Oceanic harmful algal blooms of
diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth ...conditions known to induce DA production in
, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA's diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.
In the ongoing effort to unlock the chemical potential of marine bacteria, genetic engineering of biosynthetic gene clusters (BGCs) is increasingly used to awake or improve expression of biosynthetic ...genes that may lead to discovery of novel bioactive natural products. Previously, we reported the successful capture, engineering and heterologous expression of an orphan BGC from the marine actinomycete Saccharomonospora sp. CNQ-490, which resulted in the isolation of the novel lipopeptide antibiotic taromycin A. Herein we report the isolation and structure elucidation of taromycin B, the second most abundant product of the taromycin biosynthetic series, and show that taromycins A and B exhibit complex chromatographic properties indicative of interconverting conformations. Taromycins A and B display potent activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium clinical isolates, suggestive that the taromycin molecular scaffold is a promising starting point for further derivatization to produce compounds with promising antibiotic characteristics.
Alpiniamide A is a linear polyketide produced by Streptomyces endophytic bacteria. Despite its relatively simple chemical structure suggestive of a linear assembly line biosynthetic construction ...involving a hybrid polyketide synthase-nonribosomal peptide synthetase enzymatic protein machine, we report an unexpected nonlinear synthesis of this bacterial natural product. Using a combination of genomics, heterologous expression, mutagenesis, isotope-labeling, and chain terminator experiments, we propose that alpiniamide A is assembled in two halves and then ligated into the mature molecule. We show that each polyketide half is constructed using orthogonal biosynthetic strategies, employing either cis- or trans-acyl transferase mechanisms, thus prompting an alternative proposal for the operation of this PKS-NRPS.
The disconnect between the genomic prediction of secondary metabolite biosynthetic potential and the observed laboratory production profile of microorganisms is well documented. While heterologous ...expression of biosynthetic gene clusters (BGCs) is often seen as a potential solution to bridge this gap, it is not immune to many challenges including impaired regulation, the inability to recruit essential building blocks, and transcriptional and/or translational silence of the biosynthetic genes. Here we report the discovery, cloning, refactoring, and heterologous expression of a cryptic hybrid phenazine-type BGC (spz) from the marine actinomycete Streptomyces sp. CNB-091. Overexpression of the engineered spz pathway resulted in increased production and chemical diversity of phenazine natural products belonging to the streptophenazine family, including bioactive members containing an unprecedented N-formylglycine attachment. An atypical discrete adenylation enzyme in the spz cluster is required to introduce the formylglycine moiety and represents a phylogenetically distinct class of adenylation proteins.
l‐4‐Chlorokynurenine (l‐4‐Cl‐Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a ...residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l‐tryptophan into l‐4‐Cl‐Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ‐490. We used genetic, biochemical, structural, and analytical techniques to establish l‐4‐Cl‐Kyn biosynthesis, which is initiated by the flavin‐dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3‐dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.
Two paths diverge: The biosynthetic route to neuroactive l‐4‐chlorokynurenine was revealed by using a combination of in vitro experiments, X‐ray crystallography, and CRISPR/Cas9‐mediated gene deletion. Tar13 and Tar16 are the first reported tryptophan 2,3‐dioxygenase and kynurenine formamidase homologues, respectively, that preferentially accept chlorinated substrates. The enzymes characterized in this work hold promise for use in chemoenzymatic synthesis.
L-4-Chlorokynurenine (L-4-Cl-Kyn) is a neuropharmaceutical drug candidate in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in ...the lipopeptide antibiotic taromycin. Herein we report the unprecedented conversion of L-tryptophan to L-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish L-4-Cl-Kyn biosynthesis, which is initiated by the Tar14 flavin-dependent tryptophan chlorinase and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, 14, and 16 was examined revealing intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.
Two paths diverge:
The unique enzymatic route to neuroactive
l
-4-chlorokynurenine has been revealed using a blend of in vitro experiments, X-ray crystallography, and CRISPR/Cas9-mediated gene deletion. Tar13 and Tar16 are the first tryptophan 2,3-dioxygenase and kynurenine formamidase homologs, respectively, to preferentially accept chlorinated substrates. Enzymes characterized here are promising candidates for chemoenzymatic synthesis.
Harmful cyanobacterial blooms (cyanoHABs) cause recurrent toxic events in global watersheds. Although public health agencies monitor the causal toxins of most cyanoHABs and scientists in the field ...continue developing precise detection and prediction tools, the potent anticholinesterase neurotoxin, guanitoxin, is not presently environmentally monitored. This is largely due to its incompatibility with widely employed analytical methods and instability in the environment, despite guanitoxin being among the most lethal cyanotoxins. Here, we describe the guanitoxin biosynthesis gene cluster and its rigorously characterized nine-step metabolic pathway from l-arginine in the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024. Through environmental sequencing data sets, guanitoxin (gnt) biosynthetic genes are repeatedly detected and expressed in municipal freshwater bodies that have undergone past toxic events. Knowledge of the genetic basis of guanitoxin biosynthesis now allows for environmental, biosynthetic gene monitoring to establish the global scope of this neurotoxic organophosphate.
Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin ...biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ringA is not formed. Using this metabolite invitro as a substrate analogue, SalBIII has been shown to form pyran ringA. We have determined the X-ray crystal structure of SalBIII, and structure-guided mutagenesis of putative active-site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of alpha+beta barrel fold proteins.
Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin ...biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X‐ray crystal structure of SalBIII, and structure‐guided mutagenesis of putative active‐site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+β barrel fold proteins.
It takes two: X‐ray crystallography, mutagenesis, and in vitro studies of the enzyme SalBIII in the salinomycin biosynthetic pathway identify it as a pyran synthase and highlight the role of a catalytic dyad. SalBIII is a promising candidate for chemoenzymatic synthesis.
The complex bis‐spiroacetal polyether ionophore salinomycin has been identified as a uniquely selective agent against cancer stem cells and is also strikingly effective in an animal model of latent ...tuberculosis. The basis for these important activities is unknown. We show here that deletion of the salE gene abolishes salinomycin production and yields two new analogues, in both of which the C18C19 cis double bond is replaced by a hydroxy group stereospecifically located at C19, but which differ from each other in the configuration of the bis‐spiroacetal. These results identify SalE as a novel dehydratase and demonstrate that biosynthetic engineering can be used to redirect the reaction cascade of oxidative cyclization to yield new salinomycin analogues for use in mechanism‐of‐action studies.
Redirecting polyether biosynthesis: The origin of the unusual cis double bond in the polyether ionophore salinomycin has been revealed. Deletion of a gene encoding a previously unknown dehydratase diverts oxidative cyclization to produce analogues specifically modified in the trioxaspiroacetal core.
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