Cyclic lipopeptides (CLPs), produced by diverse bacteria, including plant-associated Pseudomonas species, are bioactive metabolites with multiple functions that also benefit the host strain. Strain YL19, identified as Pseudomonas extremorientalis, showed strong antifungal activity against Bipolaris sorokiniana, the causal agent of wheat root rot. Its main secondary metabolite was purified through bioactivity-guided fractionation and identified as massetolide, marking the first report of its production by P. extremorientalis. In wheat pot experiments, the 100 mg massetolide F treatment reduced the disease index to 42.05, significantly lower than the diseased control. Massetolide F also displayed antitumor activity, with IC50 values of 24 and 22 μg/mL against HeLa and HT1080 cells. Biosynthesis of massetolde F follows the classical Gac/Rsm pathway, but novel repressor of secondary metabolism (Rsm) binding sites were found in the 5'-UTR of luxRU and luxRD mRNA and LuxR regulators recognized unique promoter sequences unreported in other Pseudomonas that were identified, indicating a species-specific regulatory mechanism in YL19.

Serratia marcescens is an opportunistic pathogen with increasing drug resistance. Serratia surfactantfaciens sp. nov. YD25T, a novel strain with multidrug resistance, shares similarities with S. marcescens. This study investigated the resistance mechanisms of YD25, with a focus on efflux pumps and their regulators. Bioinformatics analysis revealed numerous resistance genes in YD25, including five RND-type efflux pumps, SdeAB, SdeCDE, SdeGH, SdePQ-OmsA, and SdeXY. Oxyresveratrol, evernic acid, and morin were identified as efflux pump inhibitors, with oxyresveratrol showing the strongest effect targeting SdeXY and sensitizing YD25 to seven antibiotics. Among the RND pumps, SdeXY and SdeCDE are constitutively expressed and are regulated negatively by AcrR and positively by BaeSR, respectively. YD25 harbors four global regulators, including RamA, SoxS, Rob, and a novel Roblike protein. The deletion of these genes downregulated SdeXY and SdeCDE, increasing antibiotic susceptibility and confirming their role as positive regulators. Rob and Roblike exhibited functional overlap. This study elucidates the RND efflux pumps and their regulators in YD25, advancing the understanding of Serratia resistance mechanisms. Oxyresveratrol, a newly identified SdeXY inhibitor, offers potential for combination therapies against drug-resistant Serratia.IMPORTANCEThis study reveals novel insights into Serratia surfactantfaciens YD25's multidrug resistance, identifying RND efflux pumps (e.g., SdeXY and SdeCDE) as key drivers. The key discoveries include the following: (i) oxyresveratrol, a natural compound functioning as an SdeXY efflux pump inhibitor, effectively reverses bacterial resistance, thereby restoring the clinical utility of antibiotics that had been rendered nearly obsolete by drug resistance, and (ii) YD25 uniquely possesses two Rob regulators (Rob and Roblike). Despite their different origins, both positively regulate RND pump function-a finding reported for the first time in bacteria. These findings highlight the potential of natural efflux pump inhibitors against Serratia infections while uncovering an unusual regulatory redundancy in bacterial resistance.

In the special marine environment characterized by high salinity, high osmotic pressure, and oligotrophy, marine actinomycetes have evolved unique survival strategies. Some of their secondary metabolites, with novel structures and potent biological activities, hold significant research and application value in pharmaceutical development. Under conventional laboratory culture conditions, only 20% of the biosynthetic gene clusters (BGCs) in marine actinomycetes are expressed, and a large number of silent BGCs urgently need to be explored. This paper systematically reviews the silencing mechanisms and activation strategies of silent BGCs in marine actinomycetes, comprehensively summarizes recent research advances in physical activation, chemical activation, and bioengineering technologies, and deeply analyzes the advantages and limitations of various activation methods. The silencing of BGCs often resulted from the combined effects of multiple factors. Therefore, this paper proposes a multi-technology combination strategy for activating silent BGCs. This strategy simulates the original survival environment of microorganisms from multiple aspects or constructs specific expression environments for gene clusters to promote and induce the expression of silent BGCs at multiple levels, thereby obtaining novel secondary metabolites. This strategy provides theoretical references and technical pathways for exploring the metabolic potential of marine actinomycetes and promoting the discovery of new natural products.

Streptomyces sp. MJM3502 is a promising producer of rufomycins, which are a class of potent anti-tuberculosis lead compounds. Although the structure, activity, and mechanism of the main rufomycin 4/6 and its analogs have been extensively studied, a significant gap remains in our understanding of the genome sequence and biosynthetic pathway of Streptomyces sp. MJM3502, and its metabolic engineering has not yet been reported. This study established the genetic manipulation platform for the strain. Using CRISPR/Cas9-based technology to in-frame insert the strong kasO∗p promoter upstream of the rufB and rufS genes of the rufomycin BGC, we increased rufomycin 4/6 production by 4.1-fold and 2.8-fold, respectively. Furthermore, designing recombinant strains by inserting the kasO∗p promoter upstream of the biosynthetic genes encoding cytochrome P450 enzymes led to new rufomycin derivatives. These findings provide the basis for enhancing the production of valuable natural compounds in Streptomyces and offer insights into the generation of novel active natural products via synthetic biology and metabolic engineering.

Ginsenoside F2 is a protopanaxadiol saponin compound with various biological activities, including antioxidant, anti-inflammatory, and anticancer properties. Ginsenoside F2 can be found in ginseng, but in low quantities. Therefore, ginsenoside F2 production predominantly relies on the biotransformation of various ginsenosides, such as ginsenosides Rb1 and Rd. In this study, we reported the production of ginsenoside F2 by gypenoside biotransformation with Aspergillus niger JGL8, isolated from Gynostemma pentaphyllum. Ginsenoside F2 could be produced by two different biotransformation pathways, namely Gyp-V-Rd-F2 and Gyp-XVII-F2. The product exhibited antioxidant activity against free radicals (DPPH) with IC50 value of 29.54 µg/mL. Optimal biotransformation conditions were a pH of 5.0, temperature of 40 °C, and 2 mg/mL of substrate. Enzyme kinetic parameters revealed that the hydrolysis rate of Gyp-V, Rd, and Gyp-XVII was 0.625, 0.588, and 0.417 mM/h, respectively. In conclusion, we demonstrated that gypenoside is a substitutable substrate for ginsenoside F2 biotransformation.

ABSTRACT Small open reading frames (smORFs) are widely distributed in various living organisms. However, their functions remain largely unexplored. In addition, annotation and detection of smORFs are limited using existing methods and hindered by their specific properties. In this study, we systematically investigated smORFs and smORF-encoded peptides (SEPs) in Streptomyces, which are well-known bacterial producers of diverse bioactive secondary metabolites. We established a peptidogenomic workflow based on multi-integrated comprehensive database search and database-independent de novo sequencing to identify smORFs in Streptomyces xinghaiensis NRRL B-24674T (S187). In addition, we described SEPome related to the secondary metabolism, which include 68 novel SEPs and 79 common smORFs with Streptomyces coelicolor A3 (2). Functional analysis of universal smORFs revealed enrichment in biosynthetic processes, stress response, ribosomes, and nucleic acid binding. Meanwhile, 5 Cryptic smORF-encoded Peptides (CSEPs) distributed in non-annotated regions of the genome, and non-coding RNAs could encode for CSEPs. A total of 66 new RNAs, including 32 non-coding RNAs (ncRNAs) were revealed, and 4 ncRNA-encoded peptides were identified. Furthermore, an investigation of carbon metabolism showed that NagE functions in spore formation and secondary metabolism in Streptomyces. Particularly, NagE was observed to function in the biosynthesis of anti-complement agents in S. xinghaiensis, suggesting a novel role of the phosphoenolpyruvate phosphotransferase system in microbial secondary metabolism. We thus provide an effective strategy for analyzing public data sets of model strains to identify smORFs for non-model species. The ncRNAs and SEPs present rich sources for engineering streptomycetes to produce bioactive compounds. Importance Due to their small size and special chemical features, small open reading frame (smORF)-encoding peptides (SEPs) are often neglected. However, they may play critical roles in regulating gene expression, enzyme activity, and metabolite production. Studies on bacterial microproteins have mainly focused on pathogenic bacteria, which are importance to systematically investigate SEPs in streptomycetes and are rich sources of bioactive secondary metabolites. Our study is the first to perform a global identification of smORFs in streptomycetes. We established a peptidogenomic workflow for non-model microbial strains and identified multiple novel smORFs that are potentially linked to secondary metabolism in streptomycetes. Our multi-integrated approach in this study is meaningful to improve the quality and quantity of the detected smORFs. Ultimately, the workflow we established could be extended to other organisms and would benefit the genome mining of microproteins with critical functions for regulation and engineering useful microorganisms.

补体系统是先天免疫系统的重要组分之一,类风湿关节炎、自身免疫性溶血性贫血、2019冠状病毒病等多种疾病的发生与补体系统异常密切相关.目前临床常用的补体抑制剂多为化学合成药物,其选择性较差,长期使用容易导致机体免疫能力降低.天然产物来源的抗补体活性成分毒性小、易于被机体消化吸收,其中微生物来源的抗补体活性物质具有独特的优势和应用潜力,可利用基因组挖掘快速发现和鉴定,并利用代谢工程改造和发酵优化大量生产,但目前相关研究仍处于前期研究开发阶段.本综述总结了近年来国内外较常见补体抑制剂的临床应用,同时对微生物来源抗补体活性物质的研发进展进行了讨论,以期为补体抑制剂的临床研究和天然来源新型抗补体活性物质的开发提供参考.

随着信息化手段的不断丰富,新型教育理念结合线上学习平台的新信息化教学模式成为高校课堂的改革新趋势.本次教学改革利用科学(Science)、技术(Technology)、工程(Engineering)、艺术(Art)和数学(Mathematics)多学科融合的超学科教育理念(简称STEAM教育)对教师教学过程进行了整体设计,同时借助"线上+线下"教学平台对学生学习过程进行了全面优化.将原本分散的验证型、操作型实验重新整合串联成以多角度"项目式"任务为主线、以Blackboard线上平台为辅线的自主研究型实验项目.新型教学模式以学生为主体,给学生提供更多 自我展示和讨论互动的平台.从学生的课堂表现、知识测验、课后反馈、实验操作及实验报告4个方面对新型模式下的教学效果进行了分析和评价.结果表明,此模式不仅提高了学生在微生物学实验中的学习质量,增强了其学习主观能动性,而且有利于培养和提升学生的问题探究及实践创新能力.这一新型教学模式对其他生物学科实验课程的教学具有一定的借鉴意义.

海洋放线菌S187的代谢产物具有较好的抗补体活性.以抗补体活性为评价指标,在单因素实验的基础上,采用正交实验优化菌株S187产抗补体活性物质的发酵工艺.确定最佳发酵工艺为:可溶性淀粉20 g·L-1,大豆粉35 g·L-1,(NH4)2SO42g·L-1,NaCl2g·L-1,K2HPO40.5g·L-1,CaCO35g·L-1,初始pH值7.5,于28℃、220r·min-1下发酵7 d.在此条件下,菌株S187代谢产物的抗补体活性为69.35％.

Ecumicins are potent antituberculosis natural compounds produced by the rare actinomycete Nonomuraea sp. MJM5123. Here, we report an efficient genetic manipulation platform of this rare actinomycete. CRISPR/Cas9-based genome editing was achieved based on successful sporulation. Two genes in the ecumicin gene cluster were further investigated, ecuN and ecuE, which potentially encode a pretailoring cytochrome P450 hydroxylase and the core peptide synthase, respectively. Deletion of ecuN led to an enhanced ratio of the ecumicin compound EcuH16 relative to that of EcuH14, indicating that EcuN is indeed a P450 hydroxylase, and there is catalyzed hydroxylation at the C-3 position in unit 12 phenylalanine to transform EcuH16 to the compound EcuH14. Furthermore, promoter engineering of ecuE by employing the strong promoter kasO*P was performed and optimized. We found that integrating the endogenous ribosome-binding site (RBS) of ecuE together with the RBS from kasO*P led to improved ecumicin production and resulted in a remarkably high EcuH16/EcuH14 ratio. Importantly, production of the more active component EcuH16 was considerably increased in the double RBSs engineered strain EPR1 compared to that in the wild-type strain, reaching 310 mg/L. At the same time, this production level was 2.3 times higher than that of the control strain EPA1 with only one RBS from kasO*P. To the best of our knowledge, this is the first report of genome editing and promoter engineering on the rare actinomycete Nonomuraea.

Genome sequences of marine streptomycetes are valuable for the discovery of useful enzymes and bioactive compounds by genome mining. However, publicly available complete genome sequences of marine streptomycetes are still limited. Here, we present the complete genome sequence of a marine streptomyceteStreptomycessp. S063 CGMCC 14582. Species delineation based on the pairwise digital DNA-DNA hybridization and genome comparison ANI (average nucleotide identity) value showed thatStreptomycessp. S063 CGMCC 14582 possesses a unique genome that is clearly different from all of the other available genomes. Bioactivity tests showed thatStreptomycessp. S063 CGMCC 14582 produces metabolites with anti-complement activities, which are useful for treatment of numerous diseases that arise from inappropriate activation of the human complement system. Analysis of the genome reveals no biosynthetic gene cluster (BGC) which shows even low similarity to that of the known anti-complement agents was detected in the genome, indicating thatStreptomycessp. S063 CGMCC 14582 may produce novel anti-complement agents of microbial origin. Four BGCs which are potentially involved in biosynthesis of non-ribosomal peptides were disrupted, but no decrease of anti-complement activities was observed, suggesting that these four BGCs are not involved in biosynthesis of the anti-complement agents. In addition, LC-MS/MS analysis and subsequent alignment through the Global Natural Products Social Molecular Networking (GNPS) platform led to the detection of novel peptides produced by the strain.Streptomycessp. S063 CGMCC 14582 grows rapidly and is salt tolerant, which benefits efficient secondary metabolite production via seawater-based fermentation. Our results indicate thatStreptomycessp. S063 has great potential to produce novel bioactive compounds, and also is a good host for heterologous production of useful secondary metabolites for drug discovery.

Marine actinobacterium Streptomyces xinghaiensis NRRL B-24674T has been characterized as a novel species, but thus far, its biosynthetic potential remains unexplored. In this study, the high-quality genome sequence of S. xinghaiensis NRRL B-24674T was obtained, and the production of anticomplement agents, xiamycin analogs, and siderophores was investigated by genome mining. Anticomplement compounds are valuable for combating numerous diseases caused by the abnormal activation of the human complement system. The biosynthetic gene cluster (BGC) nrps1 resembles that of complestatins, which are potent microbial-derived anticomplement agents. The identification of the nrps1 BGC revealed a core peptide that differed from that in complestatin; thus, we studied the anticomplement activity of this strain. The culture broth of S. xinghaiensis NRRL B-24674T displayed good anticomplement activity. Subsequently, the disruption of the genes in the nrps1 BGC resulted in the loss of anticomplement activity, confirming the involvement of this BGC in the biosynthesis of anticomplement agents. In addition, the mining of the BGC tep5, which resembles that of the antiviral pentacyclic indolosesquiterpene xiamycin, resulted in the discovery of nine xiamycin analogs, including three novel compounds. In addition to the BGCs responsible for desferrioxamine B, neomycin, ectoine, and carotenoid, 18 BGCs present in the genome are predicted to be novel. The results of this study unveil the potential of S. xinghaiensis as a producer of novel anticomplement agents and provide a basis for further exploration of the biosynthetic potential of S. xinghaiensis NRRL B-24674T for the discovery of novel bioactive compounds by genome mining.

抗补体活性物质研究对开发相关药物、治疗多种补体过度或非正常激活引起的疾病具有重要意义,然而目前微生物来源的抗补体活性物质研究还非常有限.利用溶血法对从大连星海湾海泥样品中分离出来的42株海洋放线菌进行抗补体活性测试,并利用C18柱色谱层析及Sephadex LH-20凝胶柱层析,对具有较好抗补体活性的菌株DUTll的次生代谢产物进行分离纯化和结构鉴定.结果显示:菌株S187、M5、S088、M8、S063、DUT 11和MD16在TSB培养基中发酵后发酵液提取物和菌丝体提取物均具有较好的抗补体活性,其中菌株DUT11的抗补体活性为最强.DUTll在M33培养基中发酵后所产生的次生代谢产物的抗补体活性最强,其发酵液和菌丝体提取物的活性分别为56.5％和60.8％.进一步分离得到3个具有微弱抗补体活性的小分子化合物,分别鉴定为3-吲哚甲酸(1)、对羟基苯甲酸(2)和2-吡咯甲酸(3).本研究表明在大连星海湾海泥样品中存在多种可产生抗补体活性物质的海洋链霉菌,产生的抗补体活性化合物活性较强,结果可为进一步开发利用海洋放线菌提供新的思路,为研究新型抗补体活性化合物奠定基础.

Marine actinobacteria are potential producers of various secondary metabolites with diverse bioactivities. Among various bioactive compounds, anti-complement agents have received great interest for drug discovery to treat numerous diseases caused by inappropriate activation of the human complement system. However, marine streptomycetes producing anti-complement agents are still poorly explored. In this study, a marine-derived strain Streptomyces sp. DUT11 showing superior anti-complement activity was focused, and its genome sequence was analyzed. Gene clusters showing high similarities to that of tunicamycin and nonactin were identified, and their corresponding metabolites were also detected. Subsequently, tunicamycin I, V, and VII were isolated from Streptomyces sp. DUT11. Anti-complement assay showed that tunicamycin I, V, VII inhibited complement activation through the classic pathway, whereas no anti-complement activity of nonactin was detected. This is the first time that tunicamycins are reported to have such activity. In addition, genome analysis indicates that Streptomyces sp. DUT11 has the potential to produce novel lassopeptides and lantibiotics. These results suggest that marine Streptomyces are rich sources of anti-complement agents for drug discovery.

Serratia sp. YD25 (KCTC 42987) was originally isolated from rhizosphere soil in a continuous cropping tobacco-planting farm. Here, we show that its metabolites efficiently suppress the growth of various important pathogenic fungi and bacteria, causing infection in both plants and humans. In addition, Serratia sp. YD25 has a special trait of simultaneous production of both serrawettin W2 and prodigiosin, two important bioactive secondary metabolites produced by Serratia strains. Such co-production has not been reported in other Serratia strains. The complete genome sequence of Serratia sp. YD25 is presented, which is valuable for further exploration of its biotechnological applications in agriculture and medicine. The genome sequence reported here is also useful for understanding the unique regulatory mechanisms underlying biosynthesis of active compounds.

Background: Gram-negative bacteria of the genus Serratia are potential producers of many useful secondary metabolites, such as prodigiosin and serrawettins, which have potential applications in environmental bioremediation or in the pharmaceutical industry. Several Serratia strains produce prodigiosin and serrawettin W1 as the main bioactive compounds, and the biosynthetic pathways are co-regulated by quorum sensing (QS). In contrast, the Serratia strain, which can simultaneously produce prodigiosin and serrawettin W2, has not been reported. This study focused on analyzing the genomic sequence of Serratia sp. strain YD25(T) isolated from rhizosphere soil under continuously planted burley tobacco collected from Yongding, Fujian province, China, which is unique in producing both prodigiosin and serrawettin W2.Results: A hybrid polyketide synthases (PKS)-non-ribosomal peptide synthetases (NRPS) gene cluster putatively involved in biosynthesis of antimicrobial serrawettin W2 was identified in the genome of YD25(T), and its biosynthesis pathway was proposed. We found potent antimicrobial activity of serrawettin W2 purified from YD25(T) against various pathogenic bacteria and fungi as well as antitumor activity against Hela cells. Subsequently, comparative genomic analyses were performed among a total of 133 Serratia species. The prodigiosin biosynthesis gene cluster in YD25(T) belongs to the type I pig cluster, which is the main form of pig-encoding genes existing in most of the pigmented Serratia species. In addition, a complete autoinducer-2 (AI-2) system (including luxS, lsrBACDEF, lsrGK, and lsrR) as a conserved bacterial operator is found in the genome of Serratia sp. strain YD25(T). Phylogenetic analysis based on concatenated Lsr and LuxS proteins revealed that YD25T formed an independent branch and was clearly distant from the strains that solely produce either prodigiosin or serrawettin W2. The Fe (III) ion reduction assay confirmed that strain YD25(T) could produce an AI-2 signal molecule. Phylogenetic analysis using the genomic sequence of YD25(T) combined with phylogenetic and phenotypic analyses support this strain as a member of a novel and previously uncharacterized Serratia species.Conclusion: Genomic sequence and metabolite analysis of Serratia surfactantfaciens YD25(T) indicate that this strain can be further explored for the production of useful metabolites. Unveiling the genomic sequence of S. surfactantfaciens YD25(T) benefits the usage of this unique strain as a model system for studying the biosynthesis regulation of both prodigiosin and serrawettin W2 by the QS system.

The intestinal microbiota plays an important role in maintaining the health of its host, including human and nonhuman primates. Little is known about the intestinal bacterial composition of the Sichuan snub-nosed monkey ( Rhinopithecus roxellana ), which has been classified as Endangered on the International Union for Conservation of Nature Red List since 2003. We evaluated the fecal bacterial compositions of 11 Sichuan snub-nosed monkeys, including six young captive individuals (one sample from each), three adult captive individuals (four samples each), and two adult provisioned free-ranging individuals (four samples each). We also quantified fecal Bacteroides vulgatus , Bifidobacterium spp., and Lactobacillus spp., which are defined as probiotics in humans, using real-time polymerase chain reaction. We identified five major phyla in the collected samples, including Firmicutes (32.4 %), Bacteroidetes (14.7 %), Verrucomicrobia (8.8 %), Actinobacteria (4.4 %), and unclassified microbacteria (39.7 %). Fecal bacteria composition varied with age and different seasons. The fecal bacterial composition of the captive monkeys was less variable than that of provisioned free-ranging monkeys. B. vulgatus amounts were almost 100 times higher in the provisioned free-ranging monkeys (10 12 ) than in the captive monkeys (10 10 ). Our results provide an initial catalogue of gut microbiota in the Sichuan snub-nosed monkey, which helps to enrich our knowledge of gut microbiota in nonhuman primates.

The primers at C1 and Te region of NRPS gene were designed respectively and PCR technology was employed to find Pseudomonas fluorescens carrying cyclic lipopeptide genes from soil samples of Fujian and Shaanxi province.Then SSCP and Restriction endonuclease method were carried out to analyze the diversity of NRPS genes.Results show that the C1 and Te regions of Fujian and Shaanxi bacterial strains display 3,5,7three kind bands by SSCP electrophoresis and the positions of bands are different.C1 region has 10 types,and Te region has 10 types in total.Among them Fujian strains have 9types except SSCP-10,while Shaanxi strains have 8types except SSCP-4and SSCP-2.Cutting Te region of NRPS genes by two different restriction enzymes HinfⅠ and SphⅠ,the digestion products of Fujian strains are divided into 15 kinds,while Shaanxi strains are divided into 11 kinds.It shows that NRPS gene has a variety of forms,and the types of distribution in different regions vary.In conclusion,the NRPS genes in Fujian strains have more diversity which suggests that these strains have more potential to produce new types of lipopeptide.