Production of 3-hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans

ABSTRACT 3‐Hydroxypropionic acid (3‐HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12‐dependent glycerol dehydratase and an NAD(P)+‐dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)+ is regenerated efficiently. Hence, it is considered an ideal host for the production of 3‐HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3‐HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3‐HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3‐HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3‐HP production is that it oxidizes 3‐HP to malonate and utilizes 3‐HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3‐HP production from glycerol. Biotechnol. Bioeng. 2013;110: 3177–3187. © 2013 Wiley Periodicals, Inc. Pseudomonas denitrificans can synthesize coenzyme B12 aerobically. It showed potential to produce 3‐hydroxypropionic acid (3‐HP) from glycerol, when coenzyme B12‐dependent glycerol dehydratase (DhaB) was heterologously overexpressed. An (unknown) native or heterologous aldehyde dehydrogenase (ALDH) could catalyze the conversion of 3‐hydroxypropionaldehyde (3‐HPA) to 3‐HP. Using this recombinant strain, 37.7 mM of 3‐HP was produced with 62% (mol/mol) yield on glycerol. Unfortunately, P. denitrificans actively degraded the produced 3‐HP.