Presentation Information
[4GteX-10]In vitro reconstitution of non-oxidative glycolysis with thermophilic enzymes and its application to UDP-glucose regeneration
○Kohsuke Honda1, Gladwin Suryatin Alim1,2, Takuma Suzuki1 (1. International Center for Biotechnology, The University of Osaka (Japan), 2. Department of Chemistry, University of Basel (Switzerland))
Keywords:
enzyme cascade,thermophile,non-oxidative glycolysis,UDP-glucose
[Introduction] Enzyme cascades represent a powerful platform technology for biomanufacturing, enabling the production of target compounds with high selectivity and yield without undesired side reactions. Our research group has reported the construction of various enzyme cascades using heat-purified recombinant thermophilic enzymes as modular building blocks. In this presentation, we describe the in vitro reconstruction of non-oxidative glycolysis (NOG) using thermophilic enzymes and its extension to a UDP-glucose regeneration cascade.
[Methods] Genes encoding the enzymes constituting the NOG and UDP-glucose regeneration cascades were obtained from thermophilic microorganisms, including Thermus thermophilus. The key enzyme in NOG, phosphoketolase, was derived from Dictyoglomus thermophilum. For evaluation of the UDP-glucose regeneration cascade, trehalose synthase from Pyrococcus horikoshii was employed. All enzymes were recombinantly produced in Escherichia coli and heat-purified by incubating the E. coli cell lysates at 70 °C. The enzyme concentration ratios within the cascades were optimized using a design-of-experiments approach based on orthogonal arrays.
[Results and Discussion] The reconstructed NOG cascade was initiated by phosphorolysis of maltodextrin to glucose 1-phosphate (G1P) catalyzed by α-glucan phosphorylase. The cascade is equipped with a carbon rearrangement module that converts three molecules of erythrose 4-phosphate, generated as intermediates, into two molecules of fructose 6-phosphate. Subsequent glycolytic reactions yield three molecules of acetyl phosphate from one molecule of G1P without any carbon loss. Coupling this cascade with ATP regeneration via acetate kinase enabled the phosphorylation of three moles of ADP to ATP with one mole of C6. The ATP turnover number through this cascade exceeded 350, demonstrating its utility for ATP-dependent biomanufacturing processes. Furthermore, integration of three additional thermophilic enzymes, UDP-glucose pyrophosphorylase, nucleoside diphosphate kinase, and pyrophosphatase, into the NOG enabled construction of a UDP-glucose regeneration cascade. Compared with conventional UDP-glucose regeneration systems employing sucrose synthase, our cascade is thermodynamically favorable, with the reaction equilibrium shifted toward UDP-glucose formation. In a proof-of-concept experiment using thermophilic trehalose synthase, 10 mM aglycon (glucose) was stoichiometrically converted to trehalose with 100% conversion yield (mol/mol). By replacing the glycosyl transferase component, this cascade can serve as a versatile platform for glucosidation of a wide range of compounds.
[Methods] Genes encoding the enzymes constituting the NOG and UDP-glucose regeneration cascades were obtained from thermophilic microorganisms, including Thermus thermophilus. The key enzyme in NOG, phosphoketolase, was derived from Dictyoglomus thermophilum. For evaluation of the UDP-glucose regeneration cascade, trehalose synthase from Pyrococcus horikoshii was employed. All enzymes were recombinantly produced in Escherichia coli and heat-purified by incubating the E. coli cell lysates at 70 °C. The enzyme concentration ratios within the cascades were optimized using a design-of-experiments approach based on orthogonal arrays.
[Results and Discussion] The reconstructed NOG cascade was initiated by phosphorolysis of maltodextrin to glucose 1-phosphate (G1P) catalyzed by α-glucan phosphorylase. The cascade is equipped with a carbon rearrangement module that converts three molecules of erythrose 4-phosphate, generated as intermediates, into two molecules of fructose 6-phosphate. Subsequent glycolytic reactions yield three molecules of acetyl phosphate from one molecule of G1P without any carbon loss. Coupling this cascade with ATP regeneration via acetate kinase enabled the phosphorylation of three moles of ADP to ATP with one mole of C6. The ATP turnover number through this cascade exceeded 350, demonstrating its utility for ATP-dependent biomanufacturing processes. Furthermore, integration of three additional thermophilic enzymes, UDP-glucose pyrophosphorylase, nucleoside diphosphate kinase, and pyrophosphatase, into the NOG enabled construction of a UDP-glucose regeneration cascade. Compared with conventional UDP-glucose regeneration systems employing sucrose synthase, our cascade is thermodynamically favorable, with the reaction equilibrium shifted toward UDP-glucose formation. In a proof-of-concept experiment using thermophilic trehalose synthase, 10 mM aglycon (glucose) was stoichiometrically converted to trehalose with 100% conversion yield (mol/mol). By replacing the glycosyl transferase component, this cascade can serve as a versatile platform for glucosidation of a wide range of compounds.
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