Unveiling a CAAX Protease-Like Protein Involved in Didemnin Drug Maturation and Secretion.

The assembly line biosynthesis of the powerful anticancer-antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N-terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N-acyl-polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post-assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies.

Biosynthesis of Haloterpenoids in Red Algae via Microbial-like Type I Terpene Synthases.

Red algae or seaweeds produce highly distinctive halogenated terpenoid compounds, including the pentabromochlorinated monoterpene halomon that was once heralded as a promising anticancer agent. The first dedicated step in the biosynthesis of these natural product molecules is expected to be catalyzed by terpene synthase (TS) enzymes. Recent work has demonstrated an emerging class of type I TSs in red algal terpene biosynthesis. However, only one such enzyme from a notoriously haloterpenoid-producing red alga (Laurencia pacifica) has been functionally characterized and the product structure is not related to halogenated terpenoids. Herein, we report 10 new type I TSs from the red algae Portieria hornemannii, Plocamium pacificum, L. pacifica, and Laurencia subopposita that produce a diversity of halogenated mono- and sesquiterpenes. We used a combination of genome sequencing, terpenoid metabolomics, in vitro biochemistry, and bioinformatics to establish red algal TSs in all four species, including those associated with the selective production of key halogenated terpene precursors myrcene, trans-ß-ocimene, and germacrene D-4-ol. These results expand on a small but growing number of characterized red algal TSs and offer insight into the biosynthesis of iconic halogenated algal compounds that are not without precedence elsewhere in biology.

Extreme genome diversity and cryptic speciation in a harmful algal-bloom-forming eukaryote.

Harmful algal blooms of the toxic haptophyte Prymnesium parvum are a recurrent problem in many inland and estuarine waters around the world. Strains of P. parvum vary in the toxins they produce and in other physiological traits associated with harmful algal blooms, but the genetic basis for this variation is unknown. To investigate genome diversity in this morphospecies, we generated genome assemblies for 15 phylogenetically and geographically diverse strains of P. parvum, including Hi-C guided, near-chromosome-level assemblies for two strains. Comparative analysis revealed considerable DNA content variation between strains, ranging from 115 to 845 Mbp. Strains included haploids, diploids, and polyploids, but not all differences in DNA content were due to variation in genome copy number. Haploid genome size between strains of different chemotypes differed by as much as 243 Mbp. Syntenic and phylogenetic analyses indicate that UTEX 2797, a common laboratory strain from Texas, is a hybrid that retains two phylogenetically distinct haplotypes. Investigation of gene families variably present across the strains identified several functional categories associated with metabolic and genome size variation in P. parvum, including genes for the biosynthesis of toxic metabolites and proliferation of transposable elements. Together, our results indicate that P. parvum comprises multiple cryptic species. These genomes provide a robust phylogenetic and genomic framework for investigations into the eco-physiological consequences of the intra- and inter-specific genetic variation present in P. parvum and demonstrate the need for similar resources for other harmful algal-bloom-forming morphospecies.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Enzymatic assembly of the salinosporamide γ-lactam-ß-lactone anticancer warhead.

The marine microbial natural product salinosporamide A (marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized γ-lactam-ß-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase (KS), SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/ß-lactone synthase with roles in both transacylation and bond-forming reactions. Additionally, we present the 2.85-Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of KS enzymes and establishes a basis for future efforts toward streamlined production of a clinically relevant chemotherapeutic.

Harnessing ortho-Quinone Methides in Natural Product Biosynthesis and Biocatalysis.

The implementation of ortho-quinone methide (o-QM) intermediates in complex molecule assembly represents a remarkably efficient strategy designed by Nature and utilized by synthetic chemists. o-QMs have been taken advantage of in biomimetic syntheses for decades, yet relatively few examples of o-QM-generating enzymes in natural product biosynthetic pathways have been reported. The biosynthetic enzymes that have been discovered thus far exhibit tremendous potential for biocatalytic applications, enabling the selective production of desirable compounds that are otherwise intractable or inherently difficult to achieve by traditional synthetic methods. Characterization of this biosynthetic machinery has the potential to shine a light on new enzymes capable of similar chemistry on diverse substrates, thus expanding our knowledge of Nature's catalytic repertoire. The presently known o-QM-generating enzymes include flavin-dependent oxidases, hetero-Diels-Alderases, S-adenosyl-l-methionine-dependent pericyclases, and α-ketoglutarate-dependent nonheme iron enzymes. In this review, we discuss their diverse enzymatic mechanisms and potential as biocatalysts in constructing natural product molecules such as cannabinoids.

Marine and Anthropogenic Bromopyrroles Alter Cellular Ca2+ Dynamics of Murine Cortical Neuronal Networks by Targeting the Ryanodine Receptor and Sarco/Endoplasmic Reticulum Ca2+-ATPase.

Bromopyrroles (BrPyr) are synthesized naturally by marine sponge symbionts and produced anthropogenically as byproducts of wastewater treatment. BrPyr interact with ryanodine receptors (RYRs) and sarco/endoplasmic reticulum (SR/ER) Ca2+-ATPase (SERCA). Influences of BrPyr on the neuronal network activity remain uncharted. BrPyr analogues with differing spectra of RYR/SERCA activities were tested using RYR-null or RYR1-expressing HEK293 and murine cortical neuronal/glial cocultures (NGCs) loaded with Fluo-4 to elucidate their mechanisms altering Ca2+ dynamics. The NGC electrical spike activity (ESA) was measured from NGCs plated on multielectrode arrays. Nanomolar tetrabromopyrrole (TBP, 1) potentiated caffeine-triggered Ca2+ release independent of extracellular [Ca2+] in RYR1-HEK293, whereas higher concentrations produce slow and sustained rise in cytoplasmic [Ca2+] independent of RYR1 expression. TBP, 2,3,5-tribromopyrrole (2), pyrrole (3), 2,3,4-tribromopyrrole (4), and ethyl 4-bromopyrrole-2-carboxylate (5) added acutely to NGC showed differential potency; rank order TBP (IC50 ≈ 220 nM) > 2 ≫ 5, whereas 3 and 4 were inactive at 10 µM. TBP >2 µM elicited sustained elevation of cytoplasmic [Ca2+] and loss of neuronal viability. TBP did not alter network ESA. BrPyr from marine and anthropogenic sources are ecological signaling molecules and emerging anthropogenic pollutants of concern to environmental and human health that potently alter ER Ca2+ dynamics and warrant further investigation in vivo.

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage.

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin's DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention.

Genetic platforms for heterologous expression of microbial natural products.

Covering: 2005 up to 2019Natural products are of paramount importance in human medicine. Not only are most antibacterial and anticancer drugs derived directly from or inspired by natural products, many other branches of medicine, such as immunology, neurology, and cardiology, have similarly benefited from natural product-based drugs. Typically, the genetic material required to synthesize a microbial specialized product is arranged in a multigene biosynthetic gene cluster (BGC), which codes for proteins associated with molecule construction, regulation, and transport. The ability to connect natural product compounds to BGCs and vice versa, along with ever-increasing knowledge of biosynthetic machineries, has spawned the field of genomics-guided natural product genome mining for the rational discovery of new chemical entities. One significant challenge in the field of natural product genome mining is how to rapidly link orphan biosynthetic genes to their associated chemical products. This review highlights state-of-the-art genetic platforms to identify, interrogate, and engineer BGCs from diverse microbial sources, which can be broken into three stages: (1) cloning and isolation of genomic loci, (2) heterologous expression in a host organism, and (3) genetic manipulation of cloned pathways. In the future, we envision natural product genome mining will be rapidly accelerated by de novo DNA synthesis and refactoring of whole biosynthetic pathways in combination with systematic heterologous expression methodologies.

Synthesis, bioactivity, and enzymatic modification of antibacterial thiotetromycin derivatives.

Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli ΔtolC strain with IC50 values in a range of 1.9-36 µg mL-1. Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.

Enzymatic Cascade Reactions in Biosynthesis.

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O2 to generate high valent iron-oxo species to homolyze unactivated C-H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5'-deoxyadenosyl radical as a powerful oxidant to initiate C-H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases".

Health Literacy: Cancer Prevention Strategies for Early Adults.

Health literacy, the degree to which individuals have the capacity to obtain, process, and understand health information and services needed to make health decisions, is an essential element for early adults (aged 18-44 years) to make informed decisions about cancer. Low health literacy is one of the social determinants of health associated with cancer-related disparities. Over the past several years, a nonprofit organization, a university, and a cancer center in a major urban environment have developed and implemented health literacy programs within healthcare systems and in the community. Health system personnel received extensive health literacy training to reduce medical jargon and improve their patient education using plain language easy-to-understand written materials and teach-back, and also designed plain language written materials including visuals to provide more culturally and linguistically appropriate health education and enhance web-based information. Several sustainable health system policy changes occurred over time. At the community level, organizational assessments and peer leader training on health literacy have occurred to reduce communication barriers between consumers and providers. Some of these programs have been cancer specific, including consumer education in such areas as cervical cancer, skin cancer, and breast cancer that are targeted to early adults across the cancer spectrum from prevention to treatment to survivorship. An example of consumer-driven health education that was tested for health literacy using a comic book-style photonovel on breast cancer with an intergenerational family approach for Chinese Americans is provided. Key lessons learned from the health literacy initiatives and overall conclusions of the health literacy initiatives are also summarized.

PCR-Independent Method of Transformation-Associated Recombination Reveals the Cosmomycin Biosynthetic Gene Cluster in an Ocean Streptomycete.

The transformation-associated recombination cloning methodology facilitates the genomic capture and heterologous expression of natural product biosynthetic gene clusters (BGCs). We have streamlined this procedure by introduction of synthetic DNA gene blocks for the efficient capture of BGCs. We show the successful capture and expression of the aromatic polyketide antitumor agent cosmomycin from streptomycete bacteria and the discovery of new cosmomycin analogues by mass spectral molecular networking.

Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges.

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin.

Colibactin is an as-yet-uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886, which uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block into its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal-cancer-linked microbial chemical.

NRPquest: Coupling Mass Spectrometry and Genome Mining for Nonribosomal Peptide Discovery.

Nonribosomal peptides (NRPs) such as vancomycin and daptomycin are among the most effective antibiotics. While NRPs are biomedically important, the computational techniques for sequencing these peptides are still in their infancy. The recent emergence of mass spectrometry techniques for NRP analysis (capable of sequencing an NRP from small amounts of nonpurified material) revealed an enormous diversity of NRPs. However, as many NRPs have nonlinear structure (e.g., cyclic or branched-cyclic peptides), the standard de novo sequencing tools (developed for linear peptides) are not applicable to NRP analysis. Here, we introduce the first NRP identification algorithm, NRPquest, that performs mutation-tolerant and modification-tolerant searches of spectral data sets against a database of putative NRPs. In contrast to previous studies aimed at NRP discovery (that usually report very few NRPs), NRPquest revealed nearly a hundred NRPs (including unknown variants of previously known peptides) in a single study. This result indicates that NRPquest can potentially make MS-based NRP identification as robust as the identification of linear peptides in traditional proteomics.

Enzyme inhibition by hydroamination: design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor.

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,ß-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

Automated genome mining of ribosomal peptide natural products.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs), especially from microbial sources, are a large group of bioactive natural products that are a promising source of new (bio)chemistry and bioactivity.1 In light of exponentially increasing microbial genome databases and improved mass spectrometry (MS)-based metabolomic platforms, there is a need for computational tools that connect natural product genotypes predicted from microbial genome sequences with their corresponding chemotypes from metabolomic data sets. Here, we introduce RiPPquest, a tandem mass spectrometry database search tool for identification of microbial RiPPs, and apply it to lanthipeptide discovery. RiPPquest uses genomics to limit search space to the vicinity of RiPP biosynthetic genes and proteomics to analyze extensive peptide modifications and compute p-values of peptide-spectrum matches (PSMs). We highlight RiPPquest by connecting multiple RiPPs from extracts of Streptomyces to their gene clusters and by the discovery of a new class III lanthipeptide, informatipeptin, from Streptomyces viridochromogenes DSM 40736 to reflect that it is a natural product that was discovered by mass spectrometry based genome mining using algorithmic tools rather than manual inspection of mass spectrometry data and genetic information. The presented tool is available at cyclo.ucsd.edu.

Antileukemic activity and mechanism of drug resistance to the marine Salinispora tropica proteasome inhibitor salinosporamide A (Marizomib).

Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome ß-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit ß5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. ß-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome inhibitors.

Genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors.

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α',ß'-epoxyketone pharmacophore allows binding to the catalytic ß-subunits of the proteasome with extraordinary specificity. Here, we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anticancer drug Kyprolis, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid nonribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation.