Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant Klebsiella pneumoniae strain defective in the oxidative pathway.
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.
Luo
Lian Hua
LH
Microbe-Based Fusion Technology Research Center, Jeonbuk Branch Institute, KRIBB, Jeongeup, Jeonbuk, 580-185, South Korea.
Seo
Jeong-Woo
JW
Oh
Baek-Rock
BR
Seo
Pil-Soo
PS
Heo
Sun-Yeon
SY
Hong
Won-Kyung
WK
Kim
Dae-Hyuk
DH
Kim
Chul Ho
CH
D000429:Alcohol Oxidoreductases; D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D056804:Biofuels; D001709:Biotechnology; D005285:Fermentation; D005990:Glycerol / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism
D056804:Biofuels; D009243:NAD; D019344:Lactic Acid; C031601:hydracrylic acid; D000429:Alcohol Oxidoreductases; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; C021494:lactaldehyde reductase; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; D005990:Glycerol