Establishment of a Cre-loxP System Based on a Leaky LAC4 Promoter and an Unstable panARS Element in Kluyveromyces marxianus.

Abstract

The Cre-loxP system produces structural variations, such as deletion, duplication, inversion and translocation, at specific loci and induces chromosomal rearrangements in the genome. To achieve chromosomal rearrangements in Kluyveromyces marxianus, the positions and sequences of centromeres were identified in this species for the first time. Next, a Cre-loxP system was established in K. marxianus. In this system, the Cre recombinase was expressed from a leaky LAC4 promoter in a plasmid to alleviate the cytotoxicity of Cre, and the unstable plasmid contained a panARS element to facilitate the clearance of the plasmid from the cells. By using LAC4 as a reporter gene, the recombination frequencies between loxP sites or loxPsym sites were 99% and 73%, respectively. A K. marxianus strain containing 16 loxPsym sites in the genome was constructed. The recombination frequency of large-scale chromosomal rearrangements between 16 loxPsym sites was up to 38.9%. Our study provides valuable information and tools for studying chromosomal structures and functions in K. marxianus.