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BackgroundMetabolic 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.ResultsA 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.ConclusionsAssembly 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.