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Scientists reconstructed ancient microbial chemistry from Neanderthal dental calculus, the mineralized plaque commonly called tartar. By assembling damaged DNA into microbial genomes, finding biosynthetic gene clusters and expressing one reconstructed pathway in a laboratory host, they produced a previously unknown metabolite class called paleofurans. The result demonstrates a method for discovering natural products from archaeological material—not evidence that Neanderthals isolated or used paleofurans as medicine.
What was reconstructed from Neanderthal dental calculus?
The best-established result is a class of previously unknown microbial metabolites named paleofurans. In a 2023 Science study, Klapper and colleagues identified a biosynthetic gene cluster (BGC) preserved in ancient dental calculus. The cluster was shared by seven individuals from the Middle and Upper Paleolithic. Researchers reconstructed the pathway and used heterologous expression—putting the genetic instructions into a modern laboratory host—to produce the compounds.
A BGC is a neighboring set of genes that work together to make a specialized molecule. Such clusters can encode enzymes that assemble antibiotics, pigments, signaling molecules and other natural products. Finding a cluster in ancient DNA does not automatically reveal a finished chemical structure. The DNA may be incomplete, chemically damaged or mixed with sequences from several organisms, so the pathway must be reconstructed and tested experimentally.
The paleofuran finding therefore has two separate parts:
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- Archaeological sequence evidence: ancient calculus contained DNA fragments that could be assembled into a BGC shared among seven Paleolithic individuals.
- Modern laboratory production: a reconstructed version of that pathway functioned in a laboratory host and yielded the compounds called paleofurans.
Nothing in the study shows that Neanderthals purified, swallowed or therapeutically applied these molecules. The products are laboratory outputs guided by ancient sequence information, not recovered doses of a Neanderthal medicine.
Why mineralized plaque can preserve microbial DNA
Dental calculus forms when dental plaque becomes mineralized on a tooth surface. Unlike fresh plaque, which is routinely disturbed by eating, brushing and microbial turnover, calculus can remain attached to archaeological teeth for very long periods. Mineral deposits physically entrap cellular material and DNA fragments, creating an unusually durable archive of the oral microbiome.
That archive can preserve traces of bacteria, archaea and other organisms that lived in the mouth. A foundational 2017 Nature study showed how calculus can support research into ancient hominin behavior, health and microbial evolution. It reported evidence of an approximately 48,000-year-old Methanobrevibacter oralis genome in Neanderthal calculus. The result established that ancient oral microbial genomes could survive long enough to be reconstructed and compared with modern relatives.
Preservation is not the same as perfect recovery. Ancient DNA is usually short, chemically modified and present in small quantities. Environmental DNA, handling contamination and DNA from later occupants of a burial context can be mixed with the target material. Calculus is valuable precisely because it is durable, but its sequences still require authentication and cautious interpretation.
How researchers turn ancient DNA into testable chemistry
1. Select and document calculus samples
Researchers remove calculus from excavated teeth using contamination-controlled procedures. The 2023 study examined calculus from 12 Neanderthals and 52 anatomically modern humans, covering roughly 100,000 years to the present. The broad time span allows investigators to look for microbial lineages and pathways that appear, disappear or persist across human history.
Sample context matters. A sequence found in a Neanderthal specimen is more informative when it is consistent with the age, burial setting and other evidence from that specimen. Investigators also compare multiple individuals and include controls to identify laboratory or environmental contaminants.
2. Sequence the fragmented DNA
DNA extracted from calculus is sequenced as millions of short reads. Ancient molecules can carry characteristic chemical damage, and the reads often cover only pieces of each original genome. No single read normally contains an entire metabolic pathway; the information must be assembled from overlapping fragments.
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3. Assemble contigs and bin metagenome-assembled genomes
Computational assembly joins compatible reads into longer sequences called contigs. Those contigs are then grouped, or “binned,” according to sequence composition, coverage and relatedness to produce metagenome-assembled genomes (MAGs). A MAG is a reconstruction from a mixed microbial community, not necessarily a complete chromosome from one perfectly preserved cell.
Klapper and colleagues reconstructed 459 bacterial MAGs from the calculus material. This inventory provided the genomic framework for searching for unusual metabolic capabilities rather than treating each DNA fragment as an isolated clue.
4. Search for biosynthetic gene clusters
Specialized software scans reconstructed genomes for gene arrangements associated with natural-product biosynthesis. Researchers can compare predicted enzymes with known families, inspect whether a cluster is distributed among multiple individuals and estimate which genes may be missing or damaged.
The paleofuran-associated cluster stood out because it occurred in seven Middle and Upper Paleolithic individuals. Sharing across separate ancient samples makes a random contaminant less plausible, although it does not by itself prove that every gene in every individual was intact or active during life.
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Ancient assemblies may contain gaps, altered bases or uncertain gene boundaries. Scientists use overlapping reads, closely related genomes and biochemical logic to infer a workable pathway. Any inferred sequence remains a hypothesis until it is tested; a single incorrect base in an enzyme-coding region can stop production or change the product.
6. Express the pathway in a laboratory host
For heterologous expression, selected genes are introduced into a modern organism that can transcribe and translate them. The host supplies cellular machinery while the anciently informed pathway supplies the instructions for making the target molecules. Production in this setting demonstrates that the reconstructed genetic system is sufficiently coherent to operate, but it does not recreate the exact Neanderthal mouth or prove that the pathway was active there.
7. Identify the resulting metabolites
Researchers analyze the products made by the engineered host and determine whether they represent a distinct chemical class. In the 2023 work, this process yielded paleofurans. The naming reflects the novelty of the metabolite class, not a demonstrated medical effect.
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What the 2023 study actually established
The sample and genome scale
The study’s dataset included 12 Neanderthals and 52 anatomically modern humans. Together, the specimens spanned approximately 100,000 years to the present. From their dental calculus, the team reconstructed 459 bacterial MAGs. These figures describe the scope of the analysis; they do not mean that every specimen yielded a complete genome or a drug-producing pathway.
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One BGC was shared by seven Middle and Upper Paleolithic individuals. Its distribution across ancient samples indicated that the underlying microbial chemistry was not limited to one isolated tooth or one modern contaminant source. The team reconstructed the cluster well enough to express it in a laboratory system.
The laboratory product
Heterologous production generated paleofurans, a previously unknown class of metabolites. The result is a proof of concept that archaeological DNA can preserve actionable instructions for natural-product biosynthesis. The study did not report a clinical efficacy measurement, a therapeutic dose or evidence of use by Neanderthals.
Did Neanderthals use paleofurans as medicine?
There is no evidence that they did. A gene cluster shows that a microorganism carried biosynthetic potential; it does not reveal whether the microbe was abundant, whether the pathway was switched on, whether the compounds accumulated in the mouth or whether anyone experienced a beneficial effect.
Even if a paleofuran had antimicrobial activity in a laboratory assay, that would answer a different question from whether Neanderthals recognized, prepared or administered it. Archaeological claims about behavior require independent evidence such as material residues, repeated contextual patterns or use-wear. The paleofuran study provides a molecular reconstruction and laboratory production, not a record of medical practice.
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A 2025 Journal of the American Chemical Society study extended the natural-product approach to ancient and modern dental biofilms, including Neanderthal material. It used metagenomics, structural modeling and experimental validation to investigate Actinomyces-derived defensin-like peptides called actifensins. The work reported maintained antimicrobial activity for these peptides and evolutionary analyses connecting ancient and modern sequences.
Actifensins should not be described as the same discovery as paleofurans. The source material overlaps, but the molecular class and validation path differ.
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| Comparison point | Paleofurans (Klapper et al., Science, 2023) | Actifensins (JACS study, 2025) |
|---|---|---|
| Source material and time depth | Dental calculus from 12 Neanderthals and 52 modern humans spanning about 100,000 years to the present | Ancient and modern dental biofilms, including Neanderthal material; the cited summary does not give one combined specimen count |
| Computational approach | Metagenomic assembly, bacterial MAG reconstruction and biosynthetic-gene-cluster analysis | Metagenomics combined with structural modeling and evolutionary analysis |
| Pathway or sequence tested | A reconstructed BGC shared by seven Middle and Upper Paleolithic individuals | Actinomyces-derived defensin-like peptide sequences called actifensins |
| Product class | Paleofurans, a previously unknown metabolite class | Defensin-like antimicrobial peptides |
| Functional validation | Heterologous expression produced the metabolites in a laboratory host | Experimental testing found maintained antimicrobial activity |
| What the result supports | Ancient calculus can preserve reconstructable instructions for making novel metabolites | Ancient oral-biofilm sequences can inform peptide discovery and antimicrobial research |
How strong is the evidence, and where does it stop?
- Strongest claim: ancient calculus DNA contained enough information to reconstruct a functional biosynthetic pathway that produced paleofurans in a modern host.
- Supported but narrower: the paleofuran-associated BGC was shared by seven Middle and Upper Paleolithic individuals.
- Not established: the compounds’ clinical usefulness, safety, dose, stability in humans or use by Neanderthals.
- Not implied by sequence similarity: finding a gene resembling an antimicrobial-resistance determinant does not prove that an intact, functional resistance phenotype existed in the ancient organism.
These distinctions are essential because DNA-based discoveries can sound more direct than they are. A sequence is evidence of biological information; expression is evidence that a reconstructed version can work under chosen laboratory conditions; a therapeutic claim requires additional pharmacology, toxicology and clinical research.
Why fossil microbes could matter for drug discovery
Ancient diversity may contain chemistry absent from modern collections
Modern microbial libraries sample only the organisms and genes that still exist or can be cultured today. Ancient oral communities may preserve evolutionary lineages that have changed or disappeared. Their BGCs could encode molecules unlike those in contemporary screening collections.
DNA makes uncultivable organisms accessible
Many microbes are difficult to grow in the laboratory. Metagenomics can reveal their genes without first cultivating the organism. Heterologous expression then provides a route to test selected pathways in a manageable host.
Evolutionary comparisons can prioritize candidates
When a pathway appears in several individuals or persists across time, researchers can compare sequence variants and identify conserved catalytic features. That information can help decide which clusters deserve reconstruction, synthesis and biological testing.
The process is a discovery pipeline, not a shortcut to a drug
A promising ancient pathway still requires optimization, reproducible production, purification, activity testing against defined targets, selectivity measurements, stability studies and safety evaluation. Most candidates would not become medicines. The value of calculus is that it can expand the starting pool of natural products.
Practical limitations researchers must manage
Fragmentation and chemical damage
Short, damaged reads can create gaps or ambiguous bases. A predicted enzyme may be truncated, and a pathway may appear incomplete even when the original microbe carried a working version. Reconstruction therefore depends on transparent quality checks and comparisons with related genomes.
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Contamination
Modern human DNA, laboratory organisms and environmental microbes can enter archaeological samples. Ancient-DNA authentication, extraction blanks, independent replication and careful controls help separate authentic historical signal from contamination.
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Mixed microbial communities
Calculus contains DNA from many organisms. Binning contigs into MAGs can misassign sequences when related microbes coexist or when coverage is uneven. A BGC should be interpreted alongside genome quality, read support and its distribution across samples.
Laboratory context differs from an ancient mouth
A modern expression host may provide nutrients, oxygen levels and regulatory machinery unlike those in Neanderthal oral biofilms. Successful production proves biochemical compatibility in that host, not the exact ecological conditions under which the ancient microbe lived.
Biological activity is only one step
Antimicrobial activity in a laboratory assay does not establish effectiveness in a person. Activity can disappear at physiological concentrations, be blocked by proteins, or be accompanied by toxicity. Paleofurans currently lack a published clinical-efficacy figure.
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What to look for in future studies
Follow-up work can test whether additional paleofuran variants occur in other specimens, define the enzymes responsible for each assembly step and determine the compounds’ biological properties. Better assemblies may reveal whether the cluster was complete in individual ancient genomes rather than reconstructed by combining evidence. Researchers can also compare ancient pathways with modern oral microbes to track gains, losses and sequence changes through time.
For actifensins and related peptides, important next steps include measuring potency under physiologically relevant conditions, mapping activity against different microbial targets, assessing toxicity and determining whether the peptides can be produced and stabilized at useful scales. Those experiments would move the work from discovery toward preclinical evaluation, but they would still not establish Neanderthal use.
Why this finding changes the view of archaeological DNA
Dental calculus is more than a record of which microbes once occupied a mouth. It can preserve genetic instructions for specialized chemistry. The paleofuran study connects an archaeological specimen to a reproducible laboratory product through a chain of evidence: mineralized plaque preservation, metagenomic assembly, MAG reconstruction, BGC identification, pathway repair and heterologous expression.
That chain also defines the proper conclusion. Ancient dental calculus can function as a natural-product discovery archive, and Neanderthal-associated microbial DNA has already yielded a novel metabolite class. Whether any such molecule becomes a useful antimicrobial or other therapy remains an open biomedical question, separate from what Neanderthals themselves knew or used.
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