High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin.
High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin.
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高细胞密度培养表达 6-O-磺基转移酶的重组大肠杆菌菌株，用于生产生物工程肝素。
DOI：
10.1111/jam.12684
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发表时间：
2015
影响因子：
4
通讯作者：
Linhardt,RJ
中科院分区：
文献类型：
--
作者：
Zhang,J;Suflita,M;Fiaschetti,CM;Li,G;Li,L;Zhang,F;Dordick,JS;Linhardt,RJ
目的在大肠杆菌中克隆并表达的六种肝素生物合成酶之一是一种可溶性融合蛋白，需要大规模制备才能用于肝素的化学酶合成，肝素是一种重要的抗凝血药物。方法与结果6‐O‐硫代转移酶异构体- 3（6‐OST‐3）可在实验室中以mg/L的浓度制备。以β - D - 1 -硫代半乳糖苷异丙基诱导，光密度为0.6 - 0.8。生产大量的6 - OST - 3需要分批喂料培养e。在含有廉价碳源（如葡萄糖或甘油）的培养基上搅拌槽发酵。e的培养。研究了不同进料时间和诱导策略下不同碳源的结肠炎。建立了条件，使活性6‐OST‐3的产量（5-20 mg g‐细胞干重−1）具有优异的生产率（2-5 mg l−1h−1）。结论6 - OST - 3的分批补料发酵在廉价碳源上的生产已被证实。研究的意义和影响扩大肝素生物合成酶（如6‐OST‐3）生产的能力对于扩大肝素化学酶合成的规模至关重要。这个项目的成功可能会在某一天催生出一种商业上可行的生物工程肝素，以取代目前市场上动物来源的抗凝血产品。
AimsOne of six heparin biosynthetic enzymes, cloned and expressed inEscherichia colias a soluble fusion protein, requires large‐scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug.Methods and ResultsThe 6‐O‐sulfotransferase isoform‐3 (6‐OST‐3) can be conveniently prepared at mg/L levels in the laboratory by culturingE. colion Luria–Bertani medium in shake flasks and inducing with isopropylβ‐D‐1‐thiogalactopyranoside at an optical density of 0·6–0·8. The production of larger amounts of 6‐OST‐3 required fed‐batch cultivation ofE. coliin a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation ofE. colion various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5–20 mg g‐cell‐dry weight−1) of active 6‐OST‐3 with excellent productivity (2–5 mg l−1h−1).ConclusionsThe production of 6‐OST‐3 in a fed‐batch fermentation on an inexpensive carbon source has been demonstrated.Significance and Impact of the StudyThe ability to scale‐up the production of heparin biosynthetic enzymes, such as 6‐OST‐3, is critical for scaling‐up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal‐sourced anticoagulant product currently on the market.