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.
Highlights
Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates
Such a substrate channeling strategy was effective in channeling α -acetolactate produced by acetolactate synthase (AlsS) into 2,3-butanediol production pathway, it was not efficient to compete with the native ethanol production pathway for pyruvate availability
In S. cerevisiae, pyruvate produced by glycolysis is mainly converted to ethanol via acetaldehyde during fermentative growth (Fig. 1)
Summary
Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. 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. Three enzymes converting pyruvate to 2,3-butanediol, including α -acetolactate synthase (AlsS) and α -acetolactate decarboxylase (AlsD) from Bacillus subtilis and endogenous 2,3-butanediol dehydrogenase (Bdh1), were expressed as dockerin-fused proteins, and assembled onto a scaffold containing multiple cohesin domains[21] Such a substrate channeling strategy was effective in channeling α -acetolactate produced by AlsS into 2,3-butanediol production pathway, it was not efficient to compete with the native ethanol production pathway for pyruvate availability. By tethering heterologous pyruvate-converting enzymes to pyruvate kinase (Pyk1) using cohesin-dockerin interaction, pyruvate flux was successfully redirected to desired pathways producing lactic acid or 2,3-butanediol
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