4-羟基异亮氨酸(4-hydroxyisoleucine,4-HIL)具有促进胰岛素分泌、降低胰岛素抵抗活性功能,在治疗糖尿病方面有潜在的应用价值.在产L-异亮氨酸(L-isoleucine,Ile)的谷氨酸棒杆菌SN01菌株中过表达ido基因后可以合成4-HIL.为了促进草酰乙酸的供应和4-HIL合成,在这株重组菌中又过表达了mqo.发酵144 h后,ido-mqo过表达菌的4-HIL产量是(60.86±2.24) mmol/L,低于ido过表达菌(72.06±5.60)mmol/L;但是,Ile的积累量高达(101.39±2.20) mmol/L,显著高于ido过表达菌(5.00±1.43) mmol/L.对ido-mqo过表达菌代谢途径中部分关键基因的RT-PCR分析表明,进入TCA循环的碳流由异柠檬酸裂解酶分流,进入乙醛酸循环,导致4-HIL合成所需的另一底物α-酮戊二酸(α-ketoglutarate,α-KG)供应不足和Ile过剩.于是添加α-KG,当添加量为40 mmol/L时,4-HIL的产量提高到(77.7±10.91) mmol/L,Ile到4-HIL的转化率提高到75％.因此,过表达mqo能够促进Ile合成,添加o-KG后能够促进Ile向4-HIL转化,从而提高4-HIL产量.%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) mol/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,ot-KG was added.When 40 mmol/L of o-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.
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