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A bi-enzymatic cascade to yield pyruvate as co-substrate for l-tyrosine production

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Abstract

l-Tyrosine is a versatile compound used in the fine chemical, pharmaceutical, and functional food industries. Here, we report a bi-enzymatic cascade involving alanine racemase (ALR) and d-amino acid oxidase (DAAO) to produce pyruvate, as co-substrate for l-tyrosine production, from the cheap substrate l-alanine. The BpALR (ALR from Bacillus pseudofirmus) was used as a whole-cell biocatalyst, converting l-alanine to d, l-alanine. The FsDAAO (DAAO from Fusarium solani) was immobilized to oxidize the d-alanine generated in the first step to pyruvate. Both systems were combined as a continuous-flow reactor for maximized l-alanine-to-pyruvate conversion rates. The optimal parameters and appropriate conditions for FsDAAO immobilization were investigated. The pyruvate concentration of 86.6 g/L was achieved within 17 h. Subsequently, a whole-cell biocatalyst system for l-tyrosine production, catalyzed by the tyrosine phenol-lyase (TPL) from Erwinia herbicola (EhTPL), was developed, and a fed-batch approach was applied with phenol and the pyruvate produced with the ALR/DAAO system mentioned above. The concentration of phenol and pyruvate in the reactor should not exceed 7.5 g/L and 10 g/L, respectively. Significantly, the l-tyrosine concentration of 152.5 g/L was achieved within 10 h, demonstrating the great potential for high-efficiency production of l-tyrosine through the approach we established in this paper.

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Key points

A specific bioreactor system for pyruvate produced from l-alanine was developed

The appropriate condition for immobilization of FsDAAO was investigated

A fed-batch process was established to produce l-tyrosine with recombinant E. coli

The bi-enzymatic cascade was successfully used for l-tyrosine production at low cost

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Funding

This study was funded by the National Natural Science Foundation of China (No. 31370802 and No. 30870545).

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Contributions

X.L.G. and J.Z.H. conceived the research. X.L.G. wrote the manuscript. L.C.W. contributed important ideas in the experimental design. W.B.W. and M.L.Z. performed most of the experiments and critically reviewed the manuscript. X.L.G. and W.B.W. performed some experiments and analyzed data. All authors read and approved the manuscript.

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Correspondence to Jianzhong Huang.

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The authors declare that they have no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Guo, X., Wu, W., Zhang, M. et al. A bi-enzymatic cascade to yield pyruvate as co-substrate for l-tyrosine production. Appl Microbiol Biotechnol 104, 10005–10018 (2020). https://doi.org/10.1007/s00253-020-10975-4

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  • DOI: https://doi.org/10.1007/s00253-020-10975-4

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