Mechanism of 3-Hydroxypropionic Acid Tolerance by Deletion of a Less-characterized Transcription Factor yieP in Escherichia coli W

Thuan NGUYEN-VO,Seyoung KO,Huichang RYU,Donghyuk KIM,Jung Rae KIM,Sunghoon PARK 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.4

Toxicity of organic acids such as 3-hydroxypropionic acid (3-HP), propionic acid, lactic acid, is an important issue that limits microbial production of them at industrial levels (~ 1M). As a case study to tackle this problem, adaptive laboratory evolution has been successfully adopted to evolve Escherichia coli W for tolerance against 3-HP (1). Our previous study showed that the deletion of a less-characterised transcription factor (YieP) mainly responsible for the tolerance against 3-HP. In this current study, we exploited physiological and transcriptomics study (RNA-seq along with ChiP-exo) to study the mechanism that the deletion of yieP confers 3-HP tolerance. The growing experiment in several organic acids (C2-C4) suggested that yieP deletion conferred specific-anion tolerance toward 3-HP but not other organic acids. The transcriptomics along with deletion experiments suggested that deletion of yieP improved 3-HP tolerance via expression of two genes encoding inner membrane proteins yohJ and yohK. The role of YohJ/K was further studied using 3-HP responsive biosensor. Our results suggested that yohJK encode for (a) novel 3-HP exporter(s).

Co-production of 3-hydroxypropionic acid and 1,3-propandiol by recombinant Klebsiella pneumoniae

고연주,설은희,( Ashok Somasundar ),박성훈 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1

3-hydroxypropionic acid (3-HP) production from glycerol has gained much attention due to the importance of 3-HP as platform chemical. 3-HP production is composed of two reactions i.e. conversion of glycerol to 3-HPA oxidation to 3-HP. Klebsiella pneumoniae, one of the candidates for 3-HP production, has advantage of natural synthesis of B₁₂ under anaerobic condition. However, further reaction of 3-HPA to 3-HP involves the reduction of NAD⁺ and consequently NAD⁺ regeneration is very important for efficient production of 3-HP. On the other hand, production of 1,3-PDO by NADH-dependent oxidoredcutase of K. pneumoniae has been studied extensively. Hence, in this study, we tried co-production of 3-HP and 1,3-PDO using K. pneumoniae mutants and studied the effect of various environmental factors and genetic backgrounds on co-production yields change in K. pneumoniae.

Directed Evolution of Alpha-ketoglutaric Semialdehyde Dehydrogenase for Enhancing the Catalytic Activity toward 3-hydroxypropionaldehyde

Ye Seop PARK,Abdul NASIR,Joo Yeon SEOK,Gyoo Yeol JUNG,Sunghoon PARK,Tae Hyeon YOO 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

3-Hydroxypropionic acid (3-HP) is one of the key building blocks which is a precursor of diverse value-added chemicals and can be used as an additive or preservative agent in the food industry. The molecule can be produced by a simple biological pathway: dehydration of glycerol to 3-hydroxypropanal (3-HPA) by glycerol dehydratase and then oxidation to 3-HP by aldehyde dehydrogenase. Despite successful production of 3-HP through this pathway, several problems including the toxicity of the 3-HPA hamper the 3-HP production by industrial permissive level. To solve the issues we had engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirilum brasilense for enhancing the catalytic activity toward 3-HPA with small-size library(~2 * 10⁴). For screening better mutants with respect to catalytic activity toward 3-HPA than previous KGSADH mutants, we have made a large-size library that 5 residues were mutated simultaneously and screened the library via a 3-HP selection system followed by NADH absorbance-based 96well plate screening. After selection and screening, we got the mutant which has 4.65-fold higher activity toward 3-HPA than wild-type. Furthermore, the mutant has a higher preference toward C2-C3 carbon aldehydes than wild-type. In particular, the recombinant P. denitrificans strain harboring the mutant exhibited 1.3-fold higher 3-HP titer and specific activity than that with the wild-type. MD simulation results showed that mutations in the mutant stabilized the loop area and lowered the binding pocket entry, which could lead to an increase in catalytic activity.

An engineered aldehyde dehydrogenase for production of 3-hydroxypropionic acid from glycerol

박예섭,유태현 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0

Biological conversion of glycerol into valuable biochemicals has attracted lots of attentions because glycerol is by-product of the biodiesel production. One of these chemicals is 3-hydroxypropionic acid (3-HP) that can be produced from glycerol via two enzymatic reactions: glycerol to 3-hydroxylpropanal (3-HPA) via glycerol dehydratase and 3-HPA to 3-HP via aldehyde dehydrogenase. Biological processes using the two reactions have been developed, and the productivity of more than 0.86g/L/hr was reported. However, improvement in the productivity is needed to move to a next phase of commercialization, and some issues have been raised. One of them is the toxicity of 3-HPA. An approach to solve the problem is to develop a highly active aldehyde dehydrogenase, and the process can be operated at a low concentration of 3-HPA. In this presentation, we report the results of engineering the substrate specific of an aldehyde dehydrogenase, α-ketoglutaric semialdehyde dehydrogenase toward 3-HPA.

3-Hydroxypropionic acid Production with Optimal Pathway Rebalancing in Escherichia coli

임현규,노명현,박성훈,정규열 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0

3-Hydroxypropionic acid (3-HP) is an important platform chemical. It can be biologically produced from glycerol by two consecutive enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA, which severely reduces cell viability and 3-HP production. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer and productivity compared with the parental strain.

Optimal pathway rebalancing for 3-Hydroxypropionic acid production from glycerol in Escherichia coli

이지훈,임현규,노명현,박성훈,정규열 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.1

3-Hydroxypropionic acid (3-HP), which can be converted into many chemicals such as acrylic acid, and acrylamide, is an important platform chemical. It can be biologically produced from glycerol by two enzymatic reactions, however, imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA, which severely reduces 3-HP production. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high production of 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer (40.51 g/L) and productivity (1.35 g/L/h) compared with the parental strain. Especially, the yield on glycerol, 0.97 g 3-HP/g glycerol, in a fed-batch experiment, was the highest ever reported.

Engineering of Alpha-ketoglutaric Semialdehyde Dehydrogenase for Improving the Catalytic Activity Toward 3-HPA via 3-HP Selection Device and 96-well Microplate-based Screening

Ye Seop PARK,Sunjoo CHOI,Abdul NASIR,Joo Yeon SEOK,Gyoo yeol JUNG,Sunghoon PARK,Tae Hyeon YOO 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

3-Hydroxypropionic acid (3-HP) is one of the key building blocks which is a precursor of diverse value-added chemicals and can be used as an additive or preservative agent in the food industry. The molecule can be produced by a simple biological pathway: dehydration of glycerol to 3-hydroxypropanal (3-HPA) by glycerol dehydratase and then oxidation to 3-HP by aldehyde dehydrogenase. Despite successful production of 3-HP through this pathway, several problems including the toxicity of the 3-HPA hamper the 3-HP production by industrial permissive level. To solve the issues we had engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirilum brasilense for enhancing the catalytic activity toward 3-HPA with small-size library(~2 <SUP>*</SUP> 104). For screening better mutants with respect to catalytic activity toward 3-HPA than previous KGSADH mutants, we have made a large-size library that 5 residues were mutated simultaneously and screened the library via a 3-HP selection system followed by NADH absorbance-based 96well plate screening. After selection and screening, we got the mutant which has 4.65-fold higher activity toward 3-HPA than wild-type. Furthermore, the mutant has a higher preference toward C2-C3 carbon aldehydes than wild-type. In particular, the recombinant P. denitrificans strain harboring the mutant exhibited 1.3-fold higher 3-HP titer and specific activity than that with the wild-type. MD simulation results showed that mutations in the mutant stabilized the loop area and lowered the binding pocket entry, which could lead to an increase in catalytic activity.

Production of 3-hydroxypropionic acid by optimal pathway rebalancing from glycerol in Escherichia coli

이지훈,임현규,노명현,박성훈,정규열 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

3-Hydroxypropionic acid (3-HP), which can be converted into many chemicals such as acrylic acid, is an important platform chemical. It can be biologically produced from glycerol by two enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer (40.51 g/L) and productivity (1.35 g/L/h) compared with the parental strain.

Overexpression of Acetyl CoA Carboxylase1 and Malonyl CoA Reductase for the Production of 3-hydroxypropionic Acid by an Engineered Kluyveromyces marxianus

Kyoung-Gon KANG,Deok-Ho KWON,Suk-Jin HA 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

3-hydroxypropionic acid (3-HP) is one of the basic substances that can be synthesized various industrial compounds such as malonic acid, 1,3-propanediol, which can replace plastics, resins and adhesives produced from fossil fuel. For the production of 3-HP, an isomer of lactic acid and a C3 chemical, Kluyveromyces marxianus, known as thermo tolerance yeast was engineered. We overexpressed the acetyl CoA carboxylase1 (ACC1) gene originated from K. marxianus 17555, and then overexpressed the malonyl CoA reductase (MCR) gene originated from Chloroflexus aurantiacus to select 3-HP producing engineered strain. The engineered K. marxianus KG001 strain produced 8.28 ± 0.18 g/L of 3-HP at 100 rpm with 0.34 ± 0.00 g/L/h productivity and 0.10 ± 0.00 g/g yield for 24 h. At 200 rpm, 3-HP production, productivity, yield were improved to 10.16 ± 0.38 g/L, 0.42 ± 0.01 g/L/h, and 0.13 ± 0.00 g/g for 24 h. Through the optimization of fermentation conditions using bioreactor, the maximum 3-HP concentration was 17.51 g/L, the maximum productivity was 0.84 g/L/h, and the maximum yield was 0.22 g/g.

Optimal pathway rebalancing by 5'-UTR library for 3-hydroxypropionic acid production from glycerol in Escherichia coli

김승진,임현규,노명현,정규열 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

3-hydroxypropionic acid can be biologically produced from glycerol by two consecutive enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA, which severely reduces cell viability and 3-HP production. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer (40.51 g/L) and productivity (1.35 g/L/h) compared with the parental strain. Especially, the yield on glycerol, 0.97 g 3-HP/g glycerol.