Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Additional file 3: Figure S2. Time-dependent regulation of reporter gene expression. Data were obtained with a BioLector from the same cultures as used in Supplementary Fig. S1. Indicated strains were targeted at pHMG1 or pOLE1 as in Fig. 1C with controls (ctrl) expressing dCas9 and no gRNA. Data were collected for ~47 hrs and are presented as the average of three biological replicates. A. MFI from strains targeted with the constitutive system is presented per DCW/L (dry cell weight per liter) as a function of time. Blue; control. Green; activation (MCP-VPR). Red; repression (PCP-Mxi1). B. MFI from cultures added 250 ng/mL aTc to activate the inducible system is presented per OD (OD600) over time. Blue; control. Green; activation (dCas9-VPR). Red; repression (dCas9-Mxi1)

Engineering of synthetic, stress-responsive yeast promoters.

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

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae.

The synthetic biology toolkit for bakers yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts

A FAIR-compliant parts catalogue for genome engineering and expression control in <i>Saccharomyces cerevisiae</i>

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications

MOESM5 of Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Additional file 5: Figure S4. Transcriptional regulation vs. nucleosome occupancy. Relative MFI (deviation from ’no gRNA’ control) is shown on second axis vs. predicted nucleosome occupancy on first axis for 12 yeast promoters. Predicted nucleosome occupancies were based on Kaplan et al. [33] and are the averaged values between the 5’- and 3’-most occupancy values of gRNAs. Low predicted nucleosome occupancy scores relate to lower nucleosome densities at a given position. Dark grey symbolizes regulation with MCP-VPR (activation) and light grey with PCP-Mxi1 (repression). Results are based on at least three biological replicates