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 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.

Towards Biocatalytic Oxidation of Secondary Alcohols to Carbonyl Products of Relevance for Flavors and Fragrances

“…Addition of 0.5 or 1 mM of NADP + to the aqueous phase increased the conversion from 69% (in the absence of additional NADP + ) to more than 86% (in the presence of additional NADP + ). The increase in conversion might hint that either the NADP + amount in lyophilised E. coli cells is limited for achieving higher conversions or that during lyophilisation a part of the intracellular NADP + was degraded, as it was seen in previous studies (Zehentgruber et al, 2010;Hilberath et al, 2021).…”

Unique alcohol dehydrogenases involved in algal sugar utilization by marine bacteria

“…For instance, in Z. galactanivorans , the genes encoding the key enzymes of the ribulose monophosphate pathway are upregulated in the presence of porphyran (Brott et al 2022 ), so an accumulation of formaldehyde is unlikely. Eventually, the ADHs might have a completely different biological function, such as the regeneration of NADH (Hilberath et al 2021 ; Kokorin et al 2021 ). In the oxidative demethylation reaction, NADH is oxidized to NAD + , and a reduced growth in the ADH knockout strain due to cofactor depletion might be possible.…”

Performance of single-cell ICP-MS for quantitative biodistribution studies of silver interactions with bacteria

“…A part of the bacterial samples was subjected to lyophilisation and fixation with paraformaldehyde. 34 Samples exposed to 1 mg L −1 of silver( i ) or 2 mg L −1 of 10 nm silver nanoparticles were centrifuged at 6000 g during 3 min to remove the PBS supernatant. A 5% (w/v) glycerol solution in PBS was added to the pellet and let it work for 2 hours.…”