Engineering a Biosynthetic Pathway to Produce (+)-Brevianamides A and B.

The privileged fused-ring structure comprising the bicyclo[2.2.2]diazaoctane (BDO) core is prevalent in diketopiperazine (DKP) natural products that exhibit potent and diverse biological activities. Typically, only low yields of these compounds can be extracted from native fungal producing strains and accessing them diastereoselectively remains challenging using available synthetic routes. BDO-containing DKPs including (+)-brevianamides A and B are assembled via multi-enzyme biosynthetic pathways incorporating non-ribosomal peptide synthetases, prenyltransferases, flavin monooxygenases, cytochromes P450 and isomerases. To simplify access to this class of alkaloids, we designed an engineered biosynthetic pathway in , composed of six enzymes sourced from different kingdoms of life. The pathway includes a cyclodipeptide synthase from sp. CMB-MQ030 (NascA), cyclodipeptide oxidase from sp. F5123 (DmtD2/DmtE2), prenyltransferase from sp. MF297-2 (NotF), flavin-dependent monooxygenase from (BvnB), and kinases from and (PhoN and IPK). Cultivated in glycerol media supplemented with prenol, the engineered strain produces 5.3 mg/L of (-)-dehydrobrevianamide E , which undergoes a previously reported lithium hydroxide rearrangement cascade to yield and , with a combined 70% yield and a 94:6 diastereomeric ratio. Additionally, titers of were increased to 20.6 mg/L by enhancing NADPH pools in the engineered strain. Overall, our study combines biosynthetic pathway engineering and chemical synthesis approaches to generate complex indole alkaloids.