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A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.

Authors
  • Mans, Robert1
  • Hassing, Else-Jasmijn1
  • Wijsman, Melanie1
  • Giezekamp, Annabel1
  • Pronk, Jack T1
  • Daran, Jean-Marc1
  • van Maris, Antonius J A1
  • 1 Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands. , (Netherlands)
Type
Published Article
Journal
FEMS Yeast Research
Publisher
Oxford University Press
Publication Date
Nov 14, 2017
Identifiers
DOI: 10.1093/femsyr/fox085
PMID: 29145596
Source
Medline
Keywords
License
Unknown

Abstract

CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic-acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures, this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol (g biomass)-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.

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