Enhancement Of Substrates Supply And Ido Expression In Corynebacterium Glutamicum Ssp. Lactofermentum To Improve The Production Of 4-Hydroxyisoleucine

4-Hydroxyisoleucine（4-HIL）has potential value in treating diabetes.L-isoleucine dioxygenase（IDO）catalyzes the hydroxylation of C4 in L-isoleucine（Ile）to form 4-HIL with the concomitant oxidation ofα-ketoglutarate（α-KG）and by consuming oxygen.In our previous study,through expressing the ido gene in the Ile producer,Corynebacterium glutamicum ssp.lactofermentum SN01,4-HIL was de novo-synthesized from glucose.In this study,synergistically improving the substrates supply and IDO activity was applied to enhance the de novo biosynthesis of 4-HIL.The main contents are as follows:（1）To enhanceα-KG supply,two approaches,i.e.icd overexpression and aceA knockout to block glyoxylate pathway,were applied,resulting strains SZ08 and SZ02,respectively.Compared with the original strain SN02,SZ08 grew slowly and synthesized less 4-HIL.But in SZ02,α-KG supply was enhanced and 4-HIL production was improved to 69.47±2.18 mM,18.9%higher than in the original strain SZ02.Meanwhile,Ile synthesis was enhanced and the total amount of 4-HIL and Ile was increased by 39.7%.（2）To enhance Ile supply,mqo or lysC was co-expressed with ido in SN01ΔaceA,resulting the strains SZ03 and SZ09.Co-expression of ido with mqo or lysC further improved Ile synthesis,but decreased 4-HIL production,partially due to the inadequate activity of IDO.（3）To increase IDO activity,another Ido gene derived from other Bacillus,ido3,was co-expressed with ido-mqo and ido-lysC in SN01ΔaceA,respectively,resulting the strains SZ04 and SZ10.Co-expression of ido3 with mqo and ido efficiently promoted the IDO activity,thus improving 4-HIL production to 91.54±8.29 mM.Co-expression of ido3 with lysC and ido improved4-HIL production to 78.38±1.63 mM.Therefore,co-expression of ido3 with mqo and ido is more conducive to the synthesis of 4-HIL.（4）To increase the oxygen uptake rate of the cells,vgb gene under different promoters,i.e.P_tacM,P_dnaK and P_clgR,were co-expressed with ido-mqo-ido3 in SN01ΔaceA,resulting strains SZ05,SZ06 and SZ07,respectively.The expression of vgb did not change 4-HIL titer significantly,but increased the growth rate and 4-HIL production rate at the first 72 h of fermentation,especially when vgb was expressed by the P_dnaK promoter.At 72 h of fermentation,the yield of 4-HIL in the P_dnaK-vgb expression strain SZ06 was twice of that in SZ04.（5）For further improving the yield of 4-HIL,three vgb expression strains SZ05,SZ06 and SZ07 were cultured in the optimized fermentation medium.The yield of 4-HIL increased to102-117 mM,and the conversion ratio of Ile to 4-HIL reached over 98%.It is worth noting that the4-HIL production of SZ05 increased to 117.31±0.16 mM,which was 2-fold of that in original strain SN02.