Benzylisoquinoline alkaloid production in yeast via norlaudanosoline improves selectivity and yield

Abstract

The benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines (THIQs) comprises over 2,500 members, including the pharmaceuticals morphine, codeine, and papaverine as well as the antibiotics sanguinarine and chelerythrine used in animal husbandry. Agricultural cultivation can currently supply the demand for the BIAs that accumulate in plants, but broader access to the entire BIA family would open new avenues of research and commercialization. Microbial synthesis presents an attractive option due to cheap feedstock, genetic tractability, and ease of scale-up. Previously we reported titers of the key branch-point BIA (S)-reticuline of 4.6 g/L in yeast, which was achieved through leveraging the Ehrlich pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate 4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior route to (S)-reticuline by switching the pathway intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA) using human monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis is more selective, resolving prior issues with off-pathway THIQs synthesized due to concerted enzyme promiscuity. The new pathway is also more efficient, enabling titers of 4.8 g/L (S)-reticuline while improving yields over 40%, from 17 mg/g sucrose to 24 mg/g sucrose in fed-batch fermentations. Finally, we extend de novo (S)-reticuline synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine and sanguinarine in fed-batch fermentation the highest reported titer of these BIAs by a factor of 40. HighlightsO_LIMonoamine oxidase A (MAO) supports high-titer (S)-reticuline synthesis in yeast C_LIO_LIMAO route to (S)-reticuline improves specificity compared to Aro10 route C_LIO_LIThis work represents a 40% increase in highest reported (S)-reticuline yield C_LIO_LI653 mg/L (dihydro-) sanguinarine was produced by extending the pathway C_LI