title: Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species.
abstract: Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5' untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool.
authors.affiliation: REQUIMTE/LAQV, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , 2829-516 Caparica, Portugal.
authors.forename: Rui M C
authors.initials: RM
authors.lastname: Portela
authors.affiliation: Institute for Molecular Biotechnology, NAWI Graz University of Technology , Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Thomas
authors.identifier: 0000-0002-3892-1740
authors.initials: T
authors.lastname: Vogl
authors.affiliation: Institute for Molecular Biotechnology, NAWI Graz University of Technology , Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Kniely
authors.affiliation: Institute for Molecular Biotechnology, NAWI Graz University of Technology , Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Jasmin E
authors.initials: JE
authors.lastname: Fischer
authors.affiliation: REQUIMTE/LAQV, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , 2829-516 Caparica, Portugal.
authors.forename: Rui
authors.initials: R
authors.lastname: Oliveira
authors.affiliation: Institute for Molecular Biotechnology, NAWI Graz University of Technology , Petersgasse 14/2, 8010 Graz, Austria.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D042931:Aldehyde Oxidase / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods
chemical_list: D020121:5' Untranslated Regions; D005656:Fungal Proteins; D042931:Aldehyde Oxidase
keywords: computational design; core promoter; promoter library; synthetic biology; transcriptional fine-tuning; yeast