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Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

  • Fermentation, Cell Culture and Bioengineering
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E. coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710 mg L−1) by 2,520 mg L−1, with the extracellular enzyme activity being improved to 55.3 U mL−1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were −19.10 kcal mol−1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326 % that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.

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Acknowledgments

This work was supported by grants from the Major State Basic Research Development Program of China (973 Program, 2012CB720806), the National High Technology Research and Development Program of China (863 Program, SS2012AA023403, 2011AA100905), the Open Project Program of the Key Laboratory of Industrial Biotechnology, Ministry of Education, China (no. KLIB-KF201006), the National Natural Science Foundation of China (31000807), the Natural Science Foundation of Jiangsu Province (BK2011004), the Enterprise-university-research prospective program, Jiangsu Province (BY2009112), the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, the Key Technologies R & D Program of Jiangsu Province, China (BE2011624), and the Fundamental Research Funds for the Central Universities (JUSRP211A29).

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Authors and Affiliations

  1. National Engineering Laboratory for Cereal Fermentation Technology (NELCF), Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Hua Liu, Jianghua Li, Guocheng Du, Jingwen Zhou & Jian Chen

  2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Rd, Shanghai, 200237, China

    Jingwen Zhou

  3. School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Jingwen Zhou & Jian Chen

Authors
  1. Hua Liu
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  2. Jianghua Li
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  3. Guocheng Du
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  4. Jingwen Zhou
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  5. Jian Chen
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Corresponding authors

Correspondence to Jingwen Zhou or Jian Chen.

Electronic supplementary material

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10295_2012_1185_MOESM1_ESM.pdf

Online Resource 1: Comparison of the wild-type (wt, GenBank accession no. AAC04359.1) α-cgt and codon-optimized (co, GenBank accession no. JX412224)α-cgt (* unchanged base pairs). (PDF 87 kb)

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Liu, H., Li, J., Du, G. et al. Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization. J Ind Microbiol Biotechnol 39, 1841–1849 (2012). https://doi.org/10.1007/s10295-012-1185-y

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  • DOI: https://doi.org/10.1007/s10295-012-1185-y

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