title: Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.
abstract: Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Institute of Chemical Processes, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
authors.forename: Sujin
authors.initials: S
authors.lastname: Kim
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Institute of Chemical Processes, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
authors.forename: Sang-Jeong
authors.initials: SJ
authors.lastname: Bae
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, Institute of Chemical Processes, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
authors.forename: Ji-Sook
authors.initials: JS
authors.lastname: Hahn
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism*; D002072:Butylene Glycols / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism*; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D000097722:Cohesins
chemical_list: D000095:Acetolactate Synthase; D002072:Butylene Glycols; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D000431:Ethanol; D007770:L-Lactate Dehydrogenase; D019344:Lactic Acid; D011770:Pyruvate Kinase; D011773:Pyruvates; D029701:Saccharomyces cerevisiae Proteins; D000097722:Cohesins; C026978:2,3-butylene glycol