Samantha P. Kelly

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 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 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.

The privileged fused-ring system comprising the bicyclo[2.2.2]diazaoctane (BDO) core is prevalent in diketopiperazine (DKP) natural products with potent and diverse biological activities, with some being explored as drug candidates. Typically, only low yields of these compounds can be extracted from native fungal producing strains and the available synthetic routes remain challenging due to their structural complexity. BDO-containing DKPs including (+)-brevianamides A and B are assembled via multi-component biosynthetic pathways incorporating non-ribosomal peptide synthetases, prenyltransferases, flavin monooxygenases, cytochrome P450s and semi-pinacolases. 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 (NascA), a cyclodipeptide oxidase (DmtD2/DmtE2), a prenyltransferase (NotF), a flavin-dependent monooxygenase (BvnB), and kinases (PhoN and IPK). Cultivated in glycerol supplemented with prenol, the engineered E. coli strain produces 5.3 mg/L of (-)-dehydrobrevianamide E ( 4 ), which undergoes a terminal, ex vivo lithium hydroxide catalyzed rearrangement reaction to yield (+)-brevianamides A and B with a 46% yield and a 92:8 diastereomeric ratio. Additionally, titers of 4 were increased eight-fold by enhancing NADPH pools in the engineered E. coli strain. Our study combines synthetic biology, biocatalysis and synthetic chemistry approaches to provide a five-step engineered biosynthetic pathway for producing complex indole alkaloids in E. coli . Abstract Figure:

Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF’s substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an “induced fit docking (IFD)-guided” engineering strategy improved turnover of our largest substrates. We further demonstrated the utility of PT NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield.

The fungal indole alkaloids are a unique class of complex molecules that have a characteristic bicyclo[2.2.2]diazaoctane ring and frequently contain a spiro‐oxindole moiety. While various strains produce these compounds, an intriguing case involves the formation of individual antipodes by two unique species of fungi in the generation of the potent anticancer agents (+)‐ and (−)‐notoamide A. NotI and NotI′ have been characterized as flavin‐dependent monooxygenases that catalyze epoxidation and semi‐pinacol rearrangement to form the spiro‐oxindole center within these molecules. This work elucidates a key step in the biosynthesis of the notoamides and provides an evolutionary hypothesis regarding a common ancestor for production of enantiopure notoamides.

The paraherquamides are potent anthelmintic natural products with complex heptacyclic scaffolds. One key feature of these molecules is the spiro-oxindole moiety that lends a strained three-dimensional architecture to these structures. The flavin monooxygenase PhqK was found to catalyze spirocycle formation through two parallel pathways in the biosynthesis of paraherquamides A and G. Two new paraherquamides (K and L) were isolated from a ΔphqK strain of Penicillium simplicissimum, and subsequent enzymatic reactions with these compounds generated two additional metabolites, paraherquamides M and N. Crystal structures of PhqK in complex with various substrates provided a foundation for mechanistic analyses and computational studies. While it is evident that PhqK can react with various substrates, reaction kinetics and molecular dynamics simulations indicated that the dioxepin-containing paraherquamide L is the favored substrate. Through this effort, we have elucidated a key step in the biosynthesis of the paraherquamides and provided a rationale for the selective spirocyclization of these powerful anthelmintic agents.