Classification and Multi-Functional Use of Bacteriocins in Health, Biotechnology, and Food Industry.

Bacteriocins is the name given to products of the secondary metabolism of many bacterial genera that must display antimicrobial activity. Although there are several bacteriocins described today, it has not been possible to reach a consensus on the method of classification for these biomolecules. In addition, many of them are not yet authorized for therapeutic use against multi-drug-resistant microorganisms due to possible toxic effects. However, recent research has achieved considerable progress in the understanding, classification, and elucidation of their mechanisms of action against microorganisms, which are of medical and biotechnological interest. Therefore, in more current times, protocols are already being conducted for their optimal use, in the hopes of solving multiple health and food conservation problems. This review aims to synthetize the information available nowadays regarding bacteriocins, and their classification, while also providing an insight into the future possibilities of their usage for both the pharmaceutical, food, and biotechnological industry.

Intestinal microbiota dataset revealed by high-throughput sequencing of 16S rRNA in children with anemia in southern Peru.

Anemia is the most common hematological disorder affecting humans. In Peru, anemia is a pressing issue that present the most significant concern due to its adverse effects, such as delayed growth and psychomotor development, in addition to a deficiency in cognitive development. Anemia is a significant public health issue in Peru, which has one of the highest prevalence rates in infants in the Latin American and Caribbean (LAC) region, affecting approximately 43.6 % of children under three years nationally as of 2017, with rural areas experiencing a higher prevalence of approximately 53.3 %. In 2019, the prevalence was highest in the Sierra (48.8 %) and Selva regions (44.6 %), whereas the coast had a lower rate of 33.9 % in children under 36 months. Although the composition of the gut microbiota is relatively well described in children, there is little information on the identification of the microbiota in iron-deficiency anemia. There is evidence that diseases or health conditions can change the microbiota, or vice versa. This study aimed to identify the microbiota in children with anemia who did not recover after iron treatment. In a previous study, we found that the phylum Actinobacteria was predominant in the microbiota of children with anemia. These data will be useful for understanding the functionality of the most important bacteria found in each group at the genus or species level, especially the metabolic pathways in which they participate and their links with iron metabolism. Microbial composition data were obtained through next-generation 16S rRNA sequencing (NGS) of stool samples from children with anemia in southern Peru. Numerous studies have underscored the importance of early symbiotic development in infant health and its long-term impact on health. From infancy, modulation of the gut microbiota can promote long-term health. According to the National Institute of Health (NIH), iron-deficiency anemia may cause serious complications, such as fatigue, headaches, restless legs syndrome, heart problems, pregnancy complications, and developmental delays in children. The development of the gut microbiota is regulated by a complex interplay between host and environmental factors. The bidirectional link between the gut microbiota and anemia plays an important role in tracking the gut microbiota and will be useful in understanding the composition of the intestinal microbiota and its implications in anemia, which has now become a public health problem. Our previous study investigated the microbial composition in children with iron-deficiency anemia and revealed the presence of several bacterial groups, including Proteobacteria, Actinobacteria, Firmicutes, and Chloroflexi. In addition, these data may be useful for investigating the association between the intestinal microbiota of children with persistent anemia and those who have recovered.

Bicarbonate-binding catalysis for the enantioselective desymmetrization of keto sulfonium salts.

Herein, an enantioselective desymmetrization of cyclic keto sulfonium salts through enantioselective deprotonation/ring opening process by anion-binding catalysis is presented. We report a squaramide/HCO3- complex as catalytic active species which is able to stereo-differentiate two enantiomeric protons, triggering the ring opening event taking advantage of the great tendency of sulfonium salts to act as leaving groups. Thus, this desymmetrization methodology give rise to ß-methylsulfenylated sulfa-Michael addition type products with excellent yields and very good enantioselectivities. The bifunctional organocatalyst has been demonstrated to be capable of activating simultaneously the base and the keto sulfonium salt by DFT calculations and experimental proofs.

Volatile communication in Actinobacteria: a language for secondary metabolism regulation.

BACKGROUND: Volatile compounds are key elements in the interaction and communication between organisms at both interspecific and intraspecific levels. In complex bacterial communities, the emission of these fast-acting chemical messengers allows an exchange of information even at a certain distance that can cause different types of responses in the receiving organisms. The changes in secondary metabolism as a consequence of this interaction arouse great interest in the field of searching for bioactive compounds since they can be used as a tool to activate silenced metabolic pathways. Regarding the great metabolic potential that the Actinobacteria group presents in the production of compounds with attractive properties, we evaluated the reply the emitted volatile compounds can generate in other individuals of the same group. RESULTS: We recently reported that volatile compounds released by different streptomycete species trigger the modulation of biosynthetic gene clusters in Streptomyces spp. which finally leads to the activation/repression of the production of secondary metabolites in the recipient strains. Here we present the application of this rationale in a broader bacterial community to evaluate volatiles as signaling effectors that drive the activation of biosynthesis of bioactive compounds in other members of the Actinobacteria group. Using cocultures of different actinobacteria (where only the volatile compounds reach the recipient strain) we were able to modify the bacterial secondary metabolism that drives overproduction (e.g., granaticins, actiphenol, chromomycins) and/or de novo production (e.g., collismycins, skyllamycins, cosmomycins) of compounds belonging to different chemical species that present important biological activities. CONCLUSIONS: This work shows how the secondary metabolism of different Actinobacteria species can vary significantly when exposed in co-culture to the volatile compounds of other phylum-shared bacteria, these effects being variable depending on strains and culture media. This approach can be applied to the field of new drug discovery to increase the battery of bioactive compounds produced by bacteria that can potentially be used in treatments for humans and animals.

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Tracing of Di-Ethylhexyl Phthalate in the Tequila Production Process.

The purpose of this study was to determine the origin, presence, and fate of the endocrine disruptor di-ethylhexil phthalate (DEHP) during tequila production. For this, three tequila factories (small, medium, and large) were monitored. DEHP concentrations in water, agave, additives, lubricating greases, neoprene seals, and materials of each stage process were analyzed using gas chromatography/mass spectrometry. DEHP mass balances were performed to identify the processes with significant changes in the inputs/outputs. DEHP was detected in agave at up to 0.08 ± 0.03 mg kg-1, water 0.02 ± 0.01 mg kg-1, lubricant greases 131.05 ± 2.80 mg kg-1, and neoprene seals 369.11 ± 22.52 mg kg-1. Whereas, tequila produced in the large, medium, and small factories contained 0.05 ± 0.01, 0.24 ± 0.04, and 1.43 ± 0.48 mg kg-1 DEHP, respectively. Furthermore, in waste materials (vinasses and bagasse) released, 534.26 ± 349.02, 947.18 ± 65.84, and 5222.60 ± 2836.94 mg of DEHP was detected for every 1000 L of tequila produced. The most significant increase in DEHP occurred during the sugar extraction and distillation stages. Results demonstrate that main raw materials, such as agave and water, contain DEHP, but lubricant greases and neoprene seals are the major sources of DEHP contamination. Identification of the contamination sources can help the tequila industry to take actions to reduce it, protect consumer health and the environment, and prevent circular contamination.

Clinical examination and imaging resources in children and adolescent back pain.

Back pain is a relatively common complaint in children and adolescents. The pediatric patient presenting with back pain can often be challenging, and there are many well-known organic diagnoses that should not be missed. In younger children, an organic cause of back pain can often be found. However, back pain in older children and adolescents is often "non-specific." The differential diagnosis of back pain in children includes neoplasms, developmental, and inflammatory conditions. Basic steps should include an in-depth anamnesis, a systematic physical examination, and standard spine radiographs (anteroposterior and lateral). Nevertheless, advanced diagnostic imaging and laboratory studies should be included when indicated to avoid missing or delaying a serious diagnosis. If other types of imaging tests are necessary (magnetic resonance imaging, computed tomography, bone scan, or single photon emission computed tomography), they should be guided by diagnostic suspicion.

Mediterranean Shrub Species as a Source of Biomolecules against Neurodegenerative Diseases.

Neurodegenerative diseases are associated with oxidative stress, due to an imbalance in the oxidation-reduction reactions at the cellular level. Various treatments are available to treat these diseases, although they often do not cure them and have many adverse effects. Therefore, it is necessary to find complementary and/or alternative drugs that replace current treatments with fewer side effects. It has been demonstrated that natural products derived from plants, specifically phenolic compounds, have a great capacity to suppress oxidative stress and neutralize free radicals thus, they may be used as alternative alternative pharmacological treatments for pathological conditions associated with an increase in oxidative stress. The plant species that dominate the Mediterranean ecosystems are characterized by having a wide variety of phenolic compound content. Therefore, these species might be important sources of neuroprotective biomolecules. To evaluate this potential, 24 typical plant species of the Mediterranean ecosystems were selected, identifying the most important compounds present in them. This set of plant species provides a total of 403 different compounds. Of these compounds, 35.7% are phenolic acids and 55.6% are flavonoids. The most relevant of these compounds are gallic, vanillic, caffeic, chlorogenic, p-coumaric, and ferulic acids, apigenin, kaempferol, myricitrin, quercetin, isoquercetin, quercetrin, rutin, catechin and epicatechin, which are widely distributed among the analyzed plant species (in over 10 species) and which have been involved in the literature in the prevention of different neurodegenerative pathologies. It is also important to mention that three of these plant species, Pistacea lentiscus, Lavandula stoechas and Thymus vulgaris, have most of the described compounds with protective properties against neurodegenerative diseases. The present work shows that the plant species that dominate the studied geographic area can provide an important source of phenolic compounds for the pharmacological and biotechnological industry to prepare extracts or isolated compounds for therapy against neurodegenerative diseases.

Characterization of Pulmonary Bacteriobiota in Critically Ill Patients in Southern Peru through Next-Generation Sequencing Technology.

Sequence variation in the 16S gene is widely used to characterize diverse microbial communities. This was the first pilot study carried out in our region where the pulmonary microbiota of critically ill patients was investigated and analyzed, with the aim of finding a specific profile for these patients that can be used as a diagnostic marker. An study of critical patients mechanically ventilated for non-respiratory indications, in a polyvalent intensive care unit, was carried out; samplee were extracted by endotracheal aspiration and subsequently the microbiota was characterized through Next-Generation Sequencing Technology (NGS). The predominant phyla among the critically ill patients were Proteobacteria, Firmicutes and Bacteroidata. In the surviving patients group, the predominant phyla were Proteobacteria, Bacteroidata and Firmicutes, in the group of deceased patients thy were Firmicutes, Proteobacteria, and Bacteroidata. We found a decrease in commensal bacteria in deceased patients and a progressive increase in in-hospital germs.

Cytotoxic Activity of Saponins and Sapogenins Isolated from Chenopodium quinoa Willd. in Cancer Cell Lines.

The cytotoxic properties of two extracts from Chenopodium quinoa Willd. and three synthetic sapogenins were evaluated in different cancer cell lines (A549, SH-SY5Y, HepG2, and HeLa) to investigate their cytotoxic effects and determine if these cell lines activate the caspase pathway for apoptosis in response to saponin and sapogenin treatment. The saponin extracts were isolated from the agro-industrial waste of Chenopodium quinoa Willd., while the sapogenins were identified and quantitatively determined by High-Performance Liquid Chromatography (HPLC). Among these compounds, ursolic acid was the most active compound, with high IC50 values measured in all cell lines. In addition, hederagenin demonstrated higher caspase-3 activity than staurosporine in HeLa cells, suggesting an anti-cytotoxic activity via a caspase-dependent apoptosis pathway. HPLC analysis showed that the concentration of hederagenin was higher than that of oleanolic acid in ethanolic extracts of white and red quinoa. The ethanolic extracts of white and red quinoa did not show cytotoxic activity. On the other hand, the synthetic sapogenins such as ursolic acid, oleanolic acid, and hederagenin significantly decreased the viability of the four cell lines studied. Finally, by Caspase-3 assay, it was found that HeLa undergoes apoptosis during cell death because hederagenin produces a significant increase in PARP-1 hydrolysis in HeLa cells.

Ketorolac removal through photoelectrocatalysis using TiO2 nanotubes in water system.

Ketorolac, a highly persistent NSAID of environmental concern, was significantly removed from water (80% removal) through photoelectrocatalysis where titanium dioxide nanotubes prepared by Ti foil electrochemical anodization at 30 V were used as photoanodes. Fifteen milligrams per liter of ketorolac solutions in a 0.05 M Na2SO4 aqueous medium was subjected to irradiation from a 365-nm light with an intensity of 1 mWcm-2 and under an applied potential of 1.3 V (vs. Hg/Hg2SO4/sat.K2SO4) at pH 6.0. When each process (photo and electrocatalysis) was carried out separately, less than 20% drug removal was achieved as monitored through UV-vis spectrophotometry. Through scavenging experiments, direct oxidation on the photogenerated holes and oxidation by hydroxyl radical formation were found to play a key role on ketorolac's degradation. Chemical oxygen demand (COD) analyses also showed a significant COD decreased (68%) since the initial COD value was 31.3 mg O2/L and the final COD value was 10.1 mg O2/L. A 48% mineralization was also achieved, as shown by total organic carbon (TOC) analyses. These results showed that electrodes based on titania nanotubes are a promising alternative material for simultaneous photocatalytic and electrocatalytic processes in water remediation.

Identification of the Biosynthetic Gene Cluster of New Piperazic Acid-Containing Lipopeptides with Cytotoxic Activity in the Genome of Marine Streptomyces PHM034.

Three novel lipopeptides, PM130391 (1), PM130392 (2), and PM140293 (3) were obtained from cultures of Streptomyces tuirus PHM034 isolated from a marine sediment. Structural elucidation of the three compounds showed they belong to the nonribosomal peptides family, and they all contain an acylated alanine, three piperazic acids, a methylated glycine, and an N-hydroxylated alanine. The difference between the three compounds resides in the acyl chain bound to the alanine residue. All three compounds showed cytotoxic activity against human cancer cell lines. Genome sequence and bioinformatics analysis allowed the identification of the gene cluster responsible for the biosynthesis. Inactivation of a nonribosomal peptide synthase of this cluster abolished the biosynthesis of the three compounds, thus demonstrating the involvement of this cluster in the biosynthesis of these lipopeptides.

The Volatile Organic Compounds of Streptomyces spp.: An In-Depth Analysis of Their Antifungal Properties.

The study of volatile organic compounds (VOCs) has expanded because of the growing need to search for new bioactive compounds that could be used as therapeutic alternatives. These small molecules serve as signals to establish interactions with other nearby organisms in the environment. In this work, we evaluated the antifungal effect of VOCs produced by different Streptomyces spp. This study was performed using VOC chamber devices that allow for the free exchange of VOCs without physical contact between microorganisms or the diffusible compounds they produce. Antifungal activity was tested against Escovopsis weberi, a fungal pathogen that affects ant nest stability, and the results showed that Streptomyces spp. CS014, CS057, CS131, CS147, CS159, CS207, and CS227 inhibit or reduce the fungal growth with their emitted VOCs. A GS-MS analysis of volatiles produced and captured by activated charcoal suggested that these Streptomyces strains synthesize several antifungal VOCs, many of them produced because of the presence of E. weberi, with the accumulation of various VOCs determining the growth inhibition effect.

Co-Expression of Transcriptional Regulators and Housekeeping Genes in Streptomyces spp.: A Strategy to Optimize Metabolite Production.

The search for novel bioactive compounds to overcome resistance to current therapeutics has become of utmost importance. Streptomyces spp. are one of the main sources of bioactive compounds currently used in medicine. In this work, five different global transcriptional regulators and five housekeeping genes, known to induce the activation or overproduction of secondary metabolites in Streptomyces coelicolor, were cloned in two separated constructs and expressed in 12 different strains of Streptomyces spp. from the in-house CS collection. These recombinant plasmids were also inserted into streptomycin and rifampicin resistant Streptomyces strains (mutations known to enhance secondary metabolism in Streptomyces). Different media with diverse carbon and nitrogen sources were selected to assess the strains' metabolite production. Cultures were then extracted with different organic solvents and analysed to search for changes in their production profiles. An overproduction of metabolites already known to be produced by the biosynthesis wild-type strains was observed such as germicidin by CS113, collismycins by CS149 and CS014, or colibrimycins by CS147. Additionally, the activation of some compounds such as alteramides in CS090a pSETxkBMRRH and CS065a pSETxkDCABA or inhibition of the biosynthesis of chromomycins in CS065a in pSETxkDCABA when grown in SM10 was demonstrated. Therefore, these genetic constructs are a relatively simple tool to manipulate Streptomyces metabolism and explore their wide secondary metabolites production potential.

Uncovering the Cryptic Gene Cluster ahb for 3-amino-4-hydroxybenzoate Derived Ahbamycins, by Searching SARP Regulator Encoding Genes in the Streptomyces argillaceus Genome.

Genome mining using standard bioinformatics tools has allowed for the uncovering of hidden biosynthesis gene clusters for specialized metabolites in Streptomyces genomes. In this work, we have used an alternative approach consisting in seeking "Streptomyces Antibiotic Regulatory Proteins" (SARP) encoding genes and analyzing their surrounding DNA region to unearth cryptic gene clusters that cannot be identified using standard bioinformatics tools. This strategy has allowed the unveiling of the new ahb cluster in Streptomyces argillaceus, which had not been retrieved before using antiSMASH. The ahb cluster is highly preserved in other Streptomyces strains, which suggests a role for their encoding compounds in specific environmental conditions. By combining overexpression of three regulatory genes and generation of different mutants, we were able to activate the ahb cluster, and to identify and chemically characterize the encoded compounds that we have named ahbamycins (AHBs). These constitute a new family of metabolites derived from 3-amino-4-hydroxybenzoate (3,4-AHBA) known for having antibiotic and antitumor activity. Additionally, by overexpressing three genes of the cluster (ahbH, ahbI, and ahbL2) for the synthesis and activation of 3,4-AHBA, a new hybrid compound, AHB18, was identified which had been produced from a metabolic crosstalk between the AHB and the argimycin P pathways. The identification of this new BGC opens the possibility to generate new compounds by combinatorial biosynthesis.

In vitro release of aluminum from the geophagic clay "Chacco" in the Peruvian highlands: Chemical characterization and health risk assessment.

In the altiplano zone of Latin America, "Chacco" is one of the clays widely consumed as part of geophagy. The objectives of the study were to chemically characterize "Chacco", determine the zero charge point, evaluate the release of aluminum in vitro, perform the kinetic study and evaluate the health risk. The results by ICP-OES showed that the elements with the highest concentration were Al, Ba, Ca, Fe, K, Mg, Mn, Na, Si, Sr, Ti and Zn. ATR-FTIR analysis showed the presence of Si-O (693 and 990 cm-1), Al-O (790 cm-1), Al-Al-OH bending vibration (912 cm-1), Si-H bond stretching (2100 to 2500 cm-1) and free -OH groups (3629 cm-1). SEM-EDX results indicate that Al is one of the main constituents of "Chacco" (7.35 wt%). The pHzpc of "Chacco" was 6.83. In the dissolution profiles, the highest Al release occurred at pH 6.8 and in intestinal juice simulated with pseudo-second order dissolution kinetics. The EDIAl and EWIAl were 20.24 and 142.66 respectively, comparing EWIAl with the PTWI established by JECFA (2 mg/kg bw), it is concluded that the weekly intake of "Chacco" represents an appreciable health risk. There are no reports of the carcinogenic factor of Al, so TRAl was not calculated.

Intestinal Microbiota in Children with Anemia in Southern Peru through Next-Generation Sequencing Technology.

Knowledge of the sequencing of the 16S rRNA gene constitutes a true revolution in understanding the composition of the intestinal microbiota and its implication in health states. This study details microbial composition through next-generation sequencing (NGS) technology in children with anemia. Anemia is the most frequent hematological disorder that affects human beings. In Peru, it is one of the conditions that presents the most significant concern due to the adverse effects that cause it, such as delayed growth and psychomotor development, in addition to a deficiency in cognitive development.

Volatile Compounds in Actinomycete Communities: A New Tool for Biosynthetic Gene Cluster Activation, Cooperative Growth Promotion, and Drug Discovery.

The increasing appearance of multiresistant pathogens, as well as emerging diseases, has highlighted the need for new strategies to discover natural compounds that can be used as therapeutic alternatives, especially in the genus Streptomyces, which is one of the largest producers of bioactive metabolites. In recent years, the study of volatile compounds (VOCs) has raised interest because of the variety of their biological properties in addition to their involvement in cell communication. In this work, we analyze the implications of VOCs as mediating molecules capable of inducing the activation of biosynthetic pathways of bioactive compounds in surrounding Actinomycetes. For this purpose, several strains of Streptomyces were co-cultured in chamber devices that allowed VOC exchange while avoiding physical contact. In several of those strains, secondary metabolism was activated by VOCs emitted by companion strains, resulting in increased antibiotic production and synthesis of new VOCs. This study shows a novel strategy to exploit the metabolic potential of Actinomycetes as well as emphasizes the importance of studying the interactions between different microorganisms sharing the same ecological niche.

Combinatorial biosynthesis yields novel hybrid argimycin P alkaloids with diverse scaffolds in Streptomyces argillaceus.

Coelimycin P1 and argimycins P are two types of polyketide alkaloids produced by Streptomyces coelicolor and Streptomyces argillaceus, respectively. Their biosynthesis pathways share some early steps that render very similar aminated polyketide chains, diverging the pathways afterwards. By expressing the putative isomerase cpkE and/or the putative epoxidase/dehydrogenase cpkD from the coelimycin P1 gene cluster into S. argillaceus wild type and in argimycin mutant strains, five novel hybrid argimycins were generated. Chemical characterization of those compounds revealed that four of them show unprecedented scaffolds (quinolizidine and pyranopyridine) never found before in the argimycin family of compounds. One of these compounds (argimycin DM104) shows improved antibiotic activity. Noticeable, biosynthesis of these quinolizidine argimycins results from a hybrid pathway created by combining enzymes from two different pathways, which utilizes an aminated polyketide chain as precursor instead of lysine as it occurs for other quinolizidines.

Biosynthesis of Largimycins in Streptomyces argillaceus Involves Transient ß-Alkylation and Cryptic Halogenation Steps Unprecedented in the Leinamycin Family.

Largimycins A1 and A2 are key members of a recently identified family of hybrid nonribosomal peptide polyketides belonging to the scarcely represented group of antitumor leinamycins. They are encoded by the gene cluster lrg of Streptomyces argillaceus. This cluster contains a halogenase gene and two sets of genes for the biosynthesis and incorporation of ß branches at C3 and C9. Noticeably, largimycins A1 and A2 are nonhalogenated compounds and only contain a ß branch at C3. By generating mutants in those genes and characterizing chemically their accumulated compounds, we could confirm the existence of a chlorination step at C19, the introduction of an acetyl-derived olefinic exomethylene group at C9, and a propionyl-derived ß branch at C3 in the biosynthesis pathway. Since the olefinic exomethylene group and the chlorine atom are absent in the final products, those biosynthetic steps can be considered cryptic in the overall pathway but essential to generating keto and epoxide functionalities at C9 and C18/C19, respectively. We propose that chlorination at C19 is utilized as an activation strategy that creates the precursor halohydrin to finally yield the epoxy functionality at C18/C19. This represents a novel strategy to create such functionalities and extends the small number of natural product biosynthetic pathways that include a cryptic chlorination step.