Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.

Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0333-1) contains supplementary material, which is available to authorized users.

Transcription factors play a crucial role in the regulation of gene expression and adaptation to different environments. To better understand the underlying roles of these adaptations, we performed experiments that give us high-resolution binding of transcription factors to their targets. We investigated five transcription factors involved in lipid metabolism in yeast, and we discovered multiple novel targets and condition-specific responses that allow us to draw a better regulatory map of the lipid metabolism.

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels.

In the model eukaryote Saccharomyces cerevisiae, the transcription factor Cst6p has been reported to play important roles in several biological processes. However, the genome-wide targets of Cst6p and its physiological functions remain unknown. Here, we mapped the genome-wide binding sites of Cst6p at high resolution. Cst6p binds to the promoter regions of 59 genes with various biological functions when cells are grown on ethanol but hardly binds to the promoter at any gene when cells are grown on glucose. The retarded growth of the CST6 deletion mutant on ethanol is attributed to the markedly decreased expression of NCE103, encoding a carbonic anhydrase, which is a direct target of Cst6p. The target genes of Cst6p have a large overlap with those of stress-responsive transcription factors, such as Sko1p and Skn7p. In addition, a CST6 deletion mutant growing on ethanol shows hypersensitivity to oxidative stress and ethanol stress, assigning Cst6p as a new member of the stress-responsive transcriptional regulatory network. These results show that mapping of genome-wide binding sites can provide new insights into the function of transcription factors and highlight the highly connected and condition-dependent nature of the transcriptional regulatory network in S. cerevisiae.