Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III in Corynebacterium gl
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants III in Corynebacterium glutamicum Yali Wang 1,2,3 & Xiong Gao 4 & Xiuxia Liu 1,2,3 & Ye Li 1,2,3 & Manman Sun 1,2,3 & Yankun Yang 1,2,3 & Chunli Liu 1,2,3 & Zhonghu Bai 1,2,3 Received: 19 June 2020 / Revised: 22 July 2020 / Accepted: 14 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 in Corynebacterium glutamicum (C. glutamicum), which was applied to screen combinations of promoters and signal peptides for improved secreted rhv3 production. We first obtained three strong promoters P2252, Podhi, and PyweA from 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 of rhv3 reached 3187.5 ATU/L of PyweA-CspA-rhv3. Further increased production of rhv3 achieved large-scale fermentation using 5-L jar bioreactor, with the highest rhv3 accumulation 1.21 g/L obtained after 40 h of cultivation, which is higher than 0.95 g/L reported in E. coli. To the best of our knowledge, this is the first report of rhv3 secretory expression in C. 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. Keywords BioBrick . Corynebacterium glutamicum . Promoter . Signal peptide . rhv3 Xiong Gao is the co-first author. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10835-1) contains supplementary material, which is available to authorized users. * Xiuxia Liu [email protected]
Yankun Yang [email protected]
* Zhonghu Bai [email protected]
Chunli Liu [email protected]
Yali Wang [email protected]
1
The Key Laboratory of Industrial Biotechnology, Jiangnan University, Wuxi 214122, China
Xiong Gao [email protected]
2
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China
Ye Li [email protected]
3
Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi 214122, China
Manman Sun [email protected]
4
Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China
Appl Microbiol
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