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

摘要:

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2.38 g/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。

Abstract:

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 mdlC, and knocking out competing pathway genes including xylA, xylB, yjhE, yagH and ycdW. 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 kivD 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.