Development of a genomic engineering tool in Saccharomyces cerevisiae

• Circumvent the plasmid-incurred problems by the genomic engineering tool.
• Metabolic engineering of yeast by insertion of heterologous genes and controlled expression of genomic genes.
• Cycling improvement of yeast by repeated use of the tool based on the Cre/loxP recombination system.
• Reduce the unintentional rearrangement event using the mutant loxP sites.

Genetic manipulation of Saccharomyces cerevisiae for production of valuable biochemicals has received considerable attentions nowadays. Plasmids are a useful tool to fulfill this goal. However, problems associated with plasmids are manifest by their instability, metabolic burden, and cell–cell variation in the copy number. This issue was addressed by developing a genetic toolbox to facilitate genomic insertion of heterologous genes and in situ fusion of an artificial promoter to genomic genes. The toolbox contains integration vectors and promoter-insertion vectors, which carry the LE*-KanMX-RE* cassette that comprises a KanMX module flanked by the palindromic mutant loxP sites (i.e., LE* and RE*). This cassette confers on integrant strains the antibiotic-resistant trait. The inserted KanMX can be later removed by the Cre-mediated recombination between LE* and RE*. As proof of principle, these vectors were utilized to integrate the XYL2 gene of Pichia stipitis into yeast genome and to place the genomic XKS1 gene under the control of the PGK1 promoter. It results in a recombinant yeast strain free of markers and plasmids. After recruitment of a heterologous XYL1 gene, the recombinant strain became capable of fermenting xylose. Our result indicates that this genetic toolbox is useful for genomic engineering of S. cerevisiae.