Evaluation of TALE- and Crispr/Cas9-Based Synthetic Transcription Factors for Heterologous Gene Expression in Saccharomyces Cerevisiae

Metabolic engineering in yeast, with the goal to introduce new biochemical pathways, calls for highly specific and orthogonal regulation of heterologous gene expression. Endogenous transcription factors and promoters are not suitable for this purpose, as they would strongly affect gene expression of the host. Synthetic transcription factors (synTFs) with programmable DNA-binding specificity provide one way to overcome this challenge and to ultimately construct complete gene regulatory networks. We aim to use TALE- and CRISPR/Cas9-based transcription factors in combination with synthetic promoters to control expression of a multitude of genes on circular expression yeast artificial chromosomes, which we call xYACs. Currently, we are characterizing different synTFs using a combination of flow cytometry based reporter gene assays, growth assays and host gene expression analysis. Our approach will finally yield a library of synTFs and corresponding synthetic promoters covering a wide range of expression strength and enable us to specifically modify expression levels of individual genes. The use of synTFs and their careful characterization will reduce negative effects on the host´s gene expression and overall growth fitness. In combination with a method allowing the modular assembly of xYACs and a blue and red light-sensitive induction system, our toolbox will allow easy and fast construction of new pathways in yeast.