谷氨酸棒杆菌NAD激酶的过表达对L-异亮氨酸合成的促进作用

NAD激酶催化辅酶ⅠNAD(H)]发生磷酸化,转变成辅酶ⅡNADP(H)],而还原态辅酶Ⅱ(NADPH)是L-异亮氨酸合成的必要辅因子。为了提高NADPH的供应,首先克隆了谷氨酸棒杆菌NAD激酶基因ppnK,并利用大肠杆菌-棒状杆菌诱导型穿梭表达载体pDXW-8和组成型穿梭表达载体pDXW-9在L-异亮氨酸合成菌——乳糖发酵短杆菌JHI3-156中进行表达。摇瓶发酵后,ppnK诱导表达菌JHI3-156/pDXW-8-ppnK的NAD激酶酶活(4.33±0.74 U/g)比pDXW-8空载菌提高了83.5%,辅酶Ⅱ与辅酶Ⅰ的比例提高了63.8%,L-异亮氨酸产量(3.86±0.12 g/L)提高了82.9%;ppnK组成表达菌JHI3-156/pDXW-9-ppnK的NAD激酶酶活(7.67±0.65 U/g)比pDXW-9空载菌提高了2.20倍,辅酶Ⅱ与辅酶Ⅰ的比例提高了1.34倍,NADPH含量提高了21.7%,L-异亮氨酸产量(2.99±0.18 g/L)提高了41.7%。这说明NAD激酶有助于辅酶Ⅱ的供应和L-异亮氨酸的生物合成,这对于其他氨基酸的生产也有一定的参考依据。

Overexpression of Corynebacterium glutamicum NAD kinase improves L-isoleucine biosynthesis

NAD kinase catalyzes the phosphorylation of coenzyme Ⅰ NAD(H)] to form coenzyme Ⅱ NADP(H)],and NADPH is an important cofactor in L-isoleucine biosynthesis.In order to improve NADPH supply,ppnK,the gene encoding NAD kinase in Corynebacterium glutamicum was cloned and separately expressed in an L-isoleucine synthetic strain,Brevibacterium lactofermentum JHI3-156,by an inducible expression vector pDXW-8 and a constitutive expression vector pDXW-9.Compared with the control strain JHI3-156/pDXW-8,NAD kinase activity of the inducible ppnK-expressing strain JHI3-156/pDXW-8-ppnK was increased by 83.5%.NADP(H)/NAD(H) ratio was also increased by 63.8%.L-isoleucine biosynthesis was improved by 82.9%.Compared with the control strain JHI3-156/pDXW-9,NAD kinase activity of the constitutive ppnK-expressing strain JHI3-156/pDXW-9-ppnK was increased by 220%.NADP(H)/ NAD(H) ratio and NADPH concentration were increased by 134% and 21.7%,respectively.L-isoleucine biosynthesis was increased by 41.7%.These results demonstrate that NAD kinase can improve the coenzyme Ⅱ supply and L-isoleucine biosynthesis,which would also be useful for biosynthesis of other amino acids.