Development of a Transformation Method for Metschnikowia borealis and other CUG-Serine Yeasts

Abstract

Yeasts belonging to the Metschnikowia genus are particularly interesting for the unusual formation of only two needle-shaped ascospores during their mating cycle. Presently, the meiotic process that can lead to only two spores from a diploid zygote is poorly understood. The expression of fluorescent nuclear proteins should allow the meiotic process to be visualized in vivo; however, no large-spored species of Metschnikowia has ever been transformed. Accordingly, we aimed to develop a transformation method for Metschnikowiaborealis, a particularly large-spored species of Metschnikowia, with the goal of enabling the genetic manipulations required to study biological processes in detail. Genetic analyses confirmed that M. borealis, and many other Metschnikowia species, are CUG-Ser yeasts. Codon-optimized selectable markers lacking CUG codons were used to successfully transform M. borealis by electroporation and lithium acetate, and transformants appeared to be the result of random integration. Mating experiments confirmed that transformed-strains were capable of generating large asci and undergoing recombination. Finally, random integration was used to transform an additional 21 yeast strains, and all attempts successfully generated transformants. The results provide a simple method to transform many yeasts from an array of different clades and can be used to study or develop many species for various applications.

Keywords: CUG-Ser; DNA delivery; Metschnikowia; genetic tools; genome engineering; synthetic biology; yeasts.

Figure 1

Bioinformatic analyses of the genetic code of M. borealis. (A) Sequence alignment of URA3 (encodes Orotidine 5'-phosphate decarboxylase). The coding sequence of URA3 from M. borealis was submitted as a query sequence to BLASTx, using the standard code for the predicted protein sequence. The protein sequences of the top 30 hits were aligned in MUSCLE with the M. borealis URA3 gene, translated in both the standard code and yeast alternative code using the ExPASy translate tool. A CUG codon aligned at a highly conserved serine or threonine amino acid position, producing a leucine when URA3 was translated using the standard code (black square). (B) tRNA_CAG^Ser sequences aligned for many species of Metschnikowia and various CUG-Ser yeasts. Sequences were aligned in MEGA6 and annotated as described previously [18].

Figure 2

Electroporation of M. borealis with CaNAT1, MbKanMX, and MbShBle in standard and yeast alternative nuclear code. Electro-competent cells of M. borealis (MATa and MATα) were transformed with 1 µg of PCR-amplified CaNAT1, MbShBle, or MbKanMx, in standard or alternative code, flanked by 60 base-pair sequences of the M. borealis ADH1 promoter and terminator, using the electroporation protocol outlined in this study. After recovery, 20 µg of each transformation was plated on YPAD with the appropriate antibiotic and incubated at 30 °C for two days. NTC = nourseothricin; Zeo = zeocin; G418 = geneticin.

Figure 3

Mating experiments with marker-transformed M. borealis. (A) Photograph of a large mating ascospore. MbShBle-transformed MATa and CaNAT1-transformed MATα were mated for two days on ½-strength glucose broth lacking histidine and uracil (Teknova), collected in YPAD, and photographed under a light microscope. (B) Genotyping results of marker-transformed diploids. Recombinant colonies harbouring both selectable markers were purified by spot dilution on YPAD with 200 mg L⁻¹ zeocin and 75 mg L⁻¹ nourseothricin. Recombinant colonies were genotyped by Qiagen multiplex with primers that amplify the CaNAT1 and MbShBle genes, alongside marker-transformed haploid strains and untransformed controls. MbShBle product size (C) Spot-dilutions of mated-colonies alongside transformed haploid strains and untransformed controls on YPAD, YPAD with 200 mg L⁻¹ zeocin, YPAD with 75 mg L^-1 nourseothricin, and YPAD with 200 mg L⁻¹ zeocin and 75 mg L⁻¹ nourseothricin. Plates were incubated at 30 °C for two days. NTC = nourseothricin; Zeo = zeocin; G418 = geneticin.

Figure 4

A phylogeny of the Metschnikowiaceae, modified from Lachance (2016) [5], showing the taxonomic range of successful transformations in the family. Representative strains were successfully transformed with only the alternative-coded CaNAT1 construct (green) or with both the alternative-coded and the standard-coded CaNAT1 (blue) construct. Species in grey were not examined.