Abstract
The yeast Saccharomyces cerevisiae is 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, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double‐strand break in a specific genomic region, where multiexpression 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 of future cell factories.
Highlights
Industrial biotechnology uses cell factories to produce therapeutical proteins, antibiotics, enzymes, fuels, and chemicals
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
We decided to combine the advantages of CRISPR/Cas9, in vivo recombination, USER assembly, and RNA interference (RNAi)
Summary
Industrial biotechnology uses cell factories to produce therapeutical proteins, antibiotics, enzymes, fuels, and chemicals. We present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. We aimed to develop a method that would allow multiplex upregulation and downregulation of several genes by combining the advantages of the CRISPR/Cas9 system and RNAi. The level of upregulation and downregulation can be tuned by selecting promoters of different strengths.
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