Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Abstract Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for e...

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Autores principales:	Thomas Hilberath, Alessandra Raffaele, Leonie M. Windeln, Vlada B. Urlacher
Formato:	article
Lenguaje:	EN
Publicado:	SpringerOpen 2021
Materias:	Cytochrome P450 Whole-cell biotransformation Lyophilized cells Cofactor regeneration Biotechnology TP248.13-248.65 Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/521c7ee2410c4e4f9d27a2f6deb84766
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spelling	oai:doaj.org-article:521c7ee2410c4e4f9d27a2f6deb847662021-12-05T12:08:33ZEvaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells10.1186/s13568-021-01319-02191-0855https://doaj.org/article/521c7ee2410c4e4f9d27a2f6deb847662021-12-01T00:00:00Zhttps://doi.org/10.1186/s13568-021-01319-0https://doaj.org/toc/2191-0855Abstract Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.Thomas HilberathAlessandra RaffaeleLeonie M. WindelnVlada B. UrlacherSpringerOpenarticleCytochrome P450Whole-cell biotransformationLyophilized cellsCofactor regenerationBiotechnologyTP248.13-248.65MicrobiologyQR1-502ENAMB Express, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cytochrome P450 Whole-cell biotransformation Lyophilized cells Cofactor regeneration Biotechnology TP248.13-248.65 Microbiology QR1-502
spellingShingle	Cytochrome P450 Whole-cell biotransformation Lyophilized cells Cofactor regeneration Biotechnology TP248.13-248.65 Microbiology QR1-502 Thomas Hilberath Alessandra Raffaele Leonie M. Windeln Vlada B. Urlacher Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
description	Abstract Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.
format	article
author	Thomas Hilberath Alessandra Raffaele Leonie M. Windeln Vlada B. Urlacher
author_facet	Thomas Hilberath Alessandra Raffaele Leonie M. Windeln Vlada B. Urlacher
author_sort	Thomas Hilberath
title	Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
title_short	Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
title_full	Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
title_fullStr	Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
title_full_unstemmed	Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells
title_sort	evaluation of p450 monooxygenase activity in lyophilized recombinant e. coli cells compared to resting cells
publisher	SpringerOpen
publishDate	2021
url	https://doaj.org/article/521c7ee2410c4e4f9d27a2f6deb84766
work_keys_str_mv	AT thomashilberath evaluationofp450monooxygenaseactivityinlyophilizedrecombinantecolicellscomparedtorestingcells AT alessandraraffaele evaluationofp450monooxygenaseactivityinlyophilizedrecombinantecolicellscomparedtorestingcells AT leoniemwindeln evaluationofp450monooxygenaseactivityinlyophilizedrecombinantecolicellscomparedtorestingcells AT vladaburlacher evaluationofp450monooxygenaseactivityinlyophilizedrecombinantecolicellscomparedtorestingcells
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Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

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