Yuquan Xu

A mass spectrometry-guided genome mining approach for natural product peptidogenomics

Peptide natural products exhibit broad biological properties and are commonly produced by orthogonal ribosomal and nonribosomal pathways in prokaryotes and eukaryotes. To harvest this large and diverse resource of bioactive molecules, we introduce Natural Product Peptidogenomics (NPP), a new mass spectrometry-guided genome mining method that connects the chemotypes of peptide natural products to their biosynthetic gene clusters by iteratively matching de novo MSn structures to genomics-based structures following current biosynthetic logic. In this study we demonstrate that NPP enabled the rapid characterization of >10 chemically diverse ribosomal and nonribosomal peptide natural products of novel composition from streptomycete bacteria as a proof of concept to begin automating the genome mining process. We show the identification of lantipeptides, lasso peptides, linardins, formylated peptides and lipopeptides, many of which from well-characterized model streptomycetes, highlighting the power of NPP in the discovery of new peptide natural products from even intensely studied organisms.

Translating metabolic exchange with imaging mass spectrometry

Metabolic exchange between an organism and the environment, including interactions with neighboring organisms, is important for processes of organismal development. Here we develop and use thin-layer agar natural product MALDI-TOF imaging mass spectrometry of intact bacterial colonies grown on top of the MALDI target plate to study an interaction between two species of bacteria and provide direct evidence that a Bacillus subtilis silences the defensive arsenal of Streptomyces coelicolor.

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

Biosynthesis of the Cyclooligomer Depsipeptide Beauvericin, a Virulence Factor of the Entomopathogenic Fungus Beauveria bassiana

Beauvericin, a cyclohexadepsipeptide ionophore from the entomopathogen Beauveria bassiana, shows antibiotic, antifungal, insecticidal, and cancer cell antiproliferative and antihaptotactic (cell motility inhibitory) activity in vitro. The bbBeas gene encoding the BbBEAS nonribosomal peptide synthetase was isolated from B. bassiana and confirmed to be responsible for beauvericin biosynthesis by targeted disruption. BbBEAS utilizes D-2-hydroxyisovalerate (D-Hiv) and L-phenylalanine (Phe) for the iterative synthesis of a predicted N-methyl-dipeptidol intermediate, and forms the cyclic trimeric ester beauvericin from this intermediate in an unusual recursive process. Heterologous expression of the bbBeas gene in Escherichia coli to produce the 3189 amino acid, 351.9 kDa BbBEAS enzyme provided a strain proficient in beauvericin biosynthesis. Comparative infection assays with a BbBEAS knockout B. bassiana strain against three insect hosts revealed that beauvericin plays a highly significant but not indispensable role in virulence.

Biosynthesis of the cyclooligomer depsipeptide bassianolide, an insecticidal virulence factor of Beauveria bassiana.

Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.

Functional Characterization of the Biosynthesis of Radicicol, an Hsp90 Inhibitor Resorcylic Acid Lactone from Chaetomium chiversii

Fungal polyketides with the resorcylic acid lactone (RAL) scaffold are of interest for growth stimulation, the treatment of cancer, and neurodegenerative diseases. The RAL radicicol is a nanomolar inhibitor of the chaperone Hsp90, whose repression leads to a combinatorial blockade of cancer-causing pathways. Clustered genes for radicicol biosynthesis were identified and functionally characterized from the endophytic fungus Chaetomium chiversii, and compared to recently described RAL biosynthetic gene clusters. Radicicol production is abolished upon targeted inactivation of a putative cluster-specific regulator, or either of the two polyketide synthases that are predicted to collectively synthesize the radicicol polyketide core. Genomic evidence supports the existence of flavin-dependent halogenases in fungi: inactivation of such a putative halogenase from the C. chiversii radicicol locus yields dechloro-radicicol (monocillin I). Inactivation of a cytochrome P450 epoxidase furnishes pochonin D, a deepoxy-dihydro radicicol analog.

Connecting Chemotypes and Phenotypes of Cultured Marine Microbial Assemblages by Imaging Mass Spectrometry

From the early days of bacterial culturing over a century ago, microbiologists have known that microorganisms respond to their surroundings. Unicellular organisms rely on metabolic exchange to adapt to environmental stresses, sense colony density, occupy niches within hosts and form biofilms.1-7 For example, Bacillus subtilis utilizes metabolic exchange to lyse neighboring microbes, including siblings, during sporulation.8-10 While other forms of metabolic exchange, such as siderophores, stimulate the growth and development of Streptomyces and uncultured bacteria.11, 12 Despite the importance of chemistry in biology, studies that connect chemotypes and phenotypes to adaptive microbial behavior in Petri-dishes, including signaling and chemical warfare, have largely been disconnected and measured indirectly. To connect the chemotypes and phenotypes in this study, MALDI-based imaging mass spectrometry (IMS)13-16 was utilized to observe the chemical output and metabolic exchange of a marine microbial assemblage in two-dimensions. The capability to monitor the 2-dimensional distribution of a wide array of metabolites simultaneously from a complex mixture of distinct organisms opens the door to comprehensive analyses of interspecies signaling interactions within a microbial assemblage in a spatial fashion. Analyzing the spatial distribution of these metabolites enables one to generate a testable hypothesis, without the immediate need to know the structural characteristics, with respect to functions of the observed chemotypes. IMS provides the ability to correlate the presence of metabolites to phenotypic changes and to detect new chemotypes and/or phenotypes that cannot be observed by the naked eye. As such, IMS allows one to prioritize the molecules to be targeted for identification through proteomic and metabolomic approaches, or to be subjected to mass spectrometry guided isolation and nuclear magnetic resonance based structure elucidation. Understanding these molecular networks and interactions will illuminate how microbes respond to neighboring organisms and in turn influence and alter growth of their neighbors.

Characterization of the biosynthetic genes for 10,11-dehydrocurvularin, a heat shock response-modulating anticancer fungal polyketide from Aspergillus terreus.

ABSTRACT 10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

Rational reprogramming of fungal polyketide first-ring cyclization

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases

Significance Benzenediol lactone (BDL) polyketides are privileged structures whose various members bind to distinct receptors or modulate the heat shock response and the immune system. BDLs are biosynthesized by collaborating polyketide synthase enzyme pairs in fungi. Coexpressing random heterocombinations of these enzymes from different BDL biosynthetic pathways in yeast cells is shown here to lead to the one-pot, one-step combinatorial biosynthesis of structurally diverse polyketides in practical amounts. Combinatorial biosynthesis promises to generate a novel unexplored source of bioactive molecules in an environmentally sustainable, economical, and inherently scalable manner. Broadening the medicinally relevant chemical space of polyketides by such methods will provide unnatural products as valuable entry points for drug discovery and development. Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into “build–couple–pair” combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.