A Toolkit for Precise, Multigene Control in Saccharomyces cerevisiae

Abstract

Systems that allow researchers to precisely control the expression of genes are fundamental to biological research, biotechnology, and synthetic biology. However, few inducible gene expression systems exist that can enable simultaneous multigene control under common nutritionally favorable conditions in the important model organism and chassis Saccharomyces cerevisiae. Here we repurposed ligand binding domains from mammalian type I nuclear receptors to establish a family of up to five orthogonal synthetic gene expression systems in yeast. Our systems enable tight, independent, multigene control through addition of inert hormones and are capable of driving robust and rapid gene expression outputs, in some cases achieving up to 600-fold induction. As a proof of principle, we placed expression of four enzymes from the violacein biosynthetic pathway under independent expression control to selectively route pathway flux by addition of specific inducer combinations. Our results establish a modular, versatile, and potentially expandable toolkit for multidimensional control of gene expression in yeast that can be used to construct and control naturally occurring and synthetic gene networks.

Keywords: MoClo Toolkit; gene expression control; inducible promoters; nuclear receptors; transcription factors; yeast.

Figure 1

Design of a toolkit for multigene inducible gene expression control in yeast based on hormone-responsive type I nuclear receptors (NRs). (A) Schematic of the mechanism of action of NR-based gene regulation. Upon ligand binding, the transcriptional regulator dissociates from a chaperone, enters the nucleus, and initiates transcription of a target gene downstream of response elements. (B) Features of the NR-based synthetic transcription factor (synTF) toolkit: modularity, rapid induction in a glucose-based medium, and mutual orthogonality for simultaneous multigene control. (C) Modular synTF design enables mix-and-match construction to obtain desired properties. The synTFs are composed of interchangeable artificial DNA binding domains (DBDs), type I NR ligand binding domains (LBDs), and transactivation domains (TADs).

Figure 2

Development and characterization of a collection of hormone-inducible synthetic gene expression systems. (A) Heat maps of (top) basal and (bottom) maximum reporter expression levels for synTFs featuring different ligand binding and transactivation domains. Values are normalized to the maximum recorded fluorescence. Data were obtained by flow cytometry following induction for 16 h (see Methods) (B) Fold-change reporter induction for the five top-performing hormone-inducible synTF systems. Bars represent mean ± SEM for N = 4 biological replicates. (C) Dose–response curves and (insets) flow cytometry histograms for the five top-performing inducible systems. Data were obtained following induction for 16 h (see Methods). Points represent mean ± SEM for N = 4 biological replicates.

Figure 3

Hormone-inducible synthetic gene expression systems exhibit minimal cross-reactivity and mutual orthogonality. (A) Reporter expression for strains harboring each of the inducible systems (ER, MR, AR, and DHBR) following induction for 16 h with hormone cocktails featuring three out of the four inducers (N = 4 biological replicates per condition). The heat map represents reporter expression averages collected from three independent experiments, and the reporter outputs for each given condition were not significantly different between experiments (p > 0.05). (B) Reporter expression for a single strain quadruply integrated with all four inducible systems (ER, MR, AR, and DHBR) following induction for 16 h with the indicated hormone inducers (N = 4 biological replicates per condition). The heat map represents reporter expression averages collected from three independent experiments, and the reporter outputs for each given condition were not significantly different between experiments (p > 0.05).

Figure 4

Engineering hormone-inducible, multigene control over the violacein biosynthetic pathway to selectively route metabolic flux. (A) Schematic of the violacein pathway, wherein four enzymes (VioA, VioE, VioC, VioD) are placed under orthogonal, hormone-inducible gene expression control in a single strain. (B) HPLC chromatograms for strains in which designated enzyme combinations are either expressed constitutively (left) or induced with the corresponding hormone inducer cocktails (right), leading to the selective production of compounds in the violacein pathway.