1928 - Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain

Abstract

Kluyveromyces marxianus, a probiotic yeast, is important in industrial applications because it has a broad substrate spectrum, a rapid growth rate and high thermotolerance. To date, however, there has been little effort in its genetic engineering by the CRISPR/Cas9 system. Therefore, we aimed at establishing the CRISPR/Cas9 system in K. marxianus and creating stable haploid strains, which will make genome engineering simpler. First, we predicted the genome-wide target sites of CRISPR/Cas9 that have been conserved among the eight sequenced genomes of K. marxianus strains. Second, we established the CRISPR/Cas9 system in the K. marxianus 4G5 strain, which was selected for its high thermotolerance, rapid growth, a pH range of pH3-9, utilization of xylose, cellobiose and glycerol, and toxin tolerance, and we knocked out its MATα3 to prevent mating-type switching. Finally, we used K. marxianus MATα3 knockout diploid strains to obtain stable haploid strains with a growth rate comparable to that of the diploid 4G5 strain. In summary, we present the workflow from identifying conserved CRISPR/Cas9 targets in the genome to knock out the MATα3 genes in K. marxianus to obtain a stable haploid strain, which can facilitate genome engineering applications. Kluyveromyces marxianus is a yeast that can be isolated from dairy environments 1,2. It is a probiotic yeast 3 that is included in the list of qualified presumption of safety (QPS) biological agents by European Food Safety Authority (EFSA) 4. It has many other advantages for a cell factory host, including a broad substrate spectrum 5 , a rapid growth rate 5,6 and thermotolerance 6. We have isolated a K. marxianus strain, called 4G5, from kifer that can grow at 48 °C, has a broad pH range (pH3-pH9), has a doubling time of 1.22 ± 0.4 hour at 30 °C in YPG (2% galactose), can utilize xylose, cellobiose, and glycerol, and can tolerate 2% isobutanol, 1.5% 1-butanol, and 4% ethanol (Supplementary Figs S1-S4 and Supplementary Table S1). These traits make it a good potential cell factory host. The main aim of this study is to obtain stable haploid strains with a good fitness under common experimental conditions because a haploid strain can be genetically engineered more readily than a diploid strain. In response to harsh environmental conditions, Kluyveromyces haploids tend to become diploids and produce spores. The mating type of a cell is determined by the alleles at the MAT locus 2,7. MATa and MATα haploids can switch their mating type 2,7. The MATa locus includes the a1 and a2 genes, while the MATα locus includes the α1, α2 and α3 genes and α3 expresses a transposase that can fracture a MAT locus, switching MATα-type to MATa-type. The