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<p><i>p</i>-Coumaric acid (p-CA) is a key aromatic precursor for the biosynthesis of flavonoids, stilbenoids, and other high-value phenylpropanoids. While microbial production of p-CA typically relies on sugar-based substrates, methanol offers a sustainable and cost-effective alternative, though its use for aromatic biosynthesis remains unexplored. Here, we report the first de novo production of p-CA from methanol using engineered methylotrophic yeast <i>Pichia pastoris</i>. Through heterologous expression of a tyrosine ammonia-lyase and implementing a balanced push–pull strategy in the shikimate pathway using feedback-resistant variants of DAHP synthase (<i>ARO4</i>) and chorismate mutase (<i>ARO7</i>), carbon flux from methanol-derived C3 and C4 precursors was effectively redirected toward aromatic biosynthesis. Shake-flask studies revealed strong gene-dosage-dependent p-CA production, but strains with high-copy numbers suffered metabolic burden under high-density fermentation. Fed-batch bioreactor cultivation demonstrated that a moderate-copy strain achieved the highest titer of 704 ± 6&#xa0;mg/L, outperforming high-copy variants in robustness and scalability. This study establishes <i>P</i>. <i>pastoris</i> as a promising chassis for methanol-based aromatic production and highlights the critical trade-off between pathway amplification and cellular fitness in C1 biomanufacturing.</p> Graphical abstract <p></p>

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Methanol-based biosynthesis of p-coumaric acid by engineered Pichia pastoris

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月13日

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  • Mengyuan Chen,
  • Jiayu Fang,
  • Shuxian Wang,
  • Guoxia Liu,
  • Yanping Zhang,
  • Yin Li,
  • Kaizhi Jia,
  • Taicheng Zhu

摘要

p-Coumaric acid (p-CA) is a key aromatic precursor for the biosynthesis of flavonoids, stilbenoids, and other high-value phenylpropanoids. While microbial production of p-CA typically relies on sugar-based substrates, methanol offers a sustainable and cost-effective alternative, though its use for aromatic biosynthesis remains unexplored. Here, we report the first de novo production of p-CA from methanol using engineered methylotrophic yeast Pichia pastoris. Through heterologous expression of a tyrosine ammonia-lyase and implementing a balanced push–pull strategy in the shikimate pathway using feedback-resistant variants of DAHP synthase (ARO4) and chorismate mutase (ARO7), carbon flux from methanol-derived C3 and C4 precursors was effectively redirected toward aromatic biosynthesis. Shake-flask studies revealed strong gene-dosage-dependent p-CA production, but strains with high-copy numbers suffered metabolic burden under high-density fermentation. Fed-batch bioreactor cultivation demonstrated that a moderate-copy strain achieved the highest titer of 704 ± 6 mg/L, outperforming high-copy variants in robustness and scalability. This study establishes P. pastoris as a promising chassis for methanol-based aromatic production and highlights the critical trade-off between pathway amplification and cellular fitness in C1 biomanufacturing.

Graphical abstract