Approaches to Superefficient Yeast Transformations: DNA Phase Separation, Cell Growth State and Systematic Experimental Design.

Yeasts are model organisms and protein production hosts of choice, in part due to their ease of genetic manipulation. Yeast transformation with DNA on plasmids or integrated into the genome has been developed since the 1970s and is a routine method in many laboratories. However, transformation frequencies are surprisingly low, with common methods yielding around 1% of transformed cells in budding yeast Saccharomyces cerevisiae. In alternative species, there is lower transformation frequency and unreasonably large quantities of input DNA may be required. Surprisingly, the mechanisms by which yeast cells take up DNA are not known. Here we argue that three approaches could deliver a step-change in yeast transformation frequency and efficiency. First, investigating the mechanisms of DNA uptake. The requirement for salts and extraneous polymers-carrier DNA and PEG-in standard protocols suggests that phase separation of DNA may enhance DNA uptake into cells or subsequent nuclear delivery. Second, accounting for the physiological or growth state of the yeast including cell cycle phase and nutrient or stress conditions. Third, systematically determining the factors that affect transformation efficiency using statistical Design of Experiments. These approaches could allow superefficient transformation of S. cerevisiae, and accelerate genetic manipulation of non-model yeasts.