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

Mans, R.; Else-Hassing, J.; Wijsman, M.; Giezekamp, Annabel; Pronk, J.T.; Daran, J.G.; van Maris, A.J.A.

doi:10.1093/femsyr/fox085

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

Title

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

Author

Mans, R. (TU Delft BT/Industriele Microbiologie)
Else-Hassing, J. (TU Delft BT/Industriele Microbiologie)
Wijsman, M. (TU Delft BT/Industriele Microbiologie)
Giezekamp, Annabel (Student TU Delft)
Pronk, J.T. (TU Delft BT/Industriele Microbiologie)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)
van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie; KTH Royal Institute of Technology)

Date

2017-12-01

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.

Subject

Carboxylic acid
Cas9
CRISPR
Diffusion
Genome editing
Metabolic engineering

To reference this document use:

http://resolver.tudelft.nl/uuid:b2753a94-bd0b-49a6-a18b-885cc4ed6650

DOI

https://doi.org/10.1093/femsyr/fox085

Embargo date

2018-11-14

ISSN

1567-1356

Source

FEMS Yeast Research, 17 (8)