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Background <p>Consolidated bioprocessing (CBP) by cellulolytic fungi offers a direct route for cellulosic ethanol production, but the process still relies heavily on costly complex nitrogen sources. Replacing complex nitrogen with inorganic nitrogen is a potential strategy to reduce the cost of the medium, yet it often leads to impaired fermentation performance, and the metabolic basis for this limitation remains unclear.</p> Results <p>Replacement of yeast extract with inorganic nitrogen was evaluated in the engineered thermophilic fungus <i>Myceliophthora thermophila</i> YL913, a CBP strain capable of producing ethanol directly from cellulose. Ammonium sulfate supported higher ethanol production than nitrate, but still resulted in a lower titer than yeast extract. Under ammonium sulfate, transcriptomic and metabolomic analyses revealed altered amino acid metabolism together with reduced carbohydrate metabolic and cellulose catabolic functions, while supplementation with selected amino acids provided only limited improvement. Supplementation with 5 mg/L thiamine pyrophosphate (TPP) increased ethanol production under ammonium sulfate from 14.3 g/L to 23 g/L, reaching a level comparable to that obtained with yeast extract. Moreover, α-ketoglutarate was identified as a responsive metabolic node associated with the altered fermentation state. Stepwise strengthening of the endogenous TPP biosynthetic pathway progressively improved ammonium-based ethanol fermentation, and additional overexpression of pyruvate decarboxylase further enhanced ethanol production. In a 5-L fermenter, the final engineered strain produced 49.5 g/L ethanol under ammonium sulfate without exogenous TPP, close to the 51.2 g/L obtained with the TPP-supplemented parental strain.</p> Conclusions <p>This study establishes ammonium sulfate as a feasible low-cost alternative to yeast extract for CBP-based cellulosic ethanol production by the engineered <i>M. thermophila</i> strain. These findings provide a practical strategy for developing low-cost fungal CBP processes for cellulosic ethanol production supported by inorganic nitrogen.</p>

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Engineering thiamine pyrophosphate metabolism improves consolidated bioprocessing for cellulosic ethanol with inorganic nitrogen in Myceliophthora thermophila

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年6月30日

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  • Jiaxing Chen,
  • Jia Liu,
  • Tao Sun,
  • Rui Pang,
  • Jinyi Yu,
  • Defei Liu,
  • Jingen Li,
  • Chaoguang Tian

摘要

Background

Consolidated bioprocessing (CBP) by cellulolytic fungi offers a direct route for cellulosic ethanol production, but the process still relies heavily on costly complex nitrogen sources. Replacing complex nitrogen with inorganic nitrogen is a potential strategy to reduce the cost of the medium, yet it often leads to impaired fermentation performance, and the metabolic basis for this limitation remains unclear.

Results

Replacement of yeast extract with inorganic nitrogen was evaluated in the engineered thermophilic fungus Myceliophthora thermophila YL913, a CBP strain capable of producing ethanol directly from cellulose. Ammonium sulfate supported higher ethanol production than nitrate, but still resulted in a lower titer than yeast extract. Under ammonium sulfate, transcriptomic and metabolomic analyses revealed altered amino acid metabolism together with reduced carbohydrate metabolic and cellulose catabolic functions, while supplementation with selected amino acids provided only limited improvement. Supplementation with 5 mg/L thiamine pyrophosphate (TPP) increased ethanol production under ammonium sulfate from 14.3 g/L to 23 g/L, reaching a level comparable to that obtained with yeast extract. Moreover, α-ketoglutarate was identified as a responsive metabolic node associated with the altered fermentation state. Stepwise strengthening of the endogenous TPP biosynthetic pathway progressively improved ammonium-based ethanol fermentation, and additional overexpression of pyruvate decarboxylase further enhanced ethanol production. In a 5-L fermenter, the final engineered strain produced 49.5 g/L ethanol under ammonium sulfate without exogenous TPP, close to the 51.2 g/L obtained with the TPP-supplemented parental strain.

Conclusions

This study establishes ammonium sulfate as a feasible low-cost alternative to yeast extract for CBP-based cellulosic ethanol production by the engineered M. thermophila strain. These findings provide a practical strategy for developing low-cost fungal CBP processes for cellulosic ethanol production supported by inorganic nitrogen.