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Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution.

Authors
  • Liu, Shan1
  • Wang, Juan1
  • Zhu, Zhiguang1
  • Shi, Ting1
  • Zhang, Yi-Heng P Job1
  • 1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China. , (China)
Type
Published Article
Journal
Biotechnology and Bioengineering
Publisher
Wiley (John Wiley & Sons)
Publication Date
Oct 01, 2020
Volume
117
Issue
10
Pages
2957–2968
Identifiers
DOI: 10.1002/bit.27478
PMID: 32589796
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis. © 2020 Wiley Periodicals LLC.

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