Yeast transformation efficiency is enhanced by TORC1‐ and eisosome‐dependent signaling

Abstract

Transformation of baker's yeast ( Saccharomyces cerevisiae ) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. The addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 ( TORC 1) activated by amino acids enhances transformation via ubiquitin‐mediated endocytosis. We created mutants of the TORC 1 pathway, alpha‐arrestins, and eisosome‐related genes. Our results demonstrate that the TORC 1‐Npr1‐Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in up to a 200‐fold increase in transformation efficiency, or greater. Additionally, we suggest DNA may be taken up by domains at the membrane compartment of Can1 ( MCC ) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimize gene therapy.

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

“…However, this is of course merely speculation, and we have intentionally chosen not to attempt to decipher the exact molecular mechanism behind this effect in this work. Moreover, our results clearly support previous findings that Lithium acetate, DTT, ssDNA and elevated temperatures, are all factors of paramount importance for a successful yeast DNA transformation (2, 3, 5, 12). Although, we observe that these four parameters can, under some conditions, give rise to novel and unique transformation effects when applied in a nonconventional way, i.e., by using heat-shock and electroporation simultaneously.…”

“…Temperature (heat-shock) is also an important factor that stimulates extracellular DNA uptake during chemical transformation of yeast (2). However, unlike for bacteria, yeast chemical DNA transformation is currently understood to be mediated by endocytosis pathways (3), which is strongly enhanced by chemical agents, such as Lithium acetate, crowding agents (PEG) and nonspecific single-strand carrier DNA (ssDNA) (2, 3). In the case of bacterial and yeast DNA transformation mediated by electroporation, our current understanding suggests that a passive DNA diffusion through transiently formed pores in the membrane is the primary route of DNA uptake during electroporation (1, 4).…”

A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

“…While intensive efforts have been made to turn E. coli into a universal molecular biology tool, the power of S. cerevisiae as DNA assembly platform has so far been largely untapped. Despite the pivotal role played by S. cerevisiae in the last decade in the assembly of genomes ( 14 , 55–58 ) there is so far relatively little effort invested in turning this yeast into a universal and powerful DNA assembly platform ( 59 , 60 ), a situation that we expect will change in the future.…”

Multiplex Genome Editing in Yeast by CRISPR/Cas9 – A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways

“…The more targets to be edited, the more donor DNA and/or gRNA cassettes will be required. Transformation techniques, such as electroporation or the addition of amino acids, can be incorporated to improve the transformation efficiency ( Benatuil et al, 2010 ; Yu et al, 2019 ). Finally, the development of CRISPR/Cas9 multiplex genome integration in yeast can be improved by combining multiple methods.…”