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.

A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast

“…1), each individual gene encoding one enzyme of the biosynthetic pathway or biosynthesis gene clusters needs to be expressed from the individual promoter and terminator 27 . Recently several advanced tools such as inducible synthetic transcription factors (synTFs) and promoters 28,29 , terminators 30 , and genome editing tools 31 have been developed to facilitate regulation of heterologous protein expression in S. cerevisiae. Fortunately, these tools have been found to be functional in tuning heterologous protein expression levels in P. pastoris, for which a limited number of tools and methods are available for metabolic engineering.…”

Design of Four Small-Molecule-Inducible Systems in the Yeast Chromosome, Applied to Optimize Terpene Biosynthesis

“…56,61,63,70 Khalil and co-workers demonstrated that four hormone sensors could be incorporated into the yeast chromosome and applied to optimize a biosynthetic pathway to produce violicin. 65 S8. (b) Constructs to measure sensor outputs.…”

“…Sensors have been developed for S. cerevisiae that respond to anhydrotetracycline (aTc), xylose (Xyl), isopropyl-β- d -thiogalactoside (IPTG), vanillic acid (Van), 2-4-diacetylphloroglucinol (DAPG), salicylate, adipic acid, naringenin (Nar), cumate (CumA), 3-oxo-hexanoyl homoserine lactone (OC6), camphor, progesterone, estradiol, aldosterone, testosterone, 1,2-bis­(4-hydroxyphenyl)­ethane-1,2-dione (DHB), and dexamethasone. ,,− However, to work together in a single cell, the sensors must be orthogonal; in other words, the small molecules cannot bind to off-target regulators and the regulators must bind to unique DNA sequences. ,− Several groups have combined three sensors in a single strain. ,,, Khalil and co-workers demonstrated that four hormone sensors could be incorporated into the yeast chromosome and applied to optimize a biosynthetic pathway to produce violicin …”

A Layered Genetic Design Enables the Yeast Galactose Regulon to Respond to Cyanamide

“…Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to the GAL promoters to activate transcription . By replacing the Gal4 binding domain with synthetic zinc fingers, a suite of orthogonal systems was developed for multidimensional control of gene expression in yeast . Given that light is inexpensive and tunable, an optogenetic tool has emerged as an alternative approach to control gene expression due to its high tunability and reversibility.…”

“…18 By replacing the Gal4 binding domain with synthetic zinc fingers, a suite of orthogonal systems was developed for multidimensional control of gene expression in yeast. 19 Given that light is inexpensive and tunable, an optogenetic tool has emerged as an alternative approach to control gene expression due to its high tunability and reversibility. Zhao et al 20 created a rapid optogenetic circuit that is repressed in blue light but strongly activated in the dark by combining OptoINVRT with a modified GAL1 promoter.…”