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Background <p><i>Limosilactobacillus reuteri</i> is an important probiotic chassis for producing 3-hydroxypropionaldehyde (3-HPA), a broad-spectrum antimicrobial and biochemical. However, its production is constrained by a metabolic trilemma comprising coenzyme B₁₂ auxotrophy, redox imbalance, and carbon catabolite repression (CCR). The main bottleneck currently limiting cost-effective glucose-based production is the inability to simultaneously resolve these interconnected constraints.</p> Results <p>Through integrated multi-omics analyses of differential carbon source performance, an unexpected regulatory mechanism was obtained. Galactose orchestrates a tripartite metabolic program in which UDP-galactose may act as a signaling molecule potentially involved in alleviating CCR; redirects carbon flux through the pentose phosphate pathway to balance ATP/NADH, and transcriptionally co-activates <i>dhaB</i> (glycerol dehydratase) and <i>cobA/Q</i> (B₁₂ biosynthesis) for coordinated holoenzyme assembly. Strikingly, this native regulatory network enables 8.51&#xa0;g/L 3-HPA from galactose despite its poor support for cell growth, versus 1.67&#xa0;g/L from glucose which readily supports robust growth—a 5.1-fold advantage that highlights galactose’s metabolic priming efficacy. Guided by these insights, an engineered strain overexpressing glycerol dehydratase was combined with process optimization and a rationally designed four-factor targeted supplementation. Synergistic supplementation with coenzyme B₁₂, ATP, KCl, and MgCl₂ achieved 28.96&#xa0;g/L 3-HPA—a 17.34-fold improvement over native glucose-based performance.</p> Conclusions <p>To our knowledge, this is the first systems-level strategy for compensating the metabolic trilemma in <i>L. reuteri</i> by engineering carbon source-dependent regulatory networks and cofactor dynamics in a cost-effective glucose-based system, achieving the highest 3-HPA titer reported in lactobacilli in shake-flask fermentation to date. This work establishes a scalable platform for industrial 3-HPA production and provides a systems-level framework for reprogramming central carbon metabolism in probiotic chassis to address persistent bioproduction bottlenecks.</p>

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Multi-omics-guided metabolic engineering of Limosilactobacillus reuteri for high-level 3-hydroxypropionaldehyde production from glucose

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月22日

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  • Zuojun Liu,
  • Qiang Yin,
  • Liang Li,
  • Hang Wu,
  • Yong Fang,
  • Fuqiang Cheng,
  • Ting Guo

摘要

Background

Limosilactobacillus reuteri is an important probiotic chassis for producing 3-hydroxypropionaldehyde (3-HPA), a broad-spectrum antimicrobial and biochemical. However, its production is constrained by a metabolic trilemma comprising coenzyme B₁₂ auxotrophy, redox imbalance, and carbon catabolite repression (CCR). The main bottleneck currently limiting cost-effective glucose-based production is the inability to simultaneously resolve these interconnected constraints.

Results

Through integrated multi-omics analyses of differential carbon source performance, an unexpected regulatory mechanism was obtained. Galactose orchestrates a tripartite metabolic program in which UDP-galactose may act as a signaling molecule potentially involved in alleviating CCR; redirects carbon flux through the pentose phosphate pathway to balance ATP/NADH, and transcriptionally co-activates dhaB (glycerol dehydratase) and cobA/Q (B₁₂ biosynthesis) for coordinated holoenzyme assembly. Strikingly, this native regulatory network enables 8.51 g/L 3-HPA from galactose despite its poor support for cell growth, versus 1.67 g/L from glucose which readily supports robust growth—a 5.1-fold advantage that highlights galactose’s metabolic priming efficacy. Guided by these insights, an engineered strain overexpressing glycerol dehydratase was combined with process optimization and a rationally designed four-factor targeted supplementation. Synergistic supplementation with coenzyme B₁₂, ATP, KCl, and MgCl₂ achieved 28.96 g/L 3-HPA—a 17.34-fold improvement over native glucose-based performance.

Conclusions

To our knowledge, this is the first systems-level strategy for compensating the metabolic trilemma in L. reuteri by engineering carbon source-dependent regulatory networks and cofactor dynamics in a cost-effective glucose-based system, achieving the highest 3-HPA titer reported in lactobacilli in shake-flask fermentation to date. This work establishes a scalable platform for industrial 3-HPA production and provides a systems-level framework for reprogramming central carbon metabolism in probiotic chassis to address persistent bioproduction bottlenecks.