Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains

文献类型： 外文期刊

第一作者： Rao Ben

作者： Rao Ben;Zhou Ronghua;Dong Qing;Liao Xianqing;Liu Fang;Chen Wei;Liu Xiaoyan;Min Yong;Wang YaPing

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关键词： alpha-ketoglutaric acid; displayed enzyme; multicopy expression; Pichia pastoris

期刊名称：BIOTECHNOLOGY AND BIOPROCESS ENGINEERING （ 影响因子：2.836； 五年影响因子：2.281 ）

ISSN： 1226-8372

年卷期： 2020 年 25 卷 4 期

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收录情况： SCI

摘要： L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to alpha-aketoglutaric acid (alpha-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM inPichia pastoristo produce alpha-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed onP. pastoristo achieve the conversion. We constructed multi-copy expression plasmids using plasmid pHBM905BDM. By using this plasmid, multi-copy strains were constructed and named as PGLOD(1-3)-AG alpha 1 and PAAO(1-3)-AG alpha 1, respectively. The following results showed that expression of GLOD(1-3)-AG alpha 1 and AAO(1-3)-AG alpha 1 in multi-copy strains increased as designed and strain PGLOD3-AG alpha 1 and PAAO3-AG alpha 1 was chosen for high-density fermentation and enzyme activity experiments. By using a multi-copy expression approach and high-density fermentation, we achieved a GLOD expression yield of 688.5 U/g dry cell weight and AAO expression yield of 626.7 U/g dry cell weight. By using displayed GLOD, the average production rate of L-glutamic acid to alpha-KG was 6.22 g/L/h and the highest alpha-KG titer (124.5 g/L) was converted from 135 g/L L-glutamic acid. By using displayed AAO, the average production rate of L-glutamic acid to alpha-KG was 5.78 g/L/h and the highest alpha-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes onP. pastorisare suitable for use in industrial applications.

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