Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome

While Kluyveromyces marxianus is a promising yeast strain for biotechnological applications, genetic engineering of this strain is still challenging, especially when multiple genes are to be transformed. Sequential gene integration, which takes advantage of repetitive insertion/excision of the URA3 gene as a marker, has been the best option until now, because the URA3-deletion mutant is the only precondition for this method. However, we found that the introduced gene is co-excised during the URA3 excision step for next gene introduction, resulting in a very low cumulative probability (<1.57×10⁻⁶ % for 4 genes) of integrating all genes of interest. To overcome this extremely low probability, and to reduce labor and time, all 4 genes were simultaneously transformed. Surprisingly, the infamously high 'non-homologous end joining' activity of K. marxianus enabled simultaneous integration of all 4 genes in a single step, with a probability of 7.9%. Various K. marxianus strains could also be similarly transformed. Our finding not only reduces the labor and time required for such procedures, but also removes a number of preconditions, such as pre-made vectors, selection markers and knockout mutants, which are needed to introduce many genes into K. marxianus.