(12g) Identification of High Capacity Terminators for Improved Control of Gene Expression and Metabolic Engineering in Saccharomyces cerevisiae

Authors: 
Curran, K., The University of Texas at Austin
Karim, A., The University of Texas at Austin
Gupta, A., The University of Texas at Austin
Alper, H., The University of Texas at Austin



Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering.  While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of the 3’ regulatory region of genes, known as the terminator.  In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and identify mRNA half-life changes to be the major cause of the varied protein and transcript expression level.  We demonstrate that the difference in transcript level can be over 6.5-fold even when paired with high strength promoters.  The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between a high-capacity terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline.  Next, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept.  Through pairing a high-capacity terminator with a low-expression promoter, we were able to achieve the same phenotypic result – equal growth rates on xylose – as with a promoter an order of magnitude higher in strength.  Moreover, we can further boost the growth rate on xylose by pairing the high-strength promoter with a high-capacity terminator.  Finally, we demonstrate that minimal synthetic terminators (less than 75 bp long) can be as effective as native, full-length high-capacity terminators.  These synthetic versions possess several advantages including shorter length and less homology when compared to traditionally used elements.  Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering in the future.