Abstract
At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30 °C other than 90 °C, with a substantial decrease of 60 °C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100 g racemic vince lactam to produce 49.2 g (−) vince lactam at 30 °C within 4 h. By using chiral HPLC, the ee value of the produced (−) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (−) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.
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Acknowledgements
This work was supported by the Youth Fund of State Key Laboratory of Microbial Resources, Institute of Microbiology, CAS (To J. Wang). We thank Dr. Lihong Dou for the kind help in flow cytometry analysis and Dr. Fuquan Song for providing assistance in the NMR assay.
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J. Wang and G. Zhao contributed equally to this paper.
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Wang, J., Zhao, G., Zhang, Z. et al. Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (−) vince lactam. Appl Microbiol Biotechnol 98, 6991–7001 (2014). https://doi.org/10.1007/s00253-014-5704-9
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DOI: https://doi.org/10.1007/s00253-014-5704-9