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.
Highlights
Metabolic pathways and genetic circuits are commonly introduced into microbes such as Saccharomyces cerevisiae or Escherichia coli to produce chemicals or to implement novel functions.[1,2] Such experiments typically require the fine-tuning of gene expression to balance and optimize protein levels of metabolic enzymes or regulators
Several factors were simultaneously incorporated in the synthetic core promoter design: (i) nucleotide occurrence along the sequence of 140 strong natural S. cerevisiae core promoters, (ii) the presence and position of the TATA box, (iii) the position and number other motifs and (iv) nucleosome occupancy profiles.[28,45]
We have created a library of synthetic core promoters and 5′untranslated regions (UTRs) for generic yeast cells
Summary
Metabolic pathways and genetic circuits are commonly introduced into microbes such as Saccharomyces cerevisiae or Escherichia coli to produce chemicals or to implement novel functions.[1,2] Such experiments typically require the fine-tuning of gene expression to balance and optimize protein levels of metabolic enzymes or regulators. CRM is a general term referring to regulatory DNA sequences, named enhancers in higher eukaryotes, while in yeasts rather the terms upstream activating/repressing sequences (UAS/URS) are used.[20,21] CRMs interact with particular transcription factors conferring specific activation/repression regulatory mechanisms
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