College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
School of Food Science and Engineering, South China University of Technology, Guangzhou, 510641, China.
Biotechnol Lett. 2021 Jan;43(1):223-234. doi: 10.1007/s10529-020-03011-y. Epub 2020 Sep 29.
Glycerol-based biosynthesis of 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae involves two reactions: glycerol conversion to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase, and 3-HPA conversion to 3-HP by aldehyde dehydrogenase (ALDH). The ALDH catalysis consumes a lot of cofactor nicotinamide adenine dinucleotide (NAD), which constrains 3-HP production.
Here we report that intensifying niacin-based biosynthesis of NAD can substantially enhance 3-HP production. We constructed tac promoter-driven NAD synthesis pathway in K. pneumoniae. The strain only overexpressing nicotinate phosphoribosyltransferase (PncB) showed 14.24% increase in the production of NAD relative to the stain harboring an empty vector. When PncB was coexpressed with PuuC (one of native ALDHs), the recombinant strain exhibited increased ALDH activity but slightly reduced 3-HP production due to plasmid burden. When 30 mg niacin l (a substrate for biosynthesis of NAD) was added into shake flask, the strain produced 0.55 g 3-HP l, which was 2.75 times that of the control. In a 5-L bioreactor, replenishment of niacin led to 36.43% increase of 3-HP production.
These results indicated that intensifying niacin-based biosynthesis of NAD boosts 3-HP production.
肺炎克雷伯氏菌中基于甘油的 3-羟基丙酸（3-HP）生物合成涉及两个反应：甘油脱水酶将甘油转化为 3-羟基丙醛（3-HPA），醛脱氢酶（ALDH）将 3-HPA 转化为 3-HP。ALDH 催化消耗大量辅因子烟酰胺腺嘌呤二核苷酸（NAD），这限制了 3-HP 的生产。
在这里，我们报告了通过烟酸基生物合成大量合成 NAD 可以显著提高 3-HP 的产量。我们在肺炎克雷伯氏菌中构建了 tac 启动子驱动的 NAD 合成途径。与含有空载体的菌株相比，仅过表达烟酰胺磷酸核糖基转移酶（PncB）的菌株中 NAD 的产量增加了 14.24%。当 PncB 与 PuuC（一种天然 ALDH 之一）共表达时，由于质粒负担，重组菌株的 ALDH 活性增加，但 3-HP 的产量略有降低。当在摇瓶中添加 30mg 烟酰胺 l（NAD 生物合成的底物）时，该菌株产生 0.55g 3-HP l，是对照的 2.75 倍。在 5L 生物反应器中，补充烟酸可使 3-HP 产量增加 36.43%。
这些结果表明，通过强化基于烟酸的 NAD 生物合成可以提高 3-HP 的产量。
