细菌胞外多糖生物合成转录调控因子研究进展

细菌胞外多糖(Exopolysaccharide,EPS)因其独特的理化特性和生理活性,在食品、制药和化工等领域广泛应用。在食品行业中,黄原胶、结冷胶和热凝胶等细菌EPS备受青睐。转录调控因子能在转录水平上调控eps基因的表达,影响细菌EPS的生物合成。目前细菌EPS转录调控因子的研究报道较少,且多数已知的EPS转录因子调控机制尚未阐明。本文总结了近年来细菌EPS调控因子的研究进展,重点介绍其研究方法和调控机制,以期为细菌EPS转录调控研究提供借鉴。     

植物乳杆菌C88胞外多糖生物合成基因的克隆及序列比对

乳酸菌胞外多糖能显著改善发酵乳制品及食品的流变学和质构特性.为进一步了解乳酸菌胞外多糖的生物合成途径及调控机制,本研究对参与植物乳杆菌C88胞外多糖生物合成基因簇的部分序列进行了克隆和鉴定.根据GenBank中已报道植物乳杆菌基因序列的保守区域设计特异性引物,扩增出植物乳杆菌C88生物合成蛋白基因(cps4A)序列,并通过染色体步移方法克隆了植物乳杆菌C88 参与胞外多糖合成基因簇的部分序列(4.9 kb).利用生物信息学方法预测基因簇中6个阅读框的结构和功能,结果表明该序列与已报道的乳酸杆菌胞外多糖生物合成基因具有高度的同源性(>96%);对各阅读框功能预测分析发现,这6个基因主要编码参与胞外多糖合成中的多糖合成蛋白、糖链长度检测蛋白、UDP-葡萄糖-4-异构酶和糖基转移酶.本研究将为利用基因工程方法调控多糖的合成和产量提供理论依据.     

c-di-GMP对细菌胞外多糖合成与运输的调控

环二鸟苷酸(Cyclic diguanylate,c-di-GMP)的发现已有29年。作为重要的细菌第二信使,c-di-GMP可参与调节细菌生物膜的合成与降解、运动、毒性、细胞周期、细胞分化等多种活动过程。胞外多糖(EPS)是细菌生物膜的主要组成成分,其合成和运输主要受c-di-GMP调控。目前细菌胞外多糖在医药、食品、农业、工业和环保等多个领域均有广泛的应用,其相关研究备受关注。本文旨在论述细菌中c-di-GMP合成与降解的调控,部分合成酶(Diguanylate cyclase,DGC)与降解酶(Phosphodiesterase,PDE)及其受体分子(Receptor)晶体结构等研究成果,并结合我们研究农杆菌ATCC31749中c-di-GMP对可德胶合成调控的基础上,重点阐述c-di-GMP对纤维素、藻酸盐、多聚氮乙酰葡萄糖胺(PNAG)和可德胶等EPS合成与运输的调控机制。     

细菌胞外多糖生物活性的研究进展

细菌胞外多糖(Exopolysaccharides,EPS)是细菌产生的一类重要的生物大分子,在许多重要生命过程中起着非常关键的作用,由于其具有多种生物活性成为近年来研究的热点。目前,细菌EPS作为天然产物,可大量发酵提取,且可降低成本,特别是一些乳酸杆菌和海洋细菌的新型EPS被证实具有多样化结构多糖的、潜在的有益生物活性,如抗肿瘤、免疫调节和抗氧化等,其在食品、医学等领域被广泛应用。就细菌EPS在生物材料和药品方面的应用、免疫调节功能、抗肿瘤活性及抗氧化活性作一综述。     

细菌胞外多糖的特性及应用研究

不论是在自然或病理条件下,多数细菌均被胞外多糖所包被,胞外多糖对细菌的粘附及在竞争环境中的存活和生长都具有重要作用。近年来细菌胞外多糖以其独特的生物学活性和广阔的应用前景而备受人们关注。系统介绍了细菌胞外多糖的结构性质、特性及生理功能,重点阐述了几种多糖的应用现状,并对今后细菌胞外多糖在工业上的发展趋势进行了展望,为深入开发利用多糖功能菌资源,进一步扩展其在工业领域上的应用奠定理论基础。     

一株寡营养细菌胞外多糖的摇瓶发酵研究

从新疆的寡营养环境——古尔班通古特沙漠中分离到一株寡营养细菌Azotobacter sp．(1～15mg碳/L培养基),通过进行Azotobacter sp．菌的单因子优化培养基的试验、摇瓶培养工艺条件的优化试验(培养温度、培养时间、初始pH值、溶氧量),确定了菌种生长与营养需求等主要因子与胞外多糖产量、粘度的关系,结果表明,摇瓶发酵的最适宜条件为:以蔗糖为碳源,碳酸钙含量为2g/L,初始pH值为7左右,种龄72～84h,磷酸二氢钾、硫酸镁的含量分别为0．3g/L、0．1g/L,接种体积分数15%,于37℃摇瓶培养72h,250mL摇瓶装液量为50mL,在适宜条件下粘多糖的产量最大可达到1145．94μg/mL,粘性可达9200 mPa·s。     

嗜热链球菌IMAU20246胞外多糖基因簇及其表达分析北大核心

【目的】嗜热链球菌IMAU20246是一株具有良好发酵特性且高产胞外多糖(exopolysaccharides,EPS)的菌株,但其EPS基因簇及合成途径尚不清晰。因此可通过全基因组测序及生物信息学分析菌株基因组序列,探究EPS合成及调控机制。【方法】本实验对嗜热链球菌IMAU20246进行全基因组测序并进行生物信息学分析,解析EPS生物合成相关基因簇及EPS合成途径,同时采用实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR)对其不同时间点EPS基因簇的表达进行定量分析。【结果】嗜热链球菌IMAU20246基因组中有一个18.1 kb的EPS生物合成基因簇,编码15个与EPS生物合成相关的基因。嗜热链球菌IMAU20246通过转运葡萄糖、甘露糖、果糖、半乳糖、乳糖、海藻糖、纤维二糖及蔗糖合成UDP-葡萄糖、dTDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖、UDP-呋喃半乳糖、UDP-N-乙酰葡萄糖胺和UDP-N-乙酰半乳糖胺等7种糖核苷酸。qRT-PCR的结果表明,EPS基因簇中的基因在细胞生长阶段均能表达,特别是糖基转移酶基因epsE、epsF、epsH和epsJ在培养6 h时表达量最高,此时EPS产量达到最高。【结论】本研究从基因组解析了嗜热链球菌IMAU20246 EPS基因簇及其合成途径,为菌株的进一步开发提供了理论依据。     

假单胞菌生物被膜核心胞外多糖生物合成系统研究进展

胞外多糖是假单胞菌生物被膜的重要组成部分,能增强菌体对外界环境、抗菌剂和宿主防御的耐受性.假单胞菌能产生3种与生物被膜形成密切相关的核心胞外多糖:褐藻胶、Psl和Pel,它们在细菌细胞中的合成和转运分别依赖对应的褐藻胶、Psl和Pel生物合成系统.因此,本综述系统全面地总结了假单胞菌3种胞外多糖生物合成系统结构生物学的...     

微生物胞外多糖黄原胶的应用与研究进展

黄原胶是由植物病原菌野油菜黄单胞菌产生的一种天然胞外多糖,其结构为重复单元聚合而成的多聚体,单元结构中包含了D-葡萄糖、D-甘露糖和D-葡糖醛酸基团以及O-乙酰和丙酮酸基团。黄原胶具有优良的流变特性,作为增稠剂和稳定剂等被广泛用于食品、药品和化妆品等多个行业。本文主要综述了黄原胶的结构、性质、应用、生物合成、生物合成调控和工业生产条件,为黄原胶的后续研究提供理论基础。     

变构菌素生物合成基因簇的研究进展

变构菌素是从Streptomyces griseochromogenes菌株中分离得到的第一个高选择性的蛋白磷酸化酶-1（PP-1）抑制剂。变构菌素及其衍生物在神经系统紊乱、代谢综合症、呼吸系统及相关疾病、免疫抑制、肿瘤治疗等诸多领域都有着广泛的应用前景,因而引起了人们对其生物合成途径的研究兴趣。介绍了近年来变构菌素生物合成途径的研究进展。     

细菌O抗原基因簇的研究进展

O抗原是革兰阴性菌外膜的重要组分。它是由多糖的重复单位和一系列寡糖重复单位所组成,即O抗原基本单位,一般包括2～6个糖基。因为多糖中糖的性质不同,连接次序,连接位点都有差异,因而O抗原变异很大。染色体中有关O抗原合成的基因丛集成簇。这些基因簇的变异很大,也反映了O抗原结构的多样性。O抗原的表达受到各种因素的调节。     

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.     

Widespread Occurrence and Lateral Transfer of the Cyanobactin Biosynthesis Gene Cluster in Cyanobacteria

Cyanobactins are small cyclic peptides produced by cyanobacteria. Here we demonstrate the widespread but sporadic occurrence of the cyanobactin biosynthetic pathway. We detected a cyanobactin biosynthetic gene in 48 of the 132 strains included in this study. Our results suggest that cyanobactin biosynthetic genes have a complex evolutionary history in cyanobacteria punctuated by a series of ancient horizontal gene transfer events.     

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.     

Characterization of the Complete Zwittermicin A Biosynthesis Gene Cluster from Bacillus cereus

Bacillus cereus UW85 produces the linear aminopolyol antibiotic zwittermicin A (ZmA). This antibiotic has diverse biological activities, such as suppression of disease in plants caused by protists, inhibition of fungal and bacterial growth, and amplification of the insecticidal activity of the toxin protein from Bacillus thuringiensis. ZmA has an unusual chemical structure that includes a d amino acid and ethanolamine and glycolyl moieties, as well as having an unusual terminal amide that is generated from the modification of the nonproteinogenic amino acid β-ureidoalanine. The diverse biological activities and unusual structure of ZmA have stimulated our efforts to understand how this antibiotic is biosynthesized. Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. A nearly identical gene cluster is identified on a plasmid from B. cereus AH1134, and we show that this strain is also capable of producing ZmA. Bioinformatics and biochemical analyses of the ZmA biosynthesis enzymes strongly suggest that ZmA is initially biosynthesized as part of a larger metabolite that is processed twice, resulting in the formation of ZmA and two additional metabolites. Additionally, we propose that the biosynthesis gene cluster for the production of the amino sugar kanosamine is contained within the ZmA biosynthesis gene cluster in B. cereus UW85.Bacillus cereus strain UW85 was isolated for its ability to suppress disease in alfalfa caused by the plant pathogen Phytophthora medicaginis (17). This antiprotist activity was subsequently found to be associated with the filtrate of fully sporulated B. cereus UW85 (37). Analysis of this filtrate identified two antiprotist antibiotics, zwittermicin A (ZmA) and kanosamine (28, 37). Of the two antibiotics, ZmA has shown the more interesting biological activities, having not only antiprotist activity, but also antibiotic activity against gram-positive and gram-negative bacteria, as well as fungi (32, 38). ZmA was also found to potentiate the activity of the toxin protein of Bacillus thuringiensis against insects (3).A preliminary chemical structure of ZmA was determined by the Handelsman and Clardy groups (18). More recently, Rogers and colleagues performed a series of elegant structural studies that compared ZmA produced from B. cereus with synthetic ZmA derivatives that had varied stereocenters (32, 33). From this work, the chemical structure of ZmA with the appropriate stereocenters has been determined (Fig. ​(Fig.1).1). The antibiotic has a number of unusual structural components. First, ZmA is one of only a few linear aminopolyol natural products to be identified. Second, the core of ZmA is formed from ethanolamine and glycolyl moieties that are rarely seen in natural products. Third, the N terminus of ZmA is formed from d-serine (d-Ser), not l-Ser, as initially expected. This suggests that the amino acid either is incorporated as the d isomer or is incorporated as the l isomer and is then isomerized at some point during its biosynthesis. Finally, ZmA is the only natural product that we are aware of that contains an unusual 2-aminosuccinamide moiety. This moiety is likely to come from the amino acid β-ureidoalanine (β-Uda) that has had its carboxylic acid replaced by a terminal amide.Open in a separate windowFIG. 1.Chemical structure of ZmA. Numbers have been added to identify the sites of hydroxyl groups as discussed in the text.We have been investigating how B. cereus UW85 assembles this antibiotic to gain insights into how production of ZmA can be improved and how the unusual structural components of ZmA are formed. We previously proposed that the biosynthesis of ZmA involves the condensation of the amino acids l-Ser and l-2,3-diaminopropionate (l-Dap), along with the carboxylic acid precursors malonyl-coenzyme A (CoA), (2S)-aminomalonyl-acyl carrier protein (ACP), and (2R)-hydroxymalonyl-ACP (4, 12). The proposal for l-Ser incorporation was made prior to the full elucidation of the stereochemistry of ZmA. The condensation of amino acids and carboxylic acids suggests that ZmA is assembled via a nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) megasynthase. These megasynthases are modular enzymes with a set of catalytic domains, or modules, for each precursor incorporated into the natural product (reviewed in references 13 and 40). Support for the involvement of this type of enzymology in ZmA biosynthesis comes from a combination of genetic and biochemical studies. Transposon mutagenesis of B. cereus UW85 identified insertions in genes coding for NRPS and PKS enzymology that abolished ZmA production (12). Sequencing of a 19-kb fragment of the ZmA biosynthesis gene cluster identified genes coding for NRPS and PKS enzymology (12). Furthermore, other groups investigating ZmA biosynthesis in B. thuringiensis strains have identified genes coding for NRPS modules that are essential for ZmA biosynthesis in these strains (35, 49, 50). Finally, we used biochemistry and mass spectrometry to establish the existence of two ACP-linked PKS extender units, (2S)-aminomalonyl-ACP and (2R)-hydroxymalonyl-ACP (4). All of these data support the hypothesis that the backbone of ZmA is assembled by an NRPS/PKS megasynthase.In addition to the mixed amino acid and carboxylic acid backbone, ZmA also contains a terminal amide (Fig. ​(Fig.1).1). How these amide groups are formed was investigated by Müller and colleagues and Silakowski and colleagues as they deciphered how myxothiazole is biosynthesized (29, 36). Briefly, the NRPS/PKS megasynthase that assembles the backbone of myxothiazole forms a product that is 1 amino acid longer than myxothiazole. This results in a biosynthetic intermediate that contains a glycyl residue at the C terminus of myxothiazole, while the intermediate remains thioesterified to the peptidyl carrier protein (PCP) domain of the terminal NRPS module. The α-carbon of the glycine is hydroxylated by a flavin-dependent monooxygenase, a modification that results in an unstable intermediate that spontaneously releases the myxothiazole backbone, with the nitrogen of the terminal amide coming from the glycine. The terminal PCP domain contains the glyoxyl group left after C-N bond cleavage, and this product is released from the PCP domain by the neighboring thioesterase (Te) domain. Based on this precedent, the terminal amide of ZmA may be produced by a similar mechanism.Here, we present the identification of the complete ZmA biosynthesis gene cluster from B. cereus UW85. The biosynthesis gene cluster was identified by locating the previously reported biosynthesis genes and by mapping the locations of transposon insertions that abolished the ability of B. cereus UW85 to produce ZmA. As expected, the gene cluster codes for NRPS and PKS enzymology that is likely to be involved in ZmA assembly from its amino acid and carboxylic acid precursors. Surprisingly, we fiound that ZmA not only is likely to be processed at its C terminus to generate the terminal amide by a mechanism similar to that seen in myxothiazole biosynthesis, but it appears to also be processed at its N terminus. These two processing events potentially lead to the biosynthesis of two additional metabolites besides ZmA. Furthermore, the kanosamine biosynthesis gene cluster appears to be fully contained within the ZmA biosynthesis gene cluster. A mechanism for ZmA production is presented, along with proposals for how three additional metabolites are produced by the enzymes encoded by this unusual gene cluster.     

铁硫簇的生物合成

铁硫簇在细胞的生物学过程中起着重要的作用,可参与电子传递、代谢控制和基因调节等过程。研究显示铁硫簇具有多样性,它的合成依赖于ISC和SUF系统,固氮酶中还需要NIF系统的参与。ISC系统由iscSUA-hscBA-fdx基因串编码,合成的是一类“管家”蛋白,适于在正常条件下表达。SUF系统由基因串sufABCDSE编码,常在恶劣环境如氧化应激和铁饥饿条件下表达。NIF系统由nifSU基因编码,适于固氮酶(厌氧条件下起作用)铁硫簇的合成。