文献类型：期刊文献

英文题名：ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering

作者：Wang, Wei[1];Liu, Meixia[2];You, Chun[2];Li, Zhimin[1,3];Zhang, Yi-Heng Percival[2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Tianjin Airport Econ Area, 32 West 7th Ave, Tianjin 300308, Peoples R China;[3]Shanghai Collaborat Innovat Ctr Biomfg Technol, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2017

卷号：42

起止页码：168

外文期刊名：METABOLIC ENGINEERING

收录：;EI(收录号：20172703863373);WOS:【SCI-EXPANDED(收录号:WOS:000405854400019)】；

基金：This work was supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering and Shanghai Committee of Science and Technology (Grant no. 13DZ1930202).

语种：英文

外文关键词：Fructose 1,6-diphosphate; In vitro metabolic engineering; Sugar phosphate; Hyperthermophilic enzyme; Enzyme cocktail

摘要：Fructose 1,6-diphosphate (FDP) is a widely used medicine and is also a precursor of two important three-carbon phosphates - glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) for the biosynthesis of numerous fine chemicals. An in vitro synthetic cofactor-free enzymatic pathway comprised of four hyperthermophilic enzymes was designed to produce FDP from starch and pyrophosphate. All of four hyperthermophilic enzymes (i.e., alpha-glucan phosphorylase from Thermotaga maritima, phosphoglucomutase from Thermococcus kodakarensis, glucose 6-phosphate isomerase from Thermus thermophilus, and pyrophosphate phosphofructokinase from T. maritima) were overexpressed in E. coli BL21(DE3) and purified by simple heat precipitation. The optimal pH and temperature of one-pot biosynthesis were 7.2 and 70 degrees C, respectively. The optimal enzyme ratios of alpha GP, PGM, PGI and PFK were 2:2:1:2 in terms of units. Via step-wise addition of new substrates, up to 125 +/- 4.6 mM FDP was synthesized after 7-h reaction. This de novo ATP-free enzymatic pathway comprised of all hyperthermophilic enzymes could drastically decrease the manufacturing costs of FDP and its derivatives GA3P and DHAP, better than those catalyzed by ATP-regeneration cascade biocatalysis, the use of mesophilic enzymes, whole cell lysates, and microbial cell factories.