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Additional file 1: Table S1. List of vectors used in the study. Table S2. List of DNA BioBricks used in the study. Table S3. List of primers used in the study. Table S4. Genes with aminoacid mutations found across all strains. Table S12. Average copy numbers for the engineered xylose pathway genes in each strain.

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.