Short Synthetic Terminators for Improved Heterologous Gene Expression in Yeast

Abstract

Terminators play an important role both in completing the transcription process and impacting mRNA half-life. As such, terminators are an important synthetic component considered in applications such as heterologous gene expression and metabolic engineering. Here, we describe a panel of short (35-70 bp) synthetic terminators that can be used for modulating gene expression in yeast. The best of these synthetic terminator resulted in 3.7-fold more fluorescent protein output and 4.4-fold increase in transcript level compared to that with the commonly used CYC1 terminator. These synthetic terminators offer several advantages over native sequences, including an easily synthesized short length, minimal sequence homology to native sequences, and similar or better performance characteristics than those of commonly used longer terminators. Furthermore, the synthetic terminators are highly functional in both Saccharomyces cerevisiae and an alternative yeast, Yarrowia lipolytica, demonstrating that these synthetic designs are transferrable between diverse yeast species.

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

“…Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae , several terminator regions have shown activity in increasing the production of proteins by upstream coding genes89101112. Whereas some cis -elements within the 3′-UTR and its corresponding trans proteins downregulate gene expression via mRNA degradation or translational repression1314, in vertebrates a few promote the accumulation of protein through translation initiation by cytoplasmic polyadenylation15, stabilization of fragile mRNA16, or translational activation1718 by formation of mRNA–protein complexes.…”

Can terminators be used as insulators into yeast synthetic gene circuits?

“…They are the yeast DEG1 [15] (DEG1t) and the synthetic Tsynth8 [16] terminators. The latter, in particular, was chosen since it was described as one of the most efficient, among a collection of 30 synthetic terminators, to block RNA polymerase II read-through between two adjacent transcription units expressing different fluorescent proteins.…”

Promoter and Terminator Discovery and Engineering

“…This minimal scaffold was both diversified and enhanced using modified consensus termination elements and mRNA stability elements [128] to produce a library of rationally designed synthetic terminators (Fig. 3c) which were functional in multiple hosts and improved CAD1 expression for itaconic acid production [129]. Importantly, this technique allowed delineation of design rules based on consensus element identity and spacing, enabling potential rational design of synthetic terminators.…”

“…In S. cerevisiae, however, terminator function is much less predictable based simply on distinct sequence elements whose function is determined by biophysical models. In fact, characterization of the aforementioned rationally designed synthetic library [129] demonstrated that consensus termination motifs were not entirely additive. This suggests there is a fundamental code underlying termination in yeast which remains to be uncovered before thermodynamic prediction becomes feasible.…”