Multi-omics-guided metabolic engineering of Limosilactobacillus reuteri for high-level 3-hydroxypropionaldehyde...

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.

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.

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.