稳定表达MVA途径基因提高番茄红素产量

Integrating balanced mevalonate pathway into chromosome for improving lycopene production in Escherichia coli

摘要:

萜类化合物的直接前体物质异戊烯焦磷酸 (IPP) 和二甲基烯丙基焦磷酸酯 (DMAPP) 可以由2-甲基- D-赤藻糖醇-4-磷酸途径 (MEP途径) 和甲羟戊酸途径 (MVA途径) 合成。在已经优化MEP合成途径、番茄红素合成途径关键基因表达的重组大肠杆菌LYC101中，引入MVA途径基因，进一步提高重组大肠杆菌合成萜类化合物的能力。质粒pALV23和pALV145是本实验室在研究MVA途径基因协调表达时，用核糖体结合位点 (RBS) 文库连接MVA途径各基因构建质粒文库，而筛选到的有效提高β-胡萝卜素产量的质粒。首先比较了两个质粒分别在低产和高产番茄红素的菌株中对番茄红素合成的影响。结果表明，两个质粒在高、低产番茄红素的菌株中都可以有效提高番茄红素产量。在高产菌LYC101中pALV23比pALV145使番茄红素产量更高。然后，用CRISPR-Cas9系统辅助同源重组的方法，将MVA途经基因和启动子一共6.7 kb的条带整合到LYC101菌株的染色体上，得到遗传稳定的菌株LYC102。LYC102的番茄红素产率达40.9 mg/g，是出发菌株LYC101产率的2.19倍，比用质粒表达MVA途径基因的菌株提高了20%。在重组大肠杆菌中同时表达MVA途径和MEP途径，可以有效提高萜类化合物产率；文中构建了不含质粒的、遗传稳定的高产番茄红素菌株，为产业化合成番茄红素提供基础；同时构建平台菌株，可以用于其他萜类化合物合成。

Abstract:

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 β-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.