MATCHING SECRETION CAPACITY VIA TRANSLATIONAL CONTROL

摘要

Inducible gene expression systems commonly employed in microbial hosts are mostly composed of molecular components derived from the lactose (lac), arabinose (ara), rhamnose (rha) and tetracycline (tet) operons. These transcription-level control systems have been widely employed in research and industrial-scale protein production, however they are known to exhibit important limitations. These include; all-or-none (digital) expression profiles, stochastic transcriptional bursting, and heterogeneous expression responses at submaximal induction conditions. A new paradigm in genetic regulation emerged with the discovery of genetic regulatory elements within the 5'UTR of bacterial mRNA. Upon binding to a specific metabolite, these so-called riboswitches change conformation, permitting differential gene regulation to occur. To allow us to utilize this alternative mechanism of genetic regulation we developed and characterized a novel recombinant expression system, termed RiboTite. The system operates at both the transcriptional and translational level, using standard inducible promoters and translational-ON riboswitches respectively, collectively providing a multi-layered modular genetic circuit controlling both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The precise cellular-level tunable expression control afforded by this system offers a number of potential applications in terms of matching cellular expression rate to host synthetic and processing capacity. Here we will report one such application, where we used the RiboTite system to avoid the overload of secYEG translocon in E. coli, permitting expression/secretion attenuation of recombinant proteins into periplasmic space (Figure 1). Utilizing a library of different signal peptides that target the recombinant protein to secYEG, either via the post-translational (SecB/A) or the co-translational (SRP) pathway, we have demonstrated successful attenuation of recombinant protein reaching the periplasm. Finally under fed-batch fermentation conditions the system has been demonstrated to avoid the overload of the host secretion machinery and produce scFv antibody fragments at industrially relevant titers.