(671a) Promiscuity of Dirigent Proteins and Their Application for Lignan Analog Synthesis

Lignans are phenylpropanoid-derived phytochemicals that have drawn considerable attention for their pharmacological effects and medicinal applications. For instance, the anticancer drug etoposide is semi-synthetically produced from (–)-podophyllotoxin, a highly decorated lignan product found in the Podophyllum species. Thus, sufficient production of these compounds and their analogs is of high interest for the development of drug variants with reduced side effects or improved potency. Dirigent proteins (DIR) guide stereoselective coupling of monolignol radicals and are implicated to be involved in the first committed steps in the biosynthesis of lignans. Unfortunately, DIRs have shown high substrate specificity against homo-dimerization of non-native naturally-occurring monolignols, such as p-coumaryl alcohol and sinapyl alcohol, so the use of these proteins towards analog formation has not been fully explored. Here, we show for the first time that the (+)-pinoresinol-forming dirigent protein from Podophyllum hexandrum exhibits promiscuity towards various analogs of its native substrate, coniferyl alcohol. More specifically, subjecting both naturally-occurring and synthetic monolignol variants to coupling with coniferyl alcohol in the presence of PhDIR results in a stereoselective formation of the corresponding hetero-dimers. These findings suggest previously unrealized “half-promiscuity” of dirigent proteins with coniferyl alcohol and other monolignols in nature. Furthermore, we show that the crude plant extract obtained from PhDIR-expressing Nicotiana benthamiana leaves enable access to these non-natural heterodimers with the desired stereoselectivity at a milligram scale, a high enough yield for drug screening assays and substrate-directed biosynthesis for natural product analog formation.