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 5 and B 6 are assembled via multienzyme biosynthetic pathways incorporating nonribosomal peptide synthetases, prenyltransferases, flavin monooxygenases, cytochromes P450, and isomerases. To simplify access to this class of alkaloids, we designed an engineered biosynthetic pathway in Escherichia coli, composed of six enzymes sourced from different kingdoms of life. The pathway includes a cyclodipeptide synthase from Streptomyces sp. CMB-MQ030 (NascA), cyclodipeptide oxidase from Streptomyces sp. F5123 (DmtD2/DmtE2), prenyltransferase from Aspergillus sp. MF297–2 (NotF), flavin-dependent monooxygenase from Penicillium brevicompactum (BvnB), and kinases from Shigella flexneri and Thermoplasma acidophilum (PhoN and IPK). Cultivated in glycerol media supplemented with prenol, the engineered E. coli strain produces 5.3 mg/L of (−)-dehydrobrevianamide E 4, which undergoes a previously reported lithium hydroxide rearrangement cascade to yield 5 and 6, with a combined 70% yield and a 94:6 diastereomeric ratio. Additionally, titers of 4 were increased to 20.6 mg/L by enhancing NADPH pools in the engineered strain. Overall, our study combines de novo biosynthetic pathway engineering and chemical synthesis approaches to generate complex indole alkaloids.