Betalains are water-soluble, nitrogen-containing pigments responsible for the yellow-orange to red-violet hues in most species of the plant order Caryophyllales, which includes beetroot, quinoa, and prickly pear. Beyond their visual role, these compounds attract attention for their antioxidant, anti-inflammatory, chemopreventive, and neuroprotective properties, fueling a search for new natural sources and better production methods. Two classes exist: yellow betaxanthins and violet betacyanins, both built around a shared core molecule called betalamic acid. The enzyme that creates this core is 4,5-DOPA-extradiol-dioxygenase, known as DODA, which cleaves the amino acid L-DOPA in a way that lets betalamic acid form spontaneously before condensing with amines or amino acids to make betaxanthins, or with cyclo-DOPA to make betacyanins.

Until recently, betalain biosynthesis was documented almost exclusively in Caryophyllales plants, with scattered reports in a handful of fungi such as Amanita, Hygrocybe, and Hygrophorus, in bacteria including Gluconacetobacter diazotrophicus and Escherichia coli, in the cyanobacterium Anabaena cylindrica, and, strikingly, in the tardigrade Hypsibius henanensis. Neolecta irregularis occupies a uniquely basal position within the Ascomycota, the phylum that contains most known fungi. As one of the very few surviving members of the subphylum Taphrinomycotina and the only genus in the Neolectomycetes, it retains morphological and genomic features considered ancestral, sitting in an intermediate zone between filamentous fungi and yeasts. Its rarity and the inability to cultivate it have long blocked functional studies of its physiology.

The research team, working with two specimens preserved at the Denver Botanic Gardens fungarium, first extracted pigments from the yellow tongue-shaped fruiting bodies and analyzed them by high-performance liquid chromatography coupled to mass spectrometry. They screened for a full panel of fungal betalains, including muscaflavin and numerous amino-acid-derived betaxanthins, but none were detected. Instead, the main pigment turned out to be riboflavin, vitamin B2, identified by its retention time and exact mass of m/z 377.1464, matching the standard at m/z 377.1456. Fluorescence imaging under blue excitation confirmed the assignment, revealing the characteristic yellow-green glow of riboflavin. This finding matters because riboflavin production is a hallmark of certain fungi and bacteria, where it protects spores from ultraviolet radiation and supports flavin cofactor synthesis and redox defense.

The pigment analysis created a puzzle. RNA sequencing, performed for the first time on this species, showed that the fungus actively expresses the gene encoding a DODA enzyme, along with a cytochrome P450, yet the pathway appears incomplete. The researchers found no expression of prephenate dehydrogenase or tyrosine aminotransferase, the enzymes needed to make L-tyrosine de novo, which is the precursor to L-DOPA. Without this substrate, the betalain machinery cannot run in vivo, explaining why the fungus stays yellow from riboflavin rather than from betalains. The transcriptomic data, deposited in the NCBI Gene Expression Omnibus under accession GSE300630, revealed 24 genes related to riboflavin metabolism, underscoring the pigment’s physiological importance in this lineage.

To test whether the encoded enzyme actually works, the team cloned the NiDODA gene, optimized its sequence for expression in E. coli, and purified the resulting protein using cobalt affinity chromatography. The enzyme emerged as a monomer of 33.4 kilodaltons, confirmed by gel filtration and mass spectrometry. Structural modeling with AlphaFold showed a compact fold resembling the only crystallized betalamic-acid-forming protein, YgiD from E. coli, with which NiDODA shares just 25.2 percent sequence identity. Three conserved histidine residues, His10, His48, and His230, coordinate a metal ion at the active site, mirroring the arrangement in the bacterial enzyme. Inductively coupled plasma mass spectrometry identified iron as the likely cofactor, and supplementing purification buffers with iron boosted activity up to 8.5-fold.

Kinetic characterization revealed remarkable properties. The enzyme operates optimally at pH 6.0, notably more acidic than the pH 8.0 to 8.5 optima reported for DODAs from Amanita muscaria, beetroot, and E. coli. Most strikingly, NiDODA binds L-DOPA with a Km of 0.8 millimolar, the highest affinity ever recorded for any DODA enzyme. For comparison, the Amanita enzyme has a Km of 4.2 millimolar, beetroot 6.9, quinoa 1.5, and the cyanobacterial version a staggering 53,000 millimolar. This makes NiDODA the most efficient L-DOPA converter among all known betalain-forming dioxygenases, capable of producing betalamic acid, muscaflavin, and dopaxanthin from the same substrate.

Phylogenetic analysis placed NiDODA within a distinct fungal branch separate from plant, bacterial, and animal sequences. Ascomycota sequences clustered together, while the tardigrade enzyme branched off before the radiation of plants, fungi, and microorganisms, acting as an outgroup. The team found that Ascomycota genomes contain numerous uncharacterized proteins with similarity to NiDODA, all retaining the conserved histidine triad, suggesting that more fungal betalain-producing enzymes await discovery. This pattern supports convergent evolution, where similar catalytic solutions arise independently in distant lineages, a phenomenon already documented within Caryophyllales plants, where DODA activity emerged at least three times through gene duplication and neofunctionalization.

The broader implication is that betalain biosynthesis may have far deeper roots than previously appreciated. Rather than being a plant-specific innovation, the capacity to cleave L-DOPA into betalamic acid appears scattered across the tree of life, from fungi and bacteria to cyanobacteria and animals. Whether these enzymes represent an ancestral metabolic capacity predating eukaryotic diversification, or the result of horizontal gene transfer and convergent evolution, remains unresolved. The biological role of betalain-producing enzymes in non-plant organisms is also unknown, and the possibility that they serve functions beyond pigment formation, particularly in species that never make betalains, adds another layer of mystery.

For Neolecta irregularis itself, the study provides the first transcriptomic analysis of a species that has resisted cultivation and remains scarce in nature. It confirms that the fungus carries the genetic framework for betalain synthesis but cannot execute the pathway because it lacks the ability to produce L-tyrosine de novo. The identification of NiDODA as the second fungal betalain-producing enzyme characterized to date, and the first monomeric one, expands the known phylogenetic scope of the pathway and opens the door to exploring whether other Ascomycota species harbor similar dormant capacities. As researchers continue to probe these ancient lineages, enzymes like NiDODA may reveal how metabolic innovations emerge, persist, or vanish across evolutionary time, offering a rare glimpse into the biochemical toolkit of early fungal life.

Subject of Research: Betalain biosynthesis enzyme evolution in the early-diverging fungus Neolecta irregularis

Article Title: A functional betalain-producing dioxygenase in Neolecta irregularis reveals an expanded evolutionary origin of the pathway

Article References: Martínez-Rodríguez, P., Guerrero-Rubio, M. A., Henarejos-Escudero, P., Hernández-García, S., & Gandía-Herrero, F. (2026). A functional betalain-producing dioxygenase in Neolecta irregularis reveals an expanded evolutionary origin of the pathway. IMA Fungus, 17, Article e184549. https://doi.org/10.3897/imafungus.17.184549

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.184549

Keywords: betalains, Neolecta irregularis, DODA enzyme, Ascomycota, riboflavin, L-DOPA dioxygenase, betalamic acid, convergent evolution, Taphrinomycotina, fungal pigments, heterologous expression, enzyme kinetics

Biosurfactants are amphiphilic molecules, meaning they possess both water-loving and water-fearing regions, and this dual nature allows them to dramatically reduce surface and interfacial tension between immiscible liquids such as oil and water. Unlike their petroleum-derived synthetic counterparts, biosurfactants are biodegradable, exhibit low toxicity, and maintain high surface activity across a range of industrial conditions. These properties make them attractive for applications spanning environmental remediation of oil spills, enhanced petroleum recovery, food preservation, pharmaceutical formulations, and cosmetic products. The most extensively studied and commercialized classes are lipopeptides like surfactin, produced by Bacillus species, and glycolipids like rhamnolipids, produced by Pseudomonas species. However, the search for novel biosurfactant producers has increasingly turned toward lactic acid bacteria, a group generally recognized as safe (GRAS) and widely employed in food fermentation.

Lactobacillus species are particularly compelling candidates because they are already embedded in human food systems, carry a long history of safe use, and produce bioactive metabolites with both antimicrobial and surface-active properties. Prior research has shown that biosurfactants from Lactobacillus can inhibit pathogens, preserve foods, and support probiotic preparations. The biosynthetic machinery behind these molecules typically involves non-ribosomal peptide synthetases, which are large multidomain enzymes that assemble peptide structures without the ribosomal translation machinery. These NRPS systems contain conserved catalytic domains, including adenylation (A), condensation (C), and peptidyl carrier protein (PCP) domains, each responsible for specific steps in substrate recognition, activation, and peptide elongation. What makes the current study distinctive is its focus on whether the adenylation domain alone, without the full NRPS assembly line, could still contribute meaningfully to biosurfactant production.

To answer this question, the research team first turned to comparative bioinformatics. They retrieved the NRPS protein sequence from Bacillus velezensis FZB42, a well-characterized commercial biosurfactant producer with extensively studied NRPS domains, and used it as a query against the NCBI non-redundant protein database. The most significant hit was a non-ribosomal peptide synthetase from Lactococcus lactis subsp. lactis KF147, with 99% query coverage, a BLAST score of 1,052, and an E-value of 0.0, indicating a highly significant alignment despite only moderate sequence identity of approximately 26%. This combination of high coverage and low E-value strongly suggested functional relatedness between the NRPS systems of these taxonomically distant bacteria. Further BLASTp analysis using the Lactococcus sequence as query revealed homologous proteins across multiple Lactobacillus species, including hybrid NRPS-polyketide synthase proteins and adenylate-forming enzymes.

Domain architecture analysis using InterProScan confirmed the presence of conserved functional modules across the analyzed sequences. The team identified the NRPS domain (IPR010060), the PCP domain (IPR009081), and the PKS domain (IPR020806), confirming the hybrid modular nature of these proteins. The condensation domain (IPR001242), responsible for peptide bond formation between activated amino acid substrates, appeared at multiple positions. Critically, the adenylation AMP-binding domain (IPR000873) and the amino acid adenylation domain (IPR010071) were highly conserved across all analyzed strains, underscoring their central role in substrate recognition and activation. The AMP-binding C-terminal domain (IPR025110) was also detected, providing structural stability to the adenylation modules. Notably, the number and organization of these domains varied considerably between strains. Lactobacillus helveticus MTCC 5463 and L. helveticus DPC 4571 each carried two conserved domains, representing a relatively streamlined biosynthetic organization. In contrast, Lactococcus lactis subsp. lactis KF147 harbored seven conserved domains, suggesting a more complex biosynthetic architecture and potentially greater catalytic capacity.

With the bioinformatic foundation in place, the researchers moved to the laboratory. They extracted genomic DNA from L. helveticus MTCC 5463 and used gene-specific primers to amplify the AMP-binding domain by polymerase chain reaction. The resulting fragment, approximately 353 base pairs in length, was directionally cloned into the pET-28a expression vector using XhoI and EcoRI restriction enzymes. Successful cloning was verified through restriction digestion analysis, which released an insert fragment of the expected size in positive clones. The recombinant plasmid was then transformed into Escherichia coli BL21(DE3) for heterologous protein expression. Induction with isopropyl 2-D-1-thiogalactopyranoside (IPTG) at concentrations ranging from 0.2 to 1.0 mM, followed by time-course analysis from 1 to 7 hours, revealed a clear recombinant protein band at approximately 14 kilodaltons on SDS-polyacrylamide gel electrophoresis. Expression intensity increased progressively with both inducer concentration and induction time, reaching maximum accumulation between 5 and 7 hours post-induction.

The recombinant biosurfactant was then extracted from the E. coli culture using chloroform:methanol solvent systems. The researchers tested two ratios, 1:1 and 2:1, and found that the 2:1 ratio produced significantly more visible precipitation, with the greatest accumulation observed at the 5-hour extraction time point. This enhanced recovery is attributed to the higher chloroform content, which improves solubilization of lipid-rich amphiphilic molecules. To rule out the possibility that the observed activity originated from endogenous E. coli membrane lipids, the team conducted a critical empty-vector control experiment. An E. coli BL21(DE3) strain carrying the empty pET-28a vector was processed under identical conditions, and the resulting extract showed no measurable emulsification activity against any of the tested oils and no detectable bands on thin-layer chromatography. This control definitively linked the biosurfactant phenotype to the recombinant AMP-binding domain construct rather than host-derived lipids.

Functional evaluation of the extracted biosurfactant revealed impressive emulsification performance across multiple substrates. Using the emulsification index (E24) assay, which measures the percentage of emulsified layer height relative to total liquid column height after 24 hours, the recombinant product achieved approximately 75% to 82% emulsification against petrol and diesel oil, maintaining stability for a full week with minimal variation. Against mustard oil, the performance was even more striking, reaching 88% to 95%, far exceeding the 10% to 12% achieved by rhamnolipid and the 30% to 38% achieved by surfactin under the same conditions. For refined oil, the recombinant biosurfactant delivered 65% to 75% emulsification at 24 hours, sustaining approximately 65% to 70% even after one week, while rhamnolipid showed only 4% to 6% and surfactin showed moderate activity of 46% to 50%. These results demonstrate that the recombinant glycolipopeptide consistently outperformed both benchmark biosurfactants across hydrocarbon and vegetable oil substrates.

Analytical characterization provided detailed structural insights into the recombinant molecule. Thin-layer chromatography using iodine vapor revealed a yellow-brown spot with an Rf value of 0.80, indicating a relatively non-polar lipid-rich compound, while anisaldehyde staining produced a spot at Rf 0.75, consistent with glycolipid or lipopeptide chemistry. Fourier-transform infrared spectroscopy identified key functional groups: a broad band at 3,325 cm⁻¹ corresponding to hydrogen-bonded O-H and N-H stretching from carbohydrate and protein moieties, aliphatic C-H stretching at 2,916 cm⁻¹ confirming long hydrocarbon chains, a band at 1,649 cm⁻¹ assigned to amide I (C=O stretching) indicating peptide bonds, and C-O stretching at 1,021 cm⁻¹ pointing to carbohydrate or ester linkages. Together, these spectral features are characteristic of a glycolipopeptide structure containing lipid, peptide, and carbohydrate components.

Gas chromatography-mass spectrometry profiling further confirmed the hydrophobic lipid architecture of the biosurfactant. The most abundant compound identified was 9-octadecenamide, accounting for 20.50% of the total relative area, followed by significant amounts of C16 and C18 long-chain fatty acid derivatives and amides. These hydrophobic moieties form the structural basis for surface tension reduction, emulsification, and micelle formation. Nuclear magnetic resonance spectroscopy, conducted on a Bruker Avance NEO spectrometer at 400 MHz, provided complementary evidence. Proton NMR revealed strong signals at δ 1.23 to 1.58 ppm from methylene protons of long-chain fatty acyl groups, glyceryl and carbohydrate-associated signals at δ 3.72 ppm, and a broad singlet at δ 7.3 ppm assigned to exchangeable amide N-H protons, confirming peptide-associated functionalities. Carbon-13 NMR showed resonances at δ 18 ppm for terminal methyl carbons of branched-chain fatty acids, δ 66 and 76 ppm for O-CH₂ and CH-O carbons of glycerol or carbohydrate moieties, δ 56 and 58 ppm for alpha-methine carbons of amino acid residues, and carbonyl carbons at δ 173 to 175 ppm for amide or ester linkages. Collectively, the spectroscopic data confirm a multicomponent amphiphilic glycolipopeptide structure. The authors note that while the isolated adenylation domain alone is unlikely to catalyze complete glycolipopeptide biosynthesis, it may influence production indirectly through interactions with endogenous metabolic pathways in the E. coli host, and they plan future transcriptomic, proteomic, and metabolomic investigations to elucidate the precise molecular mechanism underlying this observation.

Subject of Research: Biosurfactant production mediated by the NRPS adenylation domain of Lactobacillus helveticus MTCC 5463

Article Title: In silico and experimental characterization of NRPS adenylation domain-mediated biosurfactant production in Lactobacillus helveticus MTCC 5463

Article References: Kumari, M., Pandey, K., Mehtab, S., & Singhal, B. (2026). In silico and experimental characterization of NRPS adenylation domain-mediated biosurfactant production in Lactobacillus helveticus MTCC 5463. Frontiers in Bacteriology, 5, Article 1895576. https://doi.org/10.3389/fbrio.2026.1895576

Image Credits: AI Generated

DOI: 10.3389/fbrio.2026.1895576

Keywords: biosurfactant, Lactobacillus helveticus, NRPS, adenylation domain, glycolipopeptide, emulsification, probiotics, non-ribosomal peptide synthetase, heterologous expression, FTIR, GC-MS, NMR

The work, published in Frontiers in Microbiology, combines two complementary strategies: carefully tuned fermentation conditions that coax more product out of the cells, and targeted genetic rewiring of the biosynthetic machinery itself. Romidepsin, also known by its laboratory designation FK228, is a complex cyclic depsipeptide whose chemical synthesis remains impractical at scale, which makes microbial fermentation the only realistic production route. That constraint has kept the drug expensive and its supply chain fragile, so any genuine improvement in titer carries weight well beyond the laboratory bench.

Romidepsin’s biological activity stems from its ability to block histone deacetylases, enzymes that remove acetyl groups from histone proteins and thereby silence stretches of chromatin. By inhibiting these enzymes, the drug reopens tightly packed DNA, reactivates tumor suppressor genes, halts proliferation and pushes cancer cells into apoptosis. That mechanism earned it FDA approval, but producing it has been an upstream headache. The molecule is assembled by a hybrid trans-acyltransferase polyketide synthase and non-ribosomal peptide synthetase assembly line that stitches together cysteine, valine, dehydrothreonine derived from threonine, and malonyl-CoA through sequential condensation, macrocyclization and oxidative modification.

Earlier attempts to improve supply followed a winding path. Researchers identified the related thailandepsin gene cluster in Burkholderia thailandensis in 2011, and fermentation optimization of that system informed later work. Heterologous expression of the complete romidepsin gene cluster in Escherichia coli achieved only about 19 milligrams per liter, comparable to the native strain. Genome mining then revealed a highly homologous cluster in B. thailandensis MSMB43, which after minor modification produced 168.5 milligrams per liter. In 2022, the Shandong group took a decisive step: they deleted the native thailandepsin cluster from B. thailandensis E264 and replaced it with an attB integration site, creating a chassis strain called KOGC1 into which a hybrid romidepsin gene cluster could be slotted. That strain yielded 581 milligrams per liter, then the record, but still short of industrial requirements.

The new study began with physiology rather than genetics. The team grew the KOGC1 strain carrying an intact romidepsin gene cluster in M9 medium and systematically varied fermentation parameters using a sequential single-factor design. Romidepsin accumulation proved strictly growth-associated: titers climbed after 24 hours, peaked near 628 milligrams per liter at 60 hours and then declined, a pattern the authors attribute to end-product inhibition or nutrient depletion. A 60-hour harvest window was therefore fixed. Inoculum size mattered too; while 0.5 to 2 percent inocula performed similarly, a 5 percent inoculum depressed production, likely through high cell density and premature metabolite accumulation, so 1 percent was adopted.

The most striking fermentation lever was an adsorbent resin. Because romidepsin can be toxic to its own producer, the team added XAD-16 macroporous resin to the culture 12 hours after inoculation, letting the resin sequester the compound extracellularly as it formed. The authors speculate that this in-situ removal alleviates end-product toxicity and feedback inhibition, though they caution that direct evidence from intracellular and extracellular quantification, adsorption kinetics or cell viability assays was not obtained. Screening resin concentrations identified 4 percent weight per volume as optimal, roughly tripling the titer relative to resin-free controls. The team then supplemented the three amino acid precursors of the assembly line. Valine alone gave a marginal 6 percent gain, but feeding cysteine, valine and threonine together produced a synergistic effect, lifting the final titer to 743.5 milligrams per liter, a 28 percent increase over the 581 milligram baseline.

Genetic engineering delivered the second leap. Earlier work had shown that the AraC-family regulator TdpR acts as a positive activator of the cluster, and that including the tdpR gene boosted romidepsin yield 1.7-fold. The Shandong team reasoned that cranking up TdpR expression with strong promoters would push the whole pathway harder. Using RedEx technology, a method for seamless DNA insertion and deletion in large multimodular gene clusters, they inserted seven strong constitutive promoters upstream of tdpR. The promoters, drawn from a library of 50 natural promoters characterized in B. thailandensis E264 by luciferase reporter assays, were ranked by apparent strength from P46 down to P11.

The results contained a instructive surprise. Although the reporter assays had ranked P16 relatively low, in the actual chromosomal context P16 drove the highest romidepsin titer of all, reaching 1,109.4 milligrams per liter, a 90 percent increase over the previous KOGC1 baseline and a 49 percent gain over the fermentation-optimized intermediate. P46 and P33 also performed well, yielding 984.6 and 870.2 milligrams per liter respectively. Quantitative PCR confirmed that transcript levels of tdpR and two biosynthetic genes, depE and tdpJ, were elevated in the best-performing strains, with P16 showing the highest mRNA levels. Notably, the chromosomal transcription ranking did not match the reporter-based promoter strength ranking, a discrepancy the authors flag as evidence that surrogate reporter data poorly predict native in-locus transcription, a lesson for anyone designing promoter swaps in secondary metabolism.

The team also probed regulatory cross-talk. The romidepsin cluster dep and the thailandepsin cluster tdp are highly conserved in gene content and share a common assembly line, yet their dedicated regulators are strikingly divergent: TdpR is an AraC-type protein while DepR from C. violaceum is LysR-type, with no significant sequence homology. When the researchers cloned depR into the romidepsin expression vector and integrated it into KOGC1, the resulting strain maintained detectable romidepsin production comparable to the TdpR control. The authors suggest DepR may support the pathway through mechanisms distinct from TdpR rather than acting as a direct functional analog, though whether it binds the cluster’s promoters directly or works through an indirect regulatory cascade remains an open, testable hypothesis.

Perhaps as important as the headline titer is what did not happen over time. Large exogenous biosynthetic gene clusters often destabilize, deleting or rearranging under the metabolic burden they impose, which has blocked many natural product platforms from industrial translation. The team passaged three representative engineered strains for five generations without any antibiotic selection and found no significant titer fluctuation from the first through the fifth generation. Colony PCR on randomly picked colonies at generations one, three and five confirmed that the full refactored cluster remained intact, with no evidence of deletion, rearrangement or cassette loss. The authors note this demonstrates short-term genotypic and phenotypic stability only, and that bioreactor-scale and extended-culture performance remain to be assessed.

Honest caveats accompany the achievement. All experiments were conducted in 65-milliliter flask cultures, so the platform must be viewed as a foundation for future scale-up rather than an immediately industrial process. Late-stage broth viscosity, worsened by the resin, complicates downstream extraction, and the team suggests that optimized cell lysis protocols or continuous-flow separation systems will be needed to minimize product loss. Quantitative yield differences among engineered strains did not reach statistical significance under the current replication scheme, although the P16 strain’s titer was consistently reproduced across multiple independent fermentation batches. The authors also emphasize that direct metabolomic evidence for precursor limitation and product toxicity is still lacking, and they propose CRISPR-Cas-based genome stabilization and multi-omics profiling as next steps. Even with those qualifications, the study delivers the most efficient romidepsin production platform yet described and a generalized framework for stabilizing large, unwieldy gene clusters, work that could eventually make this potent anticancer agent cheaper, more abundant and more accessible to the patients who need it.

Subject of Research: Metabolic engineering of the FK228 (romidepsin) biosynthetic gene cluster for high-yield production in Burkholderia thailandensis

Article Title: Engineering the FK228 biosynthetic gene clusters for high-yield production

Article References: Wang, M., Jia, X., Zhang, Y., Ma, J., Zhang, Y., Fu, J., & Li, R. (2026). Engineering the FK228 biosynthetic gene clusters for high-yield production. Frontiers in Microbiology, 17, Article 1933794. https://doi.org/10.3389/fmicb.2026.1933794

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1933794

Keywords: FK228, romidepsin, HDAC inhibitor, Burkholderia thailandensis, biosynthetic gene cluster, promoter engineering, heterologous expression, XAD-16 resin, precursor feeding, TdpR, DepR, metabolic engineering

Position effect variegation is a phenomenon familiar to geneticists working on organisms from fruit flies to humans: when a gene is moved to a different chromosomal neighborhood, its activity can change dramatically depending on the local chromatin environment, the packaging of DNA and associated proteins that determines whether a region is transcriptionally accessible or silenced. In Drosophila, relocating the white gene near heterochromatin disrupts eye pigmentation; in yeast, inserting exogenous genes into silent chromatin regions represses their transcription. Yet despite its universality across eukaryotes, the position effect had never been systematically dissected in filamentous fungi such as P. rubens, largely because the tools for doing so simply did not exist.

The research team, led by Gang Liu of the Chinese Academy of Sciences, chose P. rubens for good reason. Formerly known as Penicillium chrysogenum, this industrial workhorse has produced the world’s penicillin for decades and harbors dozens of secondary metabolite biosynthetic gene clusters in its genome, most of which remain transcriptionally silent under standard laboratory conditions. That silence, the new study suggests, may partly reflect the chromatin contexts in which these clusters sit. Understanding where in the genome a foreign pathway will be welcomed, rather than silenced, could unlock the fungus’s full potential as a chassis for making pharmaceuticals and other valuable chemicals.

To build the screening platform, the researchers adapted the piggyBac transposon system, originally isolated from the cabbage looper moth Trichoplusia ni. PiggyBac is prized among genome engineers because it inserts precisely into TTAA DNA sequences without leaving genomic scars, and in organisms such as fission yeast and Pichia pastoris it distributes itself nearly randomly across the genome. The team engineered a strain in which a piggyBac cassette carrying a selectable marker was embedded within a G418 resistance gene, then supplied a hyperactive, codon-optimized transposase called hyPBase, decorated with nuclear localization signals, on an autonomously replicating plasmid. When the transposase was expressed, the cassette excised cleanly and reinserted elsewhere, restoring G418 resistance only in cells where transposition had occurred.

Quantitative PCR confirmed that every resistant colony carried exactly one copy of the transposon, meaning that any subsequent differences in gene expression could be attributed to insertion site rather than copy number. The researchers also swapped the constitutive promoter driving the transposase for a starch-inducible promoter from P. rubens itself, giving them temporal control over transposition and stabilizing the engineered strains. Transposition efficiency peaked at around eight percent on day four of induction, a level the authors describe as sufficient for generating large-scale insertion libraries in this multicellular organism, which is far harder to manipulate than single-celled yeasts.

High-throughput sequencing of more than 13,000 independent insertions revealed that piggyBac behaves differently in P. rubens than in previously studied hosts. Only about 40 percent of insertions landed at TTAA sites, and the surrounding nucleotide preferences diverged sharply from those observed in yeast, insects, mice and Candida. The transposon showed a mild tendency toward local hopping near the donor locus but otherwise spread broadly across all chromosomes, hitting all 59 secondary metabolite gene clusters annotated in the genome and disrupting the core biosynthetic genes of 43 of them, evidence that these clusters are dispensable for growth and candidates for future genome minimization. Notably, insertion density was highest within open reading frames, a preference that should aid gene function discovery.

With the mutagenesis platform validated, the team turned it into an expression-mapping tool. They constructed a strain carrying a green fluorescent protein reporter driven by a moderately strong promoter on the mobile cassette, induced transposition, and then converted fungal mycelia into single-cell protoplasts for analysis by fluorescence-activated cell sorting. Two populations were collected: P4, comprising 14 percent of cells with fluorescence indistinguishable from untransformed controls, representing genomic loci where the reporter was silenced, and P7, the top 1.88 percent of cells with the highest fluorescence, representing hotspots of expression. Clones from both groups remained stable over five generations, confirming that the differences reflected genuine chromosomal position effects rather than transient physiological states.

To understand why some loci were hot and others cold, the researchers turned to Hi-C, a sequencing method that captures the three-dimensional folding of chromosomes. The analysis revealed that P. rubens chromosomes are organized into A and B compartments, mirroring the architecture of mammalian genomes, where compartment A corresponds to active, accessible chromatin and compartment B to compacted, inactive regions. Insertion sites from the P7 high-expression library were significantly enriched in compartment A, while P4 low-expression sites showed no particular enrichment in either compartment, hinting that silencing involves additional local features beyond gross compartmentalization. Neither P4 nor P7 sites clustered near the boundaries of topologically associating domains, suggesting that large-scale chromatin compartmentalization, rather than local boundary structure, is the primary determinant of expression potential. Genes near P7 sites were also enriched in primary metabolic pathways such as amino acid biosynthesis, consistent with those regions being constitutively active.

The decisive test came with physcion, an anthraquinone pigment produced by medicinal herbs such as Rheum and Polygonum. Physcion, the 6-O-methylated derivative of emodin, is registered as a plant-derived fungicide effective against powdery mildew, downy mildew, grey mould and anthrax, with low toxicity to humans and animals. Its biosynthetic pathway, fully elucidated in recent years, begins with malonyl-CoA condensed by a thioesterase-deficient non-reducing polyketide synthase, proceeds through atrochrysone and emodin intermediates, and ends with a critical O-methylation step catalysed by an O-methyltransferase using S-adenosylmethionine. The team first disrupted pcbAB, the gene responsible for penicillin biosynthesis, to eliminate competition from the fungus’s native flagship product, then inserted the synthetic physcion gene cluster, carried on two DNA fragments encoding the polyketide synthase, thioesterase, decarboxylase and methyltransferase, into five loci each from the P4 and P7 libraries.

The results were striking. Junction PCR and quantitative PCR confirmed correct, single-copy integration at every site, yet strains carrying the cluster at P4 loci produced an average of only 0.95 milligrams per liter of physcion, with most samples below 1.2 milligrams per liter. Strains with the identical cluster at P7 loci produced between 27 and 83 milligrams per liter, averaging 56.0 milligrams per liter, a roughly fiftyfold improvement attributable purely to chromosomal address. Quantitative RT-PCR showed markedly elevated transcription of the polyketide synthase gene AnPKS in several P7 strains, correlating with higher yields, although one P4 strain bucked the trend, indicating that transcript abundance alone does not fully explain pathway output and that further regulatory layers tied to chromosomal position remain to be explored.

The implications extend well beyond a single compound. The authors argue that P. rubens should now be regarded as a chromatin-optimized chassis for secondary metabolite production, and that rational integration-site selection, guided by expression hotspot maps, should become standard practice in fungal metabolic engineering. The methodology, from inducible piggyBac mutagenesis through FACS-based expression sorting to Hi-C-informed interpretation, offers a blueprint transferable to other industrially significant filamentous fungi, where heterologous gene silencing has long frustrated efforts to express foreign biosynthetic pathways. As synthetic biology moves into the era of three-dimensional genomics, this study makes a compelling case that in the design of microbial cell factories, the most important question may not be what you insert, but where.

Subject of Research: Position effect variegation and heterologous biosynthesis of physcion in Penicillium rubens

Article Title: Position Effect Variegation of Penicillium rubens Genome and Application in Heterologous Production of the Plant‐Derived Anthraquinone Physcion

Article References: Wu, M., Fu, Y., Tang, L., Yao, Y., Pan, Y., & Liu, G. (2026). Position Effect Variegation of Penicillium rubens Genome and Application in Heterologous Production of the Plant‐Derived Anthraquinone Physcion. Microbial Biotechnology, 19(10), Article e70449. https://doi.org/10.1111/1751-7915.70449

Image Credits: AI Generated

DOI: 10.1111/1751-7915.70449

Keywords: Penicillium rubens, position effect variegation, piggyBac transposon, physcion, heterologous expression, chromatin, Hi-C, secondary metabolites, biosynthetic gene cluster, metabolic engineering, FACS, filamentous fungi

The research team, led by Ryan Wei Kwan Wong and Thibault Mayor of the Department of Biochemistry and Molecular Biology at the Michael Smith Laboratories, set out to characterize the proteomic changes that occur when Saccharomyces cerevisiae is pushed to produce a heterologous protein. Their model target was laccase, an enzyme that is not native to yeast and therefore places distinctive demands on the cell’s protein folding and secretion machinery. By tracking protein abundance over four days of batch cultivation, the researchers were able to separate the effects of ordinary growth and nutrient depletion from the specific burden imposed by recombinant protein expression.

The scale of the survey is significant. Using quantitative mass spectrometry, the team measured the abundance of thousands of proteins across the cultivation period, capturing the ebb and flow of the cellular machinery as the culture progressed. This time-resolved approach matters because recombinant production in batch culture is not a static process: cells pass through exponential growth, nutrient exhaustion, and stress phases, and each stage imposes its own demands. A single snapshot at the end of a fermentation run would miss much of the story.

One of the clearest results is that the bulk of proteomic change during batch cultivation is driven not by the recombinant protein itself but by the shifting nutritional environment. As nutrients are depleted, a substantial portion of the proteome shifts, with ribosomal and metabolic proteins among the categories that change most noticeably. This makes biological sense: as the growth rate slows, cells downsize their protein synthesis apparatus and rewire their metabolism to survive on whatever carbon and nitrogen sources remain. Distinguishing this background remodeling from the specific response to laccase production was a central analytical challenge of the study.

When the researchers isolated the signature of laccase expression, a striking pattern emerged: only a smaller subset of proteins was specifically affected by producing the foreign enzyme. In other words, the cell does not mount a wholesale, dramatic overhaul in response to a heterologous protein. Instead, the response is comparatively targeted, involving particular classes of proteins that help the cell cope with the added biosynthetic and folding load. This finding refines the common assumption that recombinant production is a broadly destabilizing stress for the host cell.

The most consequential part of the study came from a comparison across strains. Rather than working with a single laboratory isolate, the team examined S. cerevisiae strains of different origins and with different capacities to produce active laccase. What they found was that each strain displayed a distinct proteomic response to heterologous expression, and, notably, that this individuality persisted regardless of the origin of the laccase enzyme being produced. The strain, not the foreign protein, appeared to dictate the shape of the cellular response.

Concrete examples illustrate the point. The molecular chaperones Hsp26 and Kar2 were specifically elevated in a whey-derived strain upon laccase expression. Kar2, also known as BiP, is a key chaperone of the endoplasmic reticulum, the compartment where secreted proteins fold, while Hsp26 is a cytosolic chaperone associated with stress protection. Their selective induction in one strain but not others shows that different yeast lineages deploy different quality-control strategies when confronted with the same production task. The cellular environment each strain provides is genuinely unique.

Perhaps the most provocative conclusion is that the strains with a higher capacity to produce active recombinant laccase owe their advantage not to a stronger or better-coordinated response during production, but to small groups of proteins that are constitutively expressed at different levels even before the foreign gene is switched on. In other words, the winning strains start the race with an advantage already built in. Their baseline proteome, shaped by their evolutionary history and ecological origin, happens to provide a cellular milieu that is more favorable for folding, processing, or sustaining the recombinant product.

This has practical implications for the biotechnology industry. Recombinant protein production in yeast underpins the manufacture of biopharmaceuticals, industrial enzymes, and food ingredients, and yield improvement has traditionally focused on engineering the production strain: adding gene copies, overexpressing chaperones, or optimizing promoters and secretion signals. The new results suggest a complementary and potentially underexploited strategy: screening naturally occurring yeast diversity for strains whose native proteomes are already well suited to a given product. Rather than forcing a standard laboratory strain to adapt, producers could select a strain whose inherent biology does much of the work.

The study also carries a conceptual message for the field. Because each strain responds differently to the same recombinant protein, proteomic data gathered from one background may not generalize to another, a caveat for anyone using a single reference strain to model production behavior. At the same time, the identification of constitutively expressed protein groups associated with high yields provides concrete molecular leads for future work, whether in rational engineering or in breeding programs that combine favorable natural traits. As the authors note, this is the first look at the dynamic proteome remodeling that occurs during recombinant laccase expression, and it highlights the potential of exploiting naturally occurring yeast diversity rather than relying solely on strain engineering to improve recombinant protein yields. For an organism that has served biotechnology for decades, S. cerevisiae still has surprises hidden in its wild relatives and industrial isolates, and those hidden differences may be the key to the next generation of cell biofactories.

Subject of Research: Proteomic responses of Saccharomyces cerevisiae strains during recombinant laccase production

Article Title: Alterations of the Saccharomyces cerevisiae proteome by protein production are dependent on strain origins

Article References: Wong, R. W. K., Chandhok, S., Hui, E., & Mayor, T. (2026). Alterations of the Saccharomyces cerevisiae proteome by protein production are dependent on strain origins. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14014-6

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14014-6

Keywords: Saccharomyces cerevisiae, recombinant protein production, proteomics, laccase, yeast strain diversity, chaperones, mass spectrometry, cell biofactories, heterologous expression, biotechnology, Alterations, Saccharomyces

The trouble with conventional tools is well documented. Two-dimensional cell cultures, such as the widely used B16F10 mouse melanoma line, are accessible and highly pigmented but do not capture the molecular heterogeneity of human disease. Human melanoma lines like the Sk-Mel series, MNT-1, and A375 offer more relevant genetics but remain simplified. Primary melanocytes isolated from skin biopsies behave more like cells in the body but survive only a limited number of passages before senescence. Animal models—mice, zebrafish, the frog Xenopus laevis, guinea pigs, swine, chick embryos, and fruit flies—each bring valuable features, from the optical clarity of zebrafish embryos to the tyrosinase-dependent, MITF-independent melanin deposition in Xenopus, but they suffer from divergent protein–protein interactions, species-specific genetics, high costs, and mounting ethical constraints. Most critically, identical visible pigmentation phenotypes in humans can arise from entirely distinct molecular disruptions, a depth of heterogeneity that flat cultures and cross-species models frequently miss, leading to inconsistent and unpredictable therapeutic responses.

The review, published in Bioengineering & Translational Medicine, organizes the emerging alternative into three converging categories unified by the iterative Design-Build-Test-Learn engineering cycle. The first is classical synthetic biology: direct genetic and metabolic rewiring using tools such as CRISPR-Cas9, TALENs, and heterologous microbial expression. CRISPR-Cas9, adapted from a bacterial immune system, uses a guide RNA to direct the Cas9 endonuclease to a precise DNA target, where the resulting break is repaired by non-homologous end joining or homology-directed repair. Researchers have exploited this to build exquisitely controlled disease models: knocking out SIRT1 in murine melanoma cells suppresses melanogenesis by reducing MITF expression through altered p38 and ERK signaling, creating a hypopigmentation model relevant to vitiligo, whereas ablating SIRT7 upregulates melanin production via MITF and its downstream enzymes, mimicking melasma and lentigines. In a striking proof of concept, CRISPR was used to correct the T373K missense mutation that abolishes tyrosinase activity in oculocutaneous albinism, restoring enzyme function and melanin content in an animal model and establishing a precise genotype-to-phenotype platform with implications for somatic gene therapy.

Delivery remains the central obstacle to translating these edits into clinic-ready treatments, because the stratum corneum aggressively blocks large molecular cargos and viral vectors carry limited capacity and immunogenic risks. Recent bioengineering work points toward non-viral solutions. A polyamine-modified thermosensitive hydrogel, liquid at refrigerator temperature but gelling at body temperature, has successfully delivered CRISPR-Cas9 ribonucleoproteins through the skin, achieving a 46 percent gene mutation frequency in vivo with no systemic toxicity. Complementary ex vivo strategies—correcting a patient’s own skin cells in the laboratory, expanding them as epidermal sheets, and grafting them back—offer a path to durable, safety-validated gene correction for genetic skin disorders. TALENs, which pair a DNA-binding domain with a FokI nuclease, add a second editing option with lower off-target risk, as demonstrated by disrupting the slc24a5 gene in tuna to produce hypopigmentation.

The second category, heterologous expression systems, swaps slow mammalian kinetics for engineered microbes, transforming melanin from a scarce biological product into an industrially manufacturable biomaterial. The standout example is the marine bacterium Vibrio natriegens, which doubles roughly every ten minutes; when engineered to express a prokaryotic tyrosinase from Bacillus megaterium and supplemented with L-tyrosine and copper, it generates visible melanin within fifteen minutes and saturates the culture within two hours. The same platform demonstrated optogenetic control, using blue light to derepress tyrosinase expression with reversible, spatially precise timing. Parallel efforts in Escherichia coli express tyrosinases such as melA from Rhizobium etli or route flux through the hppd gene to produce pyomelanin, while modular co-expression of multiple enzymes yields non-natural variants like melanin-diamine complexes optimized for fabric dyeing. Eukaryotic hosts fill the gap where post-translational modification matters: the yeast Yarrowia lipolytica secretes pyomelanin with photoprotective and antioxidant properties suitable for dermatological formulations, retaining the glycosylation machinery that bacteria lack.

The third and physiologically richest category comprises biomimetic systems that recreate the dynamic microenvironment of living skin. Co-culturing primary melanocytes and keratinocytes from vitiligo patients produces a patient-specific platform whose drug responses differ markedly from healthy controls—resistant to melanogenic stimulators and hypersensitive to inhibitors—making it a predictive preclinical proxy. Three-dimensional melanocyte spheroids rescue the melanin production that flat monolayers lose, and when treated with the skin-lightening compound fucoxanthin they outperform commercial tissue equivalents in sensitivity. Perhaps most impressive are microfluidic skin-on-a-chip devices: one bilayer-hydrogel platform, perfused with circulating 17β-estradiol and tuned oxygen gradients, reproduces the exact hyperpigmentation patterns of pregnancy-induced melasma, demonstrating that hormone-driven disorders can now be modeled outside the body. Skin explants preserved in gravity-driven, pumpless chips maintain viability for clinical monitoring and personalized testing.

At the macroscopic end of the spectrum, 3D bioprinting and reconstructed human pigmented skin (RHPS) and epidermis (RHPE) models bring structural fidelity to high-throughput screening. Printed bioinks containing live cells and gelatin-alginate matrices are cross-linked, cultured submerged, and then shifted to an air-liquid interface, where exposure to air forces keratinocytes to differentiate and stratify into a viable, keratinized epidermis. Commercial systems such as MelanoDerm, EpiSkin, SkinEthic, and EpiDerm, built from melanocytes and keratinocytes of diverse genetic ancestries, already support safety testing accepted by regulators for dermal irritation and phototoxicity under New Approach Methodologies aligned with the 3Rs of animal research. Pushing to the sub-cellular scale, researchers have engineered artificial melanosomes—tyrosinase encapsulated in lipid vesicles or polymersomes—that synthesize melanin only when triggered by ultraviolet light or that migrate to the perinuclear region of keratinocytes, mimicking the DNA-shielding nuclear caps of native melanosomes. Stem cell technologies extend the vision toward regenerative medicine, with induced pluripotent stem cell–derived melanocytes and MUSE cells integrating into the basal layer of skin organoids without tumorigenic risk.

Complementing all of these live systems are cell-free assays that strip melanogenesis down to its isolated biochemistry. Simple tyrosinase assays using L-DOPA remain a gold standard for rapid inhibitor screening, and they delivered a definitive mechanistic answer in the case of plumbagin, confirming it as a direct enzymatic inhibitor rather than an upstream regulator. More advanced transcription-translation (TXTL) systems can synthesize melanogenic proteins entirely outside living cells, sidestepping host toxicity and metabolic competition. These reductionist platforms are exceptionally reproducible but deliberately blind to membrane permeability and cellular feedback loops, so their findings must be validated in more complex biomimetic models. Emerging cell-free regenerative approaches—platelet-rich plasma, extracellular vesicles, and stem cell secretomes—may bypass transplantation risks altogether by stimulating resident melanocyte precursors and countering oxidative stress in disorders such as vitiligo.

The review is candid about remaining gaps. Engineered microbes are scalable but poorly represent human melanocyte biology; biomimetic constructs are physiologically faithful but expensive, hard to standardize, and still lack immune, vascular, and neural components. CRISPR faces off-target effects, immune recognition of bacterial Cas9, and delivery challenges in continuously renewing skin, prompting development of high-fidelity variants and anti-CRISPR safeguards. Engineered live therapeutics fall under strict regulatory oversight, and industrial melanin production demands biosafety containment and genetic safeguards against environmental release. Yet the trajectory is clear: by unifying programmable gene circuits, engineered microbes, printed tissue, organ-on-chip microenvironments, and synthetic organelles under shared engineering principles, researchers are replacing unpredictable and ethically constrained models with controllable biosystems. The payoff spans next-generation diagnostics, targeted therapies for vitiligo and melasma, biologically integrated sunscreens, antioxidant delivery vehicles, melanin-based radiometal sorbents for nuclear medicine, and even organic semiconductors—a breadth that positions engineered pigmentation platforms as one of the most versatile toolkits in modern dermatology.

Subject of Research: Engineered synthetic biology and biomimetic platforms for studying melanogenesis and pigmentation disorders

Article Title: Engineered platforms for melanogenesis research: Bridging synthetic biology, bioengineering, and biomimetics

Article References: Anika, B., Tarkunde, A., Swarna, B. S., Nayak, U. Y., Prabhu, V., Rao, R., & Bharath Prasad, A. S. (2026). Engineered platforms for melanogenesis research: Bridging synthetic biology, bioengineering, and biomimetics. Bioengineering & Translational Medicine, 11(5), Article e70159. https://doi.org/10.1002/btm2.70159

Image Credits: AI Generated

DOI: 10.1002/btm2.70159

Keywords: melanogenesis, melanin, synthetic biology, CRISPR-Cas9, biomimetics, 3D bioprinting, skin-on-a-chip, artificial melanosomes, heterologous expression, vitiligo, melasma, reconstructed human skin