EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains

Abstract

Saccharomyces cerevisiae is one of the key cell factories for production of chemicals and active pharmaceuticals. For large-scale fermentations, particularly in biorefinery applications, it is desirable to use stress-tolerant industrial strains. However, such strains are less amenable for metabolic engineering than the standard laboratory strains. To enable easy delivery and overexpression of genes in a wide range of industrial S. cerevisiae strains, we constructed a set of integrative vectors with long homology arms and dominant selection markers. The vectors integrate into previously validated chromosomal locations via double cross-over and result in homogenous stable expression of the integrated genes, as shown for several unrelated industrial strains. Cre-mediated marker rescue is possible for removing markers positioned on different chromosomes. To demonstrate the applicability of the presented vector set for metabolic engineering of industrial yeast, we constructed xylose-utilizing strains overexpressing xylose isomerase, xylose transporter and five genes of the pentose phosphate pathway.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-015-1684-8) contains supplementary material, which is available to authorized users.

EasyClone‐MarkerFree: A vector toolkit for marker‐less integration of genes into Saccharomyces cerevisiae via CRISPR‐Cas9

“…). This is comparable with the previous versions of EasyClone, where the targeting efficiency was 80–100% . However, we should note that wrong clones in the EasyClone‐MarkerFree system were not fluorescent and hence they did not contain the designed integration cassettes in their genomes, while for the EasyClone system with selection markers, the integration cassette would always be integrated in the genome, however sometimes it would not be targeted to the designed integration site.…”

“…Genetic manipulation of S. cerevisiae is greatly facilitated by its efficient inherent homologous recombination (HR) machinery for DNA double‐strand break (DSB) repair . As a result, many genome engineering tools have been developed that take advantage of HR for targeted integration of heterologous DNA into the yeast genome . Chromosomal gene integration has several benefits over plasmid‐based approaches, such as increased strain stability, better control of gene expression level and lower population heterogeneity .…”

“…oped that take advantage of HR for targeted integration of heterologous DNA into the yeast genome [5,6]. Chromosomal gene integration has several benefits over plasmidbased approaches, such as increased strain stability, better control of gene expression level and lower population heterogeneity [5].…”

The art of vector engineering: towards the construction of next‐generation genetic tools

“…Three years later, in 2014, Jensen and his group designed the EasyClone: a set of vectors that can integrate three cassettes at a time carrying up to two genes each and showed that this is more homogeneous and stable than expressing more than one gene in episomal vectors (Jensen et al ., ). A variation of EasyClone called EasyClone2.0, published in 2015, has the very unique feature of using dominant markers for selection instead of auxotrophy, so it can be applied to prototrophic strains (Stovicek et al ., ). A year later, the EasyCloneMulti collection was created, complementing the latter two.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…CRISPR‐Cas9 has been implemented in a variety of S. cerevisiae backgrounds, including strains used in the brewing and wine industries (Denby et al, ; Gorter de Vries, de Groot, van den Broek, & Daran, ; Vigentini, Gebbia, Belotti, Foschino, & Roth, ) and other industrially relevant yeast species such as fission yeasts (Jacobs, Ciccaglione, Tournier, & Zaratiegui, ), Kluyveromyces marxianus (Lee et al, ), Yarrowia lipolityca (Schwartz & Wheeldon, ; Shi, Huang, Kerkhoven, & Ji, ), Ogatae species (Juergens et al, ), and Pichia species (Weninger et al, ). Of particular relevance is the development of optimized CRISPR‐Cas9 methods to engineer diploid and polyploid industrial yeast strains (Stovicek, Borja, Forster, & Borodina, ; Zhang et al, ). The history and various applications of genome editing have been discussed in‐depth in the following reviews by Fraczek, Naseeb, and Delneri () and Stovicek, Holkenbrink, and Borodina ().…”