顶头孢霉高低产菌初级代谢的比较及CPC甲氧基化的探索

发布时间：2018-03-16 02:22

本文选题：顶头孢霉　切入点：初级代谢　出处：《中国医药工业研究总院》2016年博士论文　论文类型：学位论文

【摘要】：头孢菌素C(CPC)是治疗细菌感染的β-内酰胺类抗生素合成的主要前体,丝状真菌顶头孢霉是CPC的主要生产菌。在传统菌种选育过程中顶头孢霉经历多轮诱变和筛选使其CPC生产能力得到显著提高,但是发生在高产背后的遗传变化仍然未知。初级代谢通过向次级代谢提供前体和辅因子与其联系在一起,并且前体和辅因子是高产菌中抗生素产量的限制因素。在本研究中利用实验室建立的RNA测序数据和代谢物谱对高低产菌中的初级代谢进行比较分析,更好的理解了初级代谢和CPC生物合成之间的关系,有利于使用合理代谢工程手段提高CPC产量。首先对顶头孢霉中心碳代谢途径中基因转录水平进行分析,发现高产菌在发酵早期上调糖酵解途径中的基因转录水平快速消耗葡萄糖开始CPC合成,发酵后期对3-磷酸甘油酸(3PG)上下游基因转录水平的不同调控导致糖异生途径中的碳流导向3PG最终提高丝氨酸合成;在三羧酸循环中高产菌下调柠檬酸合酶基因转录水平迫使更多丙酮酸参与缬氨酸合成,而琥珀酸脱氢酶基因转录水平上调可能导致苹果酸合成增加,从而有更多苹果酸进入糖异生途径生成3PG;高产菌在乙醛酸循环中上调异柠檬酸裂解酶和苹果酸合酶基因转录水平,乙醛酸循环更加活跃生成更多琥珀酸;高产菌发酵早期戊糖磷酸途径中能生成NADPH的两个酶转录水平上调,而在发酵后期γ-氨基丁酸支路中琥珀酸半醛脱氢酶基因转录水平发生显著上调,这些都导致有更多NADPH参与CPC生物合成。将高低产菌中初级代谢中间产物胞内含量进行比较,发现葡萄糖、3PG、苹果酸和琥珀酸在高低产菌中差别比较大,其含量变化趋势与基因转录水平相一致。其次对CPC前体氨基酸合成途径基因转录水平进行分析,发现高产菌半胱氨酸合成途径中编码胱硫醚-γ-裂解酶、胱硫醚-β-合酶和S-腺苷甲硫氨酸合酶的基因转录水平上调;丝氨酸和缬氨酸生物合成途径基因转录水平在高产菌中均上调;高产菌赖氨酸合成途径中编码第一步反应酶基因转录水平上调,而编码最后一步反应酶基因转录水平下调。检测胞内丝氨酸、半胱氨酸、缬氨酸和α-氨基己二酸含量,发现高产菌中这些氨基酸含量都高于低产菌,与基因转录水平相一致。此外还对CPC进行体外和体内甲氧基化探索。体外实验从带棒链霉菌中克隆cmc I和cmc J基因,分别构建Cmc I和Cmc J蛋白表达载体并在体外纯化。将纯化后的Cmc I和Cmc J蛋白添加到变铅青链霉菌和顶头孢霉裂解液中对CPC进行转化。体内实验中构建了两个cmc I和cmc J基因的双表达载体,分别导入顶头孢霉中获得正确转化子并进行发酵产物LC-MS鉴定。
[Abstract]:Cephalosporin CPC is the main precursor of the synthesis of 尾-lactam antibiotics for the treatment of bacterial infections. The filamentous fungus cephalosporium is the main producing strain of CPC. During the breeding of traditional fungi, the production capacity of CPC was improved significantly because of repeated mutagenesis and screening of Cephalosporium apocephalus. But the genetic changes that take place behind high yields are still unknown. Primary metabolism is associated with primary metabolism by providing precursors and cofactors to secondary metabolism. Precursors and cofactors were the limiting factors of antibiotic production in high-yielding bacteria. In this study, the primary metabolism of high and low producing bacteria was compared and analyzed by using RNA sequencing data and metabolites spectrum established in laboratory. A better understanding of the relationship between primary metabolism and CPC biosynthesis is beneficial to the use of rational metabolic engineering methods to increase CPC production. Firstly, the transcription level of genes in the central carbon metabolism pathway of Cephalosporium acrocephalus was analyzed. It was found that the gene transcription level in the glycolysis pathway was upregulated by high-yielding bacteria at the early stage of fermentation. Glucose consumption was rapidly consumed to begin CPC synthesis. At the late stage of fermentation, the different regulation of gene transcription level in the upstream and downstream of 3PG3Phosphate leads to the increase of serine synthesis by the carbon-directed 3PG in the glycosylated pathway. During the tricarboxylic acid cycle, the down-regulation of citrate synthase gene transcription by high-yielding bacteria forced more pyruvate to participate in valine synthesis, while the up-regulation of succinate dehydrogenase gene transcription may lead to an increase in malic acid synthesis. Therefore, more malic acid enter the glycolytogenic pathway to produce 3PG.High-yielding bacteria up-regulate the transcription level of isocitrate lyase and malate synthase gene in glyoxylic acid cycle, and the glyoxylic acid cycle is more active to produce more succinic acid. The transcription levels of two enzymes that produce NADPH in the early pentose phosphoric acid pathway of high-yielding bacteria were upregulated, while in the 纬 -aminobutyric acid branching pathway, the transcription level of succinate hemialdehyde-dehydrogenase gene was significantly up-regulated at the late stage of fermentation. These results showed that more NADPH were involved in CPC biosynthesis. Comparing the intracellular contents of primary metabolic intermediates in high and low producing bacteria, we found that glucose 3PGs, malic acid and succinic acid were different in high and low producing bacteria. Secondly, the gene transcription level of CPC precursor amino acid synthesis pathway was analyzed, and it was found that cysteine synthesis pathway encodes cystathion-gamma lyase in high-yielding bacteria. The gene transcription level of cystathithion- 尾 synthase and S- adenosine methionine synthase was up-regulated, while that of serine and valine biosynthesis pathway was up-regulated in high-yielding bacteria. The transcription level of the first step reaction enzyme gene was up-regulated in the lysine biosynthesis pathway, while the transcription level of the last step reaction enzyme gene was down-regulated. The contents of intracellular serine, cysteine, valine and 伪 -aminoadipic acid were measured. It was found that these amino acids in high-yielding bacteria were higher than those in low-yielding strains, which were consistent with the transcription level of genes. In addition, methoxylation of CPC was investigated in vitro and in vivo. The cmc I and cmc J genes were cloned from Streptomyces rotunda in vitro. Cmc I and Cmc J protein expression vectors were constructed and purified in vitro. The purified Cmc I and Cmc J proteins were added to the lytic solution of Streptomyces lead and Cephalosporium to transform CPC. Two cmc were constructed in vivo. The double expression vector of I and cmc J gene, The correct transformants were obtained from Atracephalosporium and the fermentation products were identified by LC-MS.
【学位授予单位】：中国医药工业研究总院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R914

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