非核糖体肽类化合物的组合生物合成策略研究进展

很多微生物能够利用非核糖体肽合酶合成结构复杂、种类繁多、生物活性多样的小分子肽类化合物.组合生物合成是对控制抗生素生物合成的基因簇进行阻断、置换、重组或异源表达等遗传操作,从而达到利用生物技术和环境友好的手段构建化合物衍生物库的目的.组合生物合成在增加天然活性化合物的数量,改良天然化合物的生物学活性,提高天然化合物的产量,开发创新药物和酶制剂等领域都具有重要应用价值.近年来,非核糖体肽的组合生物合成研究取得了重要进展.本文就非核糖体肽合酶的组合生物合成研究策略,从模块定点突变、替换、插入、删除、模块“洗牌”与异源表达等角度进行了综述.     

微生物次级代谢产物生物合成基因簇异源表达研究进展

微生物天然产物的异源表达在药物的发展过程中发挥着越来越大的作用。通过基因工程技术,将目的生物合成基因簇转移至不同的异源宿主中表达,不仅能够激活沉默的生物合成基因簇,而且可将异源表达体系作为一个非常有用的工具通过组合生物合成生产更多结构新颖、功能独特的化合物。本文结合当今该领域的最新研究动态,概述了基因簇异源表达宿主的选择依据及生物合成基因簇在不同宿主表达的研究进展,以期为天然产物的研究提供一定的借鉴作用。     

血红素基因hemA和hemC对链霉菌HCCB10043产A21978C的影响

摘要：目的 考察血红素合成基因的异源表达对链霉菌HCCB10043产环脂肽类抗生素A21978C的影响。方法 应用PCR方 法扩增血红素合成基因hemA、hemC,使用属间接合转移的方法转化入链霉菌HCCB10043中,获得异源表达菌株。通过HPLC检 测突变株A21978C产量的变化。结果 通过筛选获得血红素合成基因hemA、hemC异源表达菌株,过表达后突变株A21978C产量 相比亲株增加了20%。结论 血红素合成基因的异源表达可提高链霉菌代谢产物A21978C的产量。     

新技术在聚酮组合生物合成中的应用研究进展

本文综述了生物信息学软件、光交联技术、异源表达以及底盘细胞在聚酮组合生物合成中的应用.并尝试将其整合为一粗略的开发思路,即“计算机虚拟设计→高通量技术筛选→(底盘细胞作宿主)异源表达”.希望通过引入新技术,能进一步提高聚酮组合生物合成的开发效率.     

微生物药物产生菌功能基因组学研究进展

微生物药物是一类化学结构和生物活性多样的次级代谢产物,近年来其多个产生菌基因组序列已经被测定完成,在此基础上开展的功能基因组研究方兴未艾,并在抗生素生物合成、形态分化、调控、系统发育与进化以及次级代谢产物挖掘等方面有着新的发现,展现出广阔的研究前景.本文重点阐述了四种重要抗生素产生菌功能基因组学的研究现状,集中于青霉素高产的遗传机制、红霉素产生菌红色糖多孢菌基因组与转录组分析、链霉素产生菌灰色链霉菌中A因子调控网络、阿维菌素产生菌作为次级代谢物异源表达的通用宿主与超高产菌株构建以及新型天然产物的挖掘等研究内容,同时简要介绍了当前我国微生物药物产生菌基因组学的研究概况,并从基础与应用两个角度对其未来发展趋势进行了展望.     

宏基因组学在海洋药物研究中的应用

目前,超过95%的海洋微生物不能够人工培养,这造成了资源获取上的瓶颈问题,进而限制了大量新海洋药物的开发和研究。宏基因组学是一种不依赖于人工培养,直接从海洋环境中提取DNA建立宏基因组文库的方法;如此建立的文库理论上可以覆盖环境中所有微生物的遗传信息,因此,宏基因组学的出现使获得大量生物资源成为可能。功能基因或者某一合成通路的基因簇可以用特定方法分析鉴定,进而异源表达获得异源活性物质。宏基因组学方法主要包括大片段DNA的分离、宏基因组文库的构建、基因筛选、基因簇的表达及阳性克隆的检测。本文综述了宏基因组学的研究方法及其在海洋药物开发中的地位和应用,并探讨了宏基因组学在海洋药物研究中的应用前景。     

哺乳动物细胞色素P450在大肠杆菌上表达的研究进展

细胞色素P450（cytochrome P450,CYP450）是人体内代谢各种内外源性物质最重要的酶系之一。随着分子生物学和重组技术的飞跃发展,对哺乳动物来源的CYP450cDNA进行精细操作并将使其在异源系统中表达成为可能。目前已经有多种异源系统被用来进行哺乳动物CYP450的表达,每一种系统都各具优缺点。哺乳动物CYP450在大肠杆菌上的表达是在实验室中的主要表达方式,但采用不同的方式表达的蛋白量不一样,而且对蛋白酶的活性也有着一定的影响;同时,蛋白纯化手段的选择也会影响CYP450表达方式的应用。本文拟对哺乳动物CYP450在大肠杆菌上的表达纯化现状和应用进行概述。     

哺乳动物CYP450在大肠埃希菌上表达的研究进展

［摘要］细胞色素P450（cytochrome P450，CYP450）是人体内代谢各种内外源性物质最重要的酶系之一。随着分子生物学和重组技术的快速发展，对哺乳动物来源的细胞色素P450cDNA进行精细操作并将使其在异源系统中表达成为可能。目前已经有多种异源系统被用来进行哺乳动物CYP450的表达，每一种系统都各具优缺点。哺乳动物CYP450在大肠埃希菌上的表达是在实验室中的主要表达方式，但采用不同方式表达的蛋白量不一样，而且对蛋白酶的活性也有着一定的影响；同时，蛋白纯化手段的选择也会影响CYP450表达方式的应用。该文对哺乳动物CYP450在大肠埃希菌上的表达纯化现状和应用进行概述。     

TRPV1通道异源表达体系的建立和功能的初步研究

目的构建能够表达TRPV1通道用于实验研究的HEK 293T细胞表达体系。方法采用脂质体转染方式将TRPV1质粒转入人胚胎肾HEK 293T细胞,建立TRPV1型通道瞬时异源表达体系,并对转染后的TRPV1表达体系进行了鉴定,同时对该通道功能特性进行了研究。结果经荧光显微镜观察,转染率可达40%~50%;Western Blot研究发现转染后的HEK 293T细胞在与TRPV1通道蛋白相应的位置上具有明显的条带,提示TRPV1通道蛋白在转染后的细胞中有异源性表达;共聚焦显微成像显示,转染后的HEK293T细胞在受到TRPV1通道激动剂刺激后,胞内荧光强度明显变强,提示TRPV1通道介导钙离子功能正常。结论研究结果表明,基于HEK 293T细胞的TRPV1通道瞬时转染的异源表达体系成功构建。     

产2S-甲基丙二酰辅酶A的基因工程假单胞菌的构建

目的:假单胞菌是次级代谢产物生物异源表达的优良宿主菌,然而它缺少许多次级代谢产物生物合成所需的小分子前体:2S-甲基丙二酸辅酶A(2S-mm-CoA),,因此构建产生2S-mm-CoA的宿主菌将有利于次级代谢产物的异源表达.方法:本研究将来源于大肠埃希菌的编码甲基丙二酰辅酶A变位酶的sbm基因和编码甲基丙二酰辅酶A变位...     

生物膜铜绿假单胞菌AmpC基因诱导表达的研究

目的研究铜绿假单胞菌的产酶基因AmpC在浮游和生物膜状态下的表达差异。方法改良的平板法建立铜绿假单胞菌生物膜模型,抗生素诱导浮游菌和生物膜菌AmpC基因表达,实时荧光定量聚合酶链反应测定PA01的AmpC基因表达水平。结果抗生素诱导前,PAO1浮游菌和生物膜菌的AmpC基因表达均较低,诱导后AmpC基因表达均明显上调。运用产最大AmpC酶活性浓度的抗生素诱导后,亚胺培南和头孢他啶诱导的生物膜菌的AmpC基因表达量高于其诱导的浮游菌。在浮游和生物膜状态下,亚胺培南诱导PAO1的AmpC基因表达量均高于头孢他啶。结论生物膜PA较浮游茵更易被诱导产生AmpC酶,亚胺培南的诱导能力高于头孢他啶。     

Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic Escherichia coli by using a wild-type gene fusion

S fimbrial adhesins (Sfa) represent virulence factors of E. coli wild-type strains causing urinary tract infections and meningitis of the new born. In order to determine the influence of subinhibitory concentration of antibiotics on the expression of the sfa gene cluster, a wild-type strain carrying the lacZ gene, coding for the enzyme β-galactosidase fused to the sfa determinant was used. The expression of lacZ which was under the control of the sfa wild-type promoters, was now equivalent to the sfa gene expression of wild-type strain 536. With this strain the influence of subinhibitory concentrations of 28 antibiotics on the expression of the sfa determinant was studied. The expression was strongly suppressed by a treatment of the wild-type fusion strain by aztreonam, gentamicin, clindamycin and trimethoprim; the latter had a dramatic effect on sfa expression. It was further shown for clindamycin and trimethoprim that the reduction of sfa gene expression was dependent on the concentration of the antibiotics. In contrast imipinem, amphotericin B and rifampicin weakly stimulated sfa expression. We conclude that gene fusions between virulence-associated loci and indicator genes in wild-type pathogens are useful to study virulence modulation due to subinhibitory concentration of antibiotics on the genetic level.     

Using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance

Microarray analyses reveal global changes in gene expression in response to environmental changes and, thus, are well suited to providing a detailed picture of bacterial responses to antibiotic treatment. These responses are represented by patterns of gene expression, termed expression signatures, which provide insight into the mechanism of action of antibiotics as well as the general physiological responses of bacteria to antibiotic-related stresses. The complexity of such signatures is challenging the notion that antibiotics act on single targets and this is consistent with the concept that there are multiple targets coupled with common stress responses. A more detailed knowledge of how known antibiotics act should reveal new strategies for antimicrobial drug discovery.     

Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic Escherichia coli by using a wild-type gene fusion

S fimbrial adhesins (Sfa) represent virulence factors of E. coli wild-type strains causing urinary tract infections and meningitis of the new born. In order to determine the influence of subinhibitory concentration of antibiotics on the expression of the sfa gene cluster, a wild-type strain carrying the lacZ gene, coding for the enzyme β-galactosidase fused to the sfa determinant was used. The expression of lacZ which was under the control of the sfa wild-type promoters, was now equivalent to the sfa gene expression of wild-type strain 536. With this strain the influence of subinhibitory concentrations of 28 antibiotics on the expression of the sfa determinant was studied. The expression was strongly suppressed by a treatment of the wild-type fusion strain by aztreonam, gentamicin, clindamycin and trimethoprim; the latter had a dramatic effect on sfa expression. It was further shown for clindamycin and trimethoprim that the reduction of sfa gene expression was dependent on the concentration of the antibiotics. In contrast imipinem, amphotericin B and rifampicin weakly stimulated sfa expression. We conclude that gene fusions between virulence-associated loci and indicator genes in wild-type pathogens are useful to study virulence modulation due to subinhibitory concentration of antibiotics on the genetic level.     

The biosynthetic gene clusters of aminocoumarin antibiotics

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.     

Characterization of a novel gene related to antibiotic susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen with increased intrinsic resistance to a large number of antibiotics used in clinical therapy. Therefore, understanding the mechanisms of resistance and developing therapy alternatives for P. aeruginosa are of profound importance. Previous work from our laboratory demonstrated that several mutants have isolated with altered expression of the phzA1B1C1D1E1F1G1 (phzA1) operon in the presence of sub-inhibitory concentrations (SICs) of tetracycline (TET). The present study investigates the roles of the PA0011 gene in mediating phzA1 expression at SIC of TET. The PA0011 gene encodes 2-OH-lauroytransferase by controlling the synthesis of the cell envelope and the outer membrane. We found that the PA0011 mutant strain was susceptible to several different antibiotics and environmental stresses. Complementation in the PA0011 mutant restored these phenotypes to wild-type levels. In addition, expression of the PA0011 gene, as monitored through a luciferase reporter, is increased at SICs of antibiotics. Indeed, the expression of the PA0011 gene increased about threefold in pqsR and pqsH mutants compared with the wild-type PAO1. However, the PA0011 gene negatively regulates the quorum sensing (QS) system. Taken together, these data suggest that PA0011 is involved in susceptibility to antimicrobial agents in P. aeruginosa, and that its susceptibility effect maybe partly dependent on increased QS expression.     

Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. lambda RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage PhiC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.     

亚抑菌浓度抗生素对细菌耐药性和毒力影响的研究进展

细菌的耐药性和毒力是决定抗生素治疗成败的关键。近年来研究表明亚抑菌浓度抗生素可以诱导细菌耐药并影响细菌的毒力。本文介绍了亚抑菌浓度抗生素产生的来源；阐述了亚抑菌浓度抗生素对细菌耐药性和毒力的影响。亚抑菌浓度抗生素对细菌耐药性的影响涉及了细菌的耐药选择性、生物被膜和持留菌的形成、基因突变、水平基因转移和基因表达；对细菌毒力的影响主要包括细菌的黏附性、运动性和其它毒力因子。以期为畜牧生产中减少和避免亚抑菌浓度抗菌药的产生，减缓和克服细菌耐药性，降低致病菌的毒力提供参考。     

基因研究在抗生素耐药中的应用

随着抗生素的广泛使用,诸多病原菌产生了耐药性。耐药问题已成为二十一世纪人类面临的最严峻的环境健康问题之一,受到了诸多研究者的关注。目前,新型抗生素研发、现有抗生素改良以及阐明微生物的耐药机制等研究正在不断的开展。基因组学的不断发展为病原菌耐药性研究带来了极大的便捷,高通量测序技术结合分子生物学技术构建了多种抗生素抗性基因数据库及分析工具,这极有利于研究者高效地发现各种新的耐药基因,了解耐药性产生的原因与发展过程。本综述的目的在于从基因的视角认识病原菌耐药性,为科学研究及临床工作中更好的理解和应对耐药问题提供便利。