Journal article
CRISPR/Cas9-RNAi system for combinatorial metabolic engineering of Saccharomyces cerevisiae
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Yeast Metabolic Engineering, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Technical University of Denmark4
The yeast Saccharomyces cerevisiaeis widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high-performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects.
Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, invivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double-strand break in a specific genomic region, where multi-expression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination.
We show the applicability of the method by improving cis,cis-muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction offuture cell factories.
Language: | English |
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Publisher: | John Wiley and Sons Inc. |
Year: | 2019 |
Pages: | 237-247 |
ISSN: | 10970061 and 0749503x |
Types: | Journal article |
DOI: | 10.1002/yea.3390 |
ORCIDs: | Kildegaard, Kanchana Rueksomtawin , Chekina, Ksenia , Kristensen, Mette and Borodina, Irina |