Plant-based expression of human cytochromes for oxidative biotransformation and production of novel compounds
Growing demand for easily available sources of small molecules with potential pharmacological activities as well as for feasible, inexpensive and environment-friendly regio- and stereoselective modification of known compounds could be realized via the junction of heterologous enzymes and substrates in biological synthetic systems. In this context, animal liver cytochromes P450 (CYPs) could be regarded as promising candidates because they participate in diverse reactions of xenobiotic detoxification and have broad substrate specificity.
The effectiveness of membrane-bound eukaryotic CYPs in heterologous surroundings is not always satisfactory because of low stability and activity, as well as dependence on the associated electron-transfer partner. Despite of the increasing number of applications of native or engineered CYPs for the oxidation of aromatic and aliphatic substrates, efficient expression of eukaryotic membrane-bound CYPs as biocatalysts still represents a challenging task.
Plants provide an attractive assortment of prospective host systems for foreign CYPs expression due to the abundance of potential redox partners, including members of photosynthesis-related electron transport chains. We compared constitutive (transgenic hairy roots and transplastomic plants) and transient systems for human CYPs (CYP2D6 and CYP3A4) expression for biotransformation of synthetic and plant-derived substrates. As a result, the most efficient conversion (~10-60% of the introduced substrate) was registered after Agrobacterium-mediated transient expression of the CYPs in Nicotiana benthamiana, although transplastomic N. tabacum plants displayed a higher level of the corresponding mRNA accumulation. We put forth a convenient protocol for rapid assessment of metabolites as potential substrates for human CYPs and validated it against synthetic substrate loratadine and several plant indole alkaloids with different structural scaffolds. Novel indole alkaloids were detected and isolated, confirming that the described protocol could be a promising alternative to the currently existing CYP expression systems.
The authors are grateful for financial support to Alexander von Humboldt Foundation (Georg Forster Research Fellowship (HERMES) for experienced researchers 2015 to YVS).