Yeast Synthetic Terminators: Fine Regulation of Strength through Linker Sequences.

Abstract

The design of improved synthetic components is an important research field in synthetic biology. The terminator, responsible for terminating gene transcription, is a necessary component for yeast gene expression. The efficiency element, the positioning element and the poly(A) site have been identified as the constituent parts necessary for the yeast terminator to perform its function. However, the functions of linker 1 (situated between the efficiency element and the positioning element) and linker 2 [between the positioning element and the poly(A) site] in the terminator are still controversial. Here, we have thus designed and synthesized a yeast synthetic terminator library incorporating random 10 bp linker 1 units. For indirect characterization of the strengths of 266 synthetic terminators with the aid of the enhanced green fluorescent protein (eGFP), their fluorescence intensity (FI) values were determined; they ranged from 2.3648 to 3.5270, thus indicating that the strength of yeast terminator can be finely adjusted by changing the linker 1 sequence. The strength increased with decreasing GC content in linker 1, with a T-rich linker 1 helping to enhance terminator strength further. Reducing the stem length can increase the gene expression in cases of weak and medium-strength terminators but decreases the gene expression of strong terminators. Deletion of linker 2 seems to have a positive effect on weak and medium-strength terminators. Construction of a lycopene biosynthesis pathway with synthetic terminators effectively regulated lycopene synthesis, thus indicating that it is highly feasible to use terminators for fine regulation of gene and pathway expression.