Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS.

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system.

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

Revealing Hidden Genes in Botrytis cinerea: New Insights into Genes Involved in the Biosynthesis of Secondary Metabolites.

Utilizing bioinformatics tools, this study expands our understanding of secondary metabolism in Botrytis cinerea, identifying novel genes within polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), sesquiterpene cyclase (STC), diterpene cyclase (DTC), and dimethylallyltryptophan synthase (DMATS) families. These findings enrich the genetic framework associated with B. cinerea's pathogenicity and ecological adaptation, offering insights into uncharted metabolic pathways. Significantly, the discovery of previously unannotated genes provides new molecular targets for developing targeted antifungal strategies, promising to enhance crop protection and advance our understanding of fungal biochemistry. This research not only broadens the scope of known secondary metabolites but also opens avenues for future exploration into B. cinerea's biosynthetic capabilities, potentially leading to novel antifungal compounds. Our work underscores the importance of integrating bioinformatics and genomics for fungal research, paving the way for sustainable agricultural practices by pinpointing precise molecular interventions against B. cinerea. This study sets a foundation for further investigations into the fungus's secondary metabolism, with implications for biotechnology and crop disease management.

Discovery and Biosynthesis of Streptolateritic Acids A-D: Acyclic Pentacarboxylic Acids from Streptomyces sp. FXJ1.172 with Promising Activity against Potato Common Scab.

Potato common scab (PCS) is a widespread plant disease that lacks effective control measures. Using a small molecule elicitor, we activate the production of a novel class of polyketide antibiotics, streptolateritic acids A-D, in Streptomyces sp. FXJ1.172. These compounds show a promising control efficacy against PCS and an unusual acyclic pentacarboxylic acid structure. A gene cluster encoding a type I modular polyketide synthase is identified to be responsible for the biosynthesis of these metabolites. A cytochrome P450 (CYP) and an aldehyde dehydrogenase (ADH) encoded by two genes in the cluster are proposed to catalyze iterative oxidation of the starter-unit-derived methyl group and three of six branching methyl groups to carboxylic acids during chain assembly. Our findings highlight how activation of silent biosynthetic gene clusters can be employed to discover completely new natural product classes able to combat PCS and new types of modular polyketide synthase-based biosynthetic machinery.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Biosynthetic gene clusters with biotechnological applications in novel Antarctic isolates from Actinomycetota.

Actinomycetota have been widely described as valuable sources for the acquisition of secondary metabolites. Most microbial metabolites are produced via metabolic pathways encoded by biosynthetic gene clusters (BGCs). Although many secondary metabolites are not essential for the survival of bacteria, they play an important role in their adaptation and interactions within microbial communities. This is how bacteria isolated from extreme environments such as Antarctica could facilitate the discovery of new BGCs with biotechnological potential. This study aimed to isolate rare Actinomycetota strains from Antarctic soil and sediment samples and identify their metabolic potential based on genome mining and exploration of biosynthetic gene clusters. To this end, the strains were sequenced using Illumina and Oxford Nanopore Technologies platforms. The assemblies were annotated and subjected to phylogenetic analysis. Finally, the BGCs present in each genome were identified using the antiSMASH tool, and the biosynthetic diversity of the Micrococcaceae family was evaluated. Taxonomic annotation revealed that seven strains were new and two were previously reported in the NCBI database. Additionally, BGCs encoding type III polyketide synthases (T3PKS), beta-lactones, siderophores, and non-ribosomal peptide synthetases (NRPS) have been identified, among others. In addition, the sequence similarity network showed a predominant type of BGCs in the family Micrococcaceae, and some genera were distinctly grouped. The BGCs identified in the isolated strains could be associated with applications such as antimicrobials, anticancer agents, and plant growth promoters, among others, positioning them as excellent candidates for future biotechnological applications and innovations. KEY POINTS: • Novel Antarctic rare Actinomycetota strains were isolated from soil and sediments • Genome-based taxonomic affiliation revealed seven potentially novel species • Genome mining showed metabolic potential for novel natural products.

Chalkophomycin Biosynthesis Revealing Unique Enzyme Architecture for a Hybrid Nonribosomal Peptide Synthetase and Polyketide Synthase.

Chalkophomycin is a novel chalkophore with antibiotic activities isolated from Streptomyces sp. CB00271, while its potential in studying cellular copper homeostasis makes it an important probe and drug lead. The constellation of N-hydroxylpyrrole, 2H-oxazoline, diazeniumdiolate, and methoxypyrrolinone functional groups into one compact molecular architecture capable of coordinating cupric ions draws interest to unprecedented enzymology responsible for chalkophomycin biosynthesis. To elucidate the biosynthetic machinery for chalkophomycin production, the chm biosynthetic gene cluster from S. sp. CB00271 was identified, and its involvement in chalkophomycin biosynthesis was confirmed by gene replacement. The chm cluster was localized to a ~31 kb DNA region, consisting of 19 open reading frames that encode five nonribosomal peptide synthetases (ChmHIJLO), one modular polyketide synthase (ChmP), six tailoring enzymes (ChmFGMNQR), two regulatory proteins (ChmAB), and four resistance proteins (ChmA'CDE). A model for chalkophomycin biosynthesis is proposed based on functional assignments from sequence analysis and structure modelling, and is further supported by analogy to over 100 chm-type gene clusters in public databases. Our studies thus set the stage to fully investigate chalkophomycin biosynthesis and to engineer chalkophomycin analogues through a synthetic biology approach.

Activation of secondary metabolite gene clusters in Chaetomium olivaceum via the deletion of a histone deacetylase.

Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: • Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. • Three polyketides and one asterriquinone were isolated from HDAC deleted strain. • Two different PKSs were reported in C. olivaceum SD-80A.

Structural and mechanistic insights into Quinolone Synthase to address its functional promiscuity.

Quinolone synthase from Aegle marmelos (AmQNS) is a type III polyketide synthase that yields therapeutically effective quinolone and acridone compounds. Addressing the structural and molecular underpinnings of AmQNS and its substrate interaction in terms of its high selectivity and specificity can aid in the development of numerous novel compounds. This paper presents a high-resolution AmQNS crystal structure and explains its mechanistic role in synthetic selectivity. Additionally, we provide a model framework to comprehend structural constraints on ketide insertion and postulate that AmQNS's steric and electrostatic selectivity plays a role in its ability to bind to various core substrates, resulting in its synthetic diversity. AmQNS prefers quinolone synthesis and can accommodate large substrates because of its wide active site entrance. However, our research suggests that acridone is exclusively synthesized in the presence of high malonyl-CoA concentrations. Potential implications of functionally relevant residue mutations were also investigated, which will assist in harnessing the benefits of mutations for targeted polyketide production. The pharmaceutical industry stands to gain from these findings as they expand the pool of potential drug candidates, and these methodologies can also be applied to additional promising enzymes.

Identification of novel hit molecules targeting M. tuberculosis polyketide synthase 13 by combining generative AI and physics-based methods.

In this work we investigated the Pks13-TE domain, which plays a critical role in the viability of the mycobacteria. In this report, we have used a series of AI and Physics-based tools to identify Pks13-TE inhibitors. The Reinvent 4, pKCSM, KDeep, and SwissADME are AI-ML-based tools. AutoDock Vina, PLANTS, MDS, and MM-GBSA are physics-based methods. A combination of these methods yields powerful support in the drug discovery cycle. Known inhibitors of Pks13-TE were collected, curated, and used as input for the AI-based tools, and Mol2Mol molecular optimisation methods generated novel inhibitors. These ligands were filtered based on physics-based methods like molecular docking and molecular dynamics using multiple tools for consensus generation. Rigorous analysis was performed on the selected compounds to reduce the chemical space while retaining the most promising compounds. The molecule interactions, stability of the protein-ligand complexes and the comparable binding energies with the native ligand were essential factors for narrowing the ligands set. The filtered ligands from docking, MDS, and binding energy colocations were further tested for their ADMET properties since they are among the essential criteria for this series of molecules. It was found that ligands Mt1 to Mt6 have excellent predicted pharmacokinetic, pharmacodynamic and toxicity profiles and good synthesisability.

Isolation and Structure Elucidation of JBIR-157, a Skeletally Novel Aromatic Polyketide Produced by the Heterologous Expression of a Cryptic Gene Cluster.

Heterologous expression of natural compound biosynthetic gene clusters (BGCs) is a robust approach for not only revealing the biosynthetic mechanisms leading to the compounds, but also for discovering new products from uncharacterized BGCs. We established a heterologous expression technique applicable to huge biosynthetic gene clusters for generating large molecular secondary metabolites such as type-I polyketides. As an example, we targeted concanamycin BGC from Streptomyces neyagawaensis IFO13477 (the cluster size of 99 kbp), and obtained a bacterial artificial chromosome (BAC) clone with an insert size of 211 kbp that contains the entire concanamycin BGC. Interestingly, heterologous expression for this BAC clone resulted in two additional aromatic polyketides, ent-gephyromycin, and a new compound designated as JBIR-157, together with the expected concanamycin. Bioinformatic and biochemical analyses revealed that a cryptic biosynthetic gene cluster in this BAC clone was responsible for the production of these type-II polyketide synthases (PKS) compounds. Here, we describe the production, isolation, and structure elucidation of JBIR-157, determined primarily by a series of NMR spectral analyses.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Engineered Biosynthesis and Anticancer Studies of Ring-Expanded Antimycin-Type Depsipeptides.

Respirantins are 18-membered antimycin-type depsipeptides produced by Streptomyces sp. and Kitasatospora sp. These compounds have shown extraordinary anticancer activities against a panel of cancer cell lines with nanomolar levels of IC50 values. However, further investigation has been impeded by the low titers of the natural producers and the challenging chemical synthesis due to their structural complexity. The biosynthetic gene cluster (BGC) of respirantin was previously proposed based on a bioinformatic comparison of the four members of antimycin-type depsipeptides. In this study, we report the first successful reconstitution of respirantin in Streptomyces albus using a synthetic BGC. This heterologous system serves as an accessible platform for the production and diversification of respirantins. Through polyketide synthase pathway engineering, biocatalysis, and chemical derivatization, we generated nine respirantin compounds, including six new derivatives. Cytotoxicity screening against human MCF-7 and Hela cancer cell lines revealed a unique biphasic dose-response profile of respirantin. Furthermore, a structure-activity relationship study has elucidated the essential functional groups that contribute to its remarkable cytotoxicity. This work paves the way for respirantin-based anticancer drug discovery and development.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes.

Avermectins (AVEs), a family of macrocyclic polyketides produced by Streptomyces avermitilis, have eight components, among which B1a is noted for its strong insecticidal activity. Biosynthesis of AVE "a" components requires 2-methylbutyryl-CoA (MBCoA) as starter unit, and malonyl-CoA (MalCoA) and methylmalonyl-CoA (MMCoA) as extender units. We describe here a novel strategy for increasing B1a production by enhancing acyl-CoA precursor supply. First, we engineered meilingmycin (MEI) polyketide synthase (PKS) for increasing MBCoA precursor supply. The loading module (using acetyl-CoA as substrate), extension module 7 (using MMCoA as substrate) and TE domain of MEI PKS were assembled to produce 2-methylbutyrate, providing the starter unit for B1a production. Heterologous expression of the newly designed PKS (termed Mei-PKS) in S. avermitilis wild-type (WT) strain increased MBCoA level, leading to B1a titer 262.2 µg/mL - 4.36-fold higher than WT value (48.9 µg/mL). Next, we separately inhibited three key nodes in essential pathways using CRISPRi to increase MalCoA and MMCoA levels in WT. The resulting strains all showed increased B1a titer. Combined inhibition of these key nodes in Mei-PKS expression strain increased B1a titer to 341.9 µg/mL. Overexpression of fatty acid ß-oxidation pathway genes in the strain further increased B1a titer to 452.8 µg/mL - 8.25-fold higher than WT value. Finally, we applied our precursor supply strategies to high-yield industrial strain A229. The strategies, in combination, led to B1a titer 8836.4 µg/mL - 37.8% higher than parental A229 value. These findings provide an effective combination strategy for increasing AVE B1a production in WT and industrial S. avermitilis strains, and our precursor supply strategies can be readily adapted for overproduction of other polyketides.

Molecular evolution and characterization of type III polyketide synthase gene family in Aquilaria sinensis.

2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.

Genome-guided discovery of two undescribed 6,6-spiroketal polyketides and stereochemical correction of bafilomycins P and Q from the marine-derived Streptomyces sp. SCSIO 66814.

Bafilomycins are macrocyclic polyketides with intriguing structures and therapeutic value. Genomic analysis of Streptomyces sp. SCSIO 66814 revealed a type I polyketide synthase biosynthetic gene cluster (BGC), namely blm, which encoded bafilomycins and featured rich post-modification genes. The One strain many compounds (OSMAC) strategy led to the discovery of six compounds related to the blm BGC from the strain, including two previously undescribed 6,6-spiroketal polyketides, streptospirodienoic acids D (1) and E (2), and four known bafilomycins, bafilomycins P (3), Q (4), D (5), and G (6). The structures of 1 and 2 were determined by extensive spectroscopic analysis, quantum calculation, and biosynthetic analysis. Additionally, the absolute configurations of the 6/5/5 tricyclic ring moiety containing six consecutive chiral carbons in the putative structures of 3 and 4 were corrected through NOE analysis, DP4+ calculation, and single-crystal X-ray diffraction data. Bioinformatic analysis uncovered a plausible biosynthetic pathway for compounds 1-6, indicating that both streptospirodienoic acids and bafilomycins were derived from the same blm BGC. Additionally, sequence analysis revealed that the KR domains of module 2 from blm BGC was B1-type, further supporting the configurations of 1-4. Notably, compounds 3 and 4 displayed significant cytotoxic activities against A-549 human non-small cell lung cancer cells and HCT-116 human colon cancer cells.

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Polyketide Trimming Shapes Dihydroxynaphthalene-Melanin and Anthraquinone Pigments.

Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.

Capability of a large bacterial artificial chromosome clone harboring multiple biosynthetic gene clusters for the production of diverse compounds.

The biosynthetic gene clusters (BGCs) for the macrocyclic lactone-based polyketide compounds are extremely large-sized because the polyketide synthases that generate the polyketide chains of the basic backbone are of very high molecular weight. In developing a heterologous expression system for the large BGCs amenable to the production of such natural products, we selected concanamycin as an appropriate target. We obtained a bacterial artificial chromosome (BAC) clone with a 211-kb insert harboring the entire BGC responsible for the biosynthesis of concanamycin. Heterologous expression of this clone in a host strain, Streptomyces avermitilis SUKA32, permitted the production of concanamycin, as well as that of two additional aromatic polyketides. Structural elucidation identified these additional products as ent-gephyromycin and a novel compound that was designated JBIR-157. We describe herein sequencing and expression studies performed on these BGCs, demonstrating the utility of large BAC clones for the heterologous expression of cryptic or near-silent loci.