Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates.

Antimicrobial resistance remains a significant global threat, driving up mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we report the discovery of new aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptide antibiotics through a synergistic approach combining biosynthetic rule-based omics mining and heterologous expression. We first bioinformatically identify 1172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing peptides formation from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully establish the connection between three identified BGCs and the biosynthesis of five peptide antibiotics via biosynthetic rule-guided metabolic analysis. Notably, we discover a class V lanthipeptide, massatide A, which displays excellent activity against gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration of 0.25 µg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a relatively low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing peptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.

Whole transcriptome analysis reveals that immune infiltration- lncRNAs are related to cellular apoptosis in liver transplantation.

Introduction: In most instances, liver transplantation (LT) is the only available treatment for end-stage liver diseases. However, LT could also induce serious liver diseases or injury, and the underlying mechanisms of LT-induced complications remain largely unknown, especially the mechanisms of the dysfunction of the immune system mediated by long noncoding RNAs (lncRNAs). Methods: In this study, we globally analyzed the proportion of immune cells by using the transcriptome sequencing data (RNA-seq) of needle-core liver biopsies from pre- and post-transplantation recipients. Dysregulated lncRNAs were found to be correlated with the altered fractions of immune cells. We finally explored the potential targets of dysregulated lncRNAs and analyzed their functions in LT. Results: We found that in the samples, some immune cells changed significantly after LT, including CD4 T cells, NK cells and mast cells. The proportion of macrophages in different polarization states also changed significantly, with M0 macrophages increasing and M2 macrophages decreasing. Through weighted gene co-expression network analysis (WGCNA), 7 gene expression modules related to LT were identified. These modules were related to changes in the proportion of different immune cells. The functions of these modules represent the response modes of different functional genes after LT. Among these modules, MEtan and MEyellow modules were primarily enriched in apoptosis and inflammatory pathways. Twelve immunity-related lncRNAs were identified for the first time, and the regulatory network co-changing with immune cells was also identified. The co-expressed genes of these lncRNAs were highly enriched in apoptosis-related pathways. Many apoptosis-related genes were found to be up-regulated after LT. Discussion: In summary, we speculated that the expression and regulation of these apoptotic genes may be related to the changes in the proportion of immune cells. Some of these lncRNAs and apoptosis-related genes have been reported to be related to cell proliferation and apoptosis. They are also potential biomarkers or therapeutic targets.

A systematically biosynthetic investigation of lactic acid bacteria reveals diverse antagonistic bacteriocins that potentially shape the human microbiome.

BACKGROUND: Lactic acid bacteria (LAB) produce various bioactive secondary metabolites (SMs), which endow LAB with a protective role for the host. However, the biosynthetic potentials of LAB-derived SMs remain elusive, particularly in their diversity, abundance, and distribution in the human microbiome. Thus, it is still unknown to what extent LAB-derived SMs are involved in microbiome homeostasis. RESULTS: Here, we systematically investigate the biosynthetic potential of LAB from 31,977 LAB genomes, identifying 130,051 secondary metabolite biosynthetic gene clusters (BGCs) of 2,849 gene cluster families (GCFs). Most of these GCFs are species-specific or even strain-specific and uncharacterized yet. Analyzing 748 human-associated metagenomes, we gain an insight into the profile of LAB BGCs, which are highly diverse and niche-specific in the human microbiome. We discover that most LAB BGCs may encode bacteriocins with pervasive antagonistic activities predicted by machine learning models, potentially playing protective roles in the human microbiome. Class II bacteriocins, one of the most abundant and diverse LAB SMs, are particularly enriched and predominant in the vaginal microbiome. We utilized metagenomic and metatranscriptomic analyses to guide our discovery of functional class II bacteriocins. Our findings suggest that these antibacterial bacteriocins have the potential to regulate microbial communities in the vagina, thereby contributing to the maintenance of microbiome homeostasis. CONCLUSIONS: Our study systematically investigates LAB biosynthetic potential and their profiles in the human microbiome, linking them to the antagonistic contributions to microbiome homeostasis via omics analysis. These discoveries of the diverse and prevalent antagonistic SMs are expected to stimulate the mechanism study of LAB's protective roles for the microbiome and host, highlighting the potential of LAB and their bacteriocins as therapeutic alternatives. Video Abstract.

Expanded Sequence Space of Radical S-Adenosylmethionine-Dependent Enzymes Involved in Post-translational Macrocyclization.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM-catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteine-glycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.

Constructing Microbial Hosts for the Production of Benzoheterocyclic Derivatives.

Natural products containing benzoheterocyclic skeletons are widely found in plants and exhibit various pharmacological activities. To address the current limited availability of these compounds, we herein demonstrate the production of benzopyran, furanocoumarins, and pyranocoumarins in Streptomyces xiamenensis by employing prenyltransferases and two substrate-promiscuous enzymes, XimD and XimE. To avoid the degradation in S. xiamenensis, furanocoumarins and pyranocoumarins were also successfully produced in Escherichia coli. The production of linear furanocoumarins (marmesin) and angular pyranocoumarins (decursinol) reached 3.6 and 3.7 mg/L in shake flasks, respectively. To the best of our knowledge, this is the first report of the microbial production of the plant metabolites furanocoumarins and pyranocoumarins. Our study complements the missing link in the biosynthesis of pyranocoumarins by leveraging the catalytic promiscuity of microbial enzymes.

Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives.

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

A Novel AdpA Homologue Negatively Regulates Morphological Differentiation in Streptomyces xiamenensis 318.

The pleiotropic transcriptional regulator AdpA positively controls morphological differentiation and regulates secondary metabolism in most Streptomyces species. Streptomyces xiamenensis 318 has a linear chromosome 5.96 Mb in size. How AdpA affects secondary metabolism and morphological differentiation in such a naturally minimized genomic background is unknown. Here, we demonstrated that AdpA Sx , an AdpA orthologue in S. xiamenensis, negatively regulates cell growth and sporulation and bidirectionally regulates the biosynthesis of xiamenmycin and polycyclic tetramate macrolactams (PTMs) in S. xiamenensis 318. Overexpression of the adpASx gene in S. xiamenensis 318 had negative effects on morphological differentiation and resulted in reduced transcription of putative ssgA, ftsZ, ftsH, amfC, whiB, wblA1, wblA2, wblE, and a gene encoding sporulation-associated protein (sxim_29740), whereas the transcription of putative bldD and bldA genes was upregulated. Overexpression of adpASx led to significantly enhanced production of xiamenmycin but had detrimental effects on the production of PTMs. As expected, the transcriptional level of the xim gene cluster was upregulated, whereas the PTM gene cluster was downregulated. Moreover, AdpA Sx negatively regulated the transcription of its own gene. Electrophoretic mobility shift assays revealed that AdpA Sx can bind the promoter regions of structural genes of both the xim and PTM gene clusters as well as to the promoter regions of genes potentially involved in the cell growth and differentiation of S. xiamenensis 318. We report that an AdpA homologue has negative effects on morphological differentiation in S. xiamenensis 318, a finding confirmed when AdpA Sx was introduced into the heterologous host Streptomyces lividans TK24.IMPORTANCE AdpA is a key regulator of secondary metabolism and morphological differentiation in Streptomyces species. However, AdpA had not been reported to negatively regulate morphological differentiation. Here, we characterized the regulatory role of AdpA Sx in Streptomyces xiamenensis 318, which has a naturally streamlined genome. In this strain, AdpA Sx negatively regulated cell growth and morphological differentiation by directly controlling genes associated with these functions. AdpA Sx also bidirectionally controlled the biosynthesis of xiamenmycin and PTMs by directly regulating their gene clusters rather than through other regulators. Our findings provide additional evidence for the versatility of AdpA in regulating morphological differentiation and secondary metabolism in Streptomyces.

Combinatory Biosynthesis of Prenylated 4-Hydroxybenzoate Derivatives by Overexpression of the Substrate-Promiscuous Prenyltransferase XimB in Engineered E. coli.

Prenylated aromatic compounds are important intermediates in the biosynthesis of bioactive molecules such as 3-chromanols from plants, ubiquinones from prokaryotes and meroterpenoids from sponges. Biosynthesis of prenylated aromatic compounds using prokaryotic microorganisms has attracted increasing attention in the field of synthetic biology. In this study, we demonstrated that the production of 3-geranyl-4-hydroxybenzoic acid (GBA) and a variety of GBA analogues was feasible in a metabolically engineered E. coli by using XimB, a special prenyltransferase involved in the biosynthesis of xiamenmycin A in Streptomyces xiamenensis 318. XimB exhibits broad substrate specificity and can catalyze the transfer reaction of prenyl moieties with different carbon chain lengths to both the natural substrate 4-hydroxybenzoate (4-HBA) and to different substituted 4-HBA derivatives at C-2 and C-3. Feeding 4-HBA to an engineered E. coli equipped with a hybrid mevalonate pathway increased the production of GBA up to 94.30 mg/L. Considerable amounts of other GBA derivatives, compounds 4, 5, 6, 7, and 9, can be achieved by feeding precursors. The plug-and-play design for inserting C5, C15, and C20 prenyl diphosphate synthetases under the control of the T7 promoter resulted in targeted production of 3-dimethylallyl, 3-farnesyl-, and 3-geranylgeranyl-4-hydroxybenzoic acid, respectively. Furthermore, the valuable benzopyran xiamenmycin B was successfully produced in E. coli R7-MVA by coexpression of a complete biosynthetic gene cluster, which contains ximBDE.

24-Epibrassinolide alleviates organic pollutants-retarded root elongation by promoting redox homeostasis and secondary metabolism in Cucumis sativus L.

Environmental pollution by organic pollutants (OPs) has become a global concern due to its detrimental effects on the environment and human health. As plants are used to remediate contaminated sites, understanding the responses of plants to various OPs and fortification of plant tolerance are of great significance. In this work, we studied the biochemical and molecular responses of cucumber plants to three well-known OPs, 2,4,6-trichlorophenol, chlorpyrifos and oxytetracycline in the absence or presence of 24-epibrassinolide (EBR), a potent regulator of plant growth and stress tolerance. The results showed that the selected three OPs retarded root elongation; however, the phytotoxic effects of OPs were attenuated by exogenous EBR. OPs induced accumulations of both hydrogen peroxide (H2O2) and nitric oxide (NO) in root tips and resulted in an increased malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation. Exogenous EBR reduced accumulations of H2O2, NO and MDA in the roots by increasing the expression of antioxidant and detoxification genes and the activities of the corresponding enzymes. Intriguingly, EBR not only promoted the activities of glutathione S-transferase and glutathione reductase, but also increased the content of reduced glutathione without altering the content of oxidized glutathione, which resulted in a reduced redox state under OPs stress. Furthermore, EBR increased the free radical scavenging capacity, flavonoid content and the activity and transcription of secondary metabolism related enzymes. Our results suggest that EBR treatment may fortify secondary metabolism to enhance antioxidant capacity in response to OPs treatment, which might have potential implication in phytoremediation of OPs.