Constructing an artificial short route for cell-free biosynthesis of the phenethylisoquinoline scaffold

Plant-originated natural products are important drug sources. However, total biosynthesis of these compounds is often not achievable due to their uncharacterized, lengthy biosynthetic pathways. In nature, phenethylisoquinoline alkaloids (PIAs) such as colchicine are biosynthesized via a common precursor 6,7-dihydroxy-1-(4-hydroxyphenylethyl)-1,2,3,4-tetrahydroisoquinoline (i.e., phenethylisoquinoline scaffold, PIAS). PIAS is naturally synthesized in plants by using two upstream substrates (l-phenylalanine and l-tyrosine) catalyzed by eight enzymes. To shorten this native pathway, here we designed an artificial route to synthesize PIAS with two enzymatic steps from two alternative substrates of 3-(4-hydroxyphenyl) propanol (4-HPP) and dopamine. In the two-step bioconversion, an alcohol dehydrogenase selected from yeast (i.e., ADH7) was able to oxidize its non-native alcohol substrate 4-HPP to form the corresponding aldehyde product, which was then condensed with dopamine by the (S)-norcoclaurine synthase (NCS) to synthesize PIAS. After optimization, the final enzymatic reaction system was successfully scaled up by 200 times from 50 μL to 10 mL, generating 5.4 mM of PIAS. We envision that this study will provide an easy and sustainable approach to produce PIAS and thus lay the foundation for large-scale production of PIAS-derived natural products.