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( Nguyen Vo Phu Thuan ),박성훈 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1
Coenzyme B<sub>12</sub> is a cofactor for glycerol dehydratase, an essential enzyme in biological synthesis of 3-HP. There are two pathways for B<sub>12</sub> biosynthesis, one is oxygen-dependent and the other one is oxygenindependent. Pseudomonas denitrificans can de novo synthesize B<sub>12</sub> by oxygen-dependent pathway. However, its B<sub>12</sub> biosynthesis is not sufficient. The presence of riboswitches which sense B<sub>12</sub> and repress the transcription of B<sub>12</sub> operons restricts the B<sub>12</sub> biosynthesis in P. denitrificans. In this study, we eliminated the riboswitch regulation and improved B<sub>12</sub> gene expression by systematically modifying promoter regions of B<sub>12</sub> operons in P. denitrificans. The promoter engineering could improve B<sub>12</sub> biosynthesis in P. denitrificans. Further improvement of B<sub>12</sub> biosynthesis are under studying.
Development of recombinant Pseudomonas denitrificans for enhanced coenzyme B12 production
( Nguyen Vo Phu Thuan ),( Satish Kumar Ainala ),박성훈 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0
Coenzyme B12 is an essential cofactor for the activity of glycerol dehydratase, an essential enzyme used in 3-HP production. Several recombinant Pseudomonas denitrificans strains have been developed for 3-HP production due to its capability to synthesize coenzyme B12 natively. However, the amount of native coenzyme B12 synthesized was not sufficient for glycerol dehydratase reaction in maintaining high 3-HP production rate. Genes related to Coenzyme B12 pathway are transcriptionally and/or translationally regulated by riboswitches (RS). The riboswitch mutant strains (mutation of RS2,3 and RS4) developed by homologous recombination could not show improvement in either coenzyme B12 synthesis or 3-HP production. RS4 mutant showed deteriorated B12 synthesis and sequentially led to decreased 3-HP production (13.1 mM at 35 h). Characterization and engineering these intergenic regions containing riboswitches has been studied to improve coenzyme B12 synthesis.
박예섭,Nasir Abdul,NGUYEN VO PHU THUAN,류희창,석주연,정규열,박성훈,유태현 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.6
3-Hydroxypropionic acid (3-HP) is a key building block for value-added chemicals. A biological route for synthesizing this molecule is two-step enzymatic reactions; dehydration of glycerol to 3-hydroxypropanal (3-HPA) by glycerol dehydratase and then oxidation of 3-HPA to 3-HP by aldehyde dehydrogenase. Here, we report an aldehyde dehydrogenase, an engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense. The variant, named 2C10, was obtained by applying a large KGSADH library to a selection method based on a 3-HP-responsive transcription factor and then a screening method for observing the activities of individual clones. 2C10 exhibited a 4.65-fold higher catalytic activity (kcat/Km: 100 ± 7.1 s−1mM−1) toward 3-HPA than the wild-type enzyme. The flask culture of Pseudomonas denitrificans with 2C10 resulted in an approximately 30% increase in 3-HP titer (43.2 mM) compared with that obtained using wild-type KGSADH (33.1 mM). Molecular dynamics simulations suggested that compared to the wild-type enzyme, 2C10 has a less flexible and smaller binding pocket for aldehyde substrates.