The Construction And Fermentation Of Corynebacterium Glutamicum For Violacein Production

Violacein and deoxyviolacein which was produced from gram negative bacteria like Chromobacterium violaceum and Janthinobacterium lividum have bioactivities such as antitumor and anti-pathogenic bacteria. Becourse of the limitation of low productivity and highly deathly infections in humans from the natural producers, the heterologous production of crude violacein was carried out by means of metabolic engineering, however, lots of time was usually spent on the genetic transformation only to get undesirable results. Therefore, this study adopted the food-safe Corynebacterium glutamicum that has been used to produce L-Trp for many years as the chassis to evaluate its ability of violacein production.This topic regarded the commercial Corynebacterium glutamicum ATCC 21850 that could produce L-Trp as the chassis and C. glutamicum/E. coli shuttle expression vector pEC-XK99 E as backbone to evaluate its influence on violacein production from different sources of exogenous function genes.Firstly, the constitutive expression vector pEC-vioABCDE was constructed, C. glutamicum was certified as the appropriate host to express the violacein, and fermentation results showed that 533 mg/L crude violacein was obtained. The biosynthetic pathway genes Jvio was cloned from the genome of violacein- natural-producer Janthinobacterium lividum and the expression vector pEC-J-vio1 was constructed. After electro-transformation into the chassis, the fermentation was adopted with the crude violacein production of 629 mg/L; the natural existing J-vio was found to be overlapping genes, the conservative SD sequences from C. glutamicum was added to the front of the start codon of each genes and pEC-J-vio2 was finally constructed. The recombinant ATCC 21850 with pEC-C-vio2 was fermented and the crude violacein attained 815 mg/L.Then, the functional genes related to violacein biosynthesis cloned from another natural producer Chromobacterium violaceum was codon-optimized, the conservative SD sequences were introduced again, pEC-C-vio1 was constructed and the production of crude violacein of the recombinant reached 1115 mg/L; Considering the rate-limiting and key enzymes in metabolic pathway reported, the gene order in the operon was adjusted to form pEC-C-vio2,but fermentation results weren’t ideal;Finally, the optimal induction time and concentration of the recombinant strain ATCC 21850 pEC-C-vio3 were determined. In the 3 L fermentation tank, fed batch fermentation was carried out, and the yield of crude violacein reached 5436 mg/L.