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Construction of an in vitro bypassed pyruvate decarboxylation pathway using thermostable enzyme modules and its application to N-acetylglutamate production.

Krutsakorn B, Imagawa T, Honda K, Okano K, Ohtake H - Microb. Cell Fact. (2013)

Bottom Line: One of the possible solutions for the elimination of the negative effects of natural regulatory mechanisms on artificially engineered metabolic pathway is to construct an in vitro pathway using a limited number of enzymes.Assembly of thermostable enzymes enables the flexible design and construction of an in vitro metabolic pathway specialized for chemical manufacture.This pathway is potentially applicable not only to N-acetylglutamate production but also to the production of a wide range of acetyl-CoA-derived metabolites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. honda@bio.eng.osaka-u.ac.jp.

ABSTRACT

Background: Metabolic engineering has emerged as a practical alternative to conventional chemical conversion particularly in biocommodity production processes. However, this approach is often hampered by as yet unidentified inherent mechanisms of natural metabolism. One of the possible solutions for the elimination of the negative effects of natural regulatory mechanisms on artificially engineered metabolic pathway is to construct an in vitro pathway using a limited number of enzymes. Employment of thermostable enzymes as biocatalytic modules for pathway construction enables the one-step preparation of catalytic units with excellent selectivity and operational stability. Acetyl-CoA is a central precursor involved in the biosynthesis of various metabolites. In this study, an in vitro pathway to convert pyruvate to acetyl-CoA was constructed and applied to N-acetylglutamate production.

Results: A bypassed pyruvate decarboxylation pathway, through which pyruvate can be converted to acetyl-CoA, was constructed by using a coupled enzyme system consisting of pyruvate decarboxylase from Acetobacter pasteurianus and the CoA-acylating aldehyde dehydrogenase from Thermus thermophilus. To demonstrate the applicability of the bypassed pathway for chemical production, a cofactor-balanced and CoA-recycling synthetic pathway for N-acetylglutamate production was designed by coupling the bypassed pathway with the glutamate dehydrogenase from T. thermophilus and N-acetylglutamate synthase from Thermotoga maritima. N-Acetylglutamate could be produced from an equimolar mixture of pyruvate and α-ketoglutarate with a molar yield of 55% through the synthetic pathway consisting of a mixture of four recombinant E. coli strains having either one of the thermostable enzymes. The overall recycling number of CoA was calculated to be 27.

Conclusions: Assembly of thermostable enzymes enables the flexible design and construction of an in vitro metabolic pathway specialized for chemical manufacture. We herein report the in vitro construction of a bypassed pathway capable of an almost stoichiometric conversion of pyruvate to acetyl-CoA. This pathway is potentially applicable not only to N-acetylglutamate production but also to the production of a wide range of acetyl-CoA-derived metabolites.

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Related in: MedlinePlus

Thermal stability of ApPDC (a), TtADDH (b), TtGDH (c), and TmNAGS (d). Crude lysates of the recombinant E. coli were preheated at 70°C for 30 min; an incubation temperature of 60°C was employed for preheating the lysate having ApPDC. After the removal of denatured proteins by centrifugation, the enzyme solutions were incubated at 50°C (blue diamond), 60°C (yellow square), and 70°C (green triangle) for the indicated time periods. Residual enzyme activity was determined using the standard assay conditions.
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Figure 3: Thermal stability of ApPDC (a), TtADDH (b), TtGDH (c), and TmNAGS (d). Crude lysates of the recombinant E. coli were preheated at 70°C for 30 min; an incubation temperature of 60°C was employed for preheating the lysate having ApPDC. After the removal of denatured proteins by centrifugation, the enzyme solutions were incubated at 50°C (blue diamond), 60°C (yellow square), and 70°C (green triangle) for the indicated time periods. Residual enzyme activity was determined using the standard assay conditions.

Mentions: ApPDC could retain more than 60% of its activity after incubation at 50°C for 4 h, whereas no apparent decrease was observed in the activity of TtADDH at 50°C (Figure 3a and b). Conversely, the activities of both enzymes steeply declined at an incubation temperature of 60°C or higher. Thus, a reaction temperature of 50°C was used for further studies.


Construction of an in vitro bypassed pyruvate decarboxylation pathway using thermostable enzyme modules and its application to N-acetylglutamate production.

Krutsakorn B, Imagawa T, Honda K, Okano K, Ohtake H - Microb. Cell Fact. (2013)

Thermal stability of ApPDC (a), TtADDH (b), TtGDH (c), and TmNAGS (d). Crude lysates of the recombinant E. coli were preheated at 70°C for 30 min; an incubation temperature of 60°C was employed for preheating the lysate having ApPDC. After the removal of denatured proteins by centrifugation, the enzyme solutions were incubated at 50°C (blue diamond), 60°C (yellow square), and 70°C (green triangle) for the indicated time periods. Residual enzyme activity was determined using the standard assay conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852541&req=5

Figure 3: Thermal stability of ApPDC (a), TtADDH (b), TtGDH (c), and TmNAGS (d). Crude lysates of the recombinant E. coli were preheated at 70°C for 30 min; an incubation temperature of 60°C was employed for preheating the lysate having ApPDC. After the removal of denatured proteins by centrifugation, the enzyme solutions were incubated at 50°C (blue diamond), 60°C (yellow square), and 70°C (green triangle) for the indicated time periods. Residual enzyme activity was determined using the standard assay conditions.
Mentions: ApPDC could retain more than 60% of its activity after incubation at 50°C for 4 h, whereas no apparent decrease was observed in the activity of TtADDH at 50°C (Figure 3a and b). Conversely, the activities of both enzymes steeply declined at an incubation temperature of 60°C or higher. Thus, a reaction temperature of 50°C was used for further studies.

Bottom Line: One of the possible solutions for the elimination of the negative effects of natural regulatory mechanisms on artificially engineered metabolic pathway is to construct an in vitro pathway using a limited number of enzymes.Assembly of thermostable enzymes enables the flexible design and construction of an in vitro metabolic pathway specialized for chemical manufacture.This pathway is potentially applicable not only to N-acetylglutamate production but also to the production of a wide range of acetyl-CoA-derived metabolites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. honda@bio.eng.osaka-u.ac.jp.

ABSTRACT

Background: Metabolic engineering has emerged as a practical alternative to conventional chemical conversion particularly in biocommodity production processes. However, this approach is often hampered by as yet unidentified inherent mechanisms of natural metabolism. One of the possible solutions for the elimination of the negative effects of natural regulatory mechanisms on artificially engineered metabolic pathway is to construct an in vitro pathway using a limited number of enzymes. Employment of thermostable enzymes as biocatalytic modules for pathway construction enables the one-step preparation of catalytic units with excellent selectivity and operational stability. Acetyl-CoA is a central precursor involved in the biosynthesis of various metabolites. In this study, an in vitro pathway to convert pyruvate to acetyl-CoA was constructed and applied to N-acetylglutamate production.

Results: A bypassed pyruvate decarboxylation pathway, through which pyruvate can be converted to acetyl-CoA, was constructed by using a coupled enzyme system consisting of pyruvate decarboxylase from Acetobacter pasteurianus and the CoA-acylating aldehyde dehydrogenase from Thermus thermophilus. To demonstrate the applicability of the bypassed pathway for chemical production, a cofactor-balanced and CoA-recycling synthetic pathway for N-acetylglutamate production was designed by coupling the bypassed pathway with the glutamate dehydrogenase from T. thermophilus and N-acetylglutamate synthase from Thermotoga maritima. N-Acetylglutamate could be produced from an equimolar mixture of pyruvate and α-ketoglutarate with a molar yield of 55% through the synthetic pathway consisting of a mixture of four recombinant E. coli strains having either one of the thermostable enzymes. The overall recycling number of CoA was calculated to be 27.

Conclusions: Assembly of thermostable enzymes enables the flexible design and construction of an in vitro metabolic pathway specialized for chemical manufacture. We herein report the in vitro construction of a bypassed pathway capable of an almost stoichiometric conversion of pyruvate to acetyl-CoA. This pathway is potentially applicable not only to N-acetylglutamate production but also to the production of a wide range of acetyl-CoA-derived metabolites.

Show MeSH
Related in: MedlinePlus