Resurrecting Ancient Rubiscos

By studying the history of Earth's most important protein, scientists bolster blueprints to engineer it.

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Image courtesy of Luca Schulz.

There’s a great paper about the evolutionary history of Rubisco out in Science this week. Ancient versions of the enzyme — which is about 2.4 billion years old and first arose in archaea — were ‘resurrected’ in the lab to study how it mutated and morphed over time.

The study is a collaboration between Tobias Erb and Georg Hochberg at the Max Planck Institute and was led by graduate student Luca Schulz.

Rubisco is the most populous protein on Earth and converts more than 1.25 billion tons of carbon dioxide each day. Though it evolved in a mostly anaerobic atmosphere, it now persists in an oxygen-rich one. It is thus quite slow, inadvertently mistakes oxygen for its substrate, and only converts about three molecules per enzyme per second.

Before this study, nobody really understood why Form I Rubisco, which is found in both plants and cyanobacteria, evolved to use both small and large (catalytically active) subunits. Nor did we understand how Rubisco developed its tailor-made ability to exclude oxygen in favor of its substrate, carbon dioxide — a shift that, notably, happened before Earth’s Great Oxygenation Event (more on that later).

If scientists could engineer supercharged Rubisco in the lab and put them into plants or algae, we could boost crop yields and capture more carbon dioxide from the atmosphere. In rewinding the Rubisco clock, then, this paper devises a blueprint for enhancing this important protein.

To ‘resurrect’ ancient versions of Rubisco in the lab, Schulz & Co. "computed “maximum-likelihood phylogenies” of the protein’s large and small subunits. All form I Rubiscos have eight large subunits and eight small subunits. By inferring how these subunits mutated over billions of years — based on analyses of metagenome-assembled genomes of diverse species — the researchers predicted, synthesized, and inserted ‘ancient’ Rubisco-encoding sequences into bacteria.

To their surprise, Rubisco’s exclusion of oxygen did not evolve solely via mutations in its catalytic center. Rather, the enzyme tacked on a new small subunit, even before Earth’s Great Oxygenation Event, that acted as a “modulator” for subsequent evolution, according to Schulz.

“Recruitment of the subunit changed the effect that subsequent mutations had on Rubisco's catalytic subunit,” he said in a press release. Specifically, adding on the small subunit enabled mutations in Rubisco’s catalytic center that enhanced its ability to exclude oxygen but otherwise would have ‘ruined’ the enzyme.

Large subunits of Rubisco that existed before the addition of the small subunit had much higher Michaelis constants for carbon dioxide. A Michaelis constant is the concentration of a substrate that is needed to catalyze a specific reaction at half its maximum rate. A lower number means that a smaller amount of carbon dioxide is needed to saturate Rubisco.

Tacking on the small subunit helped Rubisco to exclude oxygen, and shifted this number from 568 to 69 µM. The enzyme’s maximum rate of carboxylation — or how fast it can smash together carbon dioxide and ribulose 1,5-bisphosphate — decreased from 0.61 to 0.29. But the overall effect, with oxygen around, is that Rubisco’s catalytic efficiency more than doubled.

With these changes, the enzyme quickly became dependent upon its small subunit. But other evolutionary branches of Rubisco do not have this small subunit at all. A 2020 paper, for instance, found “a previously uncharacterized clade sister to form I Rubisco that evolved without small subunits. This clade diverged before the evolution of cyanobacteria and the origin of the small subunit.”

Rubisco’s dependence on its small subunit also predates Earth’s Great Oxygenation Event, which occurred about 2.3 billion years ago. But why would an enzyme evolve to exclude oxygen in the first place if there was so little oxygen in the atmosphere?

The first explanation could be that this change occurred during an ancient period in Earth’s history during which atmospheric carbon dioxide levels dipped. Enzymes that adapted would then be uniquely equipped to handle the upcoming oxygenation event and would multiply accordingly.

Another explanation is that our use of the term ’Great Oxygenation Event’ is misleading and oxygen was already present in the atmosphere.

Thanks to this paper, we now know more about why Form I Rubisco works as it does, and how we can engineer it.

“The enzyme is (still) surprisingly slow and can tolerate, but not fully discriminate against, oxygen,” said Tobias Erb in an interview. “We can now think about replaying the tape of evolution with our ancestral proteins [and] provide it with completely novel interaction partners to improve the enzyme’s catalysis and eventually overcome the natural limitations.”

Thanks for reading.

— Niko McCarty

Engineering Biology Index

(↑ = recommended article, * = open access, † = review, comment, etc. )

Basic Research

↑*1000 spider silkomes: Linking sequences to silk physical properties. Arakawa K…Numata K. Science Advances. Link

  • Spider hunters scoured the earth for six years, from 2014 to 2019, and looked in 9 countries, including Madagascar and Malaysia, to collect 1098 different species of spiders. They made transcriptome assemblies and measured the mechanical, thermal, and structural properties of silk from 446 species. These data provide an amazing foundation for bioengineers aiming to predict silk properties from gene sequences.

Evolution of increased complexity and specificity at the dawn of form I Rubiscos. Schulz L…Hochberg GKA. Science. Link

*The first archaeal PET-degrading enzyme belongs to the feruloyl-esterase family. Perez-Garcia P…Streit WR. bioRxiv (preprint). Link

*Maturation and circuit integration of transplanted human cortical organoids. Revah O…Pasca SP. Nature. Link

*Bacteria-on-a-bead: probing the hydrodynamic interplay of dynamic cell appendages during cell separation. Sauter N…Pfohl T. Communications Biology. Link

*Ligand-induced shifts in conformational ensembles that describe transcriptional activation. Khan SH…Okafor CD. eLife. Link

*Quorum-sensing control of matrix protein production drives fractal wrinkling and interfacial localization of Vibrio cholerae pellicles. Qin B & Bassler BL. Nature Communications. Link

*Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine. Kimura S…Waldor MK. Nature Communications. Link

*Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex. Patterson A…Bothner B. Nucleic Acids Research. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

*A genetically encoded system for oxygen generation in living cells. Markhard AL…Mootha VK. PNAS. Link

*Precisely patterned nanofibers made from extendable protein multiplexes. Bethel NP…Baker D. bioRxiv (preprint). Link

*†Carbon-negative synthetic biology: challenges and emerging trends of cyanobacterial technology. Tan C, Xu P & Tao F. Trends in Biotechnology. Link

*Upper Limit Efficiency Estimates for Electromicrobial Production of Drop-In Jet Fuels. Sheppard TJ, Specht DA & Barstow B. bioRxiv (preprint). Link

Construction of an rAAV Producer Cell Line through Synthetic Biology. Lee Z…Hu W-S. ACS Synthetic Biology. Link

Systems engineering of Escherichia coli for n-butane production. Liu Y…Mahadevan R. Metabolic Engineering. Link

A tunable metabolic valve for precise growth control and increased product formation in Pseudomonas putida. Batianis C…dos Santos VAPM. Metabolic Engineering. Link

MGCEP 1.0: A Genetic-Engineered Marine-Derived Chassis Cell for a Scaled Heterologous Expression Platform of Microbial Bioactive Metabolites. Yang Z…Ma J. ACS Synthetic Biology. Link

*Improving glycine utilization in Escherichia coli. Yuen VFK, Tan DZJ & Zhou K. bioRxiv (preprint). Link


*Humanized CB1R and CB2R yeast biosensors enable facile screening of cannabinoid compounds. Mulvihill CJ…Gardner EC. bioRxiv (preprint). Link

*Genetically Encoded Whole Cell Biosensor for Drug Discovery of HIF-1 Interaction Inhibitors. Scott LH…David F. ACS Synthetic Biology. Link


↑*Stimuli-responsive vesicles as distributed artificial organelles for bacterial activation. Gispert I…Elani Y. PNAS. Link

↑*In vitro assembly, positioning and contraction of a division ring in minimal cells. Kohyama S, Merino-Salomón A & Schwille P. Nature Communications. Link

*A self-templated route to monodisperse complex droplets as artificial extremophile-mimic from coacervate-liposome interplay. Li Q…Shum HC. bioRxiv (preprint). Link

*Engineering DNA-based synthetic condensates with programmable material properties, compositions, and functionalities. Do S…Shin Y. Science Advances. Link

Cell-Free Systems

*Characterization of Integrase and Excisionase Activity in Cell-free Protein Expression System Using a Modeling and Analysis Pipeline. Pandey A…Murray RM. bioRxiv (preprint). Link

Computational Tools & Models

*Predicting the structure of large protein complexes using AlphaFold and Monte Carlo tree search. Bryant P…Elofsson A. Nature Communications. Link

*Alignment of single-cell trajectory trees with CAPITAL. Sugihara R…Kawahara Y. Nature Communications. Link

*Process-based modelling of microbial community dynamics in the human colon. Kettle H, Louis P & Flint HJ. Journal of the Royal Society Interface. Link

*A predictive computational platform for optimizing the design of bioartificial pancreas devices. Ernst AU…Ma M. Nature Communications. Link

*MiMeDB: the Human Microbial Metabolome Database. Wishart DS…Karu N. Nucleic Acids Research. Link

*Deep mutational scanning and machine learning reveal structural and molecular rules governing allosteric hotspots in homologous proteins. Leander M…Raman S. eLife. Link

*Dynamic SARS-CoV-2 emergence algorithm for rationally-designed logical next-generation vaccines. Maison DP…Nerurkar VR. Communications Biology. Link

†Biological System Control (Book Chapter). Kim J. Wiley-IEEE Press. Link

CRISPR, Gene Editing & Control

↑*Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors. Lee S…Kim J-S. Nature Biotechnology. Link

↑*Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Durrant MG…Hsu PD. Nature Biotechnology. Link

  • Recombinases are proteins that break and rearrange DNA. A useful tool for engineering biology, most scientists tend to use Bxb1 or PhiC31 in the lab. This study now identifies thousands more recombinases and locates their DNA attachment sites. Experiments in human cells, using these newly-discovered enzymes, “achieved up to seven-fold higher recombination than Bxb1 and genome integration efficiencies of 40–75% with cargo sizes over 7 kb.” 💯

↑Engineering a precise adenine base editor with minimal bystander editing. Chen L…Li D. Nature Chemical Biology. Link

*Metagenomic discovery of novel CRISPR-Cas13 systems. Hu Y…Li W. Cell Discovery. Link

*SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome. Piñero-Lambea C…Lluch-Senar M. Nucleic Acids Research. Link

DNA Sequencing, Synthesis & Assembly

A two-residue nascent-strand steric gate controls synthesis of 2′-O-methyl- and 2′-O-(2-methoxyethyl)-RNA. Freund N…Holliger P. Nature Chemistry. Link

*basicsynbio and the BASIC SEVA collection: Software and vectors for an established DNA assembly method. Haines MC…Storch M. Synthetic Biology. Link

Direct Transfer and Consolidation of Synthetic Yeast Chromosomes by Abortive Mating and Chromosome Elimination. Guo Z…Yuan Y. ACS Synthetic Biology. Link

Gene Circuits

*Chimeric MerR-Family Regulators and Logic Elements for the Design of Metal Sensitive Genetic Circuits in Bacillus subtilis. Ghataora JS, Gebhard S & Reeksting BJ. bioRxiv (preprint). Link

Medicine & Diagnostics

↑Variants of the adeno-associated virus serotype 9 with enhanced penetration of the blood–brain barrier in rodents and primates. Yao Y…Bei F. Nature Biomedical Engineering. Link

↑*A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo. Kofink C…Farnaby W. Nature Communications. Link

*Cross-species evolution of a highly potent AAV variant for therapeutic gene transfer and genome editing. Gonzalez TJ…Asokan A. Nature Communications. Link

*Bivalent mRNA vaccine booster induces robust antibody immunity against Omicron lineages BA.2, BA.2.12.1, BA.2.75 and BA.5. Fang Z…Chen S. Cell Discovery. Link

*†Recent advances in bacteria-mediated cancer therapy. Liang S…Geng Z. Frontiers in Bioengineering and Biotechnology. Link

*Engineering a Universal Dengue Virus Vaccine using a Virus-Like Particle Scaffold. Basore DA…Bystroff C. bioRxiv (preprint). Link

*STAMP-Based Digital CRISPR-Cas13a (STAMP-dCRISPR) for Amplification-Free Quantification of HIV-1 Plasma Viral Load. Nouri R…Guan W. bioRxiv (preprint). Link

†Genome edited allogeneic donor 'universal' chimeric antigen receptor T Cells. Qasim W. Blood. Link


Dissecting the cotranscriptome landscape of plants and their microbiota. Nobori T…Tsuda K. EMBO Reports. Link

*Establishing a reproducible approach to study cellular functions of plant cells with 3D bioprinting. Van Den Broeck L…Sozzani R. Science Advances. Link

†Plant synthetic biology innovations for biofuels and bioproducts. Yang Y…Stewart Jr. CN. Trends in Biotechnology. Link

*†High-temperature stress in crops: male sterility, yield loss, and potential remedy approaches. Khan AH…Zhang X. Plant Biotechnology Journal. Link

*†Plant synthetic biology innovations for biofuels and bioproducts. Yang Y…Stewart Jr. CN. Trends in Biotechnology. Link

Tissue, Protein & Molecular Engineering

*Traceless cysteine-linchpin enables precision engineering of lysine in native proteins. Reddy NC…Rai V. Nature Communications. Link

Multistable bioinspired origami with reprogrammable self-folding. Rojas S, Riley KS & Arrieta AF. Journal of the Royal Society Interface. Link

A Modular Scaffold for Controlling Transcriptional Activation via Homomeric Protein–Protein Interactions. Lee B & Wang T. ACS Synthetic Biology. Link

*Generation of high-performance human cardiomyocytes and engineered heart tissues from extended pluripotent stem cells. Li L…Zhang D. Cell Discovery. Link

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis. Grigoryan A…Bourgine PE. Science Translational Medicine. Link

Establishment of human induced trophoblast stem cells via reprogramming of fibroblasts. Tan JP, Liu X & Polo JM. Nature Protocols. Link

Induction of human trophoblast stem cells. Castel G & David L. Nature Protocols. Link

Tools & Technology

↑*Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing. Kishi JY…Yin P. Nature Methods. Link

  • This paper reports a method for high-resolution, spatial sequencing of fixed cells. Light is first used to print DNA barcodes on cells, and this barcoding “enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation.” The best way to learn more, besides reading the paper, is probably to read the Wyss Institute’s press release or listen to the Translation podcast from Fifty Years.

*Expanding the synthetic biology toolbox with a library of constitutive and repressible promoters. Yang EJY & Nemhauser JL. bioRxiv (preprint). Link

Off-the-shelf proximity biotinylation using ProtA-TurboID. Santos-Barriopedro I, Van Mierlo G & Vermeulen M. Nature Protocols. Link

*In situ cell-type-specific cell-surface proteomic profiling in mice. Shuster SA…Luo L. Neuron. Link

Real-time dynamic single-molecule protein sequencing on an integrated semiconductor device. Reed BD…Rothberg JM. Science. Link

Genetically encoded chemical crosslinking of carbohydrate. Li S…Wang L. Nature Chemistry. Link

*Continuous biomarker monitoring with single molecule resolution by measuring free particle motion. Buskermolen AD…Prins MWJ. Nature Communications. Link

Other Interesting Stuff

↑*In vitro neurons learn and exhibit sentience when embodied in a simulated game-world. Kagan BJ…Friston KJ. Neuron. Link

  • Neurons are plated on top of a multielectrode array. A computer sends and receives signals to the cells (either mouse or human). The neurons learn how to play Pong. This, too, is a crazy paper — watch Video S2 in the supplemental materials.

The VEGAS Platform Is Unsuitable for Mammalian Directed Evolution. Denes CE…Neely GG. ACS Synthetic Biology. Link

*New Federal Incentives for Diversity in Clinical Trials. Hwang TJ & Brawley OW. The New England Journal of Medicine. Link

*Long-term life history predicts current gut microbiome in a population-based cohort study. Si J…Raes J. Nature Aging. Link

*Global airborne bacterial community—interactions with Earth’s microbiomes and anthropogenic activities. Zhao J…Li X-d. PNAS. Link