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.
Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three‐step cascade involving Cope rearrangement, 6‐exo‐trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+, to cooperatively control the stereochemistry of hapalindole natural products.
<p>The stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by the Stig cyclases through a three-step cascade involving Cope rearrangement, 6-<i>exo</i>-trig cyclization and a final electrophilic aromatic substitution. Here we report a comprehensive study of all currently annotated Stig cyclases, and reveal that these proteins can assemble into heteromeric complexes induced by Ca<sup>2+</sup> to cooperatively control the stereochemistry of hapalindole natural products.</p>
<p>The stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by the Stig cyclases through a three-step cascade involving Cope rearrangement, 6-<i>exo</i>-trig cyclization and a final electrophilic aromatic substitution. Here we report a comprehensive study of all currently annotated Stig cyclases, and reveal that these proteins can assemble into heteromeric complexes induced by Ca<sup>2+</sup> to cooperatively control the stereochemistry of hapalindole natural products.</p>
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