Functions Of Three Key Enzymes For Production Of 3-Hydroxypropionic Acid In Klebsiella Pneumoniae

3-Hydroxypropionic acid (3-HP) as an important precursor of bulk chemicals has broad applications. Klebsiella pneumoniae can metabolize glycerol to produce 3-HP via a unique dha regulon. Although 3-HP biosynthesis pathway has been reported, the roles of key enzymes in the production of 3-HP remain to be elucidated. This study focused on three enzymes in host strain K. pneumoniae, including glycerol dehydrogenase (DhaD), aldehyde dehydrogenase (AldH), and 1,3-propanediol oxidoreductase (DhaT).1. Glycerol dehydrogenase (DhaD). The redox balance rigidity of dha regulon in K. pneumoniae was firstly shown in this work by overexpressing dhaD. DhaD encodes glycerol dehydrogenase, which is the first enzyme in the glycerol oxidation pathway. In this experiment, a recombinant K. pneumoniae overexpressing dhaD was engineered and named K. p (pET-PK-dhaD). The strain K. p (pET-PK-dhaD) produced 3-HP at 1.68 g/L under shake-flask conditions, which was 60% enhancement relative to wild-type strain. Fermentation results showed that intensifying oxidative pathway simultaneously augmented the carbon flux in reduction pathway. In addition, enzyme activity assay came to the same conclusion. For instance, both glycerol dehydrogenase and glycerol dehydratase showed enhanced catalytic activities. Taken together, the aforementioned experimental results suggest a tailored redox balance rigidity of dha regulon in K. pneumoniae. Surprisingly, this conclusion has a conflict with the previous viewpoint of "trading off and taking turns" in glycerol dissimilation pathways. That is, deletion of competing pathways cannot fully enhance 3-HP production. In other words, the structural rigidity of dha regulon should not be ignored.2. Aldehyde dehydrogenase (DhaS). In this study, the aldehyde dehydrogenase dhaS from Bacillus subtilis was firstly demonstrated to benefit 3-HP production in recombinant K. p (pET-PK-dhaS), where 3-HP production increased by 3.9-fold compared with the control. Moreover, we investigated the substrate specificity and coenzyme selectivity of dhaS toward 3-HPA. We found that 3-HPA was the preferred substrate of dhaS when NAD+ was the cofactor. Fed-batch fermentation of the recombinant strain showed that dhaS expression enhanced 3-HP production. Molecular modeling of the 3D-structure of DhaS revealed that NAD+ is the appropriate cofactor for DhaS. As a newly reported aldehyde dehydrogenase, DhaS deserves an in-depth study, including basic research and industrial applications.3. 1,3-propanediol oxidoreductase(DhaT). To dissect the participation of DhaT in glycerol metabolism, three recombinants, including K. p (pET-PK-dhaT), K. p ΔdhaT(pET-PK) and K. p (pET-PK) were individually fermented at different rotation speeds aiming at determination of the cell growth and the formation of metabolites. We found that dhaT is critical to glycerol metabolism and oxygen utilization. Overall, for dhaT-containing strain, the screening of substrates, oxygen consumption conditions, as well as the control of metabolic flux need further exploration.