Engineering of Escherichia coli for D-allose fermentative synthesis from D-glucose through izumoring cascade epimerization.
Engineering of Escherichia coli for D-allose fermentative synthesis from D-glucose through izumoring cascade epimerization.
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DOI：
10.3389/fbioe.2022.1050808
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发表时间：
2022
影响因子：
5.7
通讯作者：
Zheng, Hui-Dong
中科院分区：
文献类型：
--
作者：
Zheng, Ling-Jie;Guo, Qiang;Zhang, Ya-Xing;Liu, Chen-Yang;Fan, Li-Hai;Zheng, Hui-Dong
在食品工业中，D-Allose是一种潜在的蔗糖替代品，在未来的保健产品中是一种有用的添加剂。目前，大规模生产D-allose的方法仍在探索中，大多数是基于体外酶催化的Izumoring外聚。相比之下，D-allose的发酵合成从未报道过，可能是由于缺乏可用的天然微生物。在本研究中，我们在大肠杆菌中共表达了d -半乳糖：H+同质体（GalP）、d -葡萄糖异构酶（DGI）、D-allulose 3-epimerase （DAE）和核糖-5-磷酸异构酶（RPI），从而构建了d -葡萄糖生成D-allose的体内诱导途径。通过敲除与d -果糖、d -葡萄糖和果糖-6-磷酸磷酸化通路相关的FruA、PtsG、Glk、Mak、PfkA和PfkB，可以合理调节碳通量和碳分解代谢抑制（CCR）。此外，AlsB的失活破坏了天然的D-allose转运体，从而推动了对目标产物的可逆Izumoring反应。发酵在M9培养基中进行，以甘油为碳源，d -葡萄糖为底物。结果表明，该工程大肠杆菌细胞工厂在84 h后可产生约127.35 mg/L的D-allose。本研究在D-allose发酵生产方面取得的成果可进一步促进稀有糖的绿色制造。
D-Allose is a potential alternative to sucrose in the food industries and a useful additive for the healthcare products in the future. At present, the methods for large-scale production of D-allose are still under investigation, most of which are based on in vitro enzyme-catalyzed Izumoring epimerization. In contrast, fermentative synthesis of D-allose has never been reported, probably due to the absence of available natural microorganisms. In this work, we co-expressed D-galactose: H+ symporter (GalP), D-glucose isomerase (DGI), D-allulose 3-epimerase (DAE), and ribose-5-phosphate isomerase (RPI) in Escherichia coli, thereby constructing an in vivo Izumoring pathway for yielding D-allose from D-glucose. The carbon fluxes and carbon catabolite repression (CCR) were rationally regulated by knockout of FruA, PtsG, Glk, Mak, PfkA, and PfkB involved in the pathways capable of phosphorylating D-fructose, D-glucose, and fructose-6-phosphate. Moreover, the native D-allose transporter was damaged by inactivation of AlsB, thus driving the reversible Izumoring reactions towards the target product. Fermentation was performed in the M9 medium supplemented with glycerol as a carbon source and D-glucose as a substrate. The results show that the engineered E. coli cell factory was able to produce approximately 127.35 mg/L of D-allose after 84 h. Our achievements in the fermentative production of D-allose in this work may further promote the green manufacturing of rare sugars.
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