Characterization of an Unusual α-Oxoamine Synthase Off-Loading Domain from a Cyanobacterial Type I Fatty Acid Synthase.

Type I fatty acid synthases (FASs) are known from higher eukaryotes and fungi. We report the discovery of FasT, a rare type I FAS from the cyanobacterium Chlorogloea sp. CCALA695. FasT possesses an unusual off-loading domain, which was heterologously expressed in E. coli and found to act as an α-oxoamine synthase (AOS) in vitro. Similar to serine palmitoyltransferases from sphingolipid biosynthesis, the AOS off-loading domain catalyzes a decarboxylative Claisen condensation between l-serine and a fatty acyl thioester. While the AOS domain was strictly specific for l-serine, thioesters with saturated fatty acyl chains of six carbon atoms and longer were tolerated, with the highest activity observed for stearoyl-coenzyme A (C18 ). Our findings suggest a novel route to α-amino ketones via the direct condensation of iteratively produced long-chain fatty acids with l-serine by a FAS with a cis-acting AOS off-loading domain.

Modular Oxime Formation by a trans-AT Polyketide Synthase.

Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides.

Structure of a Promiscuous Thioesterase Domain Responsible for Branching Acylation in Polyketide Biosynthesis.

Thioesterases (TEs) are fundamentally important enzymes present in all bacteria and eukaryotes, where they have conserved functions in fatty acid biosynthesis and secondary metabolism. This work provides the first structural insights into a functionally distinct group of TEs that perform diverse acylations in polyketide and peptide biosynthesis (TEB s). Structural analysis of the oocydin (OocS) TEB domain facilitated identification and engineering of the active site to modulate acyl-group acceptance. In this way, we achieved higher reactivity using a structure-based approach, building a foundation for biocatalytic development of TEB -mediated O-acylation, a modification known to improve the bioactivity of oocydin-type polyketides. Lastly, the promiscuity of the OocS TEB motivated us to investigate, and ultimately provide evidence for, the production of longer chain branched oocydins in the native host Serratia plymuthica 4Rx13. This work frames the OocS TEB and homologs as invaluable synthetic biology tools for polyketide drug development.

Modular Halogenation, α-Hydroxylation, and Acylation by a Remarkably Versatile Polyketide Synthase.

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli.

Automated structure prediction of trans-acyltransferase polyketide synthase products.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex known enzymes from secondary metabolism and are responsible for the biosynthesis of highly diverse bioactive polyketides. However, most of these metabolites remain uncharacterized, since trans-AT PKSs frequently occur in poorly studied microbes and feature a remarkable array of non-canonical biosynthetic components with poorly understood functions. As a consequence, genome-guided natural product identification has been challenging. To enable de novo structural predictions for trans-AT PKS-derived polyketides, we developed the trans-AT PKS polyketide predictor (TransATor). TransATor is a versatile bio- and chemoinformatics web application that suggests informative chemical structures for even highly aberrant trans-AT PKS biosynthetic gene clusters, thus permitting hypothesis-based, targeted biotechnological discovery and biosynthetic studies. We demonstrate the applicative scope in several examples, including the characterization of new variants of bioactive natural products as well as structurally new polyketides from unusual bacterial sources.

Single-bacterial genomics validates rich and varied specialized metabolism of uncultivated Entotheonella sponge symbionts.

Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.

Genome Mining of Oxidation Modules in trans-Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer-Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria.

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones.

Enzymatic core components from trans-acyltransferase polyketide synthases (trans-AT PKSs) catalyze exceptionally diverse biosynthetic transformations to generate structurally complex bioactive compounds. Here we focus on a group of oxygenases identified in various trans-AT PKS pathways, including those for pederin, oocydins, and toblerols. Using the oocydin pathway homologue (OocK) from Serratia plymuthica 4Rx13 and N-acetylcysteamine (SNAC) thioesters as test surrogates for acyl carrier protein (ACP)-tethered intermediates, we show that the enzyme inserts oxygen into ß-ketoacyl moieties to yield malonyl ester SNAC products. Based on these data and the identification of a non-hydrolyzed oocydin congener with retained ester moiety, we propose a unified biosynthetic pathway of oocydins, haterumalides, and biselides. By providing access to internal ester, carboxylate pseudostarter, and terminal hydroxyl functions, oxygen insertion into polyketide backbones greatly expands the biosynthetic scope of PKSs.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Heterocomplex structure of a polyketide synthase component involved in modular backbone halogenation.

Bacterial modular polyketide synthases (PKSs) generate diverse, complex and bioactive natural products that are constructed mainly based on principles of fatty acid biosynthesis. The cytotoxic oocydin-type polyketides contain a vinyl chloride moiety introduced during polyketide chain elongation. Required for modular polyketide backbone halogenation are a non-heme iron and ɑ-ketoglutarate-dependent halogenase OocP and OocQ lacking characterized homologs. This work provides structural insights into these unusual PKS components and their interactions via a high-resolution X-ray crystallography structure of the heterocomplex. By mapping the protein-protein interactions and comparison with structures of similar halogenases, we illustrate the potential of this heterodimer complex as a replacement for the conserved homodimeric structure of homologous enzymes. The OocPQ protein pair has thus evolved as a means of stabilizing the halogenase and facilitating chemical transformations with great synthetic utility.

In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids.

Tapping into the metabolic crosstalk between a host and its virus can reveal unique strategies employed during infection. Viral infection is a dynamic process that generates an evolving metabolic landscape. Gaining a continuous view into the infection process is highly challenging and is limited by current metabolomics approaches, which typically measure the average of the entire population at various stages of infection. Here, we took an innovative approach to study the metabolic basis of host-virus interactions between the bloom-forming alga Emiliania huxleyi and its specific virus. We combined a classical method in virology, the plaque assay, with advanced mass spectrometry imaging (MSI), an approach we termed 'in plaque-MSI'. Taking advantage of the spatial characteristics of the plaque, we mapped the metabolic landscape induced during infection in a high spatiotemporal resolution, unfolding the infection process in a continuous manner. Further unsupervised spatially aware clustering, combined with known lipid biomarkers, revealed a systematic metabolic shift during infection towards lipids containing the odd-chain fatty acid pentadecanoic acid (C15:0). Applying 'in plaque-MSI' may facilitate the discovery of bioactive compounds that mediate the chemical arms race of host-virus interactions in diverse model systems.

Structural and Functional Studies of a Pyran Synthase Domain from a trans-Acyltransferase Assembly Line.

trans-Acyltransferase assembly lines possess enzymatic domains often not observed in their better characterized cis-acyltransferase counterparts. Within this repertoire of largely unexplored biosynthetic machinery is a class of enzymes called the pyran synthases that catalyze the formation of five- and six-membered cyclic ethers from diverse polyketide chains. The 1.55 Å resolution crystal structure of a pyran synthase domain excised from the ninth module of the sorangicin assembly line highlights the similarity of this enzyme to the ubiquitous dehydratase domain and provides insight into the mechanism of ring formation. Functional assays of point mutants reveal the central importance of the active site histidine that is shared with the dehydratases as well as the supporting role of a neighboring semiconserved asparagine.

Synthesis and characterization of catalytically active thiazolium gold(i)-carbenes.

Thiamin analogs were used to synthesize mono gold(i)-carbene derivatives in a single step under aqueous conditions. The resulting thiazolium gold(i)-carbenes catalyze 5-endo-dig carbocyclization of an acetylenic dicarbonyl compound in organic solvents and hydroalkoxylation of an allene in aqueous buffer.