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Background <p>Mannose has wide-ranging applications but microbial fermentation remains underdeveloped compared to biotransformation for its production. The yeast <i>Komagataella phaffii</i> stands out as a premier synthetic biology platform, renowned for its safety profile and exceptional suitability for high-density fermentation. This established chassis organism is ideally positioned for large-scale mannose production through targeted rewiring of its mannose biosynthetic pathway via metabolic engineering.</p> Results <p><i>K. phaffii</i> was metabolically engineered for efficient mannose production using a dual carbon source system: glycerol for biomass generation and glucose for mannose synthesis. To redirect carbon flux toward fructose-6-phosphate (F6P) accumulation at the glycolytic node, glycolytic flux was attenuated by knocking out the phosphofructokinase II (<i>pfk2</i>) gene and downregulating phosphofructokinase I (<i>pfk1</i>). Simultaneously, pentose phosphate pathway flux was reduced by downregulating glucose-6-phosphate dehydrogenase (<i>zwf1</i>). To enhance mannose biosynthesis, conversion of F6P into mannose was promoted by suppressing phosphomannose isomerase (<i>PAS_chr3_1115</i>) and overexpressing the <i>Escherichia coli</i>-derived phosphatase gene <i>yniC</i>. Additionally, three genes involved in arabinitol and ribitol production (<i>PAS_chr2–2_0019</i>, <i>PAS_chr4_0754</i>, and <i>PAS_chr4_0988</i>) were deleted to suppress byproduct accumulation. The engineered strain achieved ~ 121.1&#xa0;g/L mannose in high-cell-density, fed-batch fermentation, representing the highest reported titer via microbial fermentation to date.</p> Conclusions <p>This study achieved efficient mannose production in <i>K. phaffii</i> by remodeling central metabolism. It not only offers a new route for mannose biosynthesis but also establishes a model framework for engineering <i>K. phaffii</i> to produce other high-value bioactive compounds.</p>

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Rewiring central metabolism in Komagataella phaffii for efficient mannose synthesis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月9日

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  • Sijia Zhao,
  • Yuheng Wang,
  • Qian Li,
  • Jiachen Xie,
  • Xinxin Xu,
  • Wei Zhang,
  • Jie Zhang,
  • Bo Liu

摘要

Background

Mannose has wide-ranging applications but microbial fermentation remains underdeveloped compared to biotransformation for its production. The yeast Komagataella phaffii stands out as a premier synthetic biology platform, renowned for its safety profile and exceptional suitability for high-density fermentation. This established chassis organism is ideally positioned for large-scale mannose production through targeted rewiring of its mannose biosynthetic pathway via metabolic engineering.

Results

K. phaffii was metabolically engineered for efficient mannose production using a dual carbon source system: glycerol for biomass generation and glucose for mannose synthesis. To redirect carbon flux toward fructose-6-phosphate (F6P) accumulation at the glycolytic node, glycolytic flux was attenuated by knocking out the phosphofructokinase II (pfk2) gene and downregulating phosphofructokinase I (pfk1). Simultaneously, pentose phosphate pathway flux was reduced by downregulating glucose-6-phosphate dehydrogenase (zwf1). To enhance mannose biosynthesis, conversion of F6P into mannose was promoted by suppressing phosphomannose isomerase (PAS_chr3_1115) and overexpressing the Escherichia coli-derived phosphatase gene yniC. Additionally, three genes involved in arabinitol and ribitol production (PAS_chr2–2_0019, PAS_chr4_0754, and PAS_chr4_0988) were deleted to suppress byproduct accumulation. The engineered strain achieved ~ 121.1 g/L mannose in high-cell-density, fed-batch fermentation, representing the highest reported titer via microbial fermentation to date.

Conclusions

This study achieved efficient mannose production in K. phaffii by remodeling central metabolism. It not only offers a new route for mannose biosynthesis but also establishes a model framework for engineering K. phaffii to produce other high-value bioactive compounds.