代谢工程改造酿酒酵母生产L-苹果酸

Metabolic Engineering of Saccharomyces cerevisiae for L-Malic Acid Production

DOI：10.3969/j.issn.1673-1689.2019.02.011

中文关键词: 酿酒酵母 胞质还原路径 L-苹果酸 代谢工程

英文关键词: Saccharomyces cerevisiae,reductive pathway,L-malic acid,metabolic engineering

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中文摘要:

为了研究胞质还原路径对酿酒酵母积累L-苹果酸的影响，通过在酿酒酵母中过量表达源于黄曲霉的丙酮酸羧化酶（Afpyc）、苹果酸脱氢酶（Afmdh）及C4-二羧酸转运蛋白（Afmae），成功构建了L-苹果酸合成的胞质还原路径。结果表明：（1） 当低水平表达Afpyc时，其丙酮酸浓度降低了42%，但不能积累L-苹果酸；（2） 当共表达Afpyc和Afmdh时，菌株W005积累了1.93 g/L的L-苹果酸，与对照菌株W004相比细胞干重提高了350%，丙酮酸降低了65.9%；（3） 当共表达Afpyc、Afmdh和Afmae时，菌株W006的L-苹果酸产量提高了21.2%，达到2.34 g/L；4） 通过提高接种量至初始OD600=2，L-苹果酸的产量提高到3.28 g/L。通过在酿酒酵母中过量表达黄曲霉胞质还原路径的关键基因，使得工程菌能够积累L-苹果酸，为目标产物的高效积累提供了一种可借鉴的思路。

英文摘要:

With the purpose of elucidating the introduction of cytoplasmic reductive pathway in Saccharomyces cerevisiae on the production of L-malic acid， the reductive pathway for L-malic acid biosynthesis is constructed in S. cerevisiae by overexpressing genes Afpyc，Afmdh and Afmae from Aspergillus flavus. When gene Afpyc is kept in low level，the pyruvate titers of W003 are decreased 42% compared with control strain W001，respectively. To further introduced gene Afmdh in strain W003，the concentration of L-malic acid in W005 is up to 1.93 g/L，the DCW is increased by 350% and the pyruvate titers is decreased by 65.9% than that of the strain W004. Moreover，the malate efflux system is enhanced by introducting the tansporter gene Afmae and the L-malic acid titers of strain W006 is increased by 21.2%（2.34 g/L）. The concentrations of L-malic acid is increased from 2.34 g/L to 3.28 g/L with the initial OD600=2. It could be summarized that introducting the reductive pathway of overproducing L-malic acid strain A. flavus in S. cerevisiae is indeed producted L-malic acid，and this successful method provides a strategy to the metabolic engineering.

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