Presentation Information
[2BRBP-11]Co-Substrate Driven Redox Engineering for Enhanced 3-Hydroxypropionic Acid in E. coli
○Kala Baskara Sathiya Singh1, Guhan Jayaraman1 (1. Indian Institute of Technology Madras (India))
Keywords:
3-Hydroxypropionic acid,dual substrate fermentation,redox imbalance,circular bioeconomy
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. Biological production of 3-HP has garnered considerable attention in recent years due to its low environmental footprint compared to the conventional chemical processes. Despite extensive research on microbial fermentation strategies, the biological production of 3-HP remains constrained by issues such as product toxicity, redox imbalance, and limited yields, which hinder its commercial feasibility.
Escherichia coli serves as a promising host for 3-HP biosynthesis due to its well-characterized metabolic network and ease of genetic manipulation. The biosynthetic pathway from glycerol to 3-HP in E. coli involves two enzymatic steps: glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA), which is subsequently oxidized to 3-HP by an NAD+-dependent aldehyde dehydrogenase. A key bottleneck in this pathway is the regeneration of NAD+. Since glycerol catabolism through glycolysis generates excess NADH, and the 3-HP biosynthetic reaction itself also produces NADH, the cellular NAD+/NADH ratio becomes skewed toward the reduced state. This imbalance restricts NAD+ availability, thereby limiting 3-HP production.To overcome this, we developed a dual-substrate feeding strategy 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 these, sucrose and xylose enabled high 3-HP titres of 61 and 50 g/L, respectively, significantly surpassing the 30 g/L titre obtained using glycerol as the sole carbon source. This strategy was successfully extended to renewable feedstocks such as molasses and pre-treated lignocellulosic liquor hydrolysate in combination with crude glycerol, achieving 3-HP titres in the range of 35- 40 g/L.
Escherichia coli serves as a promising host for 3-HP biosynthesis due to its well-characterized metabolic network and ease of genetic manipulation. The biosynthetic pathway from glycerol to 3-HP in E. coli involves two enzymatic steps: glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA), which is subsequently oxidized to 3-HP by an NAD+-dependent aldehyde dehydrogenase. A key bottleneck in this pathway is the regeneration of NAD+. Since glycerol catabolism through glycolysis generates excess NADH, and the 3-HP biosynthetic reaction itself also produces NADH, the cellular NAD+/NADH ratio becomes skewed toward the reduced state. This imbalance restricts NAD+ availability, thereby limiting 3-HP production.To overcome this, we developed a dual-substrate feeding strategy 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 these, sucrose and xylose enabled high 3-HP titres of 61 and 50 g/L, respectively, significantly surpassing the 30 g/L titre obtained using glycerol as the sole carbon source. This strategy was successfully extended to renewable feedstocks such as molasses and pre-treated lignocellulosic liquor hydrolysate in combination with crude glycerol, achieving 3-HP titres in the range of 35- 40 g/L.
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