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Comparative Proteome Analysis in Schizosaccharomyces pombe Identifies Metabolic Targets to Improve Protein Production and Secretion*

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Protein secretion in yeast is a complex process and its efficiency depends on a variety of parameters. We performed a comparative proteome analysis of a set of Schizosaccharomyces pombe strains producing the α-glucosidase maltase in increasing amounts to investigate the overall proteomic response of the cell to the burden of protein production along the various steps of protein production and secretion. Proteome analysis of these strains, utilizing an isobaric labeling/two dimensional LC-MALDI MS approach, revealed complex changes, from chaperones and secretory transport machinery to proteins controlling transcription and translation. We also found an unexpectedly high amount of changes in enzyme levels of the central carbon metabolism and a significant up-regulation of several amino acid biosyntheses. These amino acids were partially underrepresented in the cellular protein compared with the composition of the model protein. Additional feeding of these amino acids resulted in a 1.5-fold increase in protein secretion. Membrane fluidity was identified as a second bottleneck for high-level protein secretion and addition of fluconazole to the culture caused a significant decrease in ergosterol levels, whereas protein secretion could be further increased by a factor of 2.1. In summary, we show that high level protein secretion causes global changes of protein expression levels in the cell and that precursor availability and membrane composition limit protein secretion in this yeast. In this respect, comparative proteome analysis is a powerful tool to identify targets for an efficient increase of protein production and secretion in S. pombe. Data are available via ProteomeXchange with identifiers PXD002693 and PXD003016.

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Author contributions: C.H., T.K., L.C., M.B., K.S., and A.T. designed research; C.H., T.K., L.C., and S.L. performed research; C.H., T.K., L.C., D.L., U.A., M.B., E.H., K.S., and A.T. analyzed data; C.H., T.K., L.C., K.S., and A.T. wrote the paper.

Authors contributions: CWH, TK, SL, LC performed the experiments; CWH, TK, LC, DL, UA, MB, EH, KS, AT interpreted data; CWH, TK, LC, KS and AT wrote the manuscript.

*

This work was supported by the BMBF (Project: “SweePro- Sustainable and resource-efficient production of sweet proteins for health and nutrition”) and the DFG-Cluster of Excellence “Inflammation at Interfaces,” (Clusterlab CL-X).

This article contains supplemental material.

1

The abbreviations used are:

    CDW

    cell dry weight

    FDR

    false discovery rate

    GIS

    global internal standard

    GO

    gene ontology

    IP-RP

    ion pairing reversed phase

    PPP

    pentose phosphate pathway

    TCA

    tricarboxylic acid cycle

    μ

    growth rate

    2D-LC

    two-dimensional liquid chromatography.

‡‡

These authors contributed equally,

§§

contributed equally as senior authors.