该文研究了酿酒酵母中编码丙酮酸脱羧酶的基因PDC5的缺失对2-苯乙醇合成的影响,并将其应用于2-苯乙醇高产菌株的代谢改造策略。首先利用CRISPR/Cas9双质粒敲除体系构建pdc5△突变株,通过优化过的培养条件进行摇瓶发酵,发现基因PDC5的缺失能够促进酵母合成2-苯乙醇。分别表达芳香族转氨酶Aro8p和Aro9p,发现在缺失Aro9p的突变株中过表达Aro8p能够促进2-苯乙醇的合成。由此构建了重组菌株RM59-810(pdc5△aro9△ PTPI-ARO8-TTT PTPI-ARO10-TTT),在6.7 g/L L-苯丙氨酸培养基中培养120 h后2-苯乙醇的产量为3.85 g/L,摩尔转化率为0.67 mol/mol,是对照菌株的1.33倍,得率为0.5 g/g L-苯丙氨酸。该文为加强酿酒酵母合成2-苯乙醇的能力提供了一种新的代谢工程改造策略,为研究丙酮酸脱羧酶Pdc5p可能存在的调控作用提供了依据。
As an important aromatic alcohols with favorable flavor and superior property, 2-phenylethanol has been widely used in food, medicine, and chemical industry. In this study, the deletion of phenylpyruvate decarboxylase gene PDC5 was found to improve the synthesis of 2-phenylethanol, and related metabolic strategy of the Ehrlich pathway was rational reconstructed in Saccharomyces cerevisiae for further product-enhancement. Strain RM22 (pdc5△) was obtained using CRISPR/Cas9, and improved 2-phenylethanol production was observed in the optimized culture conditions. Additionally, by expressing aromatic transaminase Aro8p and Aro9p respectively, we found that overexpressing of Aro8p in aro9-deleted strain was an efficient strategy to increase 2-phenylethanol production. To further improve the production of 2-phenylethanol, metabolic engineered strain RM-59810 (pdc5△aro9△PTPI-ARO8-TTT PTPI-ARO10-TTT) was constructed, resulting in 3.85 g/L of 2-phenylethanol from 6.7 g/L phenylalanine after 120 h, and the molar conversion rate was 0.67 mol/mol, which was 1.33-fold than that of the control strain, with a yield of 0.5 g/g L-phenylalanine. In this study, a new efficient strategy was provided to improve 2-phenylethanol production in S. cerevisiae, and also provided a primary foundation for studying the possible regulatory effects of pyruvate decarboxylase Pdc5p.
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