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Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid

Zhou, Shengfang, Ashok, Somasundar, Ko, Yeounjoo, Kim, Dong-Myung, Park, Sunghoon
Applied microbiology and biotechnology 2014 v.98 no.10 pp. 4389-4398
Escherichia coli, Gram-negative bacteria, Pseudomonas denitrificans, aerobic conditions, alcohol dehydrogenase, carbon, choline, energy, glycerol, mutants
Pseudomonas denitrificans is a gram-negative bacterium that can produce vitamin B₁₂under 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.