Construction and Fermentation of Efficient Violacein-producing Corynebacterium glutamicum Strains using Low Copy Number Plasmids
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Abstract:
For high violacein production, a recombinant strain was constructed using Corynebacterium glutamicum [generally recognized as safe (GRAS)] as the host cell. The low copy number plasmid pOK12CG1 was constructed using the replication and partition elements from pTET3, a natural large plasmid of C. glutamicum. The plasmid pOK12CG1, maintained at ~6 copies per genome, was compatible with common plasmids pEC-XK99E and pXMJ19 of C. glutamicum. The violacein synthesis operon (vioABCDE)-carrying plasmid pCGvio was constructed using the low copy number plasmid pOK12CG1 as backbone. Seven violacein-producing recombinant strains were developed using the standard strain of C. glutamicum ATCC 13032 and the deletion strain of the insertion sequence (IS) element as host cells. Initial screening indicated that the recombinant strain ATCC 13032/pCGvio harboring low copy number plasmids produced more violacein (508.24 mg/L) than the recombinant strain ATCC13032/pECvio harboring medium and high copy number plasmids (376.16 mg/L). Violacein production by the IS element deletion recombinant strain ISDM023/pCGvio reached 610.13 mg/L. Fermentation conditions for the recombinant strain ISDM023/pCGvio were further optimized by orthogonal experimental design to optimize three factors: medium volume ratio (VLB:VBHIS), induction time, and IPTG inducer concentration. Under optimal conditions (VLB:VBHIS=1:2, induction time=18 h, and IPTG concentration=0.75 mmol/L) the final production yield of violacein in shake flasks was 1 007.47 mg/L. Herein, the recombinant strain ISDM023/pCGvio of C. glutamicum was successfully constructed, providing a biological system for efficient production of violacein in C. glutamicum and serving as a reference for the efficient production of other high-value compounds.
