In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD(+) is exhausted. Here we show that NAD(+) regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD(+)-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD(+). When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD(+) enhanced the activity of ALD4. More interestingly, the growth rates of all NAD(+)-regenerating strains were prolonged in comparison with the control, indicating that NAD(+) regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD(+) availability but also provides a method for regulating the dha regulon.

In Klebsiella pneumoniae, aldehyde dehydrogenases (ALDH) convert 3-hydroxypropionaldehyde (3-HPA) into 3-hydroxypropionic acid (3-HP). Although ALDHs can increase the production of 3-HP in K. pneumoniae, the substrate specificity of ALDH homologues from other microorganisms toward 3-HPA is less documented. Here we report that DhaS, a putative ALDH from Bacillus subtilis, shows high specificity toward 3-HPA when heterologously expressed in K. pneumoniae. Using NAD(+) as a cofactor, DhaS exhibited higher catalytic activity (2.3 U mg(-1)) and lower K m value (0.4 mmol l(-1)) toward 3-HPA than that toward other aldehydes. Under shake-flask conditions, the recombinant strain produced 2.1 g 3-HP l(-1) in 24 h, which is 3.9-fold of that in a control harboring a blank vector. Under non-optimized bioreactor conditions, the recombinant strain produced 18 g 3-HP l(-1) and 1,3-propanediol (1,3-PDO) at 27 g l(-1) in 24 h. The overall conversion rate from glycerol to 3-HP and 1,3-PDO reached 59.4 mol mol(-1). Homology modeling of DhaS illustrates substrate specificity and NAD(+)-binding site. DhaS is thus a 3-HPA-specific enzyme useful for production of 3-HP.

In Klebsiella pneumoniae, glycerol dissimilation involves parallel oxidation and reduction pathways. Oxidation pathway provides adenosine triphosphate (ATP) and cofactors to sustain cell growth, while reduction pathway presents 3-hydroxypropionic acid (3-HP) and 1,3-propanediol(1,3-PDO), which are commercially attractive platform chemicals. Previous metabolic engineering of K. pneumoniae focused on the intensification of reduction pathway; however, it failed to overproduce 3-HP or 1,3-PDO. Contrary to this strategy, here we show that overexpression of glycerol dehydrogenase (dhaD), the first functional enzyme in oxidation pathway, can efficiently stimulate cell growth and facilitate 3-HP accumulation. Under microaerobic conditions, although metabolic burden arising from plasmid replication, the recombinant K. pneumoniae overexpressing dhaD grew actively and showed 60% enhancement of 3-HP compared to the control. In particular, overexpression of dhaD increased the activity of glycerol dehydratase, indicating the concerted action of two enzymes and the interdependence between glycerol oxidation and reduction pathways. Moreover, the strain overexpressing dhaD produced more lactic acid yet less acetic acid than the control, implying the interplay between dhaD expression and the formation of byproducts. Together, not only showing that intensifying glycerol oxidation pathway is beneficial to 3-HP production, this study also reveals the structural rigidity of dha operon that mediates glycerol dissimilation in K. pneumoniae.