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.