Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis

Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7^P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.