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Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

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Abstract

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).

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Acknowledgements

This work was supported by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía through the Project for Excellence P06-01399 (2006) and the PAIDI group CTS569; and the Co-operation Office of European Union in the LFA program called Bioprocess: Clean Technologies in the protection and sustainability of the environment with contract no.: AML/190901/06/18414/II-0548-FC-FA. The authors wish to thank PhD J.L. Ramos of the Consejo Superior de Investigaciones Científicas (CSIC) who provided the E. coli strain AB502NemA and Guillermo Gosset from the Instituto de Biotecnología from the Universidad Nacional Autónoma de Mexico (UNAM) for providing the detailed protocol for gene inactivation. Jorge Yañéz is acknowledged by primers synthesis.

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Authors and Affiliations

  1. Department of Chemical Engineering and Food Technology, Campus de Excelencia Internacional Agroalimentario (ceiA3), University of Cádiz, Avda. República Saharaui s/n, 11510, Puerto Real, Cádiz, Spain

    Antonio Valle, Gema Cabrera & Domingo Cantero

  2. Department of Process and Technology, Autonomous Metropolitan University–Cuajimalpa, Artificios 40, 01120, México, DF, Mexico

    Sylvie Le Borgne

  3. Department of Biomedicine, Biotechnology and Public Health, Campus de Excelencia Internacional Agroalimentario (ceiA3), University of Cádiz, Avda República Saharui s/n, 11510, Puerto Real, Cádiz, Spain

    Jorge Bolívar

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  1. Antonio Valle
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  2. Sylvie Le Borgne
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  3. Jorge Bolívar
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  4. Gema Cabrera
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  5. Domingo Cantero
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Correspondence to Antonio Valle.

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A. Valle, S. Le Borgne and J. Bolívar have contributed equally to this work.

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Valle, A., Le Borgne, S., Bolívar, J. et al. Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties. Appl Microbiol Biotechnol 94, 163–171 (2012). https://doi.org/10.1007/s00253-011-3787-0

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