Heuristic Design and Engineering of pH-Inducible Yeast Promoters

While advances in synthetic biology have led to the development of synthetic promoters in eukaryotes and prokaryotes, few have been designed with environmental conditions imposed by fermentations in mind. Here, we present a set of strong, synthetic promoters for Saccharomyces cerevisiae that are inducible by low pH. By mining available transcriptome and transcription factor binding data and the relevant literature on transcriptional regulation and promoter architecture we identified transcription factor binding sites (TFBS) and other elements relevant to regulation by low pH. We then used these to modify the upstream activation sequence of a native promoter inducible by low pH, and improve its low-pH induction and overall regulatory output by a factor of six to ten. We subsequently used the same TFBS modifications to engineer a de novo response to low pH in a native promoter lacking one. After characterizing the regulatory response of the promoters at the RNA and protein level, we used the best promoter variants in industrial yeast to construct strains for the low-pH production of lactic acid. Ultimately, we achieved six-fold increased production of lactic acid with our promoters compared to titres obtained from a production strain with a popular constitutive yeast promoter. Our synthetic promoters are as strong as constitutive promoters typically used in yeast cell factories, and are a valuable addition to the toolbox of regulatory parts for metabolic engineering. Finally, our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.