Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III in Corynebacterium glutamicum

Standardized parts can be efficiently assembled into novel biological systems using the three antibiotic (3A) system, ensuring the reusability of components and repeatability of experiments. In this study, we created the 3A expression system for easy construction of gene expression cassettes in Corynebacterium glutamicum (C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secreted rhv3 production. We first obtained three strong promoters P2252, Podhi, and PyweA from all of promoters, which drive the highest expression of green fluorescent protein (egfp). The three promoters were then assembled with different signal peptides to generate a series of constructs using the 3A expression system developed in this study, from which the highest activity of rhv3 reached 3187.5 ATU/L of PyweA-CspA-rhv3. Further increased production of rhv3 achieved large-scale fermentation using 5-L jar bioreactor, with the highest rhv3 accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported in E. coli. To the best of our knowledge, this is the first report of rhv3 secretory expression in C. glutamicum, which could be applied for the production of other recombinant proteins with significant applications.