the produced tetraenes were improved by medium optimization. Our findings lay a foundation for the further development of these potent antifungal agents.

Abstract:[Background] Lexapeptide is the first member of the class V lanthipeptide family. In vitro bioassay indicated that lexapeptide has remarkable antibacterial activity against various G⁺ bacteria, and shows stronger activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) than nisin. Lexapeptide also exhibits better thermo-stability and pH-stability than nisin. The mode of actions of lexapeptide remains uncovered, hindering its potential application. [Objective] Through investigation into the antibacterial mode of action of lexapeptide to lay the foundation for its further application. [Methods] The antibacterial kinetics of lexapeptide was characterized by colony-forming unit (CFU) counting and magnesium ion assay. The pore forming ability of lexapeptide was assessed by flow cytometry (FCM) and transmission electron microscopy (TEM). High performance liquid chromatography (HPLC) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to analyze lexapeptide's effect on the accumulation of peptidoglycan biosynthetic precursor in G⁺ bacteria. [Results] Compared to nisin, the antibacterial activity of lexapeptide was less sensitive to the inhibition by magnesium ion. Bacterial cells treated with lexapeptide gained permealiability to fluorescent dye. Damaged morphology of those cells was observed with TEM. A peptidoglycan biosynthetic precursor, UDP-NAcMur-pentapeptide, was determined in the lexapeptide-treated cells.[Conclusion] Lexapeptide inhibits the growth of G⁺ bacteria by inhibiting the biosynthesis of peptidoglycan in cell wall as well as permeabilizing the cell membrane.

Abstract:[Background] Cinnamyl acetate is an important flavor compound and widely used in cosmetics and food industries. The traditional production methods include direct extraction from plants and chemical synthesis. [Objective] In this work, we aim to achieve de novo biosynthesis of cinnamyl acetate in Escherichia coli by screening benzyl alcohol O-acyl transferases from different plants and constructing biosynthetic pathway for cinnamyl acetate. [Methods] First, a biosynthetic pathway of aglycon cinnamyl alcohol from phenylalanine was constructed in the high-phenylalanine-producing E. coli strain named BPHE by expressing the enzymes phenylalanine ammonialyase (PAL), hydroxycinnamate:CoA ligase (4CL), and cinnamyl-CoA reductase and endogenous alcohol dehydrogenases or aldo-keto reductases in E. coli. Subsequently, the benzyl alcohol O-benzoyltransferase from Nicotiana tabacum (ANN09798) or benzyl alcohol O-benzoyltransferase from Clarkia breweri (ANN09796) or benzyl alcohol acetyltransferase from Clarkia breweri (BEAT) were introduced into the above recombinant E. coli strain to produce cinnamyl acetate. We further improved the acetyl-CoA production by overexpressing endogenous acetyl-CoA synthetase (ACS) in E. coli. [Results] We investigated the ability of three plant-derived benzyl alcohol acyltransferase to biosynthesize cinnamyl acetate, which were further applied for synthesizing cinnamyl acetate in E. coli. The production of cinnamyl acetate by the engineered E. coli reached 166.9±6.6 mg/L. [Conclusion] Plant derived benzyl alcohol acyltransferase demonstrate flexibility to a wide range of substrates and can catalyze the synthesis of cinnamyl acetate by using cinnamyl alcohol as substrate. This study provides a foundation for microbial production of cinnamyl acetate and its derivatives using glucose as the renewable carbon source.

Abstract:[Background] Skyllamycins are nonribosomal cyclic depsipeptides that possess inhibitory activity against the platelet-derived growth factor (PDGF) signaling pathway and antibiofilm activity. The cyclization reaction in skyllamycin biosynthesis is catalyzed by the thioesterase domain. [Objective] To characterize the substrate promiscuity of thioesterase domain, we cloned and expressed the thioesterase-encoding gene, synthesized substrate mimics and carried out the cyclization reactions in vitro. [Methods] The thioesterase domain (Skyxy-TE)-encoding gene was cloned from Streptomyces sp. PKU-MA01239 with ligation-independent cloning method, Skyxy-TE was purified by Ni-NTA affinity chromatography, and substrate mimics were synthesized by using solid-phase peptide synthesis (SPPS). [Results] Soluble Skyxy-TE was obtained and purified to homogeneity, and two substrate mimics 1 and 2 were synthesized by SPPS. Cyclization reactions were carried out in vitro, leading to the production of two cyclized peptides 3 and 4, whose structures were elucidated by NMR and HRESIMS analysis. [Conclusion] Skyxy-TE showed substrate promiscuity by catalyzing two substrate mimics to generate cyclization products, which facilitates the generation of more cyclized peptide analogues by chemoenzymatic synthesis in the future.

Abstract:[Background] Polycyclic tetramate macrolactams (PoTeMs), especially ikarugamycin and its analogs, display a wide range of antifungal, antibacterial, and cytotoxic activities. The cytochrome P450 CftA was shown to be an ikarugamycin oxidase according to the in vivo experiments, however, no in vitro evidence was available. [Objective] To study the biochemical function of ikarugamycin oxidase CftA. [Methods] CftA was purified by Ni-NTA affinity chromatography after the overexpression of synthetic gene cftA in Escherichia coli BL21(DE3), and was biochemically characterized by in vitro enzyme reaction and identification of reaction products through HPLC-HR-ESI-MS/MS. [Results] A new ikarugamycin derivative named hydroxyikarugamycin D and a known ikarugamycin derivative, clifednamide A, were observed after incubating CftA and ikarugamycin. [Conclusion] The in vitro evidence was provided to support that tandem oxidations on ikarugamycin was carried out by CftA at C29. This result paved the way for further researches to study the mechanism of ikarugamcin oxidases and the utilization to develop novel ikarugamycin type of PoTeMs.

Abstract:[Background] N-Methyl-L-phenylalanine, an N-alkylated aromatic amino acid, is a valuable chiral building block/intermediate/ingredient presented in many specialized metabolites that are very important in pharmaceutical, nutraceutical, and agrochemical industries. The synthesis and preparation of N-alkylated aromatic amino acids from aromatic α,β-unsaturated carboxylic acid remain challenging. [Objective] Herein we report a one-step biologically catalyzed N-methylamination of trans-cinnamic acid by LrPAL3, a phenylalanine ammonia lyase (PAL) from galanthamine-producing Lycoris radiata, to generate N-methyl-L-phenylalanine. [Methods] HPLC-DAD and HRESIMS analyses revealed that N-methyl-phenylalanine was produced when incubated trans-cinnamic acid and methylamine using the whole Escherichia coli BL21(DE3) cells expressing LrPAL3 as catalyst. [Results] The ¹H-NMR data and optical rotation of the enzymatic bioconversion product are in agreement with those of the authentic N-methyl-L-phenylalanine, which demonstrated the LrPAL3-catalyzed one-step regio- and enantioselective N-methylamination product of trans-cinnamic acid is N-methyl-L-phenylalanine. [Conclusion] This work provides an alternative biocatalyst for the asymmetric synthesis of valuable chiral N-methyl-L-phenylalanine. It paves a way to biologically synthesize N-alkylated amino acids through metabolic engineering and direct protein evolution of LrPAL3.

Abstract:Microbial natural products are important component of natural product derived drugs, and the excellent biological activities of natural products mainly depend on the pharmacophore groups with special structures. The biosynthesis of these special pharmacophores usually uses small molecules, like carbohydrates, amino acids and other simple primary metabolites. After specific and complex biosynthetic pathways, the simple metabolites were assembled to synthesize natural products with complex structures and diverse activities. Glutarimide containing natural products are an important class of natural products isolated from bacteria. As the potential lead compounds, they show excellent biological activity and have been developed as molecular probes. This article reviews the glutarimide containing natural products isolated from microorganisms and related biosynthetic pathways characterized in recent years, including iso-migrastatin, lactimidomycin, cycloheximide, streptimidone, gladiostatin, sesbanimide. Based on the biosynthetic study of glutarimide moiety, we expect to identify more glutarimide containing natural products through genome mining strategies.

Abstract:Microbial peptide secondary metabolites, which usually contain rare amino acids, are a series of natural products with rich chemical structures and biological activities. With the development of separation and purification technologies, a great number of new peptide products have been discovered, which play an important role in the exploration of microbial natural products. This paper reviews the microbial peptide metabolites from bacteria and fungi, as well as the new peptide products obtained by gene mining and other bioengineering methods in recent years. Combined with our team's research experiences of peptide products from entomopathogenic nematode symbiotic bacteria, this paper analyzes the existing problems and possible solutions in the research of microbial peptide products. We hope it could provide a reference for the further exploration and application of new active peptide products.

Abstract:Pyrrolizidine alkaloids (PAs) are widely distributed among higher plants, with over 650 PAs produced by more than 6 000 plants. Only a limited number of PAs of bacterial origins are discovered. For example, clazamycin A and B, reported by Umezawa in 1979 were among the firstly discovered bacterial PAs. In recent years, driven by microbial genomics and synthetic biology, the discovery and study of bacterial PAs and their biosynthetic mechanisms have made impressive progresses. To date, 12 types of PAs (~60) derived from bacteria have been identified, including bohemamines, azetidomonamides, and brabantamides, as well as polyene macrolactam ciromicins and heronamides containing PA structural units. The biosynthetic study of several PAs results in the identification of a unique pair of non-ribosomal peptide synthetase/Bayer-Villiger monooxygenase responsible for the formation of the pyrrolizidine skeleton. In contrast, the formation of the pyrrolizidine unit in β-amino acids polyene macrolactams might involve a highly diastereoselective electrocyclization process. Further, microbial genome mining reveals a lot of silent PAs biosynthetic gene clusters in bacteria, indicating that PAs play an important role in bacterial evolution and adaptation of their environment/hosts.

Abstract:Most of the antibiotics used to treat diseases are derived from natural products produced during microbial secondary metabolism. Given a strong cytotoxicity, the compound producers have to evolve effective self-resistance to avoid suicide when they synthesize these natural products. Herein, we review the recent research progress on DNA damage repair as a self-resistance strategy of natural product producing bacteria, especially those obtained from the base excision repair and nucleotide excision repair pathway in DNA damage antibiotics producers. Furthermore, the existing problems and the application perspective of the DNA damage repair mechanism in self-resistance are discussed.

Abstract:Streptomyces has great potential to produce novel natural products, but most of their biosynthetic gene clusters (BGCs) are silent or expressed in an extremely low level under laboratory conditions. Signal molecules are used to regulate phenotypic differentiation and biosynthesis of secondary metabolites. Manipulation of genes encoding the synthetases or receptors for signal molecules, or addition of exogenous signal molecules into the fermentation medium relieves the repression of signal molecule receptors on the expression of Streptomyces BGCs. This strategy can be used to activate silent BGCs to discover novel natural products or to increase the titer of known secondary metabolites. In this review we used γ-butyrolactones (GBLs) and γ-butenolides as examples to summarize the application of signal molecules in the discovery and titer improvement of Streptomyces secondary metabolites, to provide a reference for the development of microbial natural products.

Abstract:Many clinical medicines are derived from the microbial natural products of nonribosomal peptides or polyketide/nonribosomal peptide hybrids. The international literatures about nonribosomal peptides on Web of Science in the past five years were analyzed by bibliometric and statistical methods. Our results here show the hot topics and the development trend on the research field about nonribosomal peptides.