Print Email Facebook Twitter 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 acidCas9CRISPRDiffusionGenome editingMetabolic 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) Part of collection Institutional Repository Document type journal article Rights © 2017 R. Mans, J. Else-Hassing, M. Wijsman, Annabel Giezekamp, J.T. Pronk, J.G. Daran, A.J.A. van Maris Files PDF LA_Manuscript_after_rebuttal.pdf 1.11 MB Close viewer /islandora/object/uuid:b2753a94-bd0b-49a6-a18b-885cc4ed6650/datastream/OBJ/view