Tandem mass spectral metabolic profiling of 54 actinobacterial strains and their 459 mutants.

Natural products encompass a diverse range of compounds with high impact applications in consumer care, agriculture and most notably, therapeutics. However, despite the expansive chemical repertoire indicated in genomic information of microbes, only a small subset can be obtained under laboratory conditions. To increase accessible chemical space and realize Nature's full chemical potential, a multi-pronged genetic- and cultivation-based strategy has been employed to activate and upregulate natural product biosyntheses in native and heterologous strains.

Application of Cas12j for Editing.

In recent years, CRISPR-Cas toolboxes for editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of strains.

Design-rules for stapled peptides with in vivo activity and their application to Mdm2/X antagonists.

Although stapled α-helical peptides can address challenging targets, their advancement is impeded by poor understandings for making them cell permeable while avoiding off-target toxicities. By synthesizing >350 molecules, we present workflows for identifying stapled peptides against Mdm2(X) with in vivo activity and no off-target effects. Key insights include a clear correlation between lipophilicity and permeability, removal of positive charge to avoid off-target toxicities, judicious anionic residue placement to enhance solubility/behavior, optimization of C-terminal length/helicity to enhance potency, and optimization of staple type/number to avoid polypharmacology.

Exploring a general multi-pronged activation strategy for natural product discovery in Actinomycetes.

Natural products possess significant therapeutic potential but remain underutilized despite advances in genomics and bioinformatics. While there are approaches to activate and upregulate natural product biosynthesis in both native and heterologous microbial strains, a comprehensive strategy to elicit production of natural products as well as a generalizable and efficient method to interrogate diverse native strains collection, remains lacking. Here, we explore a flexible and robust integrase-mediated multi-pronged activation approach to reliably perturb and globally trigger antibiotics production in actinobacteria.

Site-selective chlorination of pyrrolic heterocycles by flavin dependent enzyme PrnC.

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking.

Expression and engineering of PET-degrading enzymes from , and .

Mismanagement of PET plastic waste significantly threatens human and environmental health. Together with the relentless increase in plastic production, plastic pollution is an issue of rising concern. In response to this challenge, scientists are investigating eco-friendly approaches, such as bioprocessing and microbial factories, to sustainably manage the growing quantity of plastic waste in our ecosystem.

De Novo Sequencing of Synthetic -cysteine Peptide Macrocycles Enabled by "Chemical Linearization" of Compound Mixtures.

A "chemical linearization" approach was applied to synthetic peptide macrocycles to enable their de novo sequencing from mixtures using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). This approach─previously applied to individual macrocycles but not to mixtures─involves cleavage of the peptide backbone at a defined position to give a product capable of generating sequence-determining fragment ions. Here, we first established the compatibility of "chemical linearization" by Edman degradation with a prominent macrocycle scaffold based on -Cys peptides cross-linked with the -xylene linker, which are of major significance in therapeutics discovery.

The Engineering, Expression, and Immobilization of Epimerases for -allulose Production.

The rare sugar -allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises -allulose 3-epimerase (DAEase) or -psicose 3-epimerase (DPEase) to convert -fructose into -allulose. Additionally, the process can also utilise -tagatose 3-epimerase (DTEase).

Further Characterization of Fungal Halogenase RadH and Its Homologs.

RadH is one of the flavin-dependent halogenases that has previously exhibited promising catalytic activity towards hydroxycoumarin, hydroxyisoquinoline, and phenolic derivatives. Here, we evaluated new functional homologs of RadH and expanded its specificities for the halogenation of non-tryptophan-derived, heterocyclic scaffolds. Our investigation revealed that RadH could effectively halogenate hydroxyquinoline and hydroxybenzothiophene.

67 million natural product-like compound database generated via molecular language processing.

Natural products are a rich resource of bioactive compounds for valuable applications across multiple fields such as food, agriculture, and medicine. For natural product discovery, high throughput in silico screening offers a cost-effective alternative to traditional resource-heavy assay-guided exploration of structurally novel chemical space. In this data descriptor, we report a characterized database of 67,064,204 natural product-like molecules generated using a recurrent neural network trained on known natural products, demonstrating a significant 165-fold expansion in library size over the approximately 400,000 known natural products.

Enhancing armeniaspirols production through multi-level engineering of a native Streptomyces producer.

Background: Nature has provided unique molecular scaffolds for applications including therapeutics, agriculture, and food. Due to differences in ecological environments and laboratory conditions, engineering is often necessary to uncover and utilize the chemical diversity. Although we can efficiently activate and mine these often complex 3D molecules, sufficient production of target molecules for further engineering and application remain a considerable bottleneck.

Cost-effective hybrid long-short read assembly delineates alternative GC-rich hosts for natural product discovery.

With the advent of rapid automated identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, , are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented intensive sequencing.

A Spirobicyclo[3.1.0]Terpene from the Investigation of Sesquiterpene Synthases from .

Milk cap mushrooms in the genus are known to produce a wide variety of terpene natural products. However, their repertoire of terpene biosynthetic enzymes has not been fully explored. In this study, several candidate sesquiterpene synthases were identified from the genome of the saffron milk cap mushroom and expressed in a sesquiterpene-overproducing strain.

A Mild and Regioselective Route to Fluoroalkyl Aromatic Compounds via Directed Cycloaddition Reactions.

The synthesis of perfluoroalkyl-substituted (hetero)arenes by benzannulation strategies is complementary to ring functionalization approaches as it obviates the need for pre-existing functionality and innate regiocontrol. We report a mild and regiospecific boron-directed benzannulation method as a vehicle for accessing a range of perfluoroalkyl-substituted (hetero)aromatic building blocks that can be readily elaborated through established C-B bond functionalization processes.

Pyrimidin-6-yl Trifluoroborate Salts as Versatile Templates for Heterocycle Synthesis.

We report a novel and general method to access a highly under-studied privileged scaffold-pyrimidines bearing a trifluoroborate at C4, and highlight the broad utility of these intermediates in a rich array of downstream functionalization reactions. This chemistry is underpinned by the unique features of the trifluoroborate group; its robustness provides an opportunity to carry out chemoselective reactions at other positions on the pyrimidine while providing a pathway for elaboration at the C-B bond when suitably activated.

Direct Deoxydehydration of Cyclic trans-Diol Substrates: An Experimental and Computational Study of the Reaction Mechanism of Vanadium(V)-based Catalysis*.

The deoxydehydration of carbohydrates represents a key target to leverage renewable biomass resources chemically. Using a vanadium(V)-based catalyst, it was possible to directly deoxydehydrate cyclic trans-diol substrates. Accompanying mechanistic characterisation of this process by density functional calculations pointed to an energetically tractable route for deoxydehydration of cyclic trans-diol substrates involving stepwise cleavage of the diol C-O bonds via the triplet state; experimentally, this was supported by light dependence of the reaction.

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin.

Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam.

Correction: Chemogenomic profiling in yeast reveals antifungal mode-of-action of polyene macrolactam auroramycin.

[This corrects the article DOI: 10.1371/journal.pone.

Chemogenomic profiling in yeast reveals antifungal mode-of-action of polyene macrolactam auroramycin.

In this study, we report antifungal activity of auroramycin against Candida albicans, Candida tropicalis, and Cryptococcus neoformans. Auroramycin, a potent antimicrobial doubly glycosylated 24-membered polyene macrolactam, was previously isolated and characterized, following CRISPR-Cas9 mediated activation of a silent polyketide synthase biosynthetic gene cluster in Streptomyces rosesporous NRRL 15998. Chemogenomic profiling of auroramycin in yeast has linked its antifungal bioactivity to vacuolar transport and membrane organization.

Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes.

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid.

Auroramycin: A Potent Antibiotic from Streptomyces roseosporus by CRISPR-Cas9 Activation.

Silent biosynthetic gene clusters represent a potentially rich source of new bioactive compounds. We report the discovery, characterization, and biosynthesis of a novel doubly glycosylated 24-membered polyene macrolactam from a silent biosynthetic gene cluster in Streptomyces roseosporus by using the CRISPR-Cas9 gene cluster activation strategy. Structural characterization of this polyketide, named auroramycin, revealed a rare isobutyrylmalonyl extender unit and a unique pair of amino sugars.

Empirical and Computational Insights into N-Arylation Reactions Catalyzed by Palladium meta-Terarylphosphine Catalyst.

An in situ generated Pd-Cy*Phine catalyst has been successfully applied to the N-arylation of primary and secondary amines, and it exhibited high performance across multiple substrate classes. The performance induced by the meta-terarylphosphine motif of the Cy*Phine ligand for C-N cross-coupling displayed only subtle differences to that of its biarylphosphine congener XPhos. DFT studies demonstrated comparable reaction energetics in the catalytic cycle steps for both Pd-Cy*Phine and Pd-XPhos, which was consistent with previous findings.

CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters.

Here we report an efficient CRISPR-Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes. This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate.

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA).

Palladium-meta-terarylphosphine catalyst for the Mizoroki-Heck reaction of (hetero)aryl bromides and functional olefins.

The evolutionary meta-terarylphosphine ligand architecture of Cy*Phine was recently shown to be a key feature that imposed outstanding performance in palladium-catalyzed copper-free Sonogashira applications. Herein, the Pd-Cy*Phine combination has similarly proven to be a powerful catalyst system for the Mizoroki-Heck reaction. Using high-throughput screening (HTS) methodology, DMF and NaHCO3 were rapidly identified as the most effective solvent and base pair for the cross-coupling catalysis of challenging and industrially valuable substrates including highly electron-rich heteroaryl bromides and unactivated olefins.