Fungal Genome Hotspots Supercharge Production of Plant-Derived Drug Physcion

In a study that could reshape how scientists engineer industrial microbes, researchers have mapped for the first time how the location of a gene within the genome of the penicillin-producing fungus Penicillium rubens dictates its expression, and then exploited that knowledge to boost production of a valuable plant-derived compound more than fiftyfold. The work, published in Microbial Biotechnology, introduces a powerful new screening platform built on the piggyBac transposon and demonstrates that chromosomal position is a decisive, and largely overlooked, variable in the design of fungal cell factories.

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