重构树干毕赤酵母合成白藜芦醇

Reconstruction of Scheffersomyces stipitis for synthesis of resveratrol

摘要:

白藜芦醇是一种有价值的植物多酚类化合物，目前主要通过植物提取法获得，受到天然原料的限制，不易长期大量获取，因此有必要开发一种更加简便、经济的白藜芦醇生产方法。本研究以树干毕赤酵母的野生型菌株为宿主，首先通过引入编码白藜芦醇合成的关键酶基因(HaTAL1、AtPAL2、AtC4H、At4CL2以及VvSTS)得到菌株Ss05，合成白藜芦醇55.28 mg/L。随后通过过表达3-脱氧-d-阿拉伯庚糖酮酸-7-磷酸(3-deoxy-d-arabino-heptulonate-7-phosphate, DAHP)合成酶突变体SsARO4^K221L及分支酸变位酶突变体SsARO7^G139S、敲除丙酮酸脱羧酶基因(PDC1)、过表达乙酰辅酶A羧化酶突变体SsACC1^S650A,S1152A，增强对香豆酸及丙二酰辅酶A的供应。在此基础上，进一步增加关键基因的拷贝数得到工程菌株Ss17，白藜芦醇产量提高到150.56 mg/L。最后将工程菌株Ss17在5 L发酵罐中通过分批补料葡萄糖发酵128 h，得到了558.40 mg/L白藜芦醇。本研究运用合成生物学思路，构建利用简单碳源合成白藜芦醇的工程菌株，同时利用生物反应器放大微生物发酵规模，为树干毕赤酵母中芳香族化合物的生物合成提供了重要参考。

Abstract:

Resveratrol is a valuable plant polyphenol. It is currently obtained primarily through plant extraction, which is difficult to obtain in large quantities over a long period of time due to the scarcity of natural raw materials. Therefore, it is necessary to develop a simple and cost-saving method for producing resveratrol. In this study, the genes encoding key enzymes for resveratrol synthesis (HaTAL1, AtPAL2, AtC4H, At4CL2, and VvSTS) were introduced into the wild-type strain of Scheffersomyces stipitis to construct strain Ss05, which achieved the resveratrol yield of 55.28 mg/L. Subsequently, the supply of p-coumaric acid and malonyl-CoA precursors was enhanced by overexpression of the feedback-insensitive 3-deoxy-d-arabino-heptulonate-7-phosphate synthase mutant SsARO4^K221L and chorismate mutase mutant SsARO7^G139S, knockout of the pyruvate decarboxylase gene (PDC1), and overexpression of the acetyl-CoA carboxylase mutant SsACC1^{S650A, S1152A}. On this basis, the copy number of key genes was increased to create the engineered strain Ss17, which achieved the resveratrol yield of 150.56 mg/L. Finally, 558.40 mg/L resveratrol was produced by fed-batch fermentation of glucose with strain Ss17 in a 5-L fermenter for 128 h. In this study, we employed synthetic biology to construct an engineered strain of S. stipitis for the synthesis of resveratrol from a simple carbon source and then scaled up the microbial fermentation in a bioreactor, providing an important reference for the biosynthesis of aromatic compounds in S. stipitis.