Taxonomic group: fungi / Ascomycota
(Phylum: Ascomycota)
NCBI PubMed ID: 30706115Publication DOI: 10.1007/s00253-019-09614-4Journal NLM ID: 8406612Publisher: Springer
Correspondence: Kang HA <hyunkang
cau.ac.kr>
Institutions: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, South Korea
The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.
α-1, conditional mutant, Saccharomyces cerevisiae, 6-mannosyltransferase, chitin synthase III, MET3 promoter
Structure type: homopolymer
Location inside paper: Fig. 1, chitin
Trivial name: chitin
Compound class: O-polysaccharide, cell wall polysaccharide, glucan, polysaccharide, chitin
Contained glycoepitopes: IEDB_135813,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_153212,IEDB_241099,IEDB_423114,IEDB_423150,SB_74,SB_85
Methods: PCR, DNA techniques, Western blotting, colorimetry, cloning, cell growth, enzymatic assay, gene expression, qRT-PCR, osmotic lysis
Related record ID(s): 49868, 49869
NCBI Taxonomy refs (TaxIDs): 4932Reference(s) to other database(s): GTC:G97099AY, CCSD:
46067, CBank-STR:5851, GenDB:KF905651
Show glycosyltransferases
There is only one chemically distinct structure:
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