Untersuchung und Optimierung von CYP450-exprimierenden Mikroorganismen für Wirkstoffderivatisierungsstudien und Produktion von Wirkstoffmetaboliten
Zusammenfassung der Projektergebnisse
The activity and stability of microbial biocatalyst containing human cytochrome P450 monooxygenases (CYP450s) is crucial for an application in screening studies for drug metabolism and discovery as well as the production of the drug metabolites. In order to finally achieve stable bioprocesses, a detailed characterization and optimization of the catalyst was performed in this study. A major focus was the establishment of a reliable method for relative and absolute quantification of CYP450s levels in the microbial host. Quantification of proteins in biological and clinical samples is a common approach in proteomics. For targeted protein quantification, peptide separation via HPLC followed by selected reaction monitoring on triple quadrupole mass spectrometers is usually used. Within this project we established a new quantification method for heterologously produced human CYP3A4 isoforms. HPLC separation was omitted by combining direct infusion with single ion monitoring (SIM) on a Q Exactive mass spectrometer. Direct-infusion SIM revealed only 14.7% (± 4.1 (s.e.m.)) deviation on average. In addition, a decreased processing and analysis time of 4.5 minutes for one sample was obtained, compare to 65 minutes for HPLC-SIM. The direct-infusion SIM approach was used to investigate the influence of different culture conditions on CYP450 expression. The successful application of direct infusion-SIM for (absolute) quantification of proteins in complex samples consisting of microbial membranes as shown in this study might in future be extended for quantification of abundant and/or pre-enriched proteins in different complex biological samples. The method developed can also be used for a fast relative quantification, e.g. to determine the influence of different expression conditions on concentration of several proteins within one sample/strain. Finally, direct infusion-SIM might be used for quantification of peptides difficult to quantify so far, such as highly hydrophilic or hydrophobic, or rather long peptides (30-50 amino acids). This study showed that E. coli is the most suitable host for the production of and catalysis with human CYP450s. Often, gene 5’-optimization for correct insertion into the bacterial membrane appeared to be necessary for protein production in E. coli. In addition, enzyme levels can be increased selection of optimal reaction conditions, such as low-dissolved oxygen levels during enzyme synthesis and the addition of tetracycline inducing a cold-shock response. This study showed that the stability of the CYP450 biocatalysts is limited to 1h and the limited stability could not be linked directly to deactivation of the enzyme or cell metabolism. However, several attempts to increase CYP450 stability were so far not successful. For testosterone biotransformation with E. coli containing CYP3A4, the specific activity of the biocatalyst could be increased by the addition of sucrose and EDTA, which relieved the substrate uptake barrier of bacterial outer membrane. Despite the increased initial activities in the first minutes of biotransformation, the stability of the biocatalyst was reduced resulting in similar final product titer as without sucrose/EDTA addition. On the other hand, genetic modification of the biocatalyst led to higher maximal whole-cell activities and to higher final product titers as observed before. An initial scale-up of the biocatalyst production and the biotransformation to bench scale bioreactors showed that the findings of the shaking flask experiments could be transferred to larger scale. However, also here the biocatalyst showed a stability of maximally 1h although active enzyme was still present and the cells still metabolically active. Altogether, we here demonstrated the potential of a fast, easy (no need for optimization of analysis parameters or data evaluation), robust, and reproducible direct infusion-SIM approach for protein quantification in complex microbial samples. E. coli can be used as a biocatalyst for human CYP450s, but the limited stability of the biocatalyst is an inherent and remaining problem of the whole-cell biocatalysts and should be taken into account for industrial application.
Projektbezogene Publikationen (Auswahl)
- (2012) Genome mining for biocatalytic oxyfunctionalizations: the oxygenase is not enough. GETGEOWEB - 1st Transnational Workshop, 29. - 30. October 2012, Freiberg, Germany
Julsing M. K., Schrewe M., Kuhn D., Cornelissen S., Bühler B. and Schmid A.
- (2012). The efficiency and productivity of P450 monooxygenases in biocatalysis. 11th International symposium on cytochrome P450. Biodiversity and Biotechnology., 22. - 26. June 2012, Turin, Italy
Schmid A., Rensmann (Debor) L., Cornelissen S., Bühler B. and Julsing M. K.
- Enzyme titer and specific activity of recombinant human CYP450 1A1 and 2D6 in function of cell physiology in a whole-cell setup. 11th International symposium on cytochrome P450: Biodiversity and Biotechnology, 22. - 26. June 2012, Turin, Italy
Rensmann (Debor) L., Maaß M., Teubner N.C., Bühler B., Julsing M. K. and Schmid A.
- Oxygenasebased catalysis of hydrophobic substrates using recombinant E. coli cells containing AlkL. European Congress of Applied Biotechnology, 21.- 25. April 2013, the Hague, the Netherlands
Julsing M.K., Schrewe M., Rensmann L., Cornelissen S., Schmid A., Bühler B.
- Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples. EuPA Open Proteomics
Volume 7, June 2015, Pages 20-26
Christina Looße, Sara Galozzi, Linde Debor, Mattijs K. Julsing, Bruno Bühler, Andreas Schmid, Katalin Barkovits, Thorsten Müller, Katrin Marcus
(Siehe online unter https://doi.org/10.1016/j.euprot.2015.03.001)
