A novel and effective Cre/lox-based genetic tool for repeated, targeted and markerless gene integration

Abstract

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise all of the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed the genetic tool was effective for integration of other genes, loci, and strains. Thus, this study significantly promotes the application of Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.Graphical abstractThe novel genetic tool comprised three plasmids, namely, Cre-Y1, Cre-Y2, and Cre-Y3. Cre-Y1 was introduced into the Upleu locus of Po1f, and then recombination between two lox71 sites could remove unnecessary plasmid elements and produce an initial strain harboring a lox71 site. Subsequently, Cre-Y2 was integrated onto the leu2 locus, forming a transitional strain with three lox sites (lox71, lox66, and rclox66). Recombination reactions among these three lox sites could excise unnecessary DNA fragments and retain a lox72 site, an rclox66 site, and a target gene expression cassette in the genome. Similarly, integration of Cre-Y3 produced strains harboring two target gene expression cassettes. Consequently, repeated, targeted, and markerless gene integration could be efficiently realized following iterative integration of Cre-Y2 and Cre-Y3.HighlightsA powerful genetic tool was developed for targeted and markerless gene integrationThe genetic tool was effective for various genes, loci, and strainsOnly a single selection marker was adequate for repeated gene integrationAll of the useless and redundant sequences would be excisedAll integration efficiencies could remain above 90%

Systems‐level approaches for understanding and engineering of the oleaginous cell factory Yarrowia lipolytica

“…For instance, a Cre/lox‐site‐specific recombination system using only a single selection marker has been developed for targeted, repeated, and marker‐less gene integration in Y. lipolytica with above 90% efficiency. Unlike similar previous methods based on Cre/lox‐site‐specific recombination, their genetic tool was also proved effective for various genes, loci, and strains which suggests a promising application of Cre/lox system and strengthens its capabilities for genome modification in Y. lipolytica (Zhou et al, 2021). Moreover, as a study to overcome the challenge with building complex pathways and multiple gene integration in Y. lipolytica , Z. Guo et al (2020) have designed a completely synthetic Y. lipolytica ‐specific artificial chromosome (ylAC) and successfully used it to introduce a complete cellobiose and xylose co‐utilization pathway introduced to the yeast in a single step within less than one week of wet lab experimentation.…”

Systems-level approaches for understanding and engineering of the oleaginous cell factory Yarrowia lipolytica

“…For instance, a Cre/lox-site-specific recombination system using only a single selection marker has been developed for targeted, repeated, and marker-less gene integration in Y. lipolytica with above 90% efficiency. Unlike similar previous methods based on Cre/lox-site-specific recombination, their genetic tool was also proved effective for various genes, loci, and strains which suggests a promising application of Cre/lox system and strengthens its capabilities for genome modification in Y. lipolytica (Zhou et al, 2021). Moreover, as a study to overcome the challenge with building complex pathways and multiple gene integration in Y. lipolytica, Z.…”