KR-20260065071-A - Method for production of 3-hydroxypropionic acid using a cell-free synthesis system

Abstract

The present invention relates to a cell-free synthesis method for 3-hydroxypropionic acid (3-HP); and a composition for synthesis. Specifically, it was confirmed that the synthesis efficiency of 3-hydroxypropionic acid is excellent when glycerol dehydratase (GDHt) and aldehyde dehydrogenase (alpha-ketoglutaric semialdehyde dehydrogenase; KGSADH) are added to glycerol in vitro , so it can be usefully utilized in various industrial fields where 3-hydroxypropionic acid is utilized.

Inventors

• 임성인
• 조한주
• 구병수
• 전민경

Assignees

• 국립부경대학교 산학협력단

Dates

Publication Date

20260508

Application Date

20241031

Claims (19)

1. A cell-free synthesis method of 3-hydroxypropionic acid (3-HP), comprising the step of treating glycerol in vitro with glycerol dehydratase (GDHt) and aldehyde dehydrogenase (alpha-ketoglutaric semialdehyde dehydrogenase; KGSADH).
2. A synthesis method in claim 1, wherein the glycerol dehydrogenase is derived from a recombinant microorganism transformed with a recombinant plasmid containing a gene encoding GDHt.
3. A synthesis method in claim 1, wherein the aldehyde dehydrogenase is derived from a recombinant microorganism transformed with a recombinant plasmid containing a gene encoding KGSADH.
4. In claim 1, the glycerol dehydrase is Klebsiella pneumoniae, Citrobacter freundii, Clostridium butyricum, Lactobacillus reuteri, Lactobacillus brevis, Mesorhizobium loti, Salmonella typhimurium A synthesis method characterized by being derived from one or more strains selected from a group of strains.
5. A synthesis method according to claim 1, wherein the aldehyde dehydrogenase is derived from one or more strains selected from the group consisting of Azospirillum brasilense, Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, Saccharomyces cerevisiae, Lactobacillus reuteri, Corynebacterium glutamicum, Bacillus subtilis, Citrobacter freundii, and Gluconobacter oxydans.
6. A synthesis method in claim 1, wherein the GDHt is composed of subunits α, β, and γ polymerized with amino acid sequences represented by SEQ ID NOs 1, 2, and 3, respectively.
7. A synthesis method in claim 1, characterized in that the KGSADH consists of an amino acid sequence represented by SEQ ID NO. 4.
8. A synthesis method according to claim 1, characterized in that the treatment is performed at 40 - 70℃.
9. A synthesis method according to claim 1, characterized in that the treatment is performed at a pH of 7.0 to 10.0.
10. A synthesis method according to claim 1, characterized in that the glycerol and glycerol dehydrogenase react to produce an intermediate, 3-hydroxypropionaldehyde (3-HPA).
11. A synthesis method according to claim 1, characterized by further adding NAD + and coenzyme B12 to the method.
12. A synthesis method according to claim 1, characterized by further adding glycerol dehydrogenase reactivator (GDHt reactivase) to the method.
13. A composition for cell-free synthesis of 3-hydroxypropionic acid (3-HP), comprising glycerol, glycerol dehydratase (GDHt) and aldehyde dehydrogenase (alpha-ketoglutaric semialdehyde dehydrogenase; KGSADH) derived from recombinant microorganisms.
14. A composition in claim 13, wherein the GDHt is composed of subunits α, β, and γ polymerized with amino acid sequences represented by SEQ ID NOs 1, 2, and 3, respectively.
15. In claim 13, the glycerol dehydrase is Klebsiella pneumoniae, Citrobacter freundii, Clostridium butyricum, Lactobacillus reuteri, Lactobacillus brevis, Mesorhizobium loti, Salmonella typhimurium A composition characterized by being derived from one or more selected from a group of strains.
16. A composition in claim 13, wherein the KGSADH is composed of an amino acid sequence represented by SEQ ID NO. 4.
17. A composition in claim 13, wherein the aldehyde dehydrogenase is derived from one or more strains selected from the group consisting of Azospirillum brasilense, Escherichia coli, Klebsiella pneumoniae, Pseudomonas putida, Saccharomyces cerevisiae, Lactobacillus reuteri, Corynebacterium glutamicum, Bacillus subtilis, Citrobacter freundii, and Gluconobacter oxydans.
18. A composition characterized by further comprising NAD + and coenzyme B12 in claim 13.
19. A composition characterized by further comprising glycerol dehydrogenase reactivase (GDHt reactivase) in claim 13.

Description

Method for production of 3-hydroxypropionic acid using a cell-free synthesis system The present invention relates, for example, to a cell-free synthesis method of 3-hydroxypropionic acid (3-HP) in vitro ; and a composition for synthesis. With growing concerns over resource depletion and the environmental impact of petroleum-based compounds, biorefinery processes are garnering attention as a sustainable alternative to chemical synthesis. Among the various bulk chemicals produced through biorefinery, 3-hydroxypropionic acid (3-HP) is particularly economical because it is derived from glycerol, an inexpensive byproduct of biodiesel production. 3-HP is used as a precursor for various useful chemicals, such as acrylic acid, acrolein, and 1,3-propanediol. Furthermore, due to its biodegradability and flexibility, 3-HP is used as a synthetic material for biodegradable plastics. There are several metabolic pathways for synthesizing 3-HP from glycerol, among which the inventors focused on a CoA-independent pathway that converts glycerol to 3-HP through a two-step enzymatic reaction (Fig. 1). In the first step, glycerol is converted to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase (GDHt). Subsequently, 3-HPA is converted to 3-HP by NAD + -dependent alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). The CoA-independent pathway is attractive for 3-HP production because it can achieve high yield and productivity compared to other biosynthetic pathways. To improve the yield and purity of 3-HP produced from glycerol, recent research has focused primarily on metabolic engineering of suitable microbial strains and the optimization of culture conditions. However, microbial approaches face challenges such as substrate or feedback inhibition and the cytotoxicity of the intermediate 3-HPA, which hinder high-density fermentation and cost-effective 3-HP biosynthesis. Although efforts have been made to manipulate metabolic pathways to address these issues, scaling up 3-HP production to an industrial level remains a difficult challenge. In order to solve these problems, the present invention has developed an in vitro catalytic system capable of avoiding the side effects of 3-HPA accumulation and feedback inhibition that occur during microbial 3-HP production. Furthermore, the in vitro catalytic system is free from intracellular impurities and cytotoxicity, thereby making 3-HP production more efficient. Through extensive condition optimization, successful expression and purification of recombinant GDHt and KGSADH were carried out in Escherichia coli (E. coli) , and it was confirmed that the original enzyme activity was not lost. Figure 1 shows the two-step enzymatic biosynthetic pathway of 3-hydroxypropionic acid (3-HP) through a CoA-independent pathway. Figures 2a and 2b show bacterial expression of GDHt and KGSADH. Figure 2a shows the GDHt expression analyzed by 15% SDS-PAGE at 28°C. Figure 2b shows the KGSADH expression analyzed by 15% SDS-PAGE at 28°C. Figures 3a to 3c illustrate the characteristics of enzyme activity. Figure 3a is a standard curve showing the change in absorbance (ΔA 305 ) at 305 nm for propionaldehyde at various concentrations reacted with MBTH reagent. Figure 3b shows the initial rate of propionaldehyde production in a series of GDHt-catalyzed reactions using 1,2-propanediol at various concentrations as a substrate. Figure 3c shows the initial rate of propionic acid production in a series of KGSADH-catalyzed reactions using propionaldehyde at various concentrations as a substrate. Figures 4a to 4c illustrate the in vitro 3-HP biosynthesis from glycerol under various physicochemical conditions. Figure 4a shows the effect of equimolar concentrations of GDHt and KGSADH on 3-HP production at 37°C and pH 8.0. Figure 4b shows the effect of reaction temperature over 20 minutes on 3-HP production when each enzyme is 100 nM and pH is 8.0. Figure 4c shows the effect of pH on 3-HP production when each enzyme is 100 nM and pH is 45°C. The inventors optimized the recombinant expression and purification of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (alpha-ketoglutaric semialdehyde dehydrogenase; KGSADH). The inventors performed in vitro synthesis of 3-hydroxypropionic acid (3-HP) using the enzyme and demonstrated efficient production of 3-HP from glycerol, and confirmed that the yield was improved, particularly under high temperature and alkaline conditions. Accordingly, the present invention provides a cell-free synthesis method for 3-hydroxypropionic acid (3-HP), comprising the step of treating glycerol in vitro with glycerol dehydratase (GDHt) and aldehyde dehydrogenase (alpha-ketoglutaric semialdehyde dehydrogenase; KGSADH). In this specification, "3-hydroxypropionic acid (3-HP)" is a bio-based chemical produced by an eco-friendly fermentation process, used as a raw material for acrylic acid, acrylonitrile, biodegradable materials, etc., and is a high-value-added material that plays an import