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Background <p>2-phenylethanol (2-PE) is a high-value aromatic alcohol. Its bioproduction is limited by pathway bottlenecks, limited by a rate-limiting decarboxylation step, product toxicity, and volatilization. We sought to build an efficient <i>Escherichia coli</i> (<i>E. coli</i>) platform by optimizing the segment of the Ehrlich route that converts L-phenylalanine (L-Phe) to 2-PE, together with a food-grade soybean-oil overlay for in situ product recovery (ISPR).</p> Results <p>Whole-cell assays showed that phenylpyruvate decarboxylation was the main bottleneck when the yeast decarboxylase Aro10 was used. A phylogeny-guided screen identified Lactococcus lactis KivD as a superior substitute in <i>E. coli</i>. After replacing Aro10 with KivD and tuning expression, leveraging endogenous glutamate dehydrogenase (GDH) for NAD(P)H recycling enabled sufficient cofactor regeneration, yielding 49.5 mM 2-PE from 50 mM L-Phe with 99.0% conversion. At the shake-flask scale, a fed-batch L-Phe feeding strategy coupled with a 2:1 soybean-oil overlay for in situ product removal reduced toxicity and volatilization losses, producing 130 mM 2-PE (15.9&#xa0;g/L). Using soybean oil as an ISPR phase effectively sequestered 2-PE from the aqueous phase, minimizing volatilization and increasing the overall recovered yield to 94.2% based on L-Phe consumption.</p> Conclusions <p>Replacing the rate-limiting decarboxylation step with KivD, balancing redox through cofactor recycling, and coupling the pathway to a mild oil-overlay recovery increased flux and enabled high-titer 2-PE in <i>E. coli</i>. The workflow of bottleneck substitution plus biocompatible in situ recovery can be transferred to related aromatic alcohols and provides a foundation for de novo routes and high-cell-density fermentations that target still higher titers.</p> Graphical Abstract <p></p>

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High-titer 2-phenylethanol production in Escherichia coli via decarboxylation-step optimization and a vegetable-oil overlay for biocompatible in situ recovery

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月27日

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  • Chang Liu,
  • Hongwei Zhang,
  • Wanbin Xing,
  • Rina Na,
  • Xuanyu Meng,
  • Sichen Huang,
  • Linlin Wang,
  • Guoqiang Cao,
  • Pengchao Wang

摘要

Background

2-phenylethanol (2-PE) is a high-value aromatic alcohol. Its bioproduction is limited by pathway bottlenecks, limited by a rate-limiting decarboxylation step, product toxicity, and volatilization. We sought to build an efficient Escherichia coli (E. coli) platform by optimizing the segment of the Ehrlich route that converts L-phenylalanine (L-Phe) to 2-PE, together with a food-grade soybean-oil overlay for in situ product recovery (ISPR).

Results

Whole-cell assays showed that phenylpyruvate decarboxylation was the main bottleneck when the yeast decarboxylase Aro10 was used. A phylogeny-guided screen identified Lactococcus lactis KivD as a superior substitute in E. coli. After replacing Aro10 with KivD and tuning expression, leveraging endogenous glutamate dehydrogenase (GDH) for NAD(P)H recycling enabled sufficient cofactor regeneration, yielding 49.5 mM 2-PE from 50 mM L-Phe with 99.0% conversion. At the shake-flask scale, a fed-batch L-Phe feeding strategy coupled with a 2:1 soybean-oil overlay for in situ product removal reduced toxicity and volatilization losses, producing 130 mM 2-PE (15.9 g/L). Using soybean oil as an ISPR phase effectively sequestered 2-PE from the aqueous phase, minimizing volatilization and increasing the overall recovered yield to 94.2% based on L-Phe consumption.

Conclusions

Replacing the rate-limiting decarboxylation step with KivD, balancing redox through cofactor recycling, and coupling the pathway to a mild oil-overlay recovery increased flux and enabled high-titer 2-PE in E. coli. The workflow of bottleneck substitution plus biocompatible in situ recovery can be transferred to related aromatic alcohols and provides a foundation for de novo routes and high-cell-density fermentations that target still higher titers.

Graphical Abstract