代谢工程改造热带假丝酵母生产1,2,4-丁三醇

Metabolic engineering of Candida tropicalis for production of 1,2,4-butanetriol

摘要:

【背景】 1,2,4-丁三醇属于手性多羟基醇，是一种重要的有机合成的化学中间体，以木糖为原料经四步酶反应是目前研究最多的生物合成路线。然而大肠杆菌的鲁棒性较弱，对发酵液中一些抑制剂的耐受性不是很好，同时存在严重的碳代谢抑制。近年来，鲁棒性较好的酵母菌成为更有吸引力的宿主，其中热带假丝酵母具有天然的木糖代谢途径，可以更好地利用木糖。【目的】在热带假丝酵母中构建从木糖到1,2,4-丁三醇的代谢途径。【方法】在热带假丝酵母中敲除木糖还原酶基因GRE3，从而阻断自身的木糖代谢途径。将来源于Caulobacter crescentus的木糖脱氢酶基因(xylB)和木糖酸脱水酶基因(xylD)及来源于Lactococcus lactis的酮酸脱羧酶基因(kdcA)克隆至C. tropicalis 207中，得到重组菌C. tropicalis BT，在此基础上考察重组菌代谢木糖合成1,2,4-丁三醇的能力，确定限速步骤，并通过增加关键基因xylD与kdcA的拷贝数提高1,2,4-丁三醇产量。【结果】在30℃、200 r/min、接种量1%、以30 g/L木糖为底物的情况下，重组菌的1,2,4-丁三醇的产量达到了1.2 g/L，在5 L发酵罐中的产量达到了3.7 g/L。【结论】在热带假丝酵母中实现以木糖为底物的1,2,4-丁三醇代谢途径，并通过在基因组上增加关键基因xylD与kdcA的拷贝数，获得了一株高产1,2,4-丁三醇的重组酵母菌株，这为后续在热带假丝酵母中进一步提高1,2,4-丁三醇产量奠定了基础。

Abstract:

[Background] 1,2,4-butanetriol (BT), a chiral polyhydric alcohol, is an important chemical intermediate in organic synthesis. Among the biosynthesis routes, four-step enzymatic reaction with xylose as raw material is popular with scholars. However, Escherichia coli shows poor robustness, with low tolerance to some inhibitors in the fermentation broth and carbon catabolite repression. In recent years, the robust yeast has attracted the interest of researchers, particularly the Candida tropicalis which harbors the xylose transporter.[Objective] To develop the metabolic pathway from xylose to BT in C. tropicalis.[Methods] The xylose reductase gene GRE3 in C. tropicalis was knocked out to block the innate xylose metabolic pathway. The genes encoding D-xylose dehydrogenase (xylB) and D-xylonate dehydratase (xylD) from Caulobacter crescentus and the gene encoding the branched-chain α-keto acid decarboxylase (kdcA) from Lactococcus lactis were cloned into C. tropicalis 207, to yield the recombinant C. tropicalis BT. Then, the ability of the recombinant C. tropicalis to synthesize BT from xylose was investigated and the rate-limiting step was identified. The production of BT was improved by increasing the copy number of key genes xylD and kdcA.[Results] Under the conditions of 30℃, 200 r/min, inoculum of 1%, and 30 g/L xylose, the yield of BT of the recombinant bacteria reached 1.2 g/L. In a 5 L fermenter, the yield was up to 3.7 g/L. [Conclusion] We developed a BT metabolic pathway in C. tropicalis with xylose as substrate, and a strain with high BT yield was constructed by increasing the copy number of key genes on the genome. The study lays a foundation for further improving the production of BT in C. tropicalis.