食品科学 ›› 2025, Vol. 46 ›› Issue (15): 128-126.doi: 10.7506/spkx1002-6630-20250122-165

• 生物工程 • 上一篇    下一篇

大肠杆菌利用葡萄糖生物合成D-阿洛酮糖的途径构建及优化

姜雅文,张苍萍,杨绍青,江正强,李延啸,闫巧娟   

  1. (1.中国农业大学食品科学与营养工程学院,中国轻工业食品生物工程重点实验室,北京 100083;2.中国农业大学工学院,北京 100083)
  • 出版日期:2025-08-15 发布日期:2025-07-22
  • 基金资助:
    “十四五”国家重点研发计划重点专项(2022YFC2104900)

Construction and Optimization of Biosynthetic Pathway for the Production of D-Allulose from Glucose in Escherichia coli

JIANG Yawen, ZHANG Cangping, YANG Shaoqing, JIANG Zhengqiang, LI Yanxiao, YAN Qiaojuan   

  1. (1. Key Laboratory of Food Bioengineering (China National Light Industry), College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China; 2. College of Engineering, China Agricultural University, Beijing 100083, China)
  • Online:2025-08-15 Published:2025-07-22

摘要: 在大肠杆菌BL21(DE3)中构建磷酸化-去磷酸化将D-葡萄糖转化为D-阿洛酮糖的途径。通过CRISPR/Cas9基因编辑系统敲除竞争途径的相关基因,探究不同来源阿洛酮糖-6-磷酸磷酸酶(allulose-6-phosphate phosphatase,A6PP)对D-阿洛酮糖合成的影响,调控合成途径基因的转录水平,并进行工程菌发酵条件优化。结果表明,敲除磷酸果糖激酶A基因pfkA、葡萄糖-6-磷酸脱氢酶基因zwf、阿洛糖-6-磷酸异构酶基因rpiB和甘露糖-6-磷酸异构酶基因manA后D-阿洛酮糖产量提升至0.95 g/L。布氏拟杆菌(Bacteroides bouchesdurhonensis)来源的A6PP(BbA6PP)合成D-阿洛酮糖的效果最好,D-阿洛酮糖的摇瓶产量提升至1.21 g/L。通过质粒拷贝数优化方式调节通路基因的表达水平得到重组菌BE-14的D-阿洛酮糖摇瓶产量最高,为2.06 g/L。经发酵条件优化后,重组菌BE-14合成D-阿洛酮糖摇瓶产量提高到2.72 g/L,在5 L发酵罐中分批补料发酵46 h的D-阿洛酮糖产量达到最高,为18.4 g/L。发酵液中仅存在微量的葡萄糖(0.7 g/L)及果糖(0.1 g/L),有利于后续分离纯化。本研究为大肠杆菌生物合成法高效生产D-阿洛酮糖提供了研究基础,对推动D-阿洛酮糖的工业化生产具有重要意义。

关键词: D-阿洛酮糖;磷酸化-去磷酸化;生物合成;代谢工程

Abstract: A pathway in Escherichia coli BL21 (DE3) was constructed to produce D-allulose from D-glucose via a phosphorylation-dephosphorylation strategy. The genes related to competitive pathways were deleted using the CRISPR/Cas9 system, and allulose-6-phosphate phosphatases from different sources were selected to evaluate their effects on the synthesis of D-allulose. The expression levels of D-allulose synthetic pathway-related genes were regulated and the fermentation conditions for the engineered strain were optimized. The results showed that the titer of D-allulose increased to 0.95 g/L after the deletion of the genes encoding phosphofructokinase A (pfkA), glucose-6-phosphate dehydrogenase (zwf), allose-6-phosphate isomerase (rpiB), and mannose-6-phosphate isomerase (manA). The allulose-6-phosphate phosphatase BbA6PP from Bacteroides bouchesdurhonensis showed the best performance in D-allulose synthesis, and its use increased the titer of D-allulose to 1.21 g/L in shake flasks. The recombinant strain BE-14, obtained by regulating the expression levels of D-allulose synthetic pathway-related genes through the optimization of plasmid copy number, achieved a maximum D-allulose yield of 2.06 g/L. After optimizing the fermentation conditions, the titer of D-allulose was improved to 2.72 g/L in shake flasks. BE-14 produced the highest titer of D-allulose of 18.4 g/L in a 5 L fermenter after 46 h fermentation. Only trace amounts of glucose (0.7 g/L) and fructose (0.1 g/L) were present in the fermentation broth, which was beneficial for subsequent separation and purification. This study provides a research basis for the efficient production of D-allulose by E. coli, which is of great significance for promoting the industrial production of D-allulose.

Key words: D-allulose; phosphorylation and dephosphorylation; biosynthesis; metabolic engineering

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