Li Ying, Liu Luo, Tian Pingfang
Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.
Biotechnol Lett. 2014 Nov;36(11):2215-21. doi: 10.1007/s10529-014-1590-6. Epub 2014 Jul 1.
The limiting step for biosynthesis of 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae is the conversion of 3-hydroxypropionaldehyde (3-HPA) to 3-HP. This reaction is catalyzed by aldehyde dehydrogenase (ALDH) with NAD(+) as a cofactor. Although NAD(+)-dependent ALDH overexpression facilitates 3-HP biosynthesis, ALDH activity decreases and 3-HP stops accumulation when NAD(+) is exhausted. Here, we show that an NAD(+)-independent aldehyde oxidase (AOX) from Pseudomonas sp. AIU 362 holds promise for cofactor-balanced 3-HP production in K. pneumoniae. The AOX coding gene, alod, was heterologously expressed in E. coli and K. pneumoniae, and their respective crude cell extracts showed 38.1 U/mg and 16.6 U/mg activities toward propionaldehyde. The recombinant K. pneumoniae expressing alod showed 13.7 U/mg activity toward 3-HPA; K m and V max were 6.7 mM and 42 μM/min/mg, respectively. In shake-flask cultures, the recombinant K. pneumoniae strain produced 0.89 g 3-HP/l, twice that of the control. Moreover, it produced 3 g 3-HP/l during 24 h fed-batch cultivation in a 5 l bioreactor. The results indicate that AOX can efficiently convert 3-HPA into 3-HP.
肺炎克雷伯菌中3-羟基丙酸（3-HP）生物合成的限速步骤是3-羟基丙醛（3-HPA）转化为3-HP。该反应由醛脱氢酶（ALDH）催化，以NAD(+)作为辅因子。虽然依赖NAD(+)的ALDH过表达促进了3-HP的生物合成，但当NAD(+)耗尽时，ALDH活性降低，3-HP停止积累。在此，我们表明来自假单胞菌属AIU 362的一种不依赖NAD(+)的醛氧化酶（AOX）有望在肺炎克雷伯菌中实现辅因子平衡的3-HP生产。AOX编码基因alod在大肠杆菌和肺炎克雷伯菌中进行了异源表达，它们各自的粗细胞提取物对丙醛显示出38.1 U/mg和16.6 U/mg的活性。表达alod的重组肺炎克雷伯菌对3-HPA显示出13.7 U/mg的活性；K m和V max分别为6.7 mM和42 μM/min/mg。在摇瓶培养中，重组肺炎克雷伯菌菌株产生了0.89 g 3-HP/l，是对照的两倍。此外，在5升生物反应器中进行24小时补料分批培养期间，它产生了3 g 3-HP/l。结果表明AOX可以有效地将3-HPA转化为3-HP。
