Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways |
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Authors: | Ying Li Mingyue Su Xizhen Ge Pingfang Tian |
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Affiliation: | 1. College of Life Science and Technology, Beijing University of Chemical Technology, 100029, Beijing, People’s Republic of China 2. College of Biochemical Engineering, Beijing Union University, 100023, Beijing, People’s Republic of China
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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. |
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