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Abstract <p>Free or protein-conjugated N-Acetylneuraminic acid (Neu5Ac) is widely distributed in organisms and possesses diverse biological functions, and for example lactoferrin with Neu5Ac-mediated sialylation exhibits significantly enhanced functional activity compared with non-sialylated lactoferrin. <i>Pichia pastoris</i> serves as an excellent host for lactoferrin; however, efficient biosynthesis of Neu5Ac is essential for efficiently expressing lactoferrin with Neu5Ac-mediated sialylation. In this study,<i> P. pastoris</i> was engineered into a high-yield Neu5Ac host for expressing highly active lactoferrin with Neu5Ac modification. Heterologous Neu5Ac biosynthesis genes (<i>gfa1</i>, <i>gna1</i>, <i>yqaB</i>, <i>age</i>, and <i>neuB</i>) were first introduced into a <i>P. pastoris</i> strain in which <i>pfk1</i> and <i>zwf</i> attenuation combined with <i>pfk2</i> deletion redirected carbon flux toward fructose-6-phosphate, yielding the Neu5Ac-producing strain <i>P. pastoris</i> N1 with Neu5Ac production of 9.19&#xa0;mg/L. Neu5Ac titers were subsequently boosted 2.8-fold via precursor enhancement through <i>pyk</i> attenuation and <i>gs</i> overexpression. Implementation of glucose–glycerol co-utilization (<i>gut1</i>, <i>gut2</i>, <i>gt2</i> overexpression) dramatically increased Neu5Ac production to 285.41&#xa0;mg/L. Optimization via increased copy number of <i>gfa1</i>, <i>age</i>, and <i>neuB</i>, coupled with their overexpression under the strong promoter P<sub><i>GAP</i></sub>, culminated in approximately 111.5-fold improvement (reaching 1.03&#xa0;g/L) over the N1 strain. The effective integration of diverse metabolic engineering strategies, including heterologous pathway introduction, precursor enhancement, and co-substrate utilization, established a valuable and transferable framework for reprogramming <i>P. pastoris</i> to synthesize Neu5Ac and other high-value compounds.</p> Key points <p>• <i>P. pastoris was engineered to synthesize Neu5Ac.</i></p> <p>• <i>Neu5Ac production achieved 1.03&#xa0;g/L via remodeling of key metabolic pathways.</i></p> <p>• <i>The strain harbored potential for synthesis of free Neu5Ac or sialylated proteins.</i></p>

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Multistep metabolic engineering of Pichia pastoris for biosynthesis of N‑acetylneuraminic acid

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月24日

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  • Xiaomin Zhao,
  • Qian Li,
  • Huiying Luo,
  • Yuan Wang,
  • Xinxin Xu,
  • Wei Zhang,
  • Jie Zhang,
  • Bo Liu

摘要

Abstract

Free or protein-conjugated N-Acetylneuraminic acid (Neu5Ac) is widely distributed in organisms and possesses diverse biological functions, and for example lactoferrin with Neu5Ac-mediated sialylation exhibits significantly enhanced functional activity compared with non-sialylated lactoferrin. Pichia pastoris serves as an excellent host for lactoferrin; however, efficient biosynthesis of Neu5Ac is essential for efficiently expressing lactoferrin with Neu5Ac-mediated sialylation. In this study, P. pastoris was engineered into a high-yield Neu5Ac host for expressing highly active lactoferrin with Neu5Ac modification. Heterologous Neu5Ac biosynthesis genes (gfa1, gna1, yqaB, age, and neuB) were first introduced into a P. pastoris strain in which pfk1 and zwf attenuation combined with pfk2 deletion redirected carbon flux toward fructose-6-phosphate, yielding the Neu5Ac-producing strain P. pastoris N1 with Neu5Ac production of 9.19 mg/L. Neu5Ac titers were subsequently boosted 2.8-fold via precursor enhancement through pyk attenuation and gs overexpression. Implementation of glucose–glycerol co-utilization (gut1, gut2, gt2 overexpression) dramatically increased Neu5Ac production to 285.41 mg/L. Optimization via increased copy number of gfa1, age, and neuB, coupled with their overexpression under the strong promoter PGAP, culminated in approximately 111.5-fold improvement (reaching 1.03 g/L) over the N1 strain. The effective integration of diverse metabolic engineering strategies, including heterologous pathway introduction, precursor enhancement, and co-substrate utilization, established a valuable and transferable framework for reprogramming P. pastoris to synthesize Neu5Ac and other high-value compounds.

Key points

• P. pastoris was engineered to synthesize Neu5Ac.

• Neu5Ac production achieved 1.03 g/L via remodeling of key metabolic pathways.

• The strain harbored potential for synthesis of free Neu5Ac or sialylated proteins.