Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts

Authors: Hector, Ronald - Ron; Mertens, Jeffrey; Nichols, Nancy

Submitted to: New Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/11/2019
Publication Date: 6/20/2019
Citation: Hector, R.E., Mertens, J.A., Nichols, N.N. 2019. Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts. New Biotechnology. 53:16-23. https://doi.org/10.1016/j.nbt.2019.06.006.
DOI: https://doi.org/10.1016/j.nbt.2019.06.006

Interpretive Summary: Optimization of metabolic pathways for the production of renewable fuels and chemicals requires control over the level of gene expression at each step in an engineered pathway. Changes in the timing of gene expression can also increase productivity. To add utility to a previously constructed xylose-regulated promoter for industrial Brewer’s yeast (Saccharomyces cerevisiae), we developed additional expression vectors by keeping the promoter constant and varying only the gene termination regions. This new vector set maintained the xylose regulation of the original promoter but showed a 26-fold range in gene expression level when induced by xylose. With a slight modification to the yeast strain, the expression vectors also provide constant expression over a 26-fold range. These new expression vectors are highly sensitive to xylose as an inducer, work with multiple carbon sources for cell growth, and thus represent an extremely versatile set of expression vectors for fine-tuning both the timing of gene expression and the expression level. The ability to control both the amount of protein produced, and when it is produced, allows fine-tuning of cellular pathways, resulting in more efficient sugar metabolism. Utilizing all of the sugars available in agricultural materials as efficiently as possible increases product yield and profitability.

Technical Abstract: Synthetic hybrid promoters for xylose-regulated gene expression in the yeast Saccharomyces cerevisiae have recently been developed. However, the narrow range of expression level from these new hybrid promoters limits their utility for pathway optimization. To expand the range of xylose-regulated gene expression, we created a series of expression vectors using a xylose derepressible promoter (PXYL) and varied termination regions from several S. cerevisiae genes. The new set of vectors showed a 26-fold range of gene expression under inducing conditions and a 13-fold average induction due to xylose. In the presence of the XylR repressor, gene expression was very sensitive to xylose concentration and full induction was observed with 0.10 g/L of xylose. In the absence of XylR, gene expression from the vector set was constitutive over a similar 26-fold range of expression. These results show that the vectors are extremely versatile for constitutive expression as well as for fine-tuning both the timing of gene expression and expression level using xylose as an inexpensive inducer.