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Directed evolution and mutagenesis of lysine decarboxylase from Hafnia alvei AS1.1009 to improve its activity toward efficient cadaverine production

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Abstract

Lysine decarboxylase (LDC) exhibits a significant role in cadaverine (1,5-pentanediamine, diaminopentane) production from lysine. In this study, an error-prone PCR and DNA shuffling were performed to improve the activity of LDC from Hafnia alvei AS1.1009 for cadaverine production. A sensitive high-throughput screening strategy based on a pH indicator was established for directed evolution of LDC. Several improved mutants were obtained from directed evolution and LDCV147F/E583G mutant showed highest activity to catalyze lysine to cadaverine. This mutant showed 1.62-fold high LDC activity when compared to wild-type. Further analysis by site-directed mutagenesis reveled that only the mutant E583G was sufficient for higher catalytic activity. Wild type LDC and mutant LDCE583G were purified by an improved method including hydrophobic chromatography. These purified enzymes were characterized and the kinetic parameters were compared between LDCE583G and WT LDC. V max of LDCE583G was 1.32-fold higher than that of WT LDC. Use of LDCE583G mutant showed 1.48-fold improved productivity of cadaverine when compared to wild type. The concentration of cadaverine in E. coli JM109/pTrc99a-ldc2-41 was 63.9 g/L with conversion yield of 93.4% during 5 h. These results indicate that the mutation has positive effects on improving LDC activity and a potential candidate for cadaverine production.

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Authors and Affiliations

  1. College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 211-816, China

    Chen Wang, Kai Zhang, Heng Cai, Wan Honggui & Pingkai Ouyang

  2. College of Food Science and Engineering, Inner Mongolia Agricultural University, Hohhot, 010-018, China

    Chen Zhongjun

Authors
  1. Chen Wang
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  2. Kai Zhang
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  3. Chen Zhongjun
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  4. Heng Cai
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  5. Wan Honggui
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  6. Pingkai Ouyang
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Correspondence to Heng Cai.

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These author’s contributed equally to the present work.

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Wang, C., Zhang, K., Zhongjun, C. et al. Directed evolution and mutagenesis of lysine decarboxylase from Hafnia alvei AS1.1009 to improve its activity toward efficient cadaverine production. Biotechnol Bioproc E 20, 439–446 (2015). https://doi.org/10.1007/s12257-014-0690-4

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  • DOI: https://doi.org/10.1007/s12257-014-0690-4

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