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Mutations of Kluyveromyces lactis dolichol kinase enhances secretion of recombinant proteins.

Authors
  • Ziogiene, Danguole1
  • Valaviciute, Monika1
  • Norkiene, Milda1
  • Timinskas, Albertas1
  • Gedvilaite, Alma1
  • 1 Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania. , (Lithuania)
Type
Published Article
Journal
FEMS Yeast Research
Publisher
Oxford University Press
Publication Date
May 01, 2019
Volume
19
Issue
3
Identifiers
DOI: 10.1093/femsyr/foz024
PMID: 30865773
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Although there are similarities in the core steps of the secretion pathway from yeast to higher eukaryotes, significant functional differences exist even among diverse yeast species. Here, we used next-generation sequencing to identify two mutations in the Kluyveromyces lactis KlSEC59 gene, encoding dolichol kinase (DK), which are responsible for an enhanced secretion phenotype in a previously isolated mutant, MD2/1-9. Compared with the temperature-sensitive Saccharomyces cerevisiae sec59-1 mutant, which exhibits reduced N-glycosylation and decreased secretory efficacy, the identified K. lactis DK mutations had fewer effects on glycosylation, as well as on survival at high temperature and cell wall integrity. Moreover, despite some glycosylation defects, double DK mutations (G405S and I419S) in the K. lactis mutant strain demonstrated three times the level of recombinant α-amylase secretion as the wild-type strain. Overexpression of potential suppressors KlMNN10, KlSEL1, KlERG20, KlSRT1, KlRER2, KlCAX4, KlLPP1 and KlDPP1 in the DK-mutant strain restored carboxypeptidase Y glycosylation to different extents and, with the exception of KISRT1, reduced α-amylase secretion to levels observed in wild-type cells. Our results suggest that enhanced secretion related to reduced activity of mutant DK in K. lactis results from mild glycosylation changes that affect activity of other proteins in the secretory pathway. © FEMS 2019.

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