ISBN 9783843950220

978-3-8439-5022-0, Reihe Biotechnologie

Lisa Marie Schmitz
Identification and application of novel P450s for biocatalytic processes

240 Seiten, Dissertation Technische Universität Dortmund (2021), Softcover, A5

Zusammenfassung / Abstract

Cytochrome P450 monooxygenases (P450s) are widely distributed heme enzymes. They selectively insert oxygen into many complex substrates catalyzing diverse chemical transformations. Due to their high catalytic diversity, P450s are considered promising biocatalysts for the oxyfunctionalization of high value compounds. Due to an increasing availability of genome sequences, sequenced microorganisms harbor huge potential to discover new P450s.

In this study, a combined approach of genome mining and activity screening was performed to identify wild-type strains with undescribed hydroxylation activity. A screening of 84 bacterial and fungal strains revealed more than 20 strains efficiently converting a broad range of pharmaceutical compounds. Ritonavir biotransformation by Actinosynnema mirum was further optimized and transferred to a preparative scale. A 90% subtrate conversion forming 13 metabolites was determined after 48 h of biotransformation with 19% yield and 97% purity for the main metabolite hydroxy ritonavir after product purification.

A transcriptome analysis of A. mirum identified highly expressed P450s under biotransformation conditions, which were selected for heterologous expression in E. coli. A subsequent activity testing revealed the P450 Amir_1302 to catalyze ritonavir hydroxylation forming four main metabolites, thereby resembling the human CYP3A4.

Another screening of additional pharmaceutical compounds and natural products, revealed the actinomycete Kutzneria albida as a promising strain for vitamin D2 and vitamin D3 hydroxylation forming among others the main human metbolites 25-OH-D and 1,25-(OH)2-D. The biotransformation was limited by low solubility of the substrates and addition of solubilizers, such as cyclodextrin derivatives resulted in up to 100-fold increased product concentrations obtaining a 14% yield in the formation of 25-OH-D3. Overall, the presented results contribute to the development of P450s for biotechnological application.