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Applied and Environmental Microbiology, December 2008, p. 7733-7739, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01936-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity ^,

Chao Yang,^1,2, Yaran Zhu,^1,2, Jijian Yang,^1,2 Zheng Liu,^1,2 Chuanling Qiao,^1* Ashok Mulchandani,^3* and Wilfred Chen³

State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China,¹ Graduate School of the Chinese Academy of Sciences, Beijing 100049, China,² Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521³

Received 20 August 2008/ Accepted 14 October 2008

Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.

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* Corresponding author. Mailing address for Chuanling Qiao: State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. Phone: 86-10-64807191. Fax: 86-10-64807099. E-mail: qiaocl{at}ioz.ac.cn. Mailing address for Ashok Mulchandani: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (951) 827-6419. Fax: (951) 827-5696. E-mail: adani{at}engr.ucr.edu

Published ahead of print on 24 October 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

C.Y. and Y.Z. contributed equally to this work.

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Applied and Environmental Microbiology, December 2008, p. 7733-7739, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01936-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.