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

AMB Express

Alessandra Raffaele¹  Vlada B. Urlacher¹  Thomas Hilberath²  Leonie M. Windeln³ 

[1] Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany;Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany;Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands;Institute of Biochemistry, Heinrich-Heine University Düsseldorf, Universitätsstraße 1, 40225, Düsseldorf, Germany;School of Chemistry, University of Southampton, B30, University Road, SO17 1BJ, Southampton, UK;

关键词: Cytochrome P450;    Whole-cell biotransformation;    Lyophilized cells;    Cofactor regeneration;   

DOI  :  10.1186/s13568-021-01319-0

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY