Development of auto-inducible gene expression system in corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacte-rium that is generally recognized as a safe organism (GRAS). Due to its safety and process perfor-mance, it has been used as a workhorse for industrial production of various proteins and biochemi-cals. As a result, many genetic manipulation tools have been developed to control the gene expres-sion systems and enhance the productivity. Among the various techniques, engineering of the pro-moter has been widely studied because it is effective in directly increasing the expression level of target genes. However, most promoters show some critical drawbacks in industrial-scale use. Consti-tutive promoters give continuous load to cells that can lower the growth rate. Also, inducible promot-ers involve inconvenient and expensive step of adding inducers. Therefore, the development of strong auto-inducible promoters is highly desired since they have great advantages in industrial-scale use by allowing auto-regulation without addition of the inducers. Here, an auto-inducible gene expression system was developed by engineering a sigma factor B (SigB)-dependent C. glutamicum native pro-moter of cg3141 gene. SigB was used as the auto-regulator to induce the gene expression during the transition between exponential growth phase and stationary phase. First, a synthetic promoter library was created by randomizing the critical promoter regions of cg3141 promoter with fixed -35 and -10 SigB binding sites. The auto-inducible promoters exhibiting high strengths and auto-inducibilities were isolated via FACS-based high-throughput screening. As a result, a strong auto-inducible pro-moter was isolated that exhibits a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the usability of the isolated promoter was verified with 5L lab-scale fed-batch cultivation of glutathione S-transferase (GST) as a target protein. Next, to im-prove the strength and inducibility of the auto-inducible promoters, the binding sequence of a re-pressor, ArnR, was added to the promoter region. A synthetic promoter library with fixed SigB and ArnR binding sites was created by randomization, and improved auto-inducible promoters were iso-lated via FACS sorting. As a result, stronger auto-inducible promoters with up to 70-fold higher strength were isolated. With ArnR over-expression, the auto-inducible promoters showed 4~6-fold inducibility. This is the first report on the development of an auto-inducible synthetic promoter in C. glutamicum, and this auto-inducible promoter will contribute to extend the usefulness of C. glutami-cum in the industrial-scale production of various recombinant proteins as well as value-added bio-chemicals.