Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III inCorynebacterium glutamicum
Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III inCorynebacterium glutamicum
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利用 BioBrick 部件构建 3A 系统，用于在谷氨酸棒杆菌中表达重组水蛭素变体 III
DOI：
10.1007/s00253-020-10835-1
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发表时间：
2020-08-25
影响因子：
5
通讯作者：
Bai，Zhonghu
中科院分区：
文献类型：
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作者：
Wang，Yali;Gao，Xiong;Bai，Zhonghu
使用三抗生素（3A）系统，标准化部件可以有效地组装成新型生物系统，确保组件的可重复使用性和实验的可重复性。在本研究中，我们构建了谷氨棒状杆菌3A表达系统，以便于构建基因表达盒。Glutamicum)，用于筛选启动子和信号肽的组合，以提高secretedrhv3的产生。我们首先从所有启动子中获得了三个强启动子P2252、Podhi和pywea，它们驱动绿色荧光蛋白（egfp）的最高表达。利用本研究开发的3A表达系统，将3个启动子与不同的信号肽进行组装，生成一系列构建体，其中PyweA-CspA-rhv3的最高活性达到3187.5 ATU/L。利用5-L罐式生物反应器进一步提高了rhv3r的产量，实现了大规模发酵，培养40 h后，rhv3r的最高积累量为1.21 g/L，高于inE报道的0.95 g/L。杆菌。据我们所知，这是第一个关于rhv3secretory expression inC. glutamicum的报道，它可以应用于其他具有重要应用价值的重组蛋白的生产。•利用3A系统对谷氨酸酵母进行遗传操作。•构建了谷氨酰胺标准序列的元素库。•通过3A系统在谷氨酸酵母中实现rhv3的分泌表达。
Standardized parts can be efficiently assembled into novel biological systems using the three antibiotic (3A) system, ensuring the reusability of components and repeatability of experiments. In this study, we created the 3A expression system for easy construction of gene expression cassettes inCorynebacterium glutamicum(C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secretedrhv3production. We first obtained three strong promoters P2252, Podhi, and PyweAfrom all of promoters, which drive the highest expression of green fluorescent protein (egfp). The three promoters were then assembled with different signal peptides to generate a series of constructs using the 3A expression system developed in this study, from which the highest activity ofrhv3reached 3187.5 ATU/L of PyweA-CspA-rhv3. Further increased production of rhv3achieved large-scale fermentation using 5-L jar bioreactor, with the highestrhv3accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported inE. coli. To the best of our knowledge, this is the first report ofrhv3secretory expression inC. glutamicum, which could be applied for the production of other recombinant proteins with significant applications.Key points• We have exploited a 3A system for the genetic manipulation in C. glutamicum.• We constructed element libraries for assembling standard sequence in C. glutamicum.• The secreted expression of rhv3 was realized by 3A system in C. glutamicum.