【作者】 周燕；

【导师】 花强；

【作者基本信息】 华东理工大学 ， 发酵工程， 2013， 硕士

【摘要】 以番茄红素为代表的类胡萝卜素是一类具有巨大商业价值的天然产物。目前,微生物生产的许多天然产物变的越来越重要。本实验通过研究大肠杆菌中的异源合成番茄红素过程中1-脱氧木酮糖-5-磷酸途径(DXP途径)及中心代谢途径一些关键基因的影响以理解大肠杆菌生产番茄红素时涉及到的代谢机制,为提高大肠杆菌合成番茄红素的产量提供一定的理论依据。本实验首先对一些关键中心代谢基因进行敲除以研究这些基因对大肠杆菌异源合成番茄红素的影响,这些基因包括6-磷酸葡萄糖异构酶基因(pgi),谷氨酸脱氢酶基因(gdhA)和6-磷酸葡萄糖脱氢酶基因(zwf)。结果显示,zwf基因的敲除使得番茄红素产量提高超过130%,而pgi和gdhA基因敲除减少了番茄红素的合成量。其次,考察了DXP途径中的关键基因,包括异戊烯焦磷酸异构酶基因(idi),1-脱氧木酮糖-5-磷酸合酶基因(dxs)和4-二磷酸胞苷-2C-甲基-D-赤藓糖醇合酶基因,2C-甲基-D-赤藓糖醇2,4-环焦磷酸合酶基因(ispDF)在大肠杆菌中过量共表达对番茄红素产量的影响。实验结果表明这些基因的共表达可得番茄红素的产量从0.89mg g-1DCW提高至5.39mg g-1DCW,进而验证了DXP途径的重要性。最后,将中心代谢基因的敲除同时结合DXP途径基因的过表达得到番茄红素产量进一步提高的工程菌(番茄红素的最高产量可达到6.85-7.55mg g-1DCW)。基因转录水平更进一步揭示了大肠杆菌产番茄红素的代谢机制。除此之外,本研究首次揭示了胞外代谢物吲哚对番茄红素产量的抑制作用。本课题以大肠杆菌合成番茄红素为例,研究构建一类有利于理解类胡萝卜素物质异源合成中的代谢调控特征的基础平台。更多还原

【Abstract】 Lycopene, as a representative of carotenoids, is a useful natural product that holds great commercial value. Currently, microbial production of many natural products is becoming more and more important. Genetic manipulation concerning knockout of crucial central metabolic genes and overexpression of1-deoxyxylulose-5-phosphate pathway (DXP pathway) genes was undertaken in order to understand the mechanism involved in the heterologous synthesis of lycopene in Escherichia coli, providing theoretical basis for higher lycopene production in E. coli.Knockout of the crucial central metabolic genes resulted in enhancement of lycopene production (above130%) with the glucose-6-phosphate dehydrogenase gene (zwf) deletion while this synthesis was regressed after the deletion of glucosephosphate isomerase gene (pgi) or glutamate dehydrogenase gene (gdhA). The amplification and overexpression of rate-limiting steps encoded by isopentenyl diphosphate isomerase (idi),1-deoxyxylulose-5-phosphate synthase gene (dxs) and4-diphosphocytidyl-2C-methyl-D-erythritol synthase gene and2C-methyl-D-erythritol2,4-cyclodiphosphate synthase gene (ispDF) improved lycopene synthesis from0.89to5.39mg g-1DCW, thus exhibiting the ultimate role of the DXP pathway. The combination of central metabolic genes knockout with the amplification of primary metabolic genes yielded best amounts of lycopene (6.85-7.55mg g-1DCW). The additional transcriptional profiles obtained permitted an extensive understanding of lycopene metabolism. In addition, the inhibitory effect of indole on lycopene production was also investigated in this study. With this example of lycopene synthesis in E. coli, a basic platform for the introduction and metabolic understanding of heterologous pathways for carotenoids production will be constructed.更多还原