A Standardized Set of MoClo-Compatible Inducible Promoter Systems for Tunable Gene Expression in Yeast

Small-molecule
control of gene expression underlies the function
of numerous engineered gene circuits that are capable of environmental
sensing, computation, and memory. While many recently developed inducible
promoters have been tailor-made for bacteria or mammalian cells, relatively
few new systems have been built for Saccharomyces cerevisiae, limiting the scale of synthetic biology work that can be done in
yeast. To address this, we created the yeast Tunable Expression Systems
Toolkit (yTEST), which contains a set of five extensively characterized
inducible promoter systems regulated by the small-molecules doxycycline
(Dox), abscisic acid (ABA), danoprevir (DNV), 1-naphthaleneacetic
acid (NAA), and 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Assembly
was made to be compatible with the modular cloning yeast toolkit (MoClo-YTK)
to enhance the ease of use and provide a framework to benchmark and
standardize each system. Using this approach, we built multiple systems
with maximal expression levels greater than those of the strong constitutive TDH3 promoter. Furthermore, each of the five classes of
systems could be induced at least 60-fold after a 6 h induction and
the highest fold change observed was approximately 300. Thus, yTEST
provides a reliable, diverse, and customizable set of inducible promoters
to modulate gene expression in yeast for applications in synthetic
biology, metabolic engineering, and basic research.