Abstract
Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B12 under aerobic conditions. Recently, recombinant strains of P. denitrificans overexpressing a vitamin B12-dependent glycerol dehydratase (DhaB) were developed to produce 3-hydroxypropionic acid (3-HP) from glycerol. The recombinant P. denitrificans could produce 3-HP successfully under aerobic conditions without an exogenous supply of vitamin B12, but the 3-HP produced disappeared during extended cultivation due to the 3-HP degradation activity in this strain. This study developed mutant strains of P. denitrificans that do not degrade 3-HP. The following eight candidate enzymes, which might be responsible for 3-HP degradation, were selected, cloned, and studied for their activity in Escherichia coli: four (putative) 3-hydroxyisobutyrate dehydrogenases (3HIBDH), a putative 3-HP dehydrogenase (3HPDH), an alcohol dehydrogenase (ADH), and two choline dehydrogenases (CHDH). Among them, 3HIBDHI, 3HIBDHIV, and 3HPDH exhibited 3-HP degrading activity when expressed heterologously in E. coli. When 3hpdh alone or along with 3hibdhIV were disrupted from P. denitrificans, the mutant P. denitrificans exhibited greatly reduced 3-HP degradation activity that could not grow on 3-HP as the sole carbon and energy source. When the double mutant P. denitrificans Δ3hpdhΔ3hibdhIV was transformed with DhaB, an improved 3-HP yield (0.78 mol/mol) compared to that of the wild-type counterpart (0.45 mol/mol) was obtained from a 24-h flask culture. This study indicates that 3hpdh and 3hibdhIV (to a lesser extent) are mainly responsible for 3-HP degradation in P. denitrificans and their deletion can prevent 3-HP degradation during its production by recombinant P. denitrificans.
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Acknowledgement
This study was supported financially by the Korean Ministry of Education, Science, and Technology through the Advanced Biomass R&D Center (ABC; grant no. 2010-0029799), KAIST, Korea. In addition, the authors are grateful for the financial assistance provided by the BK21 Plus Program for Advanced Chemical Technology at Pusan National University.
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Zhou, S., Ashok, S., Ko, Y. et al. Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid. Appl Microbiol Biotechnol 98, 4389–4398 (2014). https://doi.org/10.1007/s00253-014-5562-5
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DOI: https://doi.org/10.1007/s00253-014-5562-5