Redox-based dual-substrate fermentation enhances sustainable production of 3-hydroxypropionic acid by recombinant E. coli.

3-Hydroxypropionic acid (3-HP) is an important platform chemical for a circular bioeconomy since it is a precursor for the production of several high-value derivatives such as acrylic acid as well as biodegradable plastics. Escherichia coli is a promising host for 3-HP biosynthesis due to its well-characterized metabolic network and ease of genetic manipulation. In E. coli, 3-HP is produced from glycerol via a two-step pathway involving glycerol dehydratase and an NAD⁺-dependent aldehyde dehydrogenase. However, a major limitation in this process is redox imbalance, as both glycerol catabolism and 3-HP biosynthesis generate excess NADH, thereby restricting NAD⁺ availability and limiting product formation. In this study, we systematically evaluated the influence of cosubstrate selection to enhance 3-HP production by decoupling biomass formation from product synthesis while achieving redox balance. An engineered E. coli strain, with knockouts of glycerol kinase (glpK) and glycerol dehydrogenase (gldA) to prevent glycerol catabolism, was employed to redirect glycerol flux exclusively toward 3-HP biosynthesis. A secondary carbon source was supplied to support cell growth and promote NAD⁺ regeneration. Six different sugars were evaluated as growth substrates to assess their impact on redox balance and 3-HP production. Among the tested secondary substrates, sucrose and xylose enabled high 3-HP titers of 61 and 50 g/L, respectively, significantly surpassing the 30 g/L titer obtained using glycerol as the sole carbon source. The strategy was successfully extended to renewable feedstocks such as molasses and pretreated lignocellulosic liquor hydrolysate in combination with crude glycerol, achieving 3-HP titers in the range of ~ 35-40 g/L. KEY POINTS: • Redox imbalance limits glycerol-based 3-HP biosynthesis in E. coli. • Redox-optimized dual-substrate feeding enhances 3-HP titer, yield and productivity. • Validation with renewable substrates provides feasibility for a circular bioeconomy.