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At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small‐scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small‐scale hypoxic culture in a bench‐top multi‐gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small‐scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small‐scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes. [ABSTRACT FROM AUTHOR]

The yeast surface display system is a valuable platform for constructing cells with novel functions for various applications and high-throughput screening of protein or peptide libraries containing random mutations. Among the host microorganisms used for surface display, yeast is the most suitable microorganism for surface engineering owing to its eukaryotic features. In yeast, proper folding and glycosylation of expressed eukaryotic proteins can be performed. Furthermore, in this system, multiple kinds of proteins can be simultaneously displayed on the cell surface. This allows for a synergistic effect between the displayed enzymes, leading to an efficient multistep reaction. Alternatively, the ratio of the enzymes to be displayed can be controlled by the co-culture of surface-engineered yeasts displaying a single kind of enzyme. Therefore, yeast surface display systems have been applied to the construction of various whole-cell biocatalysts. Here, we describe methods for the simultaneous display of multiple kinds of proteins on the yeast cell surface., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of protein/peptide library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-activated cell sorting (FACS), and yeast cell chip, the cell surface display system is an effective molecular tool for high-throughput screening of mutated protein/peptide library. In this article, we describe the methods for cell surface display of proteins/peptides of interest on yeast, evaluation of display efficiency, and harvesting of the displayed proteins/peptides from cell surface., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)