顶头孢霉中噻唑合成酶的鉴定及硫胺素对CPC产量的影响

发布时间：2018-01-14 08:04

本文关键词：顶头孢霉中噻唑合成酶的鉴定及硫胺素对CPC产量的影响　出处：《上海医药工业研究院》2017年硕士论文　论文类型：学位论文

【摘要】：β-内酰胺类抗生素头孢菌素C的主要工业生产菌是丝状真菌顶头孢霉。近年来,头孢菌素C的发酵产量虽已得到大幅提升,但工业生产菌株和野生菌株中存在的代谢差异方面的分子机理仍然不是很清楚。本实验室建立了顶头孢霉高低产菌的比较蛋白质组学平台,并从中发现了具有显著差异表达的蛋白ActhiS。对顶头孢霉的总RNA序列进行Illumina sequence分析得到了总的cDNA序列,从中找到了ActhiS蛋白的编码基因Acthi。经过初步的序列比对,发现ActhiS很可能是负责硫胺素合成过程中的噻唑合成酶。通过构建沉默突变株和过表达突变株,证明了Acthi基因参与硫胺素的合成,并影响头孢菌素C的合成。本文针对ActhiS蛋白,采用体外生物化学方法对其功能进行进一步的研究,包括以下两方面的内容:一、体外表达重组菌,纯化ActhiS蛋白,进行体外酶催化,核磁鉴定终产物的结构,最终确定ActhiS的功能;二、研究顶头孢霉培养基中添加硫胺素对CPC合成的影响。主要结果如下:在进行ActhiS蛋白的活性鉴定时,发现了在LB培养基中表达纯化的ActhiS蛋白经热变性后其可以释放小分子化合物。LC-MS和H-NMR结果证实了释放的小分子化合物与之前报道的ADP加合(2-羟乙基)-4-甲基噻唑-2-羧酸(ADT)的分子量和结构相一致,ADT是噻唑环的一种衍生物。而在生物体内,正是由嘧啶环和噻唑环这两个独立的环状结构结合才生成硫胺素。采用成分简单的M9基础培养基获得了不结合该小分子化合的ActhiS蛋白,并利用该蛋白成功的进行了酶的体外催化反应,得到了上述的ADT产物。这证明了ActhiS蛋白是催化合成噻唑环的噻唑合成酶。通过定点突变,我们推测ActhiS中的Cys217为噻唑环提供硫来源,它是与其同源性蛋白THI4类似的一个自杀性酶。进一步探究硫胺素对CPC合成的影响。我们发现在顶头孢霉发酵液中添加1mg/m L硫胺素可以提高CPC产量,继续增大硫胺素浓度则抑制CPC的产量。同时研究了参与CPC的生物合成基因的转录差异,在顶头孢霉发酵培养基中添加1mg/m L和100mg/m L硫胺素,分别使得菌株中的cefEF,cefG转录水平上调和下调。这说明硫胺素可以调节CPC合成的基因转录,进而影响到CPC的产量。同时,发酵培养基中添加硫胺素对顶头孢霉的菌丝体生长和生物量也有不同程度的影响。本研究首次证实了ActhiS蛋白是顶头孢霉中的噻唑合成酶,其参与噻唑的合成,并进而影响到硫胺素的生成。硫胺素作为多种酶的辅酶,参与生物体内氨基酸代谢和碳水化合物的代谢,并影响到到CPC的合成,说明硫胺素代谢途径及Acthi基因可以作为CPC生产菌株育种的一个靶点。
[Abstract]:The main industrial producer of cephalosporin C, a 尾 -lactam antibiotic, is the filamentous fungus Cephalosporium. In recent years, the fermentation yield of cephalosporin C has been greatly increased. However, the molecular mechanism of metabolic differences between industrial and wild strains is still not clear. A comparative proteomics platform for high and low yield strains of Cephalosporium apocephalus was established in our laboratory. The protein ActhiS. the total RNA sequence of Cephalosporium was analyzed by Illumina sequence and the total cDNA sequence was obtained. The encoding gene Acthi. of ActhiS protein was found by preliminary sequence alignment. It was found that ActhiS was probably responsible for thiazole synthetase in the process of thiamine synthesis. It was proved that Acthi gene was involved in thiamine synthesis by constructing mutants of silencing and overexpression. And affect the synthesis of cephalosporin C. in this paper, ActhiS protein was further studied by biochemical method in vitro, including the following two aspects: first, the expression of recombinant bacteria in vitro. Purification of ActhiS protein, in vitro enzymatic catalysis, nuclear magnetic resonance (NMR) identification of the structure of the final product, and finally determine the function of ActhiS; Secondly, the effect of thiamine on the synthesis of CPC was studied. The main results were as follows: the activity of ActhiS protein was identified. It was found that the purified ActhiS protein expressed in LB medium could release small molecular compounds after thermal denaturation. LC-MS and H-NMR results confirmed that the released small molecular compounds were similar to those previously reported. ADP adduct. The molecular weight and structure of 2-hydroxyethyl-4-methylthiazole-2-carboxylic acid (ADT) were consistent. ADT is a derivative of thiazole ring. Thiamine was produced by the combination of pyrimidine ring and thiazolyl ring. The ActhiS protein which was not bound to this small molecule was obtained by using the simple M9 basic medium. The above ADT products were obtained by the catalytic reaction in vitro, which proved that ActhiS protein is a thiazole synthase that catalyzes the synthesis of thiazolyl ring, and by site-directed mutagenesis, it is proved that the ActhiS protein is a thiazole synthase that catalyzes the synthesis of thiazolyl ring. We speculate that Cys217 in ActhiS provides sulfur source for thiazole ring. It is a suicidal enzyme similar to its homologous protein THI4. To further explore the effect of thiamine on CPC synthesis, we found that 1 mg / m was added to the fermentation broth of Cephalosporium acrocephalus. L-thiamine could increase the yield of CPC. The production of CPC was inhibited by increasing the concentration of thiamine. The transcriptional differences of biosynthesis genes involved in CPC were also studied. 1 mg / mL and 100 mg / mL thiamine were added to the fermentation medium of Cephalosporium apocephalus, respectively, to make the cefEF in the strain. The up-regulation and down-regulation of cefG transcriptional level indicate that thiamine can regulate the transcription of genes synthesized by CPC and thus affect the production of CPC. The addition of thiamine in fermentation medium also affected the mycelium growth and biomass of Atracephalus to varying degrees. This study first confirmed that ActhiS protein is the thiazole synthase in Atracephalosporium. Thiamine is involved in the synthesis of thiazoles, which in turn affects the production of thiamine. As a coenzyme of many enzymes, thiamine is involved in the metabolism of amino acids and carbohydrates in organisms, and also in the synthesis of CPC. The results showed that thiamine metabolism pathway and Acthi gene could be used as a target for CPC producing strain breeding.
【学位授予单位】：上海医药工业研究院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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