mazF-mediated deletion system for genome engineering in budding yeast

Saccharomyces cerevisiae

Background: In the field of functional genomics and metabolic engineering in the postgenome era, the concept of minimum genome factories (MGFs) has been proposed, which can be defined as recombinant strains whose metabolism has been streamlined to the optimal minimal subset in order to maximize product formation for targeted applications (Ara et al., 2007; Giga-Hama et al., 2007; Mizoguchi et al.,

2007). Evolutionary biology imparts us that a certain inherent plasticity exists in the genomes of living organisms and has constituted a major evolution power (Trevors,
1997). The plasticity of genomes gives us hints that it is probable to streamline physiological pathways by means of genome rearrangements, as cells harbor a number of genes that only marginally contribute to cellular fitness and various dispensable activities that are not essential for survival, particularly when cells are grown in rich media. The unicellular eukaryote budding yeast Saccharomyces

cerevisiae has long been exploited for industrial and biotechnological applications and is extensively chosen as the starting platform for value-added metabolites biosynthesis and biofuel production in the wake of emerging synthetic biology and systems

biology (Ostergaard et al., 2000; Nielsen et al., 2013). Construction of a set of chassis cells with various genetic alterations provides the opportunity to obtain specific target strain(s) with desired performance through phenotype comparisons and genetic regulation analysis and thus represents the fundamental work for the generation of featured MGFs. A chassis member generally contains at least one chromosomal modification, including single gene deletion or removal of a large chromosomal region, and one unmarked chassis without undesirable foreign sequence is preferred. To this end, the employed genetic method should possess two other peculiarities: marker rescue and scarless modification, besides the ability to directly accomplish desired deletion.
Results: In the present report, we proposed a new scarless modification strategy, which evolved from a recently established mazF-based gene deletion protocol, for large-scale and scarless genome rearrangements in yeast S. cerevisiae. The main experimental process of the presented system is substantially similar to the existing gene deletion methods involving double-crossover integration and single-crossover pop-out except for the way to construct deletion cassette and to accomplish the deletion of large chromosomal targets (Fig. 1). The deletion cassette is composed of five functional parts: a URA3 marker for integrant selection, a galactose-induced mazF cassette for counter-selection and three genome segments. Each part for the deletion cassettes was individually amplified by PCR and co-transformed into yeast cells, and then the complete deletion cassettes were assembled in vivo through
homologous recombination among the 50 bp sequence overlaps introduced by primers. Homologous recombination among two flanking homology regions and their counterparts in the genome resulted in the targeted integration of deletion cassettes
and created new direct repeats of the third interior genome sequence. Integrants were selected on SC-Ura plates and successful integrations were verified by direct colony
PCR. The resultant cells were subjected to counter-selection step to accomplish the excision of genomic sequences and recovery of genetic markers resulting from the single cross-over between newly generated direct repeats. This method was demonstrated to be highly efficient for the direct deletion of a designed internal or terminal chromosomal region. Enhanced targeting efficiency of the deletion cassette constructs was achieved through extending the length of flanking homology arms. Furthermore, the developed mazF-based counter-selection system outperformed traditional URA3/5-FOA method that a greater number of resultant colonies appeared with higher percentage of desirable deletants. Despite the loss of 12 and 10 predicted ORF(s), respectively, upon the deletion of the 26.5-kb and 28.9-kb regions, the strains NK11 and NK12 still exhibited normal growth under standard laboratory conditions; and similar growth profile was also observed for mutant NK13 carrying both these
two deletions compared to that of the parental strain.
Conclusion: This mazF-based technique can be used to generate scarless and sequential chromosomal modification mutants of S. cerevisiae efficiently, using deletion cassettes assembled in vivo by means of the recombination machinery of
yeast cells. Based on the precision of its deletion, this method may provide a new way to construct genetically modified S. cerevisiae strains with large-scale genome
deletion more efficiently and advance the regulatory network study of this organism and further genome streamlining.

Keywords: Saccharomyces cerevisiae; mazF; large-scale deletion; counter-selection;

genome engineering

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Fig. 1. Schematic representation of the mazF mediated scarless deletion of Saccharomyces cerevisiae genomic region(s). HA1-3 are short segments of S. cerevisiae genome; URA3 and mazF markers are from plasmids pUG72 and pLC1, respectively. These segments were amplified by PCR, co-transformed and formed functional deletion cassette(s) through in vivo homologous recombination. The deletion cassette(s) was integrated by double crossover and the deletion of target region along with markers by single crossover.