Characterization Of L-isoleucine Dioxygenase And Production Of 4-hydroxyisoleucine In Recombinant Corynebacterium Glutamicum

4-Hydroxyisoleucine(4-HIL) is a natural nonproteinogenic amino acid that exhibits insulinotropic activity. L-isoleucine dioxygenase(IDO) catalyzes the C-4 hydroxylation of L-isoleucine(Ile) to form 4-HIL with the concomitant oxidation of α-ketoglutarate(KG) to form succinate(Suc). Here, the gene encoding IDO(ido) was cloned from Bacillus thuringiensis YBT-1520 and expressed in an Ile-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, in order to directly convert its endogenous Ile into 4-HIL through single-step fermentation. The main contents and results are as follows.1. ido and truncated idoΔ6 derived from B. thuringiensis YBT 1520 was cloned and over-expressed in Escherichia coli BL21(DE3). After purification, the enzymatic properties of Ido and IdoΔ6 were analyzed. The optimum activity of Ido and IdoΔ6 were shown at p H 6.0 and 40 oC. They were more stable at p H 5.5-7.5. But they exhibited weak thermal stability. Under optimal conditions, the activity of Ido and IdoΔ6 were 0.93±0.07 and 1.50±0.07 U/mg, respectively. For Ido, the Km values for Ile was 1.63 m M and for KG was 1.22 m M; whereas for IdoΔ6, the Km values for Ile was 2.69 m M and for KG was 2.52 m M. Compared to Ido, catalytic efficiency of IdoΔ6 for Ile increased by 31.8%, and for KG decreased by 41.3%.2. The ido-expressing C. glutamicum ssp. lactofermentum strain SN01/p JYW-4-ido was constructed. It could produce approximately 50.99±2.14 m M of 4-HIL after fermented for 144 h in shake flask. Then the effect of corn steep liquor limitation on the production of 4-HIL was investigated. The results suggested that 4-HIL production by ido-expressing strain was improved to 65.44±2.27 m M after fermented for 144 h under corn steep liquor-subsufficient(7 g/L) condition, obviously higher than that under corn steep liquor-rich and insufficient conditions. Meanwhile, the conversion ratio of Ile to 4-HIL increased to 0.85 mol/mol. Biotin limitation experiments validated that 4-HIL production by ido-expressing strain under biotin-subsufficient condition was higher than that under other biotin conditions.3. The idoΔ6-expressing C. glutamicum ssp. lactofermentum strain SN01/p JYW-4-idoΔ6 was constructed. 4-HIL biosynthesis in ido- and idoΔ6-expressing strains under optimum corn steep liquor concentration was compared. The results suggested that 4-HIL production by ido-expressing strain(65.44±2.27 m M) was higher than that by idoΔ6-expressing strain(48.91±2.92 m M). To elucidate why ido expression was more benefit for 4-HIL biosynthesis than idoΔ6 expression in recombinant C. glutamicum ssp. lactofermentum, amount of Ido and IdoΔ6 proteins expressed in recombinant C. glutamicum ssp. lactofermentum as well as the enzyme properties of Ido and IdoΔ6 at 30 oC were analyzed. However, no difference existed between Ido and IdoΔ6 in levels of protein amount and enzymatic properties. At last, one substitution in the rbs sequence of p JYW-4-idoΔ6(AGAAGGAGâ†'AGAAGGGG) was found after sequencing and the discrepancy of 4-HIL production might be caused by this variation. However, this remains to be researched in detail.In conclusion, a system for de novo biosynthesis of 4-HIL was constructed and corn steep liquor limitation was demonstrated to be a useful strategy for improving 4-HIL production in recombinant C. glutamicum ssp. lactofermentum in this research.