title: Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid.
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.
authors: School of Chemical and Biomolecular Engineering, Pusan National University, San 30, Jangeon-dong, Geumjeong-gu, Busan, 609-735, Republic of Korea.
authors: Shengfang
authors: S
authors: Zhou
authors: Somasundar
authors: S
authors: Ashok
authors: Yeounjoo
authors: Y
authors: Ko
authors: Dong-Myung
authors: DM
authors: Kim
authors: Sunghoon
authors: S
authors: Park
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005990:Glycerol / Q000378:metabolism; D006868:Hydrolysis; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D019344:Lactic Acid; C031601:hydracrylic acid; D005990:Glycerol