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

Zachary B Gordon,Marc-André Lachance,Dong Kyung Lee,Christopher Leichthammer,Bogumil J Karas,Jasmine A Therrien,Carolyn Lauzon,Rebecca S Meaney,Maximillian P.M Soltysiak,Matthew Adams,Preetam Janakirama

doi:10.3390/genes10020078

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 Metschnikowia borealis, 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.

Highlights

Yeasts have proven to be tremendously useful in the study of life’s fundamental processes
Metschnikowia borealis strains UWOPS 96-101.1 (MATα) and SUB 99-207.1 (MATa), and other strains that were used in transformation experiments were obtained from the yeast collection of the Department of Biology, University of Western Ontario, where they are kept frozen in liquid nitrogen
Before designing genetic cassettes for transformation, it was necessary to identify antibiotics that inhibit the growth of M. borealis to enable us to choose appropriate antibiotic resistance markers

Summary

Introduction

Yeasts have proven to be tremendously useful in the study of life’s fundamental processes. While the discovery of new species provides us with new opportunities, genetic tools previously developed for Saccharomyces cerevisiae and other model organisms must be re-optimized for use in newly discovered species [3,4]. Altered membrane composition, modified gene expression, and alternative codon usage can prevent the seamless application of established transformation methods onto new species. Such transformation methods are crucial in the development of targeted knockouts, changes in gene expression, and fusion proteins that allow the study of biological processes in detail.

Objectives

Methods

Results

Conclusion