Bioprocess Engineering for the Application of P450s

Biocatalytic processes are advancing because of their high selectivity and mild operating conditions, in contrast to many chemical catalyzed processes. This is a clear advantage and frequently results in improved environmental performance. Biocatalytic processes have been implemented replacing traditional chemical catalysts as well as enabling new synthesis. Regardless of the process routes, the economic feasibility is crucial for successful industrial implementation. This has also been demonstrated by implemented biocatalytic processes, showing a clear cost advantage compared to the chemical alternative.
One family of enzymes described to have a lot of potential for industrial biocatalysis is cytochrome P450 monooxygenases. The main motivation for this statement is their ability to hydroxylate nonactivated hydrocarbons in a specific manner, using molecular oxygen as oxidant. Containing more than 26 000 enzymes, this family includes diverse enzymes from all kingdoms of life. However, their dependence on cofactor, redox partners and relatively low activity and stability hinders the development of efficient processes. In this thesis, a novel systematic approach has been applied to identify bottlenecks for economically feasible whole cell P450 catalyzed processes to direct research and enable faster implementation. A methodological approach was introduced by reviewing literature based on guidance by economic metrics, followed by cases studies to confirm the initial analysis. The last part of the thesis consists of an economic assessment based on a process model using experimentally gained knowledge, including a sensitivity analysis of the biological parameters protein expression and enzyme total turnover.
Case studies of various complexities have been chosen throughout the thesis. The first case study was performed using a P450 fusion construct expressed in the well explored host Escherichia coli performing ω‐hydroxylation of dodecanoic acid. This system represents an artificial fusion construct in a non‐natural P450 expressing host. The main limitations in this case were identified to be the stability and activity of the P450, cofactor regeneration by the host cell and substrate inhibition. The latter was partially circumvented by the introduction of substrate in solid form. The second case study utilized a naturally expressing P450 host, Bacillus megaterium, expressing the steroid hydroxylase CYP106A2 for 15β‐hydroxylation of cyproterone acetate. The catalytic activity of the overexpressed CYP106A2 was dependent on the natural redox partners in the host cell. The stability of the P450 was also here identified as one of the limitations as well as product inhibition. Product inhibition was in this case addressed by introducing a modified β‐cyclodextrin, yielding 98 % conversion in the gram scale.
P450 catalyzed whole cell processes have been identified suitable for production of high value molecules. The main limitations have been shown to be P450 stability and activity, substrate and product inhibition and cofactor regeneration of heterologous expression host. Furthermore, growing cells, where fermentation and biocatalysis is performed in one step is shown to be the most economically feasible option.