Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

Abstract

Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs) and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs) with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

The Genetic Makeup and Expression of the Glycolytic and Fermentative Pathways Are Highly Conserved Within the Saccharomyces Genus

“…Since S. cerevisiae’s extremely efficient homologous recombination renders strains with repeated usage of promoter sequences genetically unstable ( Manivasakam et al, 1995 ), this shortage of promoters presents a hurdle for extensive strain construction programs. While a lot of effort is invested in the design of synthetic promoters and transcription amplifiers ( Redden and Alper, 2015 ; Rantasalo et al, 2016 ; Machens et al, 2017 ; Naseri et al, 2017 ), using slightly distant but functional orthologous promoters presents an attractive alternative ( Naesby et al, 2009 ; Harvey et al, 2018 ). Usage of especially the S. eubayanus promoters, which are slightly more distant from S. cerevisiae than the S. kudriavzevii promoters, would reduce the length of the sequences being 100% identical to the native S. cerevisiae promoters.…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…The native A. oryzae promoter of the oxidoreductase gene, kojA, which is involved in kojic acid biosynthesis successfully induced the expression of the polyketide synthase gene (wA) and production of the respective polyketide, YWA1 [36]. Whilst mainly native promoters are used for heterologous expression in filamentous fungi [37], in S. cerevisiae, universal expression systems for fungal genes comprising a set of synthetic promoters and transcription factors have been recently developed to synthesize a wide range of fungal natural products [38][39][40]. However, because A. oryzae possesses a variety of proteins and secretion systems for proteins and low-molecular-weight compounds that differ from those in S. cerevisiae [41][42][43], finding additional promoters that would be functional in A. oryzae is important for the use of this species as a heterologous expression host in addition to S. cerevisiae.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…The modularity of the functional parts allows versatile and programmable regulatory systems to be designed with desired functions and target specificities, and the modular parts can even be sourced from organisms from different taxonomic kingdoms. Examples of modular tools that can be used for synthetic transcription‐factor construction include bacterial repressor proteins (e.g., LexA; Ajo‐Franklin et al, ), zinc‐finger domains (McIsaac, Gibney, Chandran, Benjamin, & Botstein, ), transcription activator‐like effectors (Machens, Balazadeh, Mueller‐Roeber, & Messerschmidt, ), and the Cas9‐null mutant (dCas9) protein (Deaner, Mejia, & Alper, ; Machens et al, ).…”