Giant transposable elements fuel genetic diversity in fermented food fungus

Long before science could sequence a genome, brewers in Japan were unknowingly running one of the world’s longest artificial evolution experiments. For centuries, the fungus Aspergillus oryzae, known in Japan as koji mold, has been cultivated to break down starches and proteins in sake, soy sauce, and miso. Now, a research team led by Yuya Hamanaka and Jun-ichi Maruyama at the University of Tokyo has revealed a surprising engine behind this mold’s industrial versatility: giant, virus-sized chunks of DNA called Starship transposable elements, which appear to have jumped between fungal strains and even between species, carrying genes that brewers unknowingly selected for.

Transposable elements, first discovered by Barbara McClintock in maize in the mid-twentieth century, are DNA sequences that can cut themselves out of one genomic location and reinsert elsewhere. Most are modest in size, ranging from a few hundred to around ten thousand base pairs. Starships, identified in recent years in the filamentous fungal subphylum Pezizomycotina, are a different beast entirely. These elements span roughly 15,000 to 700,000 base pairs, comparable in scale to bacterial integrative and conjugative elements, which famously shuttle antibiotic resistance genes among bacteria. At one end of every Starship sits a “captain” gene encoding a tyrosine recombinase, an enzyme thought to excise the element from the chromosome and paste it elsewhere. The rest of the element carries “cargo” genes, which can confer traits as varied as heavy metal tolerance, formaldehyde resistance, and virulence in other fungal species.

What has remained unclear until now is whether Starships contribute to diversity within a single species, rather than merely between species. To address this, the team generated chromosome-level genome assemblies of six Aspergillus oryzae strains drawn from different phylogenetic clades of the mold’s industrial lineage, including the reference strain RIB40 and five so-called Tane Koji strains used in commercial fermentation. Because Starships are far too large to be resolved reliably with short-read sequencing, the researchers used long-read technologies, sequencing RIB40 with PacBio HiFi reads and the other strains with Oxford Nanopore reads, assembling genomes of 37.3 to 39.2 million base pairs in only 8 to 13 contigs, with BUSCO completeness scores of 98 percent or higher.

Comparative analysis revealed something striking. While the overall chromosome architecture was largely conserved across the strains, eight large regions of roughly 10,000 to 290,000 base pairs sat on entirely different chromosomes in different strains. Each of these regions carried a gene containing the DUF3435 domain, a molecular signature of Starship elements. Expanding their search, the researchers cataloged 66 such captain genes across the six genomes, sorting them into ten distinct classes of Starship elements, which they named SEA1 through SEA10, for Starship Element in A. oryzae. In total, 48 individual elements were identified, ranging from 6.3 to nearly 289 kilobase pairs.

Classification against starbase, a public database of fungal Starships, showed that the elements belong to known families such as Prometheus, Enterprise, Galactica, and Phoenix, while SEA10 matched nothing in the database and may represent an entirely new class. Each class carried a characteristic complement of cargo genes: some harbored patatin-like phospholipases, ferric reductases, and NUDIX domain proteins commonly found in Starships; others carried DNA-binding proteins and protein kinases suggesting roles in signal transduction, or HET-domain genes linked to heterokaryon incompatibility, the fungal self-versus-nonself recognition system that blocks fusion with genetically different individuals.

The most provocative findings concern fermentation. Several elements carried copies of alpha-amylase genes, the workhorse enzymes that koji molds use to convert starch into fermentable sugars. In strain TK-41, two divergently oriented alpha-amylase genes sat inside SEA1, flanked by jockey-like retrotransposons and other transposons that appear to have ferried the genes into the element, much as bacterial transposons load cargo into integrative and conjugative elements. When the researchers deleted these two genes using a genome-editing plasmid, both alpha-amylase expression and enzymatic activity, measured by halo formation on starch agar, dropped significantly, proving the captured genes are functional. Because alpha-amylase gene copy number varies by clade in this species, Starships may have helped tune starch-degrading capacity to the needs of different brewing applications, with soy sauce strains, which ferment soybeans rather than pure starch, needing less amylase firepower.

Other cargo genes tell an equally compelling story. A glycosyltransferase 8 gene, resembling plant galactinol synthases implicated in salt and oxidative stress tolerance, was found in SEA3 of strains used for miso and sake production, fermentations that expose the mold to high salt concentrations. Meanwhile, SEA6 of strain TK-49, used for soy sauce brewing, carried genes for a GH10 xylanase and a GH12 cellulase, enzymes that degrade soybean cell wall polysaccharides. Eight of the ten strains harboring these genes are used for soy sauce production, hinting that brewers’ selection quietly favored strains carrying these hitchhiking enzymatic toolkits. RNA sequencing and RT-qPCR confirmed that cargo genes across the elements were transcriptionally active, not silenced relics.

The phylogenetic reconstruction of how these elements spread reads like a tale of airborne gene traffic. Elements including SEA2, SEA4, and SEA9 appear to have been inherited vertically from Aspergillus flavus, the aflatoxin-producing wild ancestor from which A. oryzae was domesticated, but were then shuffled among A. oryzae strains by repeated horizontal transfer. Other elements, including SEA1, SEA3, and SEA5, are absent from A. flavus entirely but shared with Aspergillus sojae, another domesticated fermentation fungus, suggesting exchange between the two industrial species. Along the way, A. oryzae-specific cargo was gained, including NOD-like receptor genes in SEA4 that may strengthen heterokaryon incompatibility and thereby guard genomic stability during brewing, and a methyltransferase gene in SEA9 that could regulate metabolism-related transcription in clade B sake strains. Conversely, a non-ribosomal peptide biosynthesis cluster present in the A. flavus version of SEA2 was lost during domestication, mirroring the well-known loss of aflatoxin production.

The boldest claim concerns a jump across genus boundaries. SEA6 sequences with striking similarity, including the GH10 and GH12 genes, were found on chromosomes 3 and 4 of Monascus purpureus, a fungus used in East Asia to ferment foods such as tofuyo. Transcripts from the A. oryzae SEA6 showed far higher similarity to Monascus genes than any other genes did, and phylogenetic analysis of the captain genes supports a model in which the element originated in ancestral A. oryzae clades, spread among strains, and was twice transferred horizontally into M. purpureus. Although the two molds are rarely used together, both are employed in fermented food production across East Asia, and prior laboratory work has demonstrated hyphal fusion between them, plausibly providing the cellular bridge for such transfer.

Taken together, the study proposes a new conceptual model of fungal domestication, in which giant transposable elements act as vehicles for the acquisition, loss, and rearrangement of genes, generating the raw diversity upon which centuries of artificial selection acted. The researchers note limitations: only representative strains per clade were assembled, clades D and H remain unsequenced at chromosome level, and the directionality of transfers is often uncertain. But the message is clear. Just as bacteria package adaptive traits into integrative and conjugative elements, this domesticated fungus appears to have ridden Starships to fermentative success, offering a eukaryotic paradigm in which enormous mobile elements drive adaptation to a very particular niche, the human brewery.