Abstract
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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Acknowledgements
We acknowledge financial support of National Key R D Program of China (2017YFD0200900) Subject 2 (2017 YFD0200902), Joint Open Fund of National Biopesticide Engineering Research Centre and Scientific Observation and Experimental Station of Utilization of microbial resources (Central China), Ministry of Agriculture and Rural Affairs (Grant No. JF-NBCOES-1807), Natural Science Foundation of China (31300074, 21606076), Natural Science Foundation of Hubei Province (2014CFB541), Specialized Research Fund for the Doctoral Program of Higher Education (20124208120004), Key project of educational commission of Hubei province of China(D20171002).
The authors declare no conflict of interest.
Neither ethical approval nor informed consent was required for this study.
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Rao, B., Zhou, R., Dong, Q. et al. Efficient Surface Display of L-glutamate Oxidase and L-amino Acid Oxidase on Pichia pastoris Using Multi-copy Expression Strains. Biotechnol Bioproc E 25, 571–579 (2020). https://doi.org/10.1007/s12257-019-0370-5
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DOI: https://doi.org/10.1007/s12257-019-0370-5