The development and characterization of synthetic minimal yeast promoters

Abstract

Synthetic promoters, especially minimally sized, are critical for advancing fungal synthetic biology. Fungal promoters often span hundreds of base pairs, nearly ten times the amount of bacterial counterparts. This size limits large-scale synthetic biology efforts in yeasts. Here we address this shortcoming by establishing a methodical workflow necessary to identify robust minimal core elements that can be linked with minimal upstream activating sequences to develop short, yet strong yeast promoters. Through a series of library-based synthesis, analysis and robustness tests, we create a set of non-homologous, purely synthetic, minimal promoters for yeast. These promoters are comprised of short core elements that are generic and interoperable and 10 bp UAS elements that impart strong, constitutive function. Through this methodology, we are able to generate the shortest fungal promoters to date, which can achieve high levels of both inducible and constitutive expression with up to an 80% reduction in size.

Figure 1. Overview of methodology for developing minimal fungal promoters.

(a) A schematic of the promoter architecture wherein minimal synthetic promoters were assembled from a library-derived core element (blue), a neutral AT-rich spacer and a hybrid assembly of library-derived UAS elements (red). Element lengths in illustration are to scale. (b) A generic workflow for isolating minimal, core promoter elements was followed. Twenty-seven libraries totalling 15 million candidates were created to identify functional core elements. Most promising libraries (0.15%) were isolated by FACS. These sorted cells were subjected to colony analysis via flow cytometry and high-strength candidates were sequenced. Only 18 putative core elements were selected and characterized using a series of robustness tests to arrive at a final set of nine generic, functional core elements. (c) One library of 1.3 million 10-bp UAS candidates was analysed and top performers were isolated by FACS. One hundred and nineteen putative candidates were narrowed to just a pool of six UAS after colony analysis, sequencing and robustness tests. Select UAS elements were linked together to demonstrate one highly functioning triple tandem UAS that can establish strong, minimal yeast promoters when linked with a minimal core element.

Figure 2. UAS activation of synthetic core elements creates constitutive promoters.

(a) Core elements were tested with two known constitutive UAS elements, UAS_CIT and UAS_CLB. All of the final nine minimal core elements were sufficiently activated by each of these elements (n=4). Quadruplicates used in an effort to reduce error. (b) For demonstrative purposes, one core element rejected in this robustness test is shown (n=3). Error bars represent s.d. among biological replicates as indicated.

Figure 3. TFBS activation of synthetic core elements creates inducible promoters.

(a) Core elements were paired with a minimal GBS. Specifically shown here is UAS_G4BS4 to develop short, minimal galactose-inducible promoters. Lengths of promoters are illustrated to scale. Scale bar, 100 bp. (b) When linked with certain core elements, the GBS was able to establish short, galactose-inducible promoters with a fully induced strength comparable to that of the GAL1 native promoter. (c) Of the 18 core elements tested in this construct, two did not activate with both UAS_G4BS3 and UAS_G4BS4. For demonstrative purposes, one rejected core element is shown here, which does not activate with UAS_G4BS4. Error bars represent s.d. among biological triplicate.

Figure 4. Minimal core elements and UAS sequences.

TFBS are indicated by arrows with direction of arrow designating the orientation of site. Protein predicted to bind to these sites are labelled as so. (a) The final set of nine core elements established here are distinct from one another spanning a %GC content of 47–73. The quantity, quality and directionality of predicted TFBS, as determined by YEASTRACT, vary greatly. These promoters also display varying dependences to SAGA complex. In a Δspt3 strain, strengths of core elements assembled in three promoter contexts (just core, with UAS_CIT and with UAS_G4BS4) were determined. SAGA dependency score of 3 indicates that all three promoters' strengths were affected in this knockout strain, while a score of 0 indicates that none of the promoters' strengths were disrupted. High minimum E_value obtained from BLAST confirm core elements' uniqueness. (b) Six 10 bp minimal UAS sequences isolated through the library-based sorting and selection.

Figure 5. Fully synthetic minimal promoters can drive high expression.

By linking minimal UAS elements with a core promoter element, synthetic promoters approaching GPD strength can be assembled. (a) Lengths of promoters are illustrated to scale. Scale bar is provided. All synthetic UAS shown (UAS_F, UAS_E and UAS_C) are positioned upstream of the core element using a singular AT-rich neutral 30 bp spacer. Size scale bars are provided. (b) All synthetic UASs can activate core element to levels comparable to the strength of the commonly used promoters CYC1. When assembled in a hybrid fashion, tandem UAS elements can amplify the strength of these core elements with strengths approaching GPD (TDH3) in <20% of the DNA. Error bars represent s.d. among the biological triplicate.