Metabolic Engineering Of Ido-expressing Corynebacterium Glutamicum To Improve The Production Of 4-hydroxyisoleucine

4-Hydroxyisoleucine(4-HIL) exhibits unique insulinotropic and insulin-sensitizing activities and is an attractive candidate for the treatment of diabetes. In our previous study, isoleucine dioxygenase gene(ido) was cloned and overexpressed in an L-isoleucine-producing strain, Corynebacterium glutamicum ssp. lactofermentum SN01, and 4-HIL was produced from the endogenous L-isoleucine(Ile). In this study, metabolic engineering strategies are used to further enhance the 4-HIL production. The main research contents and results are as follows.(1) To improve Ile supply, lysC which encodes the first enzyme of Ile biosynthetic pathway was overexpressed and the strain Cgl/p4-ido-lysC was constructed. After 120 h of fermentation, the Cgl/p4-ido-lysC strain produced 24.00±4.32 mmol·L-1 4-HIL and the conversion ratio of Ile to 4-HIL was 32%, significantly lower than Cgl/p4-ido. But the total amount of L-aspartate family amino acids reached 134.32±6.72 mmol·L-1, 56% higher than that of Cgl/p4-ido. This indicates that overexpression of lysC is benefit for L-aspartate family amino acids biosynthesis but not for 4-HIL conversion.(2) To enhance oxaloacetate supply through anaplerotic pathway, ppc which encodes phosphoenolpyruvate carboxylase was overexpressed and the strain Cgl/p4-ido-ppc was constructed. After 144 h of fermentation, the Cgl/p4-ido-ppc strain produced 94.72±1.42 mmol·L-1 4-HIL, 29% higher than Cgl/p4-ido strain, and the conversion ratio of Ile to 4-HIL reached 90%. This suggests that overexpression of ppc contributes to 4-HIL biosynthesis.(3) Then the Cgl/p4-ido-lysC-ppc strain was onstructed. After 144 h of fermentation, the Cgl/p4-ido-lysC-ppc strain produced 30.16±2.01 mmol·L-1 4-HIL and the conversion ratio of Ile to 4-HIL was only 31%. However, the total amount of L-aspartate family amino acids of Cgl/p4-ido-lysC-ppc strain reached 181.15±43.35 mmol·L-1, 27% higher than that of Cgl/p4-ido-ppc.(4) To improve α-ketoglutarate supply, the pkn G deleted strain Cgl ΔpknG/p4 was constructed and the Ile fermentation indicated that knocking out of pkn G decreased the Ile production. To research on the effect of pknG deletion on 4-HIL production, the Cgl ΔpknG/p4-ido, Cgl Δpkn G/p4-ido-ppc and Cgl ΔpknG/p4-ido-lysC-ppc strains were constructed. After 144 h of fermentation, the 4-HIL yield of the three strains was 57.35±1.68 mmol·L-1, 86.95±4.15 mmol·L-1 and 59.71±3.27 mmol·L-1, respectively. Meanwhile, the 4-HIL yield of Cgl ΔpknG/p4-ido-ppc was 51% higher than that of Cgl ΔpknG/p4-ido, demonstrating again the beneficial effect of ppc overexpression on 4-HIL production. In addition, the 4-HIL yield(59.71±3.27 mmol·L-1) and the conversion rate of Ile to 4-HIL(76%) of Cgl ΔpknG/p4-idolysC-ppc strain were significantly higher than those of Cgl/p4-ido-lysC-ppc strain. However, the reason for this result needs to be further researched.In conclusion, overexpression of ppc was benefit for 4-HIL production, while overexpression of lysC was benefit for L-aspartate family amino acids biosynthesis but not for 4-HIL conversion. Deletion of pknG improved 4-HIL production in lysC-ppc co-overexpressing strain.