lytic functionality of recombinant P450-containing resting cells, (ii) lyophilized recombinant E. coli cells may be employed for P450-mediated biocatalysis, when (iii) metabolism-independent regeneration of NAD(P)H is ensured. The usage of these procedures illustrates interesting perspectives for practical applications of cytochrome P450s for singleor multi-step reactions.Abbreviations CYP: Cytochrome P450, Pdr:putidaredoxin reductase from Pseudomonas putida.; Pdx: Putidaredoxin from Pseudomonas putida.; Re-ADH: Alcohol dehydrogenase from Rhodococcus erythropolis.; NADH: Nicotinamide adenine dinucleotide..Supplementary InformationThe on the web version contains supplementary material obtainable at doi. org/10.1186/s13568-021-01319-0. Further file 1: Table S1: Synthetic oligonucleotides for cloning. Table S2: Plasmids made use of within this study. Table S3: Evaluation of testosterone 1 and metabolites 2-10 that have been formed for the duration of CYP105D-mediated oxidation. Figure S1: Effect of freezing and glycerol addition for the duration of lyophilization on conversion catalyzed by E. coli C43 (DE3) pET22b-cyp105D + pCOLADuet- pdx-pdr-adh. E. coli cells were after or twice frozen at – 80 and then lyophilized for either 24 h (black) or 48 h (grey). Figure S2: Exemplary LC/MS-chromatogram showing the oxidation of testosterone 1 towards the products 2-10 by the CYP105D-based E. coli whole-cell biocatalyst (pink) in comparison to a damaging manage (black). Figure S3: SDS-PAGE analysis of E. coli C43 (DE3) strains for whole-cell biocatalysis. Figure S4: Impact of NADH addition on testosterone 1 conversion mediated by the lyophilized whole-cell catalyst without the need of ADH. NADH was added up to 4 times (number in brackets) just about every two h. Acknowledgements We thank Sebastian H zel for technical assistance. Authors’ contributions TH planned, developed and carried out most experiments, analyzed all the data, and drafted the manuscript. AR performed and evaluated experiments with diverse preparations of P450 whole-cell CYP26 Inhibitor Species catalysts. LMW contributed in initial experimental design and style with regard to building of expression vectors, gene expression and activity measurements. VBU gave advices in the study function, helped in drafting the manuscript, and revised the manuscript. All authors read and approved the final manuscript. Funding Open Access funding enabled and organized by Projekt DEAL. Economic help was kindly provided by the Federal Ministry of Education and Analysis [Grant Quantity 031A223A] beneath the umbrella from the ERA-IB2 3rd call project `HyPerIn’ [Project Number EIB.12.026]. Availability of data and supplies All information generated or analyzed through this study are integrated in this published article and its Further files.DeclarationsEthics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Institute of Biochemistry, Heinrich-Heine University D seldorf, Universit sstra 1, 40225 D seldorf, Germany. 2 Present Address: Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ Delft, The Netherlands. 3 Present Address: School of Chemistry, University of Southampton, B30, University Road, SO17 1BJ Southampton, UK. Estrogen receptor Agonist Gene ID Received: 12 September 2021 Accepted: 16 NovemberHilberath et al. AMB Express(2021) 11:Web page ten ofReferences Abokitse K, Hummel W (2003) Cloning, sequence analysis, and heterologous expression on the gene encoding a (S)-specific alcohol dehydrogen