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

  • 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 rulesOrthogonal 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.show moreshow less

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Metadaten
Author:Fabian MachensORCiDGND, Salma BalazadehORCiDGND, Bernd Müller-RöberORCiDGND, Katrin Messerschmidt
DOI:https://doi.org/10.3389/fbioe.2017.00063
ISSN:2296-4185
Parent Title (English):Frontiers in Bioengineering and Biotechnology
Publisher:Frontiers
Place of publication:Lausanne
Document Type:Article
Language:English
Date of first Publication:2017/10/19
Year of Completion:2017
Publishing Institution:Universität Potsdam
Release Date:2017/11/16
Tag:JUB1; chimeric transcription factors; dead Cas9; gene expression; synthetic biology; synthetic circuits; transcriptional regulation
Volume:5
First Page:1
Last Page:11
Funder:Universität Potsdam, Publikationsfonds
Grant Number:PA 2017_57
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Peer Review:Referiert
Grantor:Publikationsfonds der Universität Potsdam
Publication Way:Open Access
Licence (German):License LogoCreative Commons - Namensnennung, 4.0 International
Notes extern:Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe ; 393