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

Leonie M Windeln,Thomas Hilberath,Vlada B Urlacher,Alessandra Raffaele

doi:10.1186/s13568-021-01319-0

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.

Highlights

Cytochromes P450 (CYPs or P450s) are versatile hemecontaining enzymes that catalyze oxidation reactions in the presence of molecular oxygen and NAD(P)H
Construction of the whole‐cell system with CYP105D and redox partners The gene coding for CYP105D from S. platensis was coexpressed with a redox partner system consisting of the Nicotinamide adenine dinucleotide (NADH)-dependent putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from two plasmids (Fig. 2A)
The gene cyp105D was cloned in the pET22b-vector, whereas pdr and pdx were integrated in the first multiple cloning site of the pCOLADuet-vector

Summary

Introduction

Cytochromes P450 (CYPs or P450s) are versatile hemecontaining enzymes that catalyze oxidation reactions in the presence of molecular oxygen and NAD(P)H Due to their ability to introduce one atom of molecular oxygen into a vast variety of organic molecules under mild reaction conditions with often high chemo- and regioselectivity, these enzymes have been recognized as attractive targets with high potential for biotechnological applications (Bernhardt 2006; Girvan and Munro 2016; Kelly and Kelly 2013; Lundemo and Woodley 2015). To increase substrate solubility organic solvents are often added, which might negatively affect the whole-cell biocatalysts either To this end, usage of lyophilized recombinant microbial cells carrying the target enzymes has been reported as an attractive alternative to both, microbial cells and isolated enzymes, because they allow working at high organic solvent concentrations and do not face the problem of substrate transport through the membrane (Jakoblinnert and Rother 2014)

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