本文選題：頂頭孢霉　切入點：初級代謝　出處：《中國醫(yī)藥工業(yè)研究總院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：頭孢菌素C(CPC)是治療細(xì)菌感染的β-內(nèi)酰胺類抗生素合成的主要前體,絲狀真菌頂頭孢霉是CPC的主要生產(chǎn)菌。在傳統(tǒng)菌種選育過程中頂頭孢霉經(jīng)歷多輪誘變和篩選使其CPC生產(chǎn)能力得到顯著提高,但是發(fā)生在高產(chǎn)背后的遺傳變化仍然未知。初級代謝通過向次級代謝提供前體和輔因子與其聯(lián)系在一起,并且前體和輔因子是高產(chǎn)菌中抗生素產(chǎn)量的限制因素。在本研究中利用實驗室建立的RNA測序數(shù)據(jù)和代謝物譜對高低產(chǎn)菌中的初級代謝進(jìn)行比較分析,更好的理解了初級代謝和CPC生物合成之間的關(guān)系,有利于使用合理代謝工程手段提高CPC產(chǎn)量。首先對頂頭孢霉中心碳代謝途徑中基因轉(zhuǎn)錄水平進(jìn)行分析,發(fā)現(xiàn)高產(chǎn)菌在發(fā)酵早期上調(diào)糖酵解途徑中的基因轉(zhuǎn)錄水平快速消耗葡萄糖開始CPC合成,發(fā)酵后期對3-磷酸甘油酸(3PG)上下游基因轉(zhuǎn)錄水平的不同調(diào)控導(dǎo)致糖異生途徑中的碳流導(dǎo)向3PG最終提高絲氨酸合成;在三羧酸循環(huán)中高產(chǎn)菌下調(diào)檸檬酸合酶基因轉(zhuǎn)錄水平迫使更多丙酮酸參與纈氨酸合成,而琥珀酸脫氫酶基因轉(zhuǎn)錄水平上調(diào)可能導(dǎo)致蘋果酸合成增加,從而有更多蘋果酸進(jìn)入糖異生途徑生成3PG;高產(chǎn)菌在乙醛酸循環(huán)中上調(diào)異檸檬酸裂解酶和蘋果酸合酶基因轉(zhuǎn)錄水平,乙醛酸循環(huán)更加活躍生成更多琥珀酸;高產(chǎn)菌發(fā)酵早期戊糖磷酸途徑中能生成NADPH的兩個酶轉(zhuǎn)錄水平上調(diào),而在發(fā)酵后期γ-氨基丁酸支路中琥珀酸半醛脫氫酶基因轉(zhuǎn)錄水平發(fā)生顯著上調(diào),這些都導(dǎo)致有更多NADPH參與CPC生物合成。將高低產(chǎn)菌中初級代謝中間產(chǎn)物胞內(nèi)含量進(jìn)行比較,發(fā)現(xiàn)葡萄糖、3PG、蘋果酸和琥珀酸在高低產(chǎn)菌中差別比較大,其含量變化趨勢與基因轉(zhuǎn)錄水平相一致。其次對CPC前體氨基酸合成途徑基因轉(zhuǎn)錄水平進(jìn)行分析,發(fā)現(xiàn)高產(chǎn)菌半胱氨酸合成途徑中編碼胱硫醚-γ-裂解酶、胱硫醚-β-合酶和S-腺苷甲硫氨酸合酶的基因轉(zhuǎn)錄水平上調(diào);絲氨酸和纈氨酸生物合成途徑基因轉(zhuǎn)錄水平在高產(chǎn)菌中均上調(diào);高產(chǎn)菌賴氨酸合成途徑中編碼第一步反應(yīng)酶基因轉(zhuǎn)錄水平上調(diào),而編碼最后一步反應(yīng)酶基因轉(zhuǎn)錄水平下調(diào)。檢測胞內(nèi)絲氨酸、半胱氨酸、纈氨酸和α-氨基己二酸含量,發(fā)現(xiàn)高產(chǎn)菌中這些氨基酸含量都高于低產(chǎn)菌,與基因轉(zhuǎn)錄水平相一致。此外還對CPC進(jìn)行體外和體內(nèi)甲氧基化探索。體外實驗從帶棒鏈霉菌中克隆cmc I和cmc J基因,分別構(gòu)建Cmc I和Cmc J蛋白表達(dá)載體并在體外純化。將純化后的Cmc I和Cmc J蛋白添加到變鉛青鏈霉菌和頂頭孢霉裂解液中對CPC進(jìn)行轉(zhuǎn)化。體內(nèi)實驗中構(gòu)建了兩個cmc I和cmc J基因的雙表達(dá)載體,分別導(dǎo)入頂頭孢霉中獲得正確轉(zhuǎn)化子并進(jìn)行發(fā)酵產(chǎn)物L(fēng)C-MS鑒定。
[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 緯 -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 偽 -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.
【學(xué)位授予單位】：中國醫(yī)藥工業(yè)研究總院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R914

【相似文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 韓姝;頂頭孢霉高低產(chǎn)菌初級代謝的比較及CPC甲氧基化的探索[D];中國醫(yī)藥工業(yè)研究總院;2016年

相關(guān)碩士學(xué)位論文 前1條

1 魏雪;根際銅綠假單胞菌M18中GacA對次級代謝、初級代謝、分泌系統(tǒng)及運動性的全局性調(diào)控[D];上海交通大學(xué);2013年

，

本文編號：1617920