[Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli].
[Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli].
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DOI：
10.13345/j.cjb.180287
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发表时间：
2019-03-25
期刊：
影响因子：
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通讯作者：
Zhang, Xueli
中科院分区：
文献类型：
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作者：
Li, Zhenxia;Chen, Qianqian;Zhang, Xueli
类异戊二烯均来源于两种名为异戊烯基二磷酸（IPP）和二甲基烯丙基二磷酸（DMAPP）的五碳结构单元，它们可通过甲羟戊酸（MVA）途径或2 - C - 甲基 - D - 赤藓糖醇 - 4 - 磷酸（MEP）途径合成。在本研究中，将MVA途径基因整合到LYC101的染色体中，通过人工调控元件优化MEP合成途径和番茄红素合成途径中关键基因的表达，以进一步提高大肠杆菌中类异戊二烯的产量。从一个利用核糖体结合位点（RBS）文库连接MVA途径基因构建的质粒文库中筛选出质粒pALV23和pALV145，这大大提高了β - 胡萝卜素的产量。分别比较了质粒pALV23和pALV145对低产和高产番茄红素菌株LYC001和LYC101中番茄红素产量的影响。在这两个菌株中，质粒pALV23和pALV145都提高了番茄红素的产量。在高产番茄红素菌株LYC101中，pALV23比pALV145产生更多的番茄红素。然后，利用CRISPR - Cas9系统辅助的同源重组方法，将pALV23的MVA基因连同启动子整合到LYC101染色体的poxB位点，得到遗传稳定的菌株LYC102。LYC102的番茄红素产量为40.9 mg/g干细胞重量（DCW），比LYC101高1.19倍，比携带pALV23的LYC101高20%。在重组大肠杆菌中同时表达MVA途径和MEP途径可有效提高萜类化合物的产量。在本研究中，构建了一种无质粒、遗传稳定、高产番茄红素的菌株，可用于工业化生产。此外，该平台菌株可用于其他萜类化合物的合成。
Isoprenoids are all derived from two five-carbon building blocks called isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are synthesized either by the mevalonate (MVA) pathway or 2-C-methyld-D-erythritol-4-phosphate (MEP) pathway. In this study, the MVA pathway genes were integrated into the chromosome of LYC101, in which the expression of key genes in the MEP synthesis pathway and lycopene synthesis pathway were optimized by artificial regulatory parts, to further improve the production of isoprenoids in Escherichia coli. The plasmids pALV23 and pALV145 were screened from a plasmid library that constructed by using the RBS library to link the genes of the MVA pathway, which greatly increased the production of beta-carotene. The effects of plasmids pALV23 and pALV145 on the lycopene production in low and high lycopene production strain, LYC001 and LYC101, were compared, respectively. The production of lycopene was increased by plasmids pALV23 and pALV145 in both strains. In high lycopene production strain LYC101, pALV23 produced more lycopene than pALV145. Then, the MVA gene together of promoter of pALV23 was integrated into the chromosome of LYC101 at poxB site using method of homologous recombination helped by CRISPR-Cas9 system, resulted in genetically stable strain, LYC102. The yield of lycopene of LYC102 was 40.9 mg/g DCW, 1.19-folds higher than that of LYC101, and 20% more than that of LYC101 with pALV23. Simultaneous expression of MVA pathway and MEP pathway in recombinant E. coli can effectively increase the yield of terpenoids. In this study, a plasmid-free, genetically stable, high-yielding lycopene strain was constructed, which could be used for industrialization. Also, the platform strain can be used for the synthesis of other terpenoids.