A protocol of rapid laboratory evolution by genome shuffling in Kluyveromyces marxianus

Genome shuffling is a process to combine advantage traits by the recombination of the entire genome and it has been successfully applied in the laboratory evolution of various industrial microorganisms. However, genome shuffling has not been described in Kluyveromyces marxianus (KM), a promising yeast host for the expression of heterologous proteins. In this protocol, genome shuffling in KM is performed by sexual reproduction and is combined with high-throughput screening to obtain high-yielding strains. Notably, the screening of diploid clones risen from one mating mixture is carried out to improve the effectiveness of evolution. Mating-sporulation-mating cycles are repeated to obtain KM strain with ideal traits. • The method combines genome shuffling with high-throughput to achieve strains displaying high yielding of heterologous proteins.• This method can be applied to the genome shuffling of other species when only a few starting strains are available for sexual reproduction.


Method details
Kluyveromyces marxianus (KM) has two mating types designated as MAT a and MAT α [1] . Upon harsh environments, haploid cells of opposite type mate to produce diploid and form spores. In KM haploid cells, the mating-type switch happens and the frequency of switching varies depending on the strains. In this protocol, genome shuffling in KM is performed by recursive mating, which can circumvent the low efficiency of protoplast fusion [2] . In order to improve the efficiency of genome recombination, parent strains have different genetic backgrounds.The feruloyl esterase Est1E is used as a marker protein to measure the capacity of KM to express a heterologous protein. The expression level of Est1E is detected by a color-based assay [3] . The HML or HMR locus in the original parental strain is removed to produce stable haploid strain. Haploid cells carry auxotrophic markers to facilitate the selection of diploid. Considering the genetic diversity in diploid cells due to mitotic recombination, we perform a high-throughput screening of diploid clones from one mating mixture to improve the effectiveness of the selection. Diploid cells sporulated and spores are screened by the same method. The mating types and auxotrophic markers of the spores displaying improved features are identified to find pairs for the next round of mating. Mating-sporulation-mating cycle can be repeated until KM strains with ideal phenotypes are obtained ( Fig. 1 ).

Construction of parental strains
The parental strain used in this study is originated from FIM-1 through UV-60 Co-γ irradiation method. The two parental stains all have hundreds of specific mutations that not isogenic. To obtain a stable haploid parental strain, the HML locus in a MAT a strain and HMR locus in a MAT α strain is deleted. This step is not necessary for some KM strains that seldom switch mating type. To provide auxotrophic markers for the selection of diploid, HIS3 in the MAT a strain and TRP1 in the MAT α strain is deleted. Both parental strains contain a deletion of URA3 to be compatible for a URA3 plasmid expressing the heterologous protein. The deletion is performed by the recombination with the aid of a CRISPR plasmid as described before [3] . Inoculate the cells by patching to produce enough cells for the next step. 2. Similar to K. lactis , the mating of KM happens in the medium containing limited nutrients [4] .
Scrape off two matchheads of MAT a and MAT α cells from YPD plates and mix them thoroughly on the ME medium by toothpicks. Spread out the mixture to make a thin layer above the medium. Culture the ME medium plates at 30 °C for 2 days.

Scrape off four matchheads of cells from the ME medium and transfer them to a 1.5 ml
Eppendorf tube by toothpicks. Serial dilute the cells by sterile water and spread them onto SD medium plate to select for diploid cells. Usually, 100 μl 10 −5 dilution produces a few hundred well-separated clones on the SD medium. 4. Inoculate clones in SD medium and grow at 30 °C for 1 day.

High-throughput screening
The purpose of this step is to select the diploid or haploid clones with ideal traits. Highthroughput screening is performed in 96-well microplates. Selection of clones yielding high-level of Est1E is described in this protocol. The protocol can be modified to screen clones expressing other heterologous proteins or clones with other ideal traits.

Identification of spores
Verify the mating type of haploids by PCR using Taq DNA polymerase. Scrape off a matchhead of cells into 30 μl 0.2% SDS and mixed them thoroughly. Boil the sample for 5 min in boiling water. Centrifuge the sample at 13.2 k rpm for 1 min and the supernatant is used as a template for PCR. MAT a locus produces a band of 1062 bp by the primer pair of YY270F (5 TGCAACCAAC CAATCCCTTCCAAATTC 3 ) and YY271F (5 TCTTCCTTGA ACCCGAAGCAAAAGATC 3 ). MAT α locus produces a band of 1515 bp by using primer pair of YY270F and YY272F (5 AACTTCAATC CCCGACCCACCGCAGTC 3 ). Identify the auxotrophic markers by inoculating haploids onto SC-Ura-Hismedium and SC-Ura-Trp-medium. Incubate haploids at 30 °C for 24 h.

Method validation
This method was successfully applied to improve the yielding of a heterologous protein in KM. After two rounds of mating and screening, a diploid strain called D 2-13 was obtained which displayed a 5-fold increase of secretory activity of Est1E compared to the parental strains [5] . Although only two parental strains were selected for mating, the method included a screening of diploid clones from one mating mixture to improve the effectiveness of evolution. Therefore, this method is useful for the genome shuffling of other species when only a few starting strains are available for sexual reproduction.