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KMID : 1094720200250040571
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Biotechnology and Bioprocess Engineering
2020 Volume.25 No. 4 p.571 ~ p.579
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Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains
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Rao Ben
Zhou Ronghua
Dong Qing
Liao Xianqing
Liu Fang
Chen Wei
Liu Xiaoyan
Min Yong
Wang Ya Ping
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Abstract
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L-glutamate oxidase (GLOD) and L-amino acid oxidase (AAO) were reported to be capable of convert L-glutamic acid to α-aketoglutaric acid (α-KG). These two enzymes gene have been successfully expressed by using pHBM905BDM in Pichia pastoris to produce α-aketoglutaric acid from L-glutamic acid in our previous studies. Here these two enzymes were displayed on P. pastoris to 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)-AGα1 and PAAO(1?3)-AGα1, respectively. The following results showed that expression of GLOD(1?3)-AGα1 and AAO(1?3)-AGα1 in multi-copy strains increased as designed and strain PGLOD3-AGα1 and PAAO3-AGα1 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 α-KG was 6.22 g/L/h and the highest α-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 α-KG was 5.78 g/L/h and the highest α-KG titer (115.6 g/L) was converted from 135 g/L L-glutamic acid. It showed that displaying enzymes on P. pastoris are suitable for use in industrial applications.
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KEYWORD
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α-ketoglutaric acid, displayed enzyme, multicopy expression, Pichia pastoris
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