后基因组时代基因组重排在微生物菌种选育中的应用及展望

基因组重排作为一种实用高效的育种技术,在缺乏遗传背景认知和可操作遗传体系等条件下,可以突破微生物种属间的限制,经过多轮递推的原生质体融合来加速其人工定向进化,在微生物菌种改良及代谢产物开发和产业化等研究领域得到了广泛应用。步入后基因组时代,快速发展的组学和生物信息学使基因组重排成为连接各种微生物育种方法的重要纽带,为我们深入探索微生物复杂的代谢网络和全局调控机制,更为精准地实施对微生物的人工调控和定向进化提供了契机。本文系统性地回顾了近年来基因组重排在微生物菌种选育中的应用研究,尤其针对围绕其开展的组学研究进行了详细阐述,并对基因组重排与组学、生物信息学和合成生物学等新兴技术的联合应用进行了展望。     

The re-emerging role of microbial natural products in antibiotic discovery

New classes of antibacterial compounds are urgently needed to respond to the high frequency of occurrence of resistances to all major classes of known antibiotics. Microbial natural products have been for decades one of the most successful sources of drugs to treat infectious diseases but today, the emerging unmet clinical need poses completely new challenges to the discovery of novel candidates with the desired properties to be developed as antibiotics. While natural products discovery programs have been gradually abandoned by the big pharma, smaller biotechnology companies and research organizations are taking over the lead in the discovery of novel antibacterials. Recent years have seen new approaches and technologies being developed and integrated in a multidisciplinary effort to further exploit microbial resources and their biosynthetic potential as an untapped source of novel molecules. New strategies to isolate novel species thought to be uncultivable, and synthetic biology approaches ranging from genome mining of microbial strains for cryptic biosynthetic pathways to their heterologous expression have been emerging in combination with high throughput sequencing platforms, integrated bioinformatic analysis, and on-site analytical detection and dereplication tools for novel compounds. These different innovative approaches are defining a completely new framework that is setting the bases for the future discovery of novel chemical scaffolds that should foster a renewed interest in the identification of novel classes of natural product antibiotics from the microbial world.     

新方法新技术与新型抗生素发现

日趋严峻的细菌耐药性问题给“抗生素传奇时代”带来了严重威胁, 传统的抗菌药物开发手段已很难应对, 因此急需新思路来寻找新型抗生素。近年来, 一些新方法新技术如新型筛选模型的构建、基因组挖掘技术以及组合生物合成等给天然产物的筛选引发了革命性的变革, 也为放线菌来源的新型抗生素的发现带来了新的希望。本文将对这些新技术及其在新型抗生素研发中的应用进展进行阐述。     

Toward steadfast growth of antibiotic research in China: From natural products to engineered biosynthesis

Antibiotics are widely used for clinical treatment and preventing or curing diseases in agriculture. Cloning and studies of their biosynthetic gene clusters are vital for yield enhancement and engineering new derivatives with new and prominent activities. In recent years, research in this aspect is impressively active in China. This article reviews biosynthetic progress on 28 antibiotics, including polyketides, nonribosomal peptides, hybrid polyketide-nonribosomal peptides, peptidyl nucleoside, nucleoside, and others. Their biosynthetic mechanisms were disclosed, and their derivatives with new structures/activities were obtained by gene inactivation, mutasynthesis and combinatorial biosynthesis.     

放线菌天然产物糖基化改造研究进展

放线菌可以产生结构多样的天然产物, 其中包括很多重要的抗菌和抗肿瘤药物。糖基化修饰在天然产物中广泛存在, 糖基侧链的变化往往会影响天然产物的生物活性。本文综述了放线菌来源天然产物糖基化改造的研究进展。糖基侧链改造的方法主要分为体内基因工程和体外酶学法。运用这两种方法已经成功对多种天然产物进行了糖基侧链改造, 获得了大量带有新糖基修饰的天然产物, 其中有些生物活性得以提高。天然产物糖基侧链改造为新药开发提供了一个重要的途径。     

植物天然产物微生物重组合成研究进展

植物天然产物是小分子药物、营养品、化妆品、香精香料等的主要来源之一,在国民经济中发挥重要的作用。目前植物天然产物主要依赖于植物提取,这种生产方式占用耕地、生长周期长,而且植物活性成分往往含量低、生产成本高。通过解析植物天然产物生物合成途径,在微生物细胞中重构,创建细胞工厂,实现利用可再生原料发酵合成,为植物天然产物的供给提供了新的路线。本文重点介绍了中国科学院天津工业生物技术研究所在萜类、黄酮类、苯丙素类等重要类型植物天然产物微生物重组合成方面的研究进展,简要探讨了当前研究面临的挑战及未来前景。     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

Phylogeny-guided (meta)genome mining approach for the targeted discovery of new microbial natural products

Genomics-based methods are now commonplace in natural products research. A phylogeny-guided mining approach provides a means to quickly screen a large number of microbial genomes or metagenomes in search of new biosynthetic gene clusters of interest. In this approach, biosynthetic genes serve as molecular markers, and phylogenetic trees built with known and unknown marker gene sequences are used to quickly prioritize biosynthetic gene clusters for their metabolites characterization. An increase in the use of this approach has been observed for the last couple of years along with the emergence of low cost sequencing technologies. The aim of this review is to discuss the basic concept of a phylogeny-guided mining approach, and also to provide examples in which this approach was successfully applied to discover new natural products from microbial genomes and metagenomes. I believe that the phylogeny-guided mining approach will continue to play an important role in genomics-based natural products research.     

抗生素抗性基因的生态风险研究进展

自抗生素被发现和使用以来,其在人类和动物疾病预防与治疗、提高动物生产等方面均发挥了重要作用。但抗生素的批量生产及大量应用,特别是在养殖业和临床医疗上的滥用,导致抗生素抗性基因(ARGs)在环境中普遍存在,其借助质粒、转座子、整合子等可移动元件通过接合、转座、转化等方式在环境中广泛传播,导致微生物药性不断增强,对人类健康和生态安全造成严重威胁。当前,ARGs对人类健康的影响已受到高度关注,但有关ARGs在环境中的生态风险研究还相对薄弱。本文综述了ARGs污染的现状及其生态风险,并对该领域中未来研究重点进行了展望,以期为今后抗性基因的研究和生态防控提供参考。     

Virulence-associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach

The bovine fecal microbiota impacts human food safety as well as animal health. Although the bacteria of cattle feces have been well characterized using culture-based and culture-independent methods, techniques have been lacking to correlate total community composition with community function. We used high throughput sequencing of total DNA extracted from fecal material to characterize general community composition and examine the repertoire of microbial genes present in beef cattle feces, including genes associated with antibiotic resistance and bacterial virulence. Results suggest that traditional 16S sequencing using “universal” primers to generate full-length sequence may under represent Acitinobacteria and Proteobacteria. Over eight percent (8.4%) of the sequences from our beef cattle fecal pool sample could be categorized as virulence genes, including a suite of genes associated with resistance to antibiotic and toxic compounds (RATC). This is a higher proportion of virulence genes found in Sargasso sea, chicken cecum, and cow rumen samples, but comparable to the proportion found in Antarctic marine derived lake, human fecal, and farm soil samples. The quantitative nature of metagenomic data, combined with the large number of RATC classes represented in samples from widely different habitats indicates that metagenomic data can be used to track relative amounts of antibiotic resistance genes in individual animals over time. Consequently, these data can be used to generate sample-specific and temporal antibiotic resistance gene profiles to facilitate an understanding of the ecology of the microbial communities in each habitat as well as the epidemiology of antibiotic resistant gene transport between and among habitats.     

甲基转移酶在微生物合成天然产物中的应用

甲基转移酶(Methyltransferases,MTs)普遍存在于所有生物有机体中,通常以S-腺苷甲硫氨酸作为甲基供体催化底物的甲基化反应,在基因的表达调控和许多天然化合物的合成中起着至关重要的作用。近年来,在微生物中异源表达MTs以实现一些重要天然产物的生物合成取得了巨大的进步,但迄今为止这方面的研究还没有得到详细和全面的总结。文中综述了MTs在微生物合成苯丙烷类化合物、香料类化合物、激素和抗生素等重要天然产物的最新研究进展,重点阐述了应用代谢工程策略高效合成这些甲基化的天然产物,以及利用MTs拓展天然产物分子多样性的研究进展。最后,探讨了MTs应用于微生物合成天然产物所面临的挑战,并对利用MTs进一步高效生产结构和生物活性多样化的天然产物进行了展望。     

基于全基因组关联分析研究粪肠球菌发酵食品分离株的耐药性

【目的】研究15株分离自自然发酵食品的粪肠球菌(Enterococcus faecalis)和1株粪肠球菌模式株对15种(1种β-内酰胺类和14种非β-内酰胺类)抗生素的耐药性,并通过菌株耐药表型与基因型的关联分析找到粪肠球菌潜在的耐药基因。【方法】利用微量肉汤稀释法检测试验菌株对15种抗生素的耐药性,运用Scoary软件进行表型与基因型的关联分析。【结果】16株粪肠球菌对卡那霉素、万古霉素、利奈唑胺和红霉素100%敏感;对其余11种抗生素均表现出不同程度的耐药,其中对克林霉素为100%耐药。基因组关联分析发现了9个功能基因与5种抗生素(氯霉素、环丙沙星、甲氧苄啶、新霉素和四环素)存在显著相关关系,其中基因FAM000296和FAM005768与氯霉素、甲氧苄啶和环丙沙星的耐药性有关。进一步分析发现基因FAM000296和FAM005768同被注释为Sec G,但基因FAM005768与基因FAM000296相比在3′端丢失了21个碱基。【结论】分离自自然发酵食品的粪肠球菌对多种抗生素具有耐药性,其基因组中携带有潜在的耐药基因。因此,对分离自自然发酵食品的粪肠球菌需要进行全面的安全性评估后方可考虑应用。     

后基因组时代的真菌天然产物发现

真菌产生的次级代谢产物是新药发现的重要来源之一,然而近年来传统的真菌天然产物发现方法在大量真菌基因组测序完成的时代遇到了很大的挑战。如何利用这些基因组数据来发现真菌中新的天然产物已成为后基因组时代天然产物发现的研究重点和热点。本综述先后介绍了真菌天然产物的类型及其相应基因簇和骨架酶的特征,基于基因组挖掘技术发展而来的天然产物发现新策略,以及利用合成生物学理念和技术在真菌天然产物发现中的应用现状,最后展望了后基因组时代中的天然产物发现研究前沿及基因组数据在后基因组时代对真菌天然产物发现的应用前景。     

Evolution of Streptomyces pristinaespiralis for resistance and production of pristinamycin by genome shuffling

Improvement of pristinamycin production by Streptomyces pristinaespiralis was performed by using recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-mug/ml pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/l, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable. It is concluded that genome shuffling improves the production of pristinamycin by enhancing product-resistance in a stepwise manner. Pristinamycin fermentation experiments by recombinant G 4-17 were carried out in a 5-l fermentor, and its production of pristinamycin reached 0.90 g/l after 60 h of fermentation.     

宏基因组学在微生物抗生素抗性基因检测中的应用

抗生素广泛应用于人类和动物疾病的治疗等过程中。不合理利用和滥用抗生素导致耐药细菌、抗性基因的产生和传播。宏基因组学能够分析不同环境中抗生素抗性基因的多样性,并且完善目前已有的或构建新的宏基因组文库,从而为将来进行基因比对提供有力的参考。本文将综述宏基因组学在人类、动物和环境中微生物抗生素抗性基因检测的应用,以期为未来评估抗性基因风险和解决抗生素耐药性问题提供技术支持。     

常见变质水性工业产品中的微生物种类和耐药性分析

为了科学治理常见变质水性工业产品中的腐败微生物,对其进行分离、鉴定和分类,同时以卡松、布罗波尔、甲基异噻唑啉酮和苯并异噻唑啉酮四种杀菌防腐剂对6种标准细菌菌株的最小抑菌浓度(MIC)作为耐药性标准,对腐败细菌进行耐药性评估分析.结果显示,日化用品变质样中革兰氏阴性细菌约占80.00％(克雷伯氏菌属、假单胞菌属、伯克霍尔...     

Recent advances in the chemoenzymatic synthesis of bioactive natural products

The field of organic chemistry has recently witnessed a rapid rise in the use of chemoenzymatic strategies for the synthesis of complex molecules. Under this paradigm, biocatalytic methods and contemporary synthetic methods are used synergistically in a multistep approach toward a target molecule. In light of the unparalleled regioselectivity and stereoselectivity of enzymatic transformations and the reaction diversity of contemporary organic chemistry, chemoenzymatic strategies hold enormous potential for streamlining access to important bioactive molecules. This review covers recent demonstrations of chemoenzymatic approaches in chemical synthesis, with special emphasis on the preparation of medicinally relevant natural products.     

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

Due to the worldwide prevalence of multidrug-resistant pathogens and high incidence of diseases such as cancer, there is an urgent need for the discovery and development of new drugs. Nearly half of the FDA-approved drugs are derived from natural products that are produced by living organisms, mainly bacteria, fungi, and plants. Commercial development is often limited by the low yield of the desired compounds expressed by the native producers. In addition, recent advances in whole genome sequencing and bioinformatics have revealed an abundance of cryptic biosynthetic gene clusters within microbial genomes. Genetic manipulation of clusters in the native host is commonly used to awaken poorly expressed or silent gene clusters, however, the lack of feasible genetic manipulation systems in many strains often hinders our ability to engineer the native producers. The transfer of gene clusters into heterologous hosts for expression of partial or entire biosynthetic pathways is an approach that can be used to overcome this limitation. Heterologous expression also facilitates the chimeric fusion of different biosynthetic pathways, leading to the generation of “unnatural” natural products. The genus Streptomyces is especially known to be a prolific source of drugs/antibiotics, its members are often used as heterologous expression hosts. In this review, we summarize recent applications of Streptomyces species, S. coelicolor, S. lividans, S. albus, S. venezuelae and S. avermitilis, as heterologous expression systems.