Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae

Abstract

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production

“…A recent and innovative study used engineered TFs with yeast, virus, and plant activation domains that were fused to a nuclear localization signal and a full-length TF or its DNA binding domain. In this case, the authors noticed that transcription of a yeast-enhanced green fluorescent protein reporter gene under the control of a yeast promoter was differentially modulated by each domain (Naseri et al, 2017 ). The authors also discussed the influence of the number of binding sites embedded in such promoters and noticed that some artificial TFs derived from Arabidopsis thaliana could also modulate the transcription of the fluorescence pattern in the presence/absence of an inducer (Naseri et al, 2017 ).…”

“…In this case, the authors noticed that transcription of a yeast-enhanced green fluorescent protein reporter gene under the control of a yeast promoter was differentially modulated by each domain (Naseri et al, 2017 ). The authors also discussed the influence of the number of binding sites embedded in such promoters and noticed that some artificial TFs derived from Arabidopsis thaliana could also modulate the transcription of the fluorescence pattern in the presence/absence of an inducer (Naseri et al, 2017 ). This approach expands the possibilities for the future application of synthetic biology in other eukaryotic cells.…”

Synthetic biology strategies toward heterologous phytochemical production

“…In addition, orthogonal regulation of gene expression has been attempted in S. cerevisiae through introducing NAC transcription factors from A. thaliana, activation domains from yeast or plant, and cognate binding sites into the yeast transcription system, resulting in even higher transcription activation output than the commonly utilized constitutive promoter TDH3. 156…”

Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors

“…In metabolic engineering and synthetic biology, the expression of multiple genes commonly needs to be regulated either simultaneously, consecutively or even in an inverse mode. Acquiring control over the natural regulation of promoters, the design of synthetic promoters and artificial transcription factors (TFs) have proven imperative for optimizing protein production, metabolic engineering, and synthetic biology applications (Blazeck, Garg, Reed, & Alper, 2012;Da Silva & Srikrishnan, 2012;Gardner, Cantor, & Collins, 2000;Hartner et al, 2008;Khalil et al, 2012;Leavitt, Tong, Tong, Pattie, & Alper, 2016;Naseri et al, 2017;Nevozhay, Adams, Murphy, Josic, & Balázsi, 2009;Nevozhay, Zal, & Balázsi, 2013;Ottoz, Rudolf, & Stelling, 2014;Rantasalo et al, 2016;Ryo et al, 2017;Weinhandl, Winkler, Glieder, & Camattari, 2014).…”