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.

One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers. Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes. The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.

Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site. In this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker’s yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG. Taken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.