Thermostablein vitrotranscription-translation for enzyme screening in microdroplets

Abstract

BackgroundIn vitroexpression involves the utilization of the transcription and translation machinery derived from the cell to produce one or more proteins of interest and has found widespread application in the optimization of gene circuits or metabolic pathways in synthetic biology but also in pharmaceutical manufacturing. Mostin vitroexpression systems available are active at moderate temperatures but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. Moreover, given the fact that the main barrier toward the general use ofin vitroexpression is its high price compared with host-based recombinant expression, there is a need to develop alternativein vitroexpression systems operating at high temperatures and compatible with technologies that enable ultrahigh-throughput screening in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.ResultsTo this end, we produced high-expression cell-free extracts fromThermus thermophilusforin vitrotranslation and supplemented them with thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable to other thermostablein vitroexpression systems, while the preparation procedure is simpler and can be suited to differentThermus thermophilusstrains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. Although the composition of these extracts showed a high background in carboxyl esterase assays, β-glucosidase and cellobiose hydrolase activities could be measured with minimal background.ConclusionsCell-free extracts fromThermus thermophilusrepresent a simpler alternative to heavily optimized or pure component thermostablein vitroexpression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.