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<p>Phenylpyruvate (PPA) is a valuable organic acid with broad applications in the food, feed, pharmaceutical, and chemical industries. Although current industrial production depends mainly on chemical synthesis, growing emphasis on green manufacturing has accelerated the development of biosynthetic routes using enzymatic or microbial platforms. Notably, the <span>l</span>-amino acid deaminase from <i>Proteus mirabilis</i> (<i>Pmi</i>LAAD) represents a promising biocatalyst for PPA biosynthesis due to its broad substrate range and high catalytic efficiency. However, the inherent substrate inhibition of <i>Pmi</i>LAAD at high concentrations severely limits PPA productivity and economic viability in whole-cell systems, posing a major bottleneck for industrial application. To address this, the substrate-binding pocket of <i>Pmi</i>LAAD was engineered through semi-rational design and random saturation mutagenesis, obtaining a beneficial mutant <i>Pmi</i>LAAD<sup>M440V</sup> (M4). Compared to the wild-type, the M4 variant increased product concentrations by 2.17-fold and 16.69-fold at substrate concentrations of 45&#xa0;g/L and 60&#xa0;g/L, respectively, demonstrating significantly enhanced catalytic efficiency and high-substrate tolerance. The whole-cell biocatalyst in a 10 mL system exhibited remarkable performance, reaching a PPA space-time yield of 2.90&#xa0;g/L/h with a molar yield of 77.7%. This study demonstrates that reshaping the substrate-binding pocket of <i>Pmi</i>LAAD effectively alleviates substrate inhibition, providing a strategic foundation for overcoming efficiency bottlenecks in industrial PPA production.</p>

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Enhanced substrate tolerance of l-amino acid deaminase by reshaping substrate binding pocket for phenylpyruvic acid production

  • 期刊论文

  • 发表日期：

    2026年3月17日

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  • Kai Chu,
  • Yingjia Tong,
  • Qingbo Deng,
  • Xiaomei Zhang,
  • Jinsong Shi,
  • Zhenghong Xu,
  • Hui Li

摘要

Phenylpyruvate (PPA) is a valuable organic acid with broad applications in the food, feed, pharmaceutical, and chemical industries. Although current industrial production depends mainly on chemical synthesis, growing emphasis on green manufacturing has accelerated the development of biosynthetic routes using enzymatic or microbial platforms. Notably, the l-amino acid deaminase from Proteus mirabilis (PmiLAAD) represents a promising biocatalyst for PPA biosynthesis due to its broad substrate range and high catalytic efficiency. However, the inherent substrate inhibition of PmiLAAD at high concentrations severely limits PPA productivity and economic viability in whole-cell systems, posing a major bottleneck for industrial application. To address this, the substrate-binding pocket of PmiLAAD was engineered through semi-rational design and random saturation mutagenesis, obtaining a beneficial mutant PmiLAADM440V (M4). Compared to the wild-type, the M4 variant increased product concentrations by 2.17-fold and 16.69-fold at substrate concentrations of 45 g/L and 60 g/L, respectively, demonstrating significantly enhanced catalytic efficiency and high-substrate tolerance. The whole-cell biocatalyst in a 10 mL system exhibited remarkable performance, reaching a PPA space-time yield of 2.90 g/L/h with a molar yield of 77.7%. This study demonstrates that reshaping the substrate-binding pocket of PmiLAAD effectively alleviates substrate inhibition, providing a strategic foundation for overcoming efficiency bottlenecks in industrial PPA production.