Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways

Abstract

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.

Enhanced Promoter Activity by Replenishment of Sigma Factor rpoE in Klebsiella pneumoniae

“…Previously, pk promoter was harnessed to drive the expression of ALDH, however, 3-HP failed to be overproduced [3,8]. One major reason is that pk is a constitutive promoter and its intrinsic activity is lower than T7 promoter.…”

“…S. cerevisiae was grown in Yeast Extract Peptone Dextrose medium (g/L): yeast extract, 10; peptone, 20; glucose, 20. The medium for producing 3-HP by recombinant K. pneumoniae was the same as the previously reported [3]. The recombinant strain was microaerobically grown in 50 mL Erlenmeyer flasks containing 25 mL medium and 50 lg kanamycin/mL at 37°C and 150 rpm continuous shaking.…”

“…Klebsiella pneumoniae can naturally convert glycerol into a series of economically important chemicals, including 1,3-propanediol (1,3-PD) [1], 2,3-butanediol (2,3-BD) [2], and 3-hydroxypropionic acid (3-HP) [3]. In K. pneumoniae, glycerol metabolism is mediated by the dha regulon which governs the parallel oxidation and reduction pathways [4].…”

“…One reason is the inherent low activity of pk because it is a constitutive promoter tailored for governing glycerol metabolism in K. pneumoniae. The second reason may be cofactor imbalance caused by AldH overexpression [3]. Apart from above reasons, RNA polymerases especially sigma factors (r factors) may be related to pk activity, because sigma factors recognize and bind promoters to initiate Electronic supplementary material The online version of this article (doi:10.1007/s12088-016-0576-6) contains supplementary material, which is available to authorized users.…”

Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae

“…Cofactor availability is crucial for aldehyde dehydrogenase (ALDH) activity and 3-HP biosynthesis [24]. In glycerol reductive pathway, ALDH together with NAD ?…”