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

Overexpression of Corynebacterium glutamicum NAD kinase improves L-isoleucine biosynthesis

摘要:

NAD激酶催化辅酶Ⅰ[NAD(H)]发生磷酸化，转变成辅酶Ⅱ[NADP(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。

Abstract:

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.