【作者】 朱敬；

【导师】 沈月毛； 王浩鑫；

【作者基本信息】 山东大学 ， 微生物， 2014， 博士

【摘要】 对三联苯类(p-terphenyl)化合物是一类具有显著结构特征但比较“稀有”的天然产物,仅发现分布于真菌和细菌中,该类化合物结构多样,且具有抗氧化、抗肿瘤和免疫抑制等多种生物活性。尽管如此,直到最近才有真菌对三联苯骨架生物合成的少量研究报道,因此,深入开展对三联苯化合物的生物合成研究,预期可能发现新颖的酶学机制,从而为通过进化生物学或／和合成生物学方法制造各方面性能更优异的对三联苯类化合物奠定基础和提供指导。在前期的工作中,本实验室从一株海洋链霉菌LZ35中分离获得了一类新颖的对三联苯糖苷类化合物(Echoside A-E)。根据真菌中对三联苯骨架合成基因的信息,我们在LZ35基因组中定位了两个含有A-T-TE结构域组成的单模块非核糖体肽合成酶(NRPS)基因的基因簇SM4和SM25,初步的基因敲除和代谢谱比较显示,SM4基因簇的敲除并不影响echosides的产生,而SM25的敲除会终止echosides的合成,初步明确了echosides的合成基因簇。ech基因簇含有一个无缩合(Condensation)结构域的单模块NRPS合酶基因(echA),七个氧化还原酶或脱水酶基因(echB, echC, orf4-6,9,12),一个甲基转移酶基因(echD),三个转运蛋白相关基因(orf1-3)和四个调控因子基因(orf7,10,11,13)。在基因簇周围没有找到催化苯丙氨酸形成苯丙酮酸的转氨酶及加载葡萄糖醛酸的糖基转移酶基因,推测可能位于基因组其它位置。本论文结合体内的遗传学证据和体外生化证据,对SM25基因簇核心基因echABC的功能进行了研究。对ech基因敲除突变株的代谢谱分析表明只有echABC三个基因的敲除会明显影响echoside苷元骨架的合成,其中敲除echB和echC均积累了多个中间产物。通过echBC双敲除实验我们证实EchB负责催化的反应位于EchC之前。通过对echB和echC敲除突变株中间产物的结构分析,我们推测了EchB和EchC的天然底物。Echosides可能的合成途径是：两分子苯丙酮酸经过EchA催化缩合形成polyporic acid,在NAD(P)H辅酶存在下EchB催化polyporic acid的还原形成中间体DP-A,随后由EchC催化脱去一分子水形成echoside C的苷元,接下来经糖基化,甲基化,杂环形成等一系列后修饰过程形成一系列的echosides化合物。苯醌合成酶EchA为缺少缩合结构域的单模块NRPS,其组成不同于典型NRPS,缺少负责肽键形成C结构域,只含有A结构域、T结构域和TE结构域,其A结构域负责活化加载α-芳香酮酸单体,由TE结构域介导分子间的Dieckmann缩合形成C-C键,这是一种新颖的NRPS催化与释放机制,显著区别于传统NRPS中的TE结构域,后者水解释放线性多肽链时形成的C-O键,环化释放多肽链时形成C-N键。参考同源蛋白TdiA和AtrA的研究,我们首先使用Sfp对EchA进行磷酸泛酰巯基乙胺化获得holo-EchA,再使用holo-EchA构建invitro体系,成功检测到了polyporic acid的产生。将不同浓度下苯丙酮酸发生反应的初速度与浓度拟合,获得EchA的Km值为4.5±1.1μM,kca,值为0.35±0.02min-1。这是首个放线菌来源的苯醌合成酶在生物化学水平上得到鉴定,也是首次报道的以苯丙酮酸为底物的苯醌合成酶,我们将EchA酶命名为polyporic acid合成酶。数据库搜索显示EchB的同源物均缺少功能验证,保守结构域搜索显示EchB属于短链脱氢酶/还原酶(SDR)超家族,是已知最大的蛋白超家族之一,广泛分布于各生物界。SDR超家族成员之间一级序列的相似度只有15-30%,三维结构上存在保守的Rossmann折叠,为辅酶结合位点和酶活中心。不同于其它的脱氢酶超家族,SDR并不依赖金属离子,仅利用辅酶NAD(H)和NADP(H)行使其催化功能。我们分别使用NADPH和NADH为辅酶,以polyporic acid为底物,在异源表达的重组EchB催化下得到了特定立体构型的化合物DP-A,与echC敲除突变株获得的中间产物一致,证实了EchB的功能。我们将EchB命名为polyporic acid还原酶。数据库中EchC的同源物功能也缺乏验证,保守结构域搜索EchC含有SnoaL样结构域,属核转运因子2样(NTF2-like)超家族,该家族成员包括异构酶如△5-3-酮类固醇异构酶,水解酶如柠檬烯-1,2-环氧水解酶,脱水酶如scytalone脱水酶(scytalone dehydratases),以及很多信号转导相关的非催化蛋白如NTF2等。根据中间产物的结构我们推测EchC为脱水酶,出人意料的是,在EchC蛋白表达可溶性良好的前提下,体外生物化学实验却没有检测到任何催化活性。我们对EchC进行了蛋白结晶,希望通过三维结构寻找线索。EchC蛋白晶体为二聚体,其结构与同家族蛋白结构相似。从电子密度图上,我们可以看到位于EchC圆锥体结构中央空腔的酶活中心被未知配体占据,因此,我们推测在异源表达过程中EchC结合了大肠杆菌来源的小分子抑制剂,导致体外检测不到活性。据此,我们拟在后续实验中采用变性-透析-复性手段除去活性中心结合的抑制剂再进行酶活测定,或者在原产生菌LZ35中表达并纯化有活性的蛋白用于酶活测定。综上,本论文成功克隆了echosides的生物合成基因簇,在体内,我们通过体内遗传学实验证实三种酶(EchA, EchB, EchC)依次催化从苯丙酮酸合成echoside的对三联苯骨架；在体外,我们成功表征了EchA, EchB的活性,并通过晶体学手段推测了异源表达的重组EchC没有检测到酶活的原因,为后续实验指明了方向。更多还原

【Abstract】 The para-terphenyl natural products have unique structural features and diverse biological activities, including antioxidant, antitumor and immunosuppressive activities. Most of these compounds were isolated from fungi, and only a dozen members have been reported from Streptomyces species. The biosynthesis mechanism of this group of metabolites has not been reported until recent years. Therefore, the studies on the biosynthesis of p-terphenyls will lead to the discovery of novel enzymatic mechanism, and facilitate bioengineering novel p-terphenyls with better properties.Previously, five novel p-terphenyls bearing glucuronic acid moiety, namely echosides A-E, were isolated from Streptomyces sp. LZ35. Base on the biosynthetic genes of p-terphenyls in fungi, two putative p-terphenyls biosynthetic gene clusters (SM4and SM25) were identified from the genome of strain LZ35. Both of them have a single-module NRPS-like enzyme, which is proposed to be responsible for the biosynthesis of the core carbon skeleton of p-terphenyls. In order to identify which one is involved in the formation of echosides, the gene disruption experiments were performed. The HPLC analysis of the metabolites from the mutants and wild type revealed that SM25is responsible for the biosynthesis of echosides. The SM25cluster consists of a single-module NRPS gene (echA), which lacks a condensation (C) domain, seven redox genes (echB, echC, orf4-6,9, and12), and a methyltransferase gene (echD). In addition, the ech cluster harbors genes postulated to encode transporter genes (orf1-3) and regulatory factor genes (orf7,10,11, and13). Surprisingly, there is no gene encoding aminotransferase, which is responsible for the formation of phenylpyruvic acid from L-phenylalanine, and a gene encoding glycosyltransferase, which is responsible for the addition of the sugar residue to the echosides, located in the gene cluster. Both of them could be located elsewhere in the genome of strain LZ35. In this study, the "core" genes in SM25, echABC, were characterized by genetic and biochemical means.The gene disruption experiments indicated that only three genes in SM25, echABC, were involved in the biosynthesis of echosides. The metabolic profiling of single and double mutant of echB and echC revealed that the reaction catalyzed by EchB is before that by EchC. In addition, the intermediates accumulated in the echB and echC mutant were identified. Base on the above results, the biosynthetic pathway of echosides was proposed: two molecules of phenylpyruvate were condensed into polyporic acid by EchA, which was then reduced to DP-A, in the presence of NAD(P)H, by EchB, followed by dehydration catalyzed by EchC to produce the aglycone of echoside C, which was further processed by glycosylation, methylation and heterocyclization to give echosides.The quinone synthetase EchA is a single tri-domain (adenylation-thiolation-thioesterase, A-T-TE) NRPS module, which lacks the condensation domain for peptide bond formation. The a-aromatic keto acid was activated and loaded by A domain, followed by Dieckmann condensation to form C-C bond and released from the NRPS catalyzed by TE domain, which presents a novel catalytic mechanism of NRPS. Previously, the TE domain of NRPS can only release linear polypeptide chain by forming C-O bond, or cyclized polypeptide chain by forming C-N bond. According to the studies on TdiA and AtrA, we prepared holo-EchA through enzymatic phosphopantetheinyl (Ppant) transfer, catalyzed by the Ppant transferase Sfp. When the holo-EchA was incubated with phenylpyruvate and ATP, it was able to convert phenylpyruvate to polyporic acid. By measuring the conversion of phenylpyruvate to polyporic acid, the maintenance of good activity of EchA was presented in a linear reaction phase, and we determined that EchA had a KM of4.5±1.1μM and a kcat of0.35±0.02min-1. We therefore conclude that EchA represents the first quinone synthetase of the Streptomyces genus that has been biochemically characterized. Since this is the first report that a quinone synthetase can use phenylpyruvate as a native substrate, EchA was named polyporic acid synthetase.Bioinformation analysis revealed that EchB belongs to short-chain dehydrogenases/reductases (SDRs) superfamily, which is one of largest protein superfamily widely distributed in the biosphere. Members of SDR superfamily typically shared only15-30%sequence identity, and the most conserved feature is a classical Rossmann fold for nucleotide binding. SDRs are different from other dehydrogenase by using NAD(H) or NADP(H) as co-factor, and no metal is required. When EchB was incubated with polyporic acid, in the presence of NADPH or NADH, DP-A was produced in the reaction mixture, which is the intermediate accumulated in the echC disruption mutant. Therefore, EchB was named polyporic acid reductase.Bioinformation analysis revealed that EchB has a SnoaL-like domain, which belongs to NTF2-like superfamily. Members of this superfamily include isomerase, hydrolase, dehydratase and many signal transduction related proteins. According to the intermediate identified from echC disruption mutant, we proposed that EchC is a dehydratase. However, the in vitro enzymatic assay got no positive results. In order to identify the reason, an experimental three dimensional structure of EchC will be much helpful. The crystal structure of EchC is a dimer, which is similar to other members of its family. Base on the electron density map, we observed that an unknown ligand occupied the active site pocket of EchC, which may affect the catalytic activity of EchC. Therefore, we will remove the ligand by means of degeneration/dialysis/refolding or express EchC in strain LZ35in the subsequent experiment, and then test the activity of EchC.In conclusion, the echosides (ech) biosynthetic gene cluster was identified and characterized. The gene knockout and intermediates identification experiments revealed that EchA, EchB and EchC were responsible for the formation of echosides from phenylpyruvate. In addition, the functions of EchA and EchB were biochemically characterized in vitro. Finally, the crystal structure of EchC has been resolved, which will facilitate the subsequent biochemically study of EchC.更多还原