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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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Effect of NAG concentration on TtGDH. The enzyme activity was determined under the standard assay conditions at the indicated concentrations of NAG.
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Figure 6: Effect of NAG concentration on TtGDH. The enzyme activity was determined under the standard assay conditions at the indicated concentrations of NAG.

Mentions: In this study, we constructed an in vitro bypassed pathway for pyruvate decarboxylation by employing an enzyme couple of ApPDC and TtADDH. The bypassed pathway was integrated into the newly designed synthetic pathway for NAG production, in which consumption and regeneration rates of both NAD(H) and CoA were essentially balanced. The highly traceable nature of the in vitro synthetic pathway enabled us to quantify the intermediate pool sizes without using special equipment and to identify the rate-limiting of the synthetic pathway. The time-course analysis of the intermediates in the NAG-producing pathway indicated that the decrease in the catalytic ability of TtGDH and TtADDH caused by the thermal decomposition of NAD(H) was the bottleneck. In fact, by continuously supplying NADH to the reaction mixture, 5.4 mM NAG could be produced with a molar yield of 55% and a CoA-recycling number of 27 could be achieved. The operational stability of the in vitro production system would be improved by the screening and employment of a thermostable glutamate dehydrogenase with lower Km for NAD(H). However, the intermediate analysis indicated that accumulation of α-ketoglutarate was still not insignificant even when NADH was supplied (Figure 5c). This can be explained by the inhibitory effect of NAG on TtGDH (Figure 6). Although in vitro synthetic pathways are independent of transcriptional and translational regulatory mechanisms of living cells, allosteric regulation cannot be necessarily eliminated. Protein engineering approaches or the substitution with another enzyme module that is less sensitive to allosteric effects would be a straightforward way to eliminate the effect of allosteric inhibition. In fact, we previously demonstrated that the inhibitory effect of NAD+ on lactate production through the chimeric glycolytic pathway can be eliminated by changing the NAD+-sensitive lactate dehydrogenase to malate/lactate dehydrogenase [6]. Use of a NAG-insensitive thermophilic glutamate dehydrogenase would be needed to achieve a higher titer of NAG production through the synthetic pathway.


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)

Effect of NAG concentration on TtGDH. The enzyme activity was determined under the standard assay conditions at the indicated concentrations of NAG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of NAG concentration on TtGDH. The enzyme activity was determined under the standard assay conditions at the indicated concentrations of NAG.
Mentions: In this study, we constructed an in vitro bypassed pathway for pyruvate decarboxylation by employing an enzyme couple of ApPDC and TtADDH. The bypassed pathway was integrated into the newly designed synthetic pathway for NAG production, in which consumption and regeneration rates of both NAD(H) and CoA were essentially balanced. The highly traceable nature of the in vitro synthetic pathway enabled us to quantify the intermediate pool sizes without using special equipment and to identify the rate-limiting of the synthetic pathway. The time-course analysis of the intermediates in the NAG-producing pathway indicated that the decrease in the catalytic ability of TtGDH and TtADDH caused by the thermal decomposition of NAD(H) was the bottleneck. In fact, by continuously supplying NADH to the reaction mixture, 5.4 mM NAG could be produced with a molar yield of 55% and a CoA-recycling number of 27 could be achieved. The operational stability of the in vitro production system would be improved by the screening and employment of a thermostable glutamate dehydrogenase with lower Km for NAD(H). However, the intermediate analysis indicated that accumulation of α-ketoglutarate was still not insignificant even when NADH was supplied (Figure 5c). This can be explained by the inhibitory effect of NAG on TtGDH (Figure 6). Although in vitro synthetic pathways are independent of transcriptional and translational regulatory mechanisms of living cells, allosteric regulation cannot be necessarily eliminated. Protein engineering approaches or the substitution with another enzyme module that is less sensitive to allosteric effects would be a straightforward way to eliminate the effect of allosteric inhibition. In fact, we previously demonstrated that the inhibitory effect of NAD+ on lactate production through the chimeric glycolytic pathway can be eliminated by changing the NAD+-sensitive lactate dehydrogenase to malate/lactate dehydrogenase [6]. Use of a NAG-insensitive thermophilic glutamate dehydrogenase would be needed to achieve a higher titer of NAG production through the synthetic pathway.

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