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

• Published in: AMB Express Vol. 11; no. 1; p. 162
• Main Authors: Hilberath, Thomas, Raffaele, Alessandra, Windeln, Leonie M., Urlacher, Vlada B.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 04-12-2021; Springer Nature B.V; SpringerOpen
• Subjects: Alcohol dehydrogenase; Biocatalysts; Biomedical and Life Sciences; Biotechnology; Catalysis; Catalysts; Cofactor regeneration; Cofactors; Conversion; Cytochrome P450; Cytochromes P450; E coli; Enzymes; Life Sciences; Lyophilized cells; Microbial Genetics and Genomics; Microbiology; Microorganisms; Monooxygenase; Nicotinamide; Original; Original Article; Oxidation; Regeneration; Rhodococcus; Substrates; Testosterone; Whole-cell biotransformation; Whole-cell biotransformation; Lyophilized cells; Cofactor regeneration; Cytochrome P450
• ISSN: 2191-0855; 2191-0855
• DOI: 10.1186/s13568-021-01319-0

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.