EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9
Artikel i vetenskaplig tidskrift, 2016

Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90–100% and 60–70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.


Mathew M. Jessop-Fabre

Danmarks Tekniske Universitet (DTU)

T. Jakociunas

Danmarks Tekniske Universitet (DTU)

Vratislav Stovicek

Danmarks Tekniske Universitet (DTU)

Zongijie Dai

Chalmers, Biologi och bioteknik, Systembiologi

M. K. Jensen

Danmarks Tekniske Universitet (DTU)

J.D. Keasling

Danmarks Tekniske Universitet (DTU)

Lawrence Berkeley National Laboratory

Joint BioEnergy Institute, California

University of California

I. Borodina

Danmarks Tekniske Universitet (DTU)

Biotechnology journal

1860-6768 (ISSN) 1860-7314 (eISSN)

Vol. 11 8 1110-1117


Bioinformatik och systembiologi



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