How Scientists Are Harnessing Mesozoic Laccases
Imagine holding a biological blueprint from the age of dinosaurs—a molecular relic that could revolutionize modern biotechnology. This isn't science fiction; through ancestral sequence reconstruction, scientists are resurrecting ancient enzymes called laccases from fungi that thrived millions of years ago. These resurrected catalysts are now being engineered to tackle some of today's most pressing industrial and environmental challenges.
Laccases are remarkable natural oxidizers found in fungi, plants, and bacteria. Known as "green catalysts," they can break down numerous compounds using only oxygen from the air and releasing water as their only by-product 2 3 .
Their potential applications span from textile manufacturing and biofuel production to environmental cleanup and food processing 1 2 . However, modern laccases often struggle to withstand industrial conditions, and producing them efficiently has been a persistent challenge 1 .
By combining palaeontology and protein engineering, researchers have discovered that ancient laccases from the Mesozoic era (approximately 250 to 500 million years ago) possess unique advantages over their modern counterparts, including superior stability and easier production 1 4 5 . This article explores how scientists are resurrecting these ancient enzymes and refining them through directed evolution to create powerful biocatalysts for a sustainable future.
Laccases belong to the multicopper oxidase family of enzymes. They are typically extracellular glycoproteins with a complex molecular structure featuring three cupredoxin-type domains arranged in a Greek-key barrel pattern 3 .
Begins at the Type 1 copper site where substrates are oxidized, with electrons traveling to a trinuclear copper cluster for oxygen reduction 3 .
The broad substrate specificity of laccases, coupled with their environmentally friendly reaction requirements, makes them invaluable across numerous industries:
Despite this tremendous potential, industrial applications have been limited because many modern laccases are difficult to produce in large quantities and may lack the robustness required for industrial processes 1 .
Ancestral sequence reconstruction represents a fascinating intersection of bioinformatics, molecular biology, and evolutionary theory. The process begins with comparing modern laccase sequences to identify their evolutionary relationships. Using sophisticated computational models, scientists work backward along evolutionary trees to infer the most likely sequences of ancient proteins 1 .
Once these ancestral sequences are predicted, they're synthesized in the laboratory and introduced into host organisms—typically yeast species like Saccharomyces cerevisiae—for production and testing 1 2 . This process, known as "resurrection," allows researchers to study properties of enzymes that haven't existed for millions of years.
When scientists resurrected three ancestral fungal laccase nodes (LacAnc95, LacAnc98, and LacAnc100) dating back 250-500 million years, they made a remarkable discovery. Unlike many modern laccases that are difficult to produce in laboratory hosts, these ancient versions showed strikingly high heterologous expression in yeast systems 1 .
| Laccase Variant | Estimated Age (Million Years) | Expression Level | pH Stability | Key Features |
|---|---|---|---|---|
| LacAnc95 | ~500 | Not expressed | Not determined | Unable to produce in yeast system |
| LacAnc98 | ~300 | High | Broad | Similar kinetics, distinct pH profile |
| LacAnc100 | ~250 | High | Broad | 136 ancestral mutations, suitable for engineering |
The resurrected Mesozoic laccases also displayed:
One particular resurrected enzyme, the Agaricomycetes laccase, carried 136 ancestral mutations—a molecular testimony to its ancient origin 1 . These mutations apparently contributed to enhanced stability and expressibility, suggesting that ancient enzymes might have been generalists capable of functioning under diverse conditions 1 .
In a groundbreaking study published in 2020, researchers embarked on an ambitious project to resurrect and improve ancient laccases 1 . Their methodology provides a fascinating blueprint for combining paleoenzymology with modern protein engineering:
The team first reconstructed three ancestral laccase nodes (LacAnc95, LacAnc98, and LacAnc100) using the TimeTree of Life database, dating them back to the Mesozoic era (500-252 million years ago) 1 .
The inferred ancestral sequences were synthesized and cloned into Saccharomyces cerevisiae yeast expression systems. The researchers tested different signal sequences (including αPM1, αPcL, and pre-α-prokiller) to optimize secretion 1 .
Culture conditions were systematically optimized, revealing that supplying ethanol and maintaining temperatures at 30°C significantly enhanced secretion—possibly by enhancing membrane permeability and triggering stress responses related to protein folding 1 .
The successfully expressed ancestral laccases (LacAnc98 and LacAnc100) were purified and their biochemical properties were thoroughly analyzed, including kinetic parameters, pH stability, and temperature tolerance 1 .
The resurrected Agaricomycetes laccase was subjected to directed evolution to enhance its ability to oxidize 1,3-cyclopentanedione—a β-diketone initiator used in vinyl polymerization reactions and a poor natural substrate for laccases 1 .
The experimental results demonstrated the tremendous potential of combining ancestral resurrection with directed evolution:
| Evolution Round | Improvement in 1,3-cyclopentanedione oxidation | Key Mutations Accumulated |
|---|---|---|
| Initial ancestral | Baseline | None (ancestral sequence) |
| Early rounds | Moderate improvement | Not specified in study |
| Later rounds | Significant improvement | Not specified in study |
The study revealed that unlike modern laccases that often require extensive engineering for adequate expression, the Mesozoic laccases were readily secreted by yeast—a significant practical advantage for industrial applications 1 . When researchers compared the ancestral sequences to a modern laboratory-evolved laccase (OB-1) that had undergone eight generations of directed evolution, they discovered that one of the key beneficial mutations in the modern engineered enzyme (S224G) was actually an ancestral resurrection—it was naturally present in the ancient laccases 1 .
Most importantly, the research demonstrated that ancestral laccases could serve as excellent starting points for further engineering. The directed evolution campaign successfully enhanced the enzyme's ability to oxidize 1,3-cyclopentanedione, proving that these ancient scaffolds are highly engineerable 1 .
| Tool/Reagent | Function in Research | Application Examples |
|---|---|---|
| Saccharomyces cerevisiae | Heterologous expression host | Secretes laccases with proper folding and post-translational modifications 2 |
| α-factor prepro-leader | Signal sequence for secretion | Enhances laccase secretion in yeast; variants include αPM1, αPcL 1 |
| ABTS, 2,6-dimethoxy phenol | Substrates for activity screening | Colorimetric detection of laccase activity in high-throughput assays 2 |
| In vivo DNA recombination | Generating genetic diversity | Methods like IVOE (In Vivo Overlap Extension) create mutant libraries 2 |
| Episomal vectors | Maintaining foreign DNA in yeast | Allows easy recovery of mutant genes without genomic integration 2 |
The resurrection of Mesozoic laccases represents more than just a scientific curiosity—it demonstrates a powerful new paradigm in biotechnology. By looking to the ancient past, researchers are discovering superior molecular scaffolds that combine robust stability with enhanced expressibility.
The alliance between ancestral resurrection and directed evolution creates a virtuous cycle: ancient enzymes provide optimized starting points for engineering, while modern laboratory evolution tailors them for specific contemporary applications. This approach is already being extended to other enzyme families, suggesting we may be at the dawn of a new era of biocatalyst development 1 9 .
As research progresses, these resurrected ancient enzymes may lead to more sustainable industrial processes, effective environmental remediation strategies, and innovative biotechnological applications we have yet to imagine. The molecular ghosts of dinosaurs' fungal contemporaries are finding new life in the modern world, proving that sometimes the path forward begins by looking backward—250 million years backward.