In vitro enzymatic production of 3-hydroxypropionic acid from glycerol via CoA-independent pathway

Abstract

3-Hydroxypropionic acid (3-HP) is a C3-based versatile platform chemical offering significant industrial and environmental advantages over petrochemical derivatives. Microbial cell factories are commonly employed to produce 3-HP from various carbon sources, with glycerol being a key substrate via CoA-dependent and -independent pathways. However, challenges such as inhibited growth and reduced productivity, often due to substrate inhibition and toxic byproducts like 3-hydroxypropionaldehyde (3-HPA), limit the efficiency of glycerol-based microbial 3-HP production and its industrial scalability. To address these challenges, we propose an alternative in vitro approach for 3-HP production from glycerol through a coupled enzymatic reaction. We optimized the recombinant production of glycerol dehydratase (GDHt) and alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), two key enzymes from Escherichia coli. After purification, GDHt and KGSADH showed 23.4 ± 4.6 s⁻¹ mM⁻¹ and 28.7 ± 5.0 s⁻¹ mM⁻¹ of k_cat/K_m, respectively. We then systematically varied enzyme concentrations, reaction temperature, and pH to determine the optimal conditions for 3-HP biosynthesis. The chain reaction, conducted with 100 nM of each enzyme, produced 43.5 μg/mL of 3-HP within 20 min under optimal conditions of 45 ºC and pH 9.0. This approach demonstrated the potential for a clean and efficient in vitro system for 3-HP production from renewable sources.