A conserved vdcBCD cluster encoding a vanillate/p-hydroxybenzoate decarboxylase catalyzes the formation of phenol from p-hydroxybenzoate in the cork-associated Streptomyces graminifolii B37 strain.
Aug 2026· Frontiers in Microbiology· Vol 17, pp.
1913779
· 0 citations· 53 references
Medicine
TL;DR
Results identify the B37 vdcBCD cluster as a bifunctional aromatic acid decarboxylation module and link this reaction to the formation of phenolic intermediates relevant to yellow-stained cork and cork taint-associated chemistry, including the de novo formation of chlorophenols and chloroanisoles.
Abstract
Cork affected by yellow stain contains aromatic acids and volatile phenols, although the microbial reactions contributing to their formation are still poorly characterized. Streptomyces sp. B37, isolated from yellow-stained cork, was previously shown to convert p-hydroxybenzoate into phenol, but both its precise taxonomic placement and the genetic basis of this bioconversion were unknown. We combined whole-genome comparison, pangenome analysis, and heterologous expression to identify and validate the aromatic acid decarboxylation system underlying this phenotype. Phylogenomic analysis assigned strain B37 to Streptomyces graminifolii species. Comparative genomics showed that B37 belongs to a subclade of closely related Vanillate Decarboxylase (VDC)-positive genomes with an accessory repertoire enriched in functions related to aromatic compound uptake, regulation, redox metabolism and ring-cleavage pathways. In contrast, functions associated with primary cork-polymer degradation were not overrepresented. A conserved vdcBCD cluster was present in the genomes of Streptomyces species comprising this group and embedded in a partially conserved chromosomal neighborhood containing regulatory, oxidative and transport-related genes. Heterologous expression of the B37 vdcBCD cluster in Streptomyces lividans JI66 conferred the ability to decarboxylate p-hydroxybenzoate into phenol and vanillic acid into guaiacol, whereas the empty-vector control S. lividans JI66(pIJ699) showed no detectable decarboxylase activity. These results identify the B37 vdcBCD cluster as a bifunctional aromatic acid decarboxylation module and link this reaction to the formation of phenolic intermediates relevant to yellow-stained cork and cork taint-associated chemistry, including the de novo formation of chlorophenols and chloroanisoles.
Genome mining of the marine-derived Streptomyces sp. S42 uncovered a type II polyketide synthetase (T2 PKS) biosynthetic gene cluster (BGC) harboring a gene for 3-ketoacyl-ACP synthase III (KAS III), a hallmark of non-acetate starter unit incorporation, suggesting that the BGC may produce previously unidentified aromatic polyketides. Heterologous expression and promoter engineering of this prioritized BGC in host Streptomyces albus J1074 activated the biosynthetic pathway, leading to the isolation of eight new polycyclic aromatic derivatives, verrucones A–H (1–8). Comprehensive structural elucidation via NMR and HRESIMS revealed that these compounds feature either a 2-methylbutyryl or an isobutyryl starter unit and can be classified into three distinct skeletal types. Based on these findings and bioinformatic analysis, a plausible biosynthetic pathway for 1–8 involving divergent spontaneous cyclization from a common nascent polyketide intermediate was proposed. Among the isolated compounds, 1–5 exhibited inhibitory activity against several protein tyrosine phosphatases (PTPs) with IC50 values ranging from 1.84 μM to 24.82 μM. This study presents a successful case study demonstrating that combining KAS III-targeted genome mining with heterologous expression is a viable approach for discovering non-acetate-primed aromatic polyketides.
Xingkun Hao, Ming Yang, Ping Yan et al.· Microorganisms· 0 citations
The genus
Streptomyces
is one of the richest sources of bioactive natural products; however, a substantial proportion of its biosynthetic gene clusters (BGCs) remain cryptic and their metabolic products are unresolved. Advances in genome mining and computational prediction now enable comprehensive exploration of this hidden biosynthetic repertoire. In this study, whole-genome sequencing and comparative genomic analyses were performed on three three newly isolated
Streptomyces
strains to evaluate their specialized metabolic potential. Genome assemblies were annotated and systematically analyzed using antiSMASH, DeepBGC, GECCO, and PRISM to identify, cross-validate, and functionally characterize BGCs while predicting their associated secondary metabolite scaffolds. Taxonomic analyses based on Average Nucleotide Identity (ANI), phylogenomics, and BLAST identified the isolates as
Streptomyces thinghirensis, Streptomyces novocaesareae
, and
Streptomyces griseorubens
. Applying the consensus framework across the three
Streptomyces
genomes yielded 43 cryptic BGCs, lacking close similarity to reference BGCs in the MIBiG database, of which 26 were classified as HIGH, 10 as MEDIUM, and 7 as LOW confidence. Notably, numerous BGCs exhibited low abundance to characterized reference clusters, indicating a high potential for previously undescribed biosynthetic pathways and novel metabolite scaffolds. Comparative analyses further revealed strain-specific biosynthetic architectures together with putative metal-responsive regulatory systems;
Fur, Zur
, and
Nur
, which were frequently associated with specialized metabolite biosynthetic loci. Collectively, these findings demonstrate the effectiveness of integrated genome-mining strategies for prioritizing cryptic biosynthetic gene clusters and highlight the remarkable biosynthetic potential of newly identified
Streptomyces
isolates as a source of novel natural products.
Nada S. Al-Theyab, Haila M. Alnassar, Mohanad A. Ibrahim et al.· Frontiers in Microbiology· 0 citations
Ferredoxin–NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron–sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min−1—a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186–199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.
Weigao Wang, Qianqiao Liu, James R. Swartz· bioRxiv· 0 citations
Microbes produce bioactive secondary metabolites as toxins, pigments, or virulence factors. These specialized compounds are produced by nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), or hybrid NRPS/PKS pathways. The genes encoding NRPS and PKS reside in biosynthetic gene clusters (BGCs), some of which have no identified metabolite associated with them. Characterization of these orphan BGCs could provide insights into potential bioactive compounds that have yet to be discovered. Here, we characterize PA1216, a putative methyltransferase embedded within an NRPS BGC in Pseudomonas aeruginosa strain PAO1. We cloned, expressed, and purified PA1216, and developed an optimized differential scanning fluorimetry assay to measure its thermal stability, demonstrating concentration‐dependent stabilization in the presence of established methyltransferase cofactors and inhibitors. We then adapted this assay for high‐throughput screening of potential PA1216 substrates, identifying destabilizing compounds, including glycyl‐glycine dipeptides, amino esters with aromatic or basic side chains, and N‐Boc‐protected amino acids. In contrast, sodium salts of organic acids stabilized PA1216. Lastly, we employed AlphaFold to construct a predictive model, revealing that PA1216 contains a Rossmann‐like fold and a glycine‐rich loop, typical of class I methyltransferases, and we corroborated these secondary structural elements using circular dichroism spectroscopy. Overall, these studies illuminate PA1216 function and establish a platform for characterizing cryptic gene clusters within secondary metabolic pathways.
Amaan Fruitwala, Jade X Tiszler, Daniel Watson et al.· Protein Science· 0 citations
Lignin depolymerization by white-rot fungi generates diverse aromatic compounds derived from hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Although the metabolic pathways for G- and H-unit-derived aromatics have been studied, the enzymatic step responsible for the oxidative decarboxylation of the S-unit intermediate syringic acid (SA) has remained unknown. Here, we identify PcMNX1, a group A flavoprotein monooxygenase (FPMO) from the white-rot fungus Phanerochaete chrysosporium, as the enzyme catalyzing this missing step. Recombinant PcMNX1 catalyzed the NAD(P)H-dependent oxidative decarboxylation of SA to dimethoxyhydroquinone (DMHQ) and also converted other lignin-derived aromatics, including vanillic acid and 4-hydroxybenzoic acid, with markedly higher catalytic efficiency than the closely related enzyme GsMNX1 from Gelatoporia (Ceriporiopsis) subvermispora. The crystal structure of PcMNX1 was determined at 2.00 Å resolution, revealing a typical group A FPMO fold with FAD bound in the "out" conformation. Structure-guided mutagenesis demonstrated that His247 functions as the catalytic base required for decarboxylative hydroxylation. Comparative structural analysis with bacterial 3-hydroxybenzoate 6-hydroxylase (3HB6H) indicated that subtle substitutions in active-site residues alter substrate positioning and reaction outcomes. Consistent with this hypothesis, introduction of PcMNX1-type residues into 3HB6H conferred decarboxylation activity toward lignin-derived aromatics. Furthermore, enlargement of the PcMNX1 active-site cavity through the L264A substitution markedly enhanced SA conversion. Together, these findings demonstrate that PcMNX1 catalyzes the oxidative decarboxylation of SA and reveal how subtle active-site remodeling diversifies the catalytic repertoire of closely related group A FPMOs involved in lignin-derived aromatic metabolism.
Reini Mori, Hiromitsu Suzuki, T. Ishida et al.· Journal of Biological Chemis...· 0 citations
Microbial biotransformation of para-hydroxycinnamic acids (pHCAs) such as para-coumaric, caffeic, ferulic and sinapic acids into vinylphenols is catalyzed by phenolic acid decarboxylases (PADs), while reduction to their corresponding aldehydes and alcohols is mediated by carboxylic acid reductases (CARs) and alcohol dehydrogenases (ADHs), respectively. The present study systematically evaluated a diverse set of endophytic and basidiomycetes fungi as whole-cell biocatalysts for the transformation of pHCAs into their corresponding vinylphenols and/or aldehydes and alcohols. Twenty-three fungal strains were screened for their PAD, CAR and ADH activities. Based on ultra-high-performance liquid chromatography–diode array detector (UHPLC-DAD) analysis, fourteen strains were selected for preparative-scale biotransformations across all four substrates. Previous literature largely emphasizes enzyme activity or single substrates, seldom covering all four pHCAs. The strain Umbelopsis sp. JAR-T demonstrated promising biotransformation of para-coumaric acid and ferulic acid to 4-vinylphenol (28% isolated yield) and 4-vinylguaiacol (40% isolated yield), respectively, with minimal by-product formation. The results highlight endophytic fungi as largely untapped and versatile biocatalysts for pHCA biotransformation and establish whole-cell fungal systems as robust, non-recombinant alternatives to engineered platforms. This integrated screening-to-preparative workflow provides a scalable framework for the production of value-added compounds with potential applications in the food, cosmetic and pharmaceutical industries.
A. Baskaran, Stefano Serra, El-Sayed R. El-Sayed et al.· Molecules· 0 citations