4-hydroxyisoleucine(4-HIL),which can exhibit unique insulinotropic and insulin-sensitizing activities,has potential medical value in treating diabetes.An L-isoleucine(Ile)-producer,Corynebacterium glutamicum ssp.lactofermentum SN01 can synthesize 4-HIL when ido gene.In order to promote the supply of oxaloacetate and synthesis of 4-HIL,mqo was overexpressed in this recombinant strain.After 144 h of fermentation,4-HIL production of ido-mqo-expressing strain was(60.86±2.24) mmol/L,lower than that of ido-expressing strain(72.06±5.60)mmol/L.However,its Ile concentration was(101.39±2.20) mmol/L,significantly higher than that of ido-expressing strain(5.00±1.43) mmol/L.The RT-PCR analysis of some key genes in the metabolic pathway of ido-mqo-expressing strain showed that the carbon flow entering the TCA cycle was diverted by the isocitrate lyase to the glyoxylic acid cycle,resulting in the insufficient supply of another substrate for 4-HIL synthesis,i.e.α-ketoglutarate(α-KG)and excessive Ile concentration.Therefore,α-KG was added.When 40 mmol/L of α-KG was added,the yield of 4-HIL increased to(77.7±10.91) mmol/L and the conversion ratio of Ile to 4-HIL increased to 75%.Therefore,overexpression of mqo can promote Ile synthesis,and addition of α-KG can promote the conversion of Ile to 4-HIL,thereby increasing the yield of 4-HIL.