Cyanobacteria produce numerous valuable bioactive secondary metabolites (natural products) including alkaloids, isoprenoids, nonribosomal peptides, and polyketides. However, the genomic organization of the biosynthetic gene clusters, complex gene expression patterns, and low compound yields synthesized by the native producers currently limits access to the vast majority of these valuable molecules for detailed studies. Molecular cloning and expression of such clusters in heterotrophic hosts is often precarious owing to genetic and biochemical incompatibilities. Production of such biomolecules in photoautotrophic hosts analogous to the native producers is an attractive alternative that has been under-explored. Here, we describe engineering of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce key compounds of the hapalindole family of indole–isonitrile alkaloids. Engineering of the 42-kbp “fam” hapalindole pathway from the cyanobacterium Fischerella ambigua UTEX 1903 into S2973 was accomplished by rationally reconstructing six to seven core biosynthetic genes into synthetic operons. The resulting Synechococcus strains afforded controllable production of indole–isonitrile biosynthetic intermediates and hapalindoles H and 12-epi-hapalindole U at a titer of 0.75–3 mg/L. Exchanging genes encoding fam cyclase enzymes in the synthetic operons was employed to control the stereochemistry of the resulting product. Establishing a robust expression system provides a facile route to scalable levels of similar natural and new forms of bioactive hapalindole derivatives and its structural relatives (e.g., fischerindoles, welwitindolinones). Moreover, this versatile expression system represents a promising tool for exploring other functional characteristics of orphan gene products that mediate the remarkable biosynthesis of this important family of natural products.

This review covers isolation, biological activity, an overview of total synthesis efforts and recent biosynthetic discoveries related to hapalindole-type indole alkaloids.

Genome sequencing and bioinformatics tools have facilitated the identification and expression of an increasing number of cryptic biosynthetic gene clusters (BGCs). However, functional analysis of all components of a metabolic pathway to precisely determine biocatalytic properties remains time-consuming and labor intensive. One way to speed this process involves microscale cell-free protein synthesis (CFPS) for direct gene to biochemical function analysis, which has rarely been applied to study multicomponent enzymatic systems in specialized metabolism. We sought to establish an in vitro transcription/translation (TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type natural products (cNPs) because of their diverse bioactivity profiles and complex structural diversity. Using a CFPS system including a plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the central prenylated intermediate (3GC) in the presence of exogenous geranyl-pyrophosphate (GPP) and cis-indole isonitrile. Further addition of a plasmid bearing the famC1 Stig cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases (FamC1−C4) produced hapalindole U and hapalindole H, while FisC identified from Fischerella sp. SAG46.79 generated 12-epifischerindole U. The CFPS system was further employed to screen six unnatural halogenated cis-indole isonitrile substrates using FamC1 and FisC, and the reactions were scaled-up using chemoenzymatic synthesis and identified as 5-and 6-fluoro-12-epihapalindole U, and 5-and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents an effective, high throughput strategy to determine the functional role of biosynthetic enzymes from diverse natural product BGCs.

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonematales order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, l-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process, including a Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution, to create a polycyclic core. The formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to a wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward the core structural diversification of this family of indole alkaloids. Through the synthesis of cis-indole isonitrile subunit derivatives, and with the aid of protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives and gained insights into the basis for substrate flexibility in this system.

3-Aryl- and 3-heteroaryloxazolidin-2-ones, by virtue of the diverse pharmacologic activities exhibited by them after subtle changes to their appended substituents, are becoming increasingly important and should be considered privileged chemical structures. The iodocyclocarbamation reaction has been extensively used to make many 3-alkyl-5-(halomethyl)­oxazolidin-2-ones, but the corresponding aromatic congeners have been relatively underexplored. We suggest that racemic 3-aryl- and 3-heteroaryl-5-(iodomethyl)­oxazolidin-2-ones, readily prepared by the iodocyclocarbamation reaction of N-allylated N-aryl or N-heteroaryl carbamates, may be useful intermediates for the rapid preparation of potential lead compounds with biological activity. We exemplify this point by using this approach to prepare racemic linezolid, an antibacterial agent. Herein, we report the results of our systematic investigation into the scope and limitations of this process and have identified some distinguishing characteristics within the aryl/heteroaryl series. We also describe the first preparation of 3-aryloxazolidin-2-ones bearing new functionalized C-5 substituents derived from conjugated 1,3-dienyl and cumulated 1,2-dienyl carbamate precursors. Finally, we describe the utility of the iodocyclocarbamation reaction for making six-membered tetrahydro-3-aryl-1,3-oxazin-2-ones.