通过代谢工程改造酿酒酵母L-苯丙氨酸合成相关途径,强化L-苯丙氨酸合成并实现胞外积累,为后续深入挖掘酿酒酵母芳香族氨基酸合成和转运机制,利用酿酒酵母生产芳香族氨基酸及其高价值衍生物提供参考。首先对酿酒酵母中心代谢途径和莽草酸途径进行代谢改造,获得1株合成L-苯丙氨酸初步强化菌株,测得胞外L-苯丙氨酸和L-酪氨酸产量分别为2.49和6.54 mg/L。为了进一步增强L-苯丙氨酸的积累,敲除L-苯丙氨酸消耗途径基因ARO10和PDC5。最后,敲除TYR1阻断竞争性L-酪氨酸合成途径,胞外L-苯丙氨酸产量提高至45.40 mg/L,这是目前研究酿酒酵母L-苯丙氨酸从头合成的最高胞外产量。
As one of the important aromatic amino acids, L-phenylalanine has been widely used in food, chemical industry, medicine and other industrial products. In this study, the central metabolic pathway and shikimic acid pathway of Saccharomyces cerevisiae were rational reconstructed, and a preliminary enhanced strain of L-phenylalanine synthesis was obtained. The production of extracellular L-phenylalanine and L-tyrosine was 2.49 mg/L and 6.54 mg/L, respectively. In order to further enhance the accumulation of L-phenylalanine, ARO10 and PDC5 genes were knocked out. Furthermore, TYR1 was knocked out to block the competitive L-tyrosine synthesis pathway, and the extracellular L-phenylalanine production increased to 45.40 mg/L, which is the highest extracellular production for de novo synthesis of L-phenylalanine in S. cerevisiae.
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