Notable Improvement of 3‑Hydroxypropionic Acid and 1,3-Propanediol Coproduction Using Modular Coculture Engineering and Pathway Rebalancing

Sustainable bioconversion of glycerol into 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) is often bottlenecked by the accumulation of a toxic metabolic intermediate, 3-hydroxypropanaldehyde (3-HPA), mostly due to the imbalanced expression of the two key enzymes, namely, aldehyde dehydrogenase (AldH) and 1,3-PDO oxidoreductase (PDOR). Herein, we attempted to overcome it by modular coculture engineering using Lactobacillus reuteri and recombinant Escherichia coli strains through overexpressing an AldH (GabD4) and a PDOR (PduQ) in E. coli and pathway rebalancing via 5′-untranslated region (5′-UTR) engineering in the gabD4 gene. We then used the L. reuteri–E. coli coculture systems in resting cell biocatalysis of glycerol under several technological configurations to produce 3-HP and 1,3-PDO. The conventional approach, which is a two-step process comprising resting cell production (first step) and biotransformation of glycerol with resting cells (second step), produced 51.36 g/L 3-HP and 34.27 g/L 1,3-PDO from 120 g/L glycerol, leaving more than 30 g/L glycerol unconverted. Subsequently, we did experiments in a modified approach, including one more biotransformation step with the two-step approach to convert the residual glycerol. This three-step approach increased glycerol consumption to 140 g/L and produced 71.62 g/L 3-HP and 47.68 g/L 1,3-PDO. Finally, the sequential feeding of glycerol and fresh cells notably improved glycerol consumption to 240 g/L and titers of 3-HP and 1,3-PDO to 125.93 and 88.46 g/L, respectively, with a net titer of 214.39 g/L, which was the highest titer ever reported. Therefore, modular coculture engineering with an appropriate technological configuration could have the potential for improved production of value-added chemicals, particularly 3-HP and 1,3-PDO.