Brian J. Curtis

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.

Diversification of structurally complex natural products remains a key challenge in the discovery of next-generation therapeutics. Premarineosin A, a potent and selective antimalarial natural product, is a promising yet underexplored scaffold due to limited availability and synthetic complexity. In this work, we overcome both barriers by coupling metabolic engineering with late-stage derivatization, enabling the first systematic exploration of the premarineosin A scaffold. Rational engineering of Streptomyces eitanensis , encoding a (−)-premarineosin A biosynthetic gene cluster, increased titers over 200-fold. Sustainable production of (−)-premarineosin A enabled a unique semi-synthetic and biocatalytic derivatization campaign. In this first structure-activity relationship study of premarineosin A, we accessed a suite of novel analogs, including a C12-brominated derivative with nanomolar potency (EC50 < 5 nM). This work establishes (−)-premarineosin A as a tractable and evolvable antimalarial scaffold, demonstrating how chemical biology approaches can unlock new structural and pharmacological space from complex microbial metabolites.

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:

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression ^1–4 , but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans . Untargeted metabolomics of a β-oxidation mutant, acdh-11 , in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a β- cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli . Screening for structurally related endogenous metabolites revealed a β-methyl fatty acid, bemeth#1, whose activity mimics that of microbiota-dependent becyp#1, but is derived from a methyltransferase, fcmt-1 , that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated β-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation.