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Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of d-amino Acid Oxidase Expressed in Escherichia coli


d-amino acid oxidase (DAAO) is widely used in the industrial preparation of l-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of l-phosphinothricin (l-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous d-alanine (d-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L d-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.

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Data Availability

The authors declare that the data and materials are transparent.

Code Availability

All data, models, and code generated or used during the study appear in the submitted article.


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This work was financially supported by the National Key R & D Program of China (2018YFA0901400).

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Authors and Affiliations



Jian-Miao Xu, Hui-Ting Cao, and Kai Zhang made substantial contributions to the design of the work, performed the most analysis, experiments, and wrote the manuscript. Ming Wang, Bao-Jian Ma, and Liu-Yu Wang participate in the experiment. Jian-Miao Xu, Feng Cheng, Ya-Ping Xue, and Yu-Guo Zheng revised it critically for important intellectual content and approved the version to be published.

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Correspondence to Ya-Ping Xue.

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Xu, JM., Cao, HT., Wang, M. et al. Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of d-amino Acid Oxidase Expressed in Escherichia coli. Appl Biochem Biotechnol 193, 2029–2042 (2021).

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  • E. coli
  • d-amino acid oxidase
  • Fed-batch fermentation
  • Hydrogen peroxide
  • d-alanine