Development of novel and efficient Corynebacterium glutamicum gene expression systems for industrial enzyme...

An ideal microbial gene expression system for industrial enzyme production should possess a function complementary selection marker, a cost-effective inducer, and the ability for extracellular protein secretion. In this study，we employed CRISPR-Cpf1 gene editing technology to construct an engineered Corynebacterium glutamicum host strain, C. glutamicum Δalr∷araE ΔmurI, by deleting the alanine racemase-encoding gene alr and the glutamate racemase-encoding gene murI, and by integrating the Bacillus subtilis arabinose-related compounds permease-encoding gene araE into the chromosome. The two secretion-type expression vectors pAU30S (Sec-type) and pAU30T (Tat-type) that both employ alr as selection marker, the T7 transcription system to transcribe target genes, and the B. subtilis AraR-OR_A1/OR_A2 negative control system to control gene transcription, were constructed. The α-amylase AmyF from Geobacillus stearothermophilus was used as reporter protein to test the applicability of the L-arabinose-induced gene expression systems C. glutamicum/pAU30S and C. glutamicum Δalr∷araE ΔmurI/pAU30T. The results of transparent circle investigation, SDS-PAGE and amylase activity analysis demonstrated that the recombinant AmyF was efficiently expressed and completely secreted into the culture medium in its active form. The C. glutamicum Δalr∷araE ΔmurI/pAU30S and C. glutamicum Δalr∷araE ΔmurI/pAU30T systems represent highly efficient gene expression platforms suitable for the secretory production of industrial enzymes.