过表达mqo对重组谷氨酸棒杆菌L-异亮氨酸和4-羟基异亮氨酸合成的影响

Effects of mqo overexpression on the synthesis of L-isoleucine and 4-hydroxyisoleucine in recombinant Corynebacterium glutamicum

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α-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 activi- ties, 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） retool/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-express- ing strain （5.00 ± 1.43） mmol/L. The RT-PCR analysis of some key genes in the metabolic pathway of ido-mqo-ex- pressing 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 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 ct-KG can promote the conversion of Ile to 4-HIL, thereby increasing the yield of 4-HIL.