反向筛选标记基因upp在杀真菌链霉菌遗传改造中的应用

为进一步简化放线菌的遗传操作流程,缩短重组菌株的筛选周期,在对杀真菌链霉菌(Streptomyces fungicidicus ATCC 21013)进行遗传操作的过程中引入了一个反向筛选标记基因——尿嘧啶磷酸核糖转移酶基因(upp)。并通过原始菌株中upp基因的敲除,以及带有upp基因的自杀型基因敲除载体的构建,开发了一套完整的针对杀真菌链霉菌的无痕敲除系统。通过载体的整合、二次交换及反向筛选实现了杀真菌链霉菌基因组中StrR基因的快速无痕敲除。upp反向筛选标记基因的引入使得链霉菌重组菌株的平均筛选周期缩短了2周左右,并进一步减少了假阳性重组菌株出现的概率,可实现放线菌中目的基因的连续无痕敲除,因此值得进一步推广和应用。     

大肠杆菌基因无痕敲除技术及策略

基因敲除技术是大肠杆菌基因组减小和代谢途径改造的有效手段,其中基于同源重组原理的基因无痕敲除技术显现出其他技术所不具备的应用优势和发展潜力。该技术可以快速敲除大肠杆菌基因组中的目标基因,并且在基因组中不残留任何外源片段,所以不会干扰后续的基因操作。我们分类介绍了无痕敲除技术中所涉及载体的结构、功能及其相应的敲除策略,着重介绍了无痕敲除技术的原理及载体构建方法。     

非定向激活沉默基因簇挖掘链霉菌活性次级代谢产物研究进展

链霉菌(Streptomyces spp.)是活性次级代谢产物的主要生产菌,在发现结构新颖化合物上具有重大潜力。随着对链霉菌基因组数据的深入分析,发现大量产次级代谢产物合成基因簇处于沉默状态,因此利用非定向和定向策略激活链霉菌中沉默基因簇、挖掘结构新颖化合物成为当前的主要手段。本文主要综述了培养基组成改变或培养条件优化、共培养、添加化学激发子及全局性调控等非定向策略在挖掘链霉菌次级代谢产物中的应用以及取得的进展,以期为链霉菌次级代谢产物高效开发提供参考。     

放线菌基因组与生物技术

天蓝色链霉菌全基因组序列的公布 ,对目前工业生产生物活性代谢产物菌株的遗传改造 ,构建生产高价值药物的超级菌 ,以及由微生物资源去寻找新的生物活性代谢产物将产生巨大影响。文中就基因组时代如何发展由基因组信息和化合物库预测次生代谢路径、研究功能基因组学时代的放线菌次生代谢调控、基因工程技术在放线菌抗生素生产中的应用以及体外分子定向进化与分子育种等生物技术问题进行文献综述。     

除虫链霉菌10个聚酮类抗生素生物合成基因簇的敲除对阿维菌素产量的影响

【目的】考察除虫链霉菌基因组中其它聚酮合成酶类(Polyketide synthase,PKS)抗生素生物合成基因簇的敲除突变对于阿维菌素产量的影响。【方法】构建了11个PKS基因簇的打靶Cosmid和质粒载体,导入除虫链霉菌中筛选突变株。【结果】在工业菌株MMR630中成功敲除了10个PKS基因簇。发酵结果显示7个PKS基因簇敲除突变株中阿维菌素的产量均有不同程度的提高,而2个突变株不能产生阿维菌素。然而,在3个连续敲除2个PKS基因簇的突变株中阿维菌素产量没有能够超过单个PKS敲除突变株的提升幅度。【结论】除虫链霉菌基因组的一些PKS基因簇的敲除可以提高阿维菌素的产量,同时暗示同一类次生代谢产物的代谢流之间存在复杂的相互作用关系。     

信号分子在链霉菌天然产物发现和开发中的应用研究进展

链霉菌具有巨大的合成次级代谢产物的潜力,但在实验室常规培养条件下链霉菌中大部分生物合成基因簇是沉默的,或者表达量极低。链霉菌中信号分子可调节形态分化和代谢产物的生物合成。通过对编码这些信号分子合成酶或受体的基因进行操作,或在发酵液中添加信号分子,可以激活链霉菌中的沉默生物合成基因簇,发现新的天然产物,或者提升已发现的天然产物产量。本文以γ-丁内酯和γ-丁烯内酯两类信号分子为例总结了过去十余年中信号分子在链霉菌天然产物发现和产量提升中的应用,以期为微生物天然产物的开发提供借鉴。     

卡伍尔氏链霉菌NA4的Ⅱ型聚酮基因簇的靶向激活及其产物鉴定

【目的】以基因组信息为导向,定向激活海洋来源卡伍尔氏链霉菌（Streptomyces cavourensis） NA4中沉默的Ⅱ型聚酮类次级代谢产物生物合成基因簇,鉴定新产生的次级代谢产物的结构和抑菌活性。【方法】通过添加启动子和敲除负调控基因的方法激活实验室培养条件下沉默或低表达的生物合成基因簇,并完成目标化合物的分离与纯化,通过电喷雾质谱（electrospray ionization-mass spectrometry,ESI-MS）和核磁共振（nuclear magnetic resonance,NMR）数据分析鉴定目标化合物结构,对目标化合物进行抑菌活性鉴定,基于生物信息学信息推导化合物的生物合成途径。【结果】根据基因组生物信息学分析,从海洋来源链霉菌Streptomyces cavourensis NA4中选取一个编码PKSⅡ型次级代谢产物的生物合成基因簇开展研究,成功激活目标基因簇,从中分离到1个PKSⅡ型化合物,推导了其生物合成途径并进行了抑菌活性鉴定。【结论】基因组导向下的天然产物挖掘,可以目标明确地分离产物,充分挖掘链霉菌编码次级代谢产物的潜力。     

链霉菌负调控因子突变筛选系统的构建

摘要:【目的】构建用于阻遏链霉菌隐性次级代谢基因簇表达的负调控因子筛选的报告系统。【方法】通过“REDIRECT (Rapid Efficient Directed Recombination Time Saving)”技术结合链霉菌温和噬菌体BT1整合酶的体内位点特异性重组技术,对链霉菌中多基因进行无痕敲除。以链霉菌隐性次级代谢基因簇中受阻遏的启动子驱动链霉菌中保守的inoA 构建报告质粒,针对阻遏次级代谢基因簇表达的负调控基因的突变进行检测,以验证报告系统的可行性。【结果】本研究首先通过对天蓝色链霉菌的肌醇从头合成途径关键酶基因inoA,及合成黄色聚酮类隐性抗生素(yellow cryptic polyketide,yCPK)的途径特异性负调控基因scbR2依次进行了无痕敲除,以构建进一步筛选所用的受体菌,再以scbR2阻遏的cpkO启动子控制inoA 的表达构建了报告质粒pIJ8660::PcpkO::inoA。结果显示沉默的cpkO 启动子在突变的受体菌中被激活并使inoA得到了表达,可以使inoA的光秃型突变表型在不添加肌醇的培养基上恢复到产孢的野生型表型。【结论】inoA可以作为新的链霉菌普遍适用的报告基因,可方便地通过表型变化的观察进行筛选,同时可针对性对负调控基因的突变进行检测,可应用于链霉菌隐性抗生素激活的研究。     

链霉菌的全局调控蛋白DasR

链霉菌能够产生多种次级代谢产物,在临床、农牧业、生物技术等领域具有重要应用价值;对链霉菌的调控网络进行深入研究有助于提高次级代谢产物产量并发现新的次级代谢产物.链霉菌中次级代谢产物生物合成按调控通路分为全局调控与途径特异性调控,其中全局调控蛋白可靶向多种通路特异调控基因和生物合成基因,在链霉菌的生命活动中发挥着更为普遍...     

Genome sequence of Streptomyces sp. strain Tü6071

Streptomyces sp. Tü6071 is a soil-dwelling bacterium which has a highly active isoprenoid biosynthesis. Isoprenoids are important precursors for biopharmaceutical molecules such as antibiotics or anticancer agents, e.g., landomycin. Streptomyces sp. Tü6071 produces the industrially important terpene glycosides phenalinolactones, which have antibacterial activity against several Gram-positive bacteria. The availability of the genome sequence of Streptomyces sp. Tü6071 allows for understanding the biosynthesis of these pharmaceutical molecules and will facilitate rational genome modification to improve industrial use.     

乳酸菌的基因组编辑技术研究进展

乳球菌(Lactococcussp.)和乳杆菌(Lactobacillussp.)是工业上常用的乳酸菌(lacticacid bacteria,LAB),长期应用于食品和饮料的发酵。近年来,随着分子操作及遗传改造技术的不断完善,推动了乳球菌和乳杆菌的基础和应用研究,其作为功能菌株和工业微生物细胞工厂的重要潜能也不断突显出来。本文综述了工业常用乳酸菌的基因组编辑技术研究进展,着重介绍了基于整合质粒的敲除、敲入,基于基因组重组工程的精细修饰和敲除、敲入,以及基于成簇的规律性间隔的短回文重复序列及其相关蛋白9[(clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)/CRISPR-associated nuclease9 (Cas9), CRISPR/Cas9]系统的基因组编辑技术。     

2013合成生物学专刊序言

合成生物学目前在全球得到迅猛发展。在此专刊中,综述了一些相关技术在合成生物学领域的进展,其中有:链霉菌无痕敲除方法、基因合成技术、DNA组装新方法、最小化基因组的方法及分析、合成生物系统的组合优化。也讨论了应用合成生物学策略优化光合蓝细菌底盘、产溶剂梭菌分子遗传操作技术、蛋白质预算(Protein budget)作为合成生物学的成本标尺。最后,用几个例子说明了合成生物学的应用,包括复杂天然产物合成人工生物系统的设计与构建、微生物木糖代谢途径改造制备生物基化学品以及构建酿酒酵母工程菌合成香紫苏醇。     

基因打靶技术的原理及操作

基因打靶是近几年发展起来的一种通过同源重组定点改变小鼠基因组特定位点的技术,其诞生是分子生物学与实验胚胎学方法相结合的产物,它的出现又导致了体内研究与体外研究、分子生物学与临床病理学的有机结合,为研究基因的体内功能和疾病的致病机理提供了一种有力的实验手段。本文以基因打靶的实验过程为主线,介绍该技术的原理、操作、进展和应用。     

Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338

Saccharopolyspora erythraea is used for the industrial-scale production of the antibiotic erythromycin A, derivatives of which play a vital role in medicine. The sequenced chromosome of this soil bacterium comprises 8,212,805 base pairs, predicted to encode 7,264 genes. It is circular, like those of the pathogenic actinomycetes Mycobacterium tuberculosis and Corynebacterium diphtheriae, but unlike the linear chromosomes of the model actinomycete Streptomyces coelicolor A3(2) and the closely related Streptomyces avermitilis. The S. erythraea genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, at least 72 genes predicted to confer resistance to a range of common antibiotic classes and many sets of duplicated genes to support its saprophytic lifestyle. The availability of the genome sequence of S. erythraea will improve insight into its biology and facilitate rational development of strains to generate high-titer producers of clinically important antibiotics.     

Impact of the first <Emphasis Type="Italic">Streptomyces</Emphasis> genome sequence on the discovery and production of bioactive substances

An important addition to the field of bacterial genomics is the recent publication of the complete genome sequence of Streptomyces coelicolor. This strain has been for some decades the model organism for streptomycetes and other filamentous actinomycetes, Gram-positive bacteria highly valuable for their ability to produce thousands of bioactive metabolites, many of which have found important applications in medicine and agriculture. We discuss here the impacts that the S. coelicolor genome sequence is likely to have on the production of bioactive metabolites by current industrial strains, on the possible development of future superhost(s) for the production of valuable drugs, and on the search for new bioactive substances from microbial sources.     

东乡野生稻内生放线菌Streptomyces sp. PRh5的全基因组测序及序列分析

【目的】Streptomyces sp. PRh5是从东乡野生稻(Oryza rufipogon Griff.)中分离获得的一株对细菌和真菌都具有较强抗菌活性的内生放线菌。为深入研究PRh5菌株抗菌机制及挖掘次级代谢产物基因资源,有必要解析PRh5菌株的基因组序列信息。【方法】采用高通量测序技术对PRh5菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、直系同源簇(COG)聚类分析、共线性分析及次级代谢产物合成基因簇预测等。【结果】基因组组装获得290 contigs,整个基因组大小约11.1 Mb,GC含量为71.1%,序列已提交至GenBank数据库,登录号为JABQ00000000。同时,预测得到50个次级代谢产物合成基因簇。【结论】将为Streptomyces sp. PRh5的功能基因组学研究及相关次级代谢产物的生物合成途径与异源表达研究提供基础。     

基于CRISPR/Cas系统的黑曲霉基因组编辑技术

黑曲霉Aspergillus niger是有机酸与酶制剂的重要工业生产菌株,以极端环境耐受性、高生产经济性、强发酵鲁棒性与高食品安全性等优势成为不可多得的细胞工厂。合成生物学与系统生物学的快速发展,不仅拉开了全面揭示黑曲霉细胞工厂高效运转机制的序幕,而且为高效黑曲霉细胞工厂的创建优化提供了新技术体系。作为新一代的基因组编辑技术,基于CRISPR/Cas系统的基因组编辑技术为黑曲霉基因组定向改造与基因表达调控带来了革命性突破。本文重点综述该技术在黑曲霉中的最新进展及其在黑曲霉基因编辑与表达调控中的应用,并对其未来发展方向进行展望。     

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

Genetic modification of large DNA fragments(gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity.In this study,we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction(PCR) targeting combined with Gibson assembly.In this strategy,the biosynthetic genes for peptidyl moieties(HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid(CPOAA)from the polyoxin biosynthetic gene cluster to generate a~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette.The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic,polynik A,was obtained and verified.This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.     

Metabolic engineering: techniques for analysis of targets for genetic manipulations

Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement-random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated.