Cytochrome P450-mediated biotransformation of noscapine
PAR-Chemical Engineering 1-G20
Chemical and Biomolecular Engineering Building 1
Abstract: Cytochrome P450 enzymes (P450s) are promising enzymes for industrial and pharmaceutical biotechnology due to their ability to catalyse selective oxidations that are often difficult to achieve with synthetic organic chemistry. In the context of pharmaceutical development, the regio- and stereo-selective modification of natural products or other drug candidates by P450s can lead to new classes of drug analogues with improved pharmacological properties. The ability of P450s to catalyse reactions under aqueous conditions at ambient temperatures is also an attractive property for the manufacture of drugs and their intermediates at process scale. This class of enzymes, however, is currently underrepresented in industrial biocatalysis due to issues with enzyme stability and cofactor requirements, as well as the poor aqueous solubility and mass transfer properties of many P450 substrates. The opium poppy alkaloid noscapine is a suitable candidate for P450-mediated functionalisation, as various modifications to the noscapine scaffold have been shown to improve its natural anticancer properties. This project aimed to investigate biocatalytic pathways to noscapine analogues using cytochrome P450s and to determine their scale-up potential using whole-cell biotransformation systems. First, a library of enzyme mutants based on the bacterial P450 enzyme P450BM3 was constructed using site-directed mutagenesis and screened for noscapine biotransformation activity. This identified several enzyme mutants capable of N-demethylation of noscapine with high selectivity. The best enzyme candidate was then implemented in a whole-cell biotransformation system. Mass transfer of substrate into cells was identified as a major process limitation and was overcome by selection of an alternative microbial host. To address the poor aqueous solubility of noscapine, a two-liquid-phase biotransformation process was developed using a systematic solvent selection process and in silico solubility screening with Hansen solubility parameters. The use of cyclodextrins and aqueous polymers as solubility enhancers was also explored. Enzyme stability, cell viability and cofactor supply were also identified as potential process bottlenecks and investigated.
Luke Richards, The University of Melbourne