Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant Klebsiella pneumoniae strain defective in the oxidative pathway |
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Authors: | Lian Hua Luo Jeong-Woo Seo Baek-Rock Oh Pil-Soo Seo Sun-Yeon Heo Won-Kyung Hong Dae-Hyuk Kim Chul Ho Kim |
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Affiliation: | (1) Microbe-Based Fusion Technology Research Center, Jeonbuk Branch Institute, KRIBB, Jeongeup, Jeonbuk, 580-185, South Korea;(2) Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University, Jeonju, Chonbuk, 561-756, South Korea;(3) Interdisciplinary Program of Graduate School for Bioenergy and Biochemicals, Chonnam National University, Gwangju, 500-757, South Korea; |
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Abstract: | Previously, we constructed a glycerol oxidative pathway-deficient mutant strain of Klebsiella pneumoniae by inactivation of glycerol dehydrogenase (dhaD) to eliminate by-product synthesis during production of 1,3-propanediol (1,3-PD) from glycerol. Although by-product formation was successfully blocked in the resultant strain, the yield of 1,3-PD was not enhanced, probably because dhaD disruption resulted in insufficient regeneration of the cofactor NADH essential for the activity of 1,3-PD oxidoreductase (DhaT). To improve cofactor regeneration, in the present study we overexpressed an NAD+-dependent aldehyde dehydrogenase in the recombinant strain. To this end, an aldehyde dehydrogenase AldHk homologous to E. coli AldH but with NAD+-dependent propionaldehyde dehydrogenase activity was identified in K. pneumoniae. Functional analysis revealed that the substrate specificity of AldHk embraced various aldehydes including propionaldehyde, and that NAD+ was preferred over NADP+ as a cofactor. Overexpression of AldHk in the glycerol oxidative pathway-deficient mutant AK/pVOTHk resulted in a 3.6-fold increase (0.57 g l−1 to 2.07 g l−1) in the production of 3-hydroxypropionic acid (3-HP), and a 1.1-fold enhancement (8.43 g l−1 to 9.65 g l−1) of 1,3-PD synthesis, when glycerol was provided as the carbon source, compared to the levels synthesized by the control strain (AK/pVOT). Batch fermentation using AK/pVOTHk showed a significant increase (to 70%, w/w) in conversion of glycerol to the reductive metabolites, 1,3-PD and 3-HP, with no production of by-products except acetate. |
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