Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae

Abstract

3-Hydroxypropionic acid (3-HP) can be produced from glycerol via two enzymatic reactions catalyzed by a coenzyme B12-dependent glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) in Klebsiella pneumoniae. As the intracellular GDHt activity in K. pneumoniae is high, the overall rate of 3-HP production is controlled by the ALDH activity. To examine the effect of different ALDH activity on 3-HP production, three different ALDHs, AldH from Escherichia coli (EaldH), PuuC from K. pneumoniae (PuuC) and KGSADH from Azospirillum brasilense (KGSADH), were overexpressed and compared in various recombinant K. pneumoniae strains. In addition, the genes encoding DhaT and YqhD, which are responsible for the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PDO), were disrupted individually from K. pneumoniae to enhance the carbon flux from 3-HPA to 3-HP. When the ALDH activity was measured in various recombinant K. pneumoniae, KGSADH showed the highest crude cell activity of 8.0U/mg protein, which was 2 and 4 times higher than that of PuuC and EaldH, respectively. The different ALDH activities had a significant effect on 3-HP production in a flask culture containing 100mM glycerol, and K. pneumoniae ΔdhaT (KGSADH) resulted in the highest titer (64mM) among the nine recombinant strains (three ALDH×three host strains; one wild type and two mutants). In glycerol fed-batch bioreactor cultivation, K. pneumoniae ΔdhaT (KGSADH) exhibited 3-HP production at >16g/L in 48h with a glycerol carbon yield of >40%. In comparison, K. pneumoniae ΔdhaT (PuuC) produced only 11g/L 3-HP in 48h with a yield of >23%. This study demonstrates that a high ALDH activity is essential for the effective production of 3-HP from glycerol with recombinant K. pneumoniae.