大肠杆菌合成1,2,4-丁三醇的途径优化

Optimization of 1,2,4-butanetriol synthetic pathway in Escherichia coli

1,2,4-丁三醇(BT)是一种在工业中有多种用途的重要的非天然化合物。文中通过将外源基因xdh和mdlC导入大肠杆菌BW25113表达,并敲除了xylA、xylB、yagE、yjhH、yiaE和ycdW等木糖和中间产物代谢旁路基因,构建了能够将D-木糖转化为BT的重组菌株。为优化BT合成途径,针对BT合成途径中的限速步骤——3-脱氧-D-甘油-戊酮糖酸的脱羧反应,进行了新酶的筛选和评价,获得了可显著提高反应效率的新的2-酮酸脱羧酶——KivD,并构建了表达该酶的重组菌株BW-025。在此基础上,通过初步条件优化,将BT产量提高至2.38g/L;进一步调节途径中各个酶的表达量,探究了它们对BW-025合成BT的影响,最终获得了BT产量较BW-025提高了48.62%的重组菌株BW-074。

1,2,4-Butanetriol（BT） is an important non-natural chemical with a variety of industrial applications. A recombinant Escherichia coli biosynthesizing BT from D-xylose was constructed by heterologously expressing xdh and mdl C, and knocking out competing pathway genes including xyl A, xyl B, yjh E, yag H and ycd W. To optimize BT synthesis pathway, the third catalytic step that catalyzes the decarboxylation reaction of 3-deoxy-D-glycero-pentulosonic acid was identified as a potential bottleneck. Consequently, 2-keto acid decarboxylases from three different microorganisms were screened, and the kiv D gene from Lactococcus lactis was found to increase BT titer by 191%. The improved strain BW-025 reached a final BT titer of 2.38 g/L under optimized transformation conditions. Attempts on synthetic pathway optimization were also made by fine-tuning the expression levels of each enzyme involved in the whole pathway based on BW-025. As a result, an xdh overexpressed recombinant strain, BW-074 was finally generated, with 48.62% higher BT production than that of BW-025.