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<p>To address waste management challenges, lignocellulosic industrial co-products can be valorized microbially to propose sustainable and economically viable alternatives to fossil routes. This study advances a bio-based ethyl acetate microbial production by the yeast <i>Kluyveromyces marxianus</i> by investigating glucose availability under iron-limiting fed-batch conditions. Two feeding strategies were compared: one that maintained an excess of glucose and one that operated at zero residual glucose to understand their respective effects on ethyl acetate synthesis dynamics. Metabolite productions and kinetics were quantified across both conditions, enabling the evaluation of metabolic flux distributions in <i>K. marxianus</i>, rarely explored in the literature. Our results demonstrate that EA production rates observed under iron deficiency conditions cannot be attributed solely to iron limitation. As this study demonstrates, EA synthesis is multifactorial and depends on respiratory chain efficiency, pyruvate flux distribution and acetyl-CoA management. Herein, ethyl acetate synthesis was modelled via mitochondrial Eat1 enzyme and intracellular fluxes were analyzed under both iron and glucose-controlled culture conditions using a compartmented metabolic model of <i>K. marxianus.</i> Despite iron limitation, excess glucose preserves electron transport chain and tricarboxylic acid cycle activities, favoring metabolic balance over biomass. In contrast, glucose limitation promotes growth, consequently leading to downregulation of tricarboxylic acid cycle flux, constrained oxaloacetate synthesis and mitochondrial acetyl-CoA accumulation, thereby activating EA synthesis. These findings refine existing hypotheses and underscore the necessity of finely tuning electron transport chain and tricarboxylic acid cycle fluxes to induce mitochondrial acetyl-CoA overflow to optimize ethyl acetate production from lignocellulosic substrates.</p>

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Analyzing the effect of glucose availability on the ethyl acetate production by Kluyveromyces marxianus under iron limitation conditions by metabolic flux analysis approach

  • 期刊论文

  • 发表日期：

    2025年12月6日

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  • Elise Viau,
  • Alicia Huesca,
  • Carine Bideaux,
  • Sandrine Alfenore

摘要

To address waste management challenges, lignocellulosic industrial co-products can be valorized microbially to propose sustainable and economically viable alternatives to fossil routes. This study advances a bio-based ethyl acetate microbial production by the yeast Kluyveromyces marxianus by investigating glucose availability under iron-limiting fed-batch conditions. Two feeding strategies were compared: one that maintained an excess of glucose and one that operated at zero residual glucose to understand their respective effects on ethyl acetate synthesis dynamics. Metabolite productions and kinetics were quantified across both conditions, enabling the evaluation of metabolic flux distributions in K. marxianus, rarely explored in the literature. Our results demonstrate that EA production rates observed under iron deficiency conditions cannot be attributed solely to iron limitation. As this study demonstrates, EA synthesis is multifactorial and depends on respiratory chain efficiency, pyruvate flux distribution and acetyl-CoA management. Herein, ethyl acetate synthesis was modelled via mitochondrial Eat1 enzyme and intracellular fluxes were analyzed under both iron and glucose-controlled culture conditions using a compartmented metabolic model of K. marxianus. Despite iron limitation, excess glucose preserves electron transport chain and tricarboxylic acid cycle activities, favoring metabolic balance over biomass. In contrast, glucose limitation promotes growth, consequently leading to downregulation of tricarboxylic acid cycle flux, constrained oxaloacetate synthesis and mitochondrial acetyl-CoA accumulation, thereby activating EA synthesis. These findings refine existing hypotheses and underscore the necessity of finely tuning electron transport chain and tricarboxylic acid cycle fluxes to induce mitochondrial acetyl-CoA overflow to optimize ethyl acetate production from lignocellulosic substrates.