Chengzhang Fu

Myxoglucamides, natural products recently isolated from Cystobacterineae sp., are featured by an unprecedented vinyl‐substituted α‐keto‐γ‐amino acid that is linked to a glycosylated 14‐methyl‐pentadecanoic acid. Thus, they unite elements from three biomolecular classes in a compact glycolipopeptide. To elucidate the biological relevance of this arrangement, we searched for molecular targets by activity‐based protein profiling (ABPP)—although a phenotypic bioactivity has not been reported. An access to the compound class was established through the first total synthesis of myxoglucamide A in 11 steps. A proteome‐wide ABPP study led to the identification of aldo‐keto reductase 1C3 (AKR1C3) as the primary target of myxoglucamides in human cells. AKR1C3 is an oncogenic factor involved in prostaglandin and steroid synthesis, promoting the growth, proliferation, and metastasis of carcinoma cells. The functional inhibition of AKR1C3 by a competitive mechanism (IC ₅₀ = 1.61 µ m ) was validated in vitro, and 20 analogs provided structure‐activity relationships and more potent analogs (IC ₅₀ = 181 n m ). Biophysical interactions were quantified by thermal shift assays, and essential molecular protein‐ligand interactions were characterized by X‐ray crystallography at 2.0 Å resolution. The study implies that the search for targets of natural products is rewarding even in the absence of an initial phenotypic activity.

ZUSAMMENFASSUNG: Die Naturstoffklasse der Myxoglucamide, die kürzlich aus Cystobacterineae sp . isoliert wurden, zeichnet sich durch eine bislang beispiellose vinylsubstituierte α‐Keto‐γ‐Aminosäure aus, die mit einer glykosylierten 14‐Methylpentadecansäure verknüpft ist. Dadurch vereinen sie Elemente aus drei Biomolekülklassen in einem kompakten Glykolipopeptid. Um die biologische Bedeutung dieser Struktur aufzuklären, suchten wir mittels aktivitätsbasierter Proteinprofilierung (Activity‐Based Protein Profiling, ABPP) nach molekularen Zielstrukturen—obwohl bislang keine phänotypische biologische Aktivität beschrieben worden war. Der Zugang zur Verbindungsklasse wurde durch die erste Totalsynthese von Myxoglucamid A in 11 Syntheseschritten geschaffen. Eine proteomweite ABPP‐Studie führte zur Identifizierung der Aldo‐Keto‐Reduktase 1C3 (AKR1C3) als primäres Zielprotein der Myxoglucamide in menschlichen Zellen. AKR1C3 ist ein onkogener Faktor, der an der Prostaglandin‐ und Steroidsynthese beteiligt ist und Wachstum, Proliferation sowie Metastasierung von Karzinomzellen fördert. Die funktionelle Hemmung von AKR1C3 durch einen kompetitiven Mechanismus (IC ₅₀ = 1,61 µ m ) wurde in vitro bestätigt. Darüber hinaus lieferten 20 Analoga Erkenntnisse zu Struktur‐Wirkungs‐Beziehungen und führten zur Identifizierung wirksamerer Verbindungen (IC ₅₀ = 181 n m ). Biophysikalische Wechselwirkungen wurden mittels Thermal‐Shift‐Assays quantifiziert, und die wesentlichen molekularen Protein‐Ligand‐Interaktionen wurden durch Röntgenkristallographie mit einer Auflösung von 2,0 Å charakterisiert. Die Studie zeigt, dass die Suche nach molekularen Zielstrukturen von Naturstoffen selbst dann lohnenswert ist, wenn zunächst keine phänotypische biologische Aktivität bekannt ist.

Condensation (C) domains in nonribosomal peptide synthetase (NRPS) pathways exhibit versatile functions that drive biosynthetic and chemical novelty. Through genome mining for atypical C domains, we identified a hybrid NRPS/polyketide synthase (PKS) biosynthetic gene cluster ( mxg ) from Cystobacterineae sp. MCy9003 and discovered myxoglucamides, a family of glycolipopeptides featuring an unprecedented vinyl‐substituted γ‐amino acid bearing an α‐hydroxy/α‐ketoamide functionality. Heterologous expression of the promoter‐refactored pathway revealed new O ‐acylated myxoglucamides, and subsequent studies unveiled the C domain‐like enzyme MxgH as a promiscuous O ‐acyltransferase decorating the glucose moiety with short‐chain acyl groups. Biosynthetic investigations demonstrated that the unusual γ‐amino acid originates from l ‐glutamate. Completion of the cryptic β‐hydroxylation of peptidyl carrier protein‐tethered glutamate by the α‐ketoglutarate‐dependent dioxygenase Ox _MxgA occurs only concomitantly with upstream chain extension, revealing a bidirectional checkpoint for substrate fidelity. Unexpectedly, the C‐domain‐like interface domain I _MxgB is dispensable for this coupled transformation. Mutational analysis of the FMN‐dependent monooxygenase encoded by mxgE , together with characterization of a shunt metabolite, supported its role in α‐oxidation for α‐hydroxy/α‐ketoamide formation during γ‐amino acid assembly. Together, these findings uncover an unrecognized biosynthetic logic for generating vinyl‐substituted, α‐oxidized γ‐amino acids and substantially expand the functional repertoire of NRPS/PKS assembly lines.

Condensation (C) domains in nonribosomal peptide synthetase (NRPS) pathways exhibit versatile functions that drive biosynthetic and chemical novelty. Through genome mining for atypical C domains, we identified a hybrid NRPS/polyketide synthase (PKS) biosynthetic gene cluster ( mxg ) from Cystobacterineae sp. MCy9003 and discovered myxoglucamides, a family of glycolipopeptides featuring an unprecedented vinyl‐substituted γ‐amino acid bearing an α‐hydroxy/α‐ketoamide functionality. Heterologous expression of the promoter‐refactored pathway revealed new O ‐acylated myxoglucamides, and subsequent studies unveiled the C domain‐like enzyme MxgH as a promiscuous O ‐acyltransferase decorating the glucose moiety with short‐chain acyl groups. Biosynthetic investigations demonstrated that the unusual γ‐amino acid originates from l ‐glutamate. Completion of the cryptic β‐hydroxylation of peptidyl carrier protein‐tethered glutamate by the α‐ketoglutarate‐dependent dioxygenase Ox _MxgA occurs only concomitantly with upstream chain extension, revealing a bidirectional checkpoint for substrate fidelity. Unexpectedly, the C‐domain‐like interface domain I _MxgB is dispensable for this coupled transformation. Mutational analysis of the FMN‐dependent monooxygenase encoded by mxgE , together with characterization of a shunt metabolite, supported its role in α‐oxidation for α‐hydroxy/α‐ketoamide formation during γ‐amino acid assembly. Together, these findings uncover an unrecognized biosynthetic logic for generating vinyl‐substituted, α‐oxidized γ‐amino acids and substantially expand the functional repertoire of NRPS/PKS assembly lines.

Streptavidin, the well‐known biotin‐binding protein extensively used in biotechnology, is naturally co‐produced in Streptomyces avidinii alongside stravidins—inhibitors of biotin biosynthesis. Here, we uncover and activate a conserved genomic region flanked by two streptavidin genes, revealing multiple biosynthetic gene clusters that produce diverse biotin‐related metabolites, including stravidins, acidomycin, α‐methyl‐KAPA, α‐methyldesthiobiotin, and the novel non‐proteinogenic amino acid 2‐aminonona‐5,7‐dienedioic acid (ANDA). We show α‐methyldesthiobiotin arises from α‐methyl‐KAPA, illustrating how methylated analogues interfere with distinct steps of biotin biosynthesis. Contrary to the view that streptavidin functions solely by sequestering biotin, our biochemical, structural, and bioactivity analyses demonstrate that it also binds acidomycin, neutralizing its antibacterial activity to protect the producer while likely facilitating compound secretion. The crystal structure of the streptavidin–acidomycin complex reveals the molecular basis for this dual functionality. Our findings establish a multifunctional streptavidin that integrates biotin sequestration and self‐resistance to balance an antibiotic network targeting the biotin pathway in microbial competition.

Inspired by natural gene transfer systems, we developed ACTIMOT, a CRISPR-Cas9–based in vivo DNA mobilization and multiplication platform that transfers large biosynthetic gene clusters (BGCs) from bacterial chromosomes onto replicative plasmids within the same cell. This strategy amplifies and activates cryptic BGCs efficiently, greatly enhancing product yields and enabling the discovery of four new natural product families, thereby unlocking hidden microbial biosynthetic potential.

Streptavidin, the well-known biotin-binding protein extensively used in biotechnology, is naturally co-produced in Streptomyces avidinii alongside stravidins—inhibitors of biotin biosynthesis. Here, we uncover and activate a conserved genomic region flanked by two streptavidin genes, revealing multiple biosynthetic gene clusters that produce diverse biotin-related metabolites, including stravidins, acidomycin, α-methyl-KAPA, α-methyldesthiobiotin, and the novel non-proteinogenic amino acid ANDA. We show α-methyldesthiobiotin arises from α-methyl-KAPA, illustrating how methylated analogues interfere with distinct steps of biotin biosynthesis. Contrary to the view that streptavidin functions solely by sequestering biotin, our biochemical, structural, and bioactivity analyses demonstrate that it also binds acidomycin, neutralizing its antibacterial activity to protect the producer while likely facilitating compound secretion. The crystal structure of the streptavidin–acidomycin complex reveals the molecular basis for this dual functionality. Our findings establish a multifunctional streptavidin that integrates biotin sequestration and self-resistance to balance an antibiotic network targeting the biotin pathway in microbial competition.

Tuberkulose (TB), die durch Mycobacterium tuberculosis verursacht wird , ist nach wie vor eine der gefährlichsten Infektionskrankheiten, wobei die zunehmende Antibiotikaresistenz den dringenden Bedarf an neuen Therapeutika verschärft. Wir berichten hier nun über die Entdeckung der Crossiguanipyrazine A‐I, einer seltenen Familie von potenten Anti‐TB‐Alkylpyrazinen aus dem wenig erforschten Actinobakterium Crossiella cryophila DSM 44230. Crossiguanipyrazine (CGPs) weisen ein ungewöhnliches 3‐Methoxyl‐2,5,6‐Trialkyl‐Pyrazin‐Gerüst mit variabler Hydroxylierung und N ‐Prenylierung auf. Die Konfiguration der stereogenen Zentren der CGPs A‐G wurde durch Mosher‐Analyse und elektronische Zirkulardichroismusspektroskopie bestimmt. Die Markierung stabiler Isotope ergab einen einzigartigen biosynthetischen Ursprung aus Arginin und Acetat. Eine Hemmung des Biosyntheseweges mit einem Cytochrom‐P450‐Inhibitor unterdrückte die Hydroxylierung, während Fütterungsexperimente mit stabilem Isotopen‐markierten CGP I seine Rolle als Vorläufer für andere CGPs zeigten, was auch P450‐Enzyme in die CGP‐Diversifikation einbezieht. Die CGPs C und H zeigten im Gegensatz zu den zuvor beschriebenen zytotoxischen Naturstoffen des Trialkylpyrazin‐Typs eine starke Anti‐TB‐Aktivität im niedrigen mikromolaren Bereich ohne Zytotoxizität gegenüber Säugetierzellen. Studien zur Struktur‐Aktivitäts‐Beziehung (SAR) zeigten die entscheidende Rolle von Hydroxylierung und N ‐Prenylierung bei der Modulation der biologischen Aktivität. Darüber hinaus gelang uns die erste Totalsynthese von CGP I, die die chemische Struktur validierte und eine Grundlage für die weitere Optimierung dieser Verbindungsklasse bildet. Diese Ergebnisse deuten darauf hin, dass CGPs nützliche Ausgangsstoffe für die Entwicklung von Medikamenten gegen TB sein könnten.

Tuberculosis (TB), caused by Mycobacterium tuberculosis , remains one of the most devastating infectious diseases, with rising antimicrobial resistance exacerbating the urgent need for new therapeutic agents. Here, we report the discovery of crossiguanipyrazines A‐I, a rare family of potent anti‐TB alkylpyrazines from the underexplored actinobacterium Crossiella cryophila DSM 44230. Crossiguanipyrazines (CGPs) feature an unusual 3‐methoxyl‐2,5,6‐trialkyl‐pyrazine scaffold with variable hydroxylation and N ‐prenylation. The stereochemistry of CGPs A‐G was determined by Mosher's analysis and electronic circular dichroism spectroscopy. Stable isotope‐labeling revealed a unique biosynthetic origin from arginine and acetate. Inhibition with a cytochrome P450 inhibitor suppressed hydroxylation, while feeding experiments with stable isotope‐labeled CGP I demonstrated its role as a precursor for other CGPs, implicating P450 enzymes in CGP diversification. CGPs C and H exhibited potent anti‐TB activity in the low micromolar range without mammalian cell cytotoxicity, unlike previously reported cytotoxic trialkylpyrazine‐type natural products. Structure‐activity relationship (SAR) studies highlighted the critical roles of hydroxylation and N ‐prenylation in modulating biological activity. Furthermore, we achieved the first total synthesis of CGP I, which validated the chemical structure and provided a foundation for further optimization of this compound class. These findings suggest that CGPs can serve as useful starting points for anti‐TB drug development.

Pepstatins are potent inhibitors of aspartic proteases, featuring two statine residues crucial for target binding. However, the biosynthesis of pepstatins, especially their statine substructure, remains elusive. Here, we discover and characterize an unconventional gene cluster responsible for pepstatin biosynthesis, comprising discrete nonribosomal peptide synthetase and polyketide synthase genes, highlighting its trans-acting and iterative nature. Central to this pathway is PepI, an F₄₂₀H₂-dependent oxidoreductase. The biochemical characterization of PepI reveals its role in the tandem reduction of β-keto pepstatin intermediates. PepI first catalyzes the formation of the central statine, then produces the C-terminal statine moiety. The post-assembly-line formation of statine by PepI contrasts with the previously hypothesized biosynthesis involving polyketide synthase ketoreductase domains. Structural studies, site-directed mutagenesis, and deuterium-labeled enzyme assays probe the mechanism of F₄₂₀H₂-dependent oxidoreductases and identify critical residues. Our findings uncover a unique statine biosynthetic pathway employing the only known iterative F₄₂₀H₂-dependent oxidoreductase to date.