Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time

Abstract

Uniformitarian assumptions underlie the oldest evidence for living organisms on Earth, the distinct isotope fractionation between inorganic and organic carbon. Aside from a handful of compelling deviations, the 13C/12C isotopic mean of preserved organic carbon (δ13Corg) has remained remarkably unchanged through time. RuBisCO is the principal carboxylase/oxygenase biomolecular component that is thought to primarily account for the generation of these distinct carbon isotopic signals. However, it is difficult to reconcile a mostly unchanging mean δ13Corg with several known factors that can affect the isotope fractionation of RuBisCO, such as atmospheric composition and the amino acid composition of the enzyme itself, which have each changed markedly over Earth history. Here we report the resurrection and genetic incorporation of a Precambrian-age, Form IB RuBisCO in a modern cyanobacterial host. The isotopic composition of biomass relative to CO2 (ϵp) in ancestral and control strains were much greater when grown under Precambrian CO2 concentrations compared to modern ambient levels, but displaying values within a nominal envelope of modern-day RuBisCO IB enzyme variants. We infer that these isotopic differences derive indirectly from the decreased fitness of the AncIB strain, which includes diminished growth capacity and total cell RuBisCO activity. We argue that to answer the greatest questions of deep-time paleobiology, ancient biogeochemical signals should be reproduced in the laboratory through the synthesis of the geologic record with experimentally-derived constraints on underlying ancient molecular biology.

Carbon isotope fractionation by an ancestral rubisco suggests biological proxies for CO2through geologic time should be re-evaluated

“…Prior studies also worked to account for issues related to growth physiology— specifically growth rate, cell shape and size— to adapt the C3 plant model to unicellular algae (25). However, that landmark study measured ε p in wild-type Cyanobacteria—the same wild-type strain studied here—and, unlike this study and (39), found ε p to be roughly constant independent of environmental pCO 2 and growth rate. Popp and coauthors hypothesized that this observed independence stems from the large surface area to volume ratio (SA/V) of Cyanobacteria, which was taken to imply much faster passive CO 2 uptake (scaling with SA) than fixation (scaling with V).…”

“…This suggested that CO2 availability is a growth-limiting factor for ANC but not WT, and further suggested that the differences in their growth kinetics are related to the efficacy of their CCMs. Consistent with our results, a similar ancestral Form 1B analogue displayed total carboxylase activity roughly half that of the modern Form 1B (39).…”

“…It is readily apparent that ε Rubisco does not set an upper bound on ε p , nor does it predict which strains will have larger ε p values in vivo (Figure 3). This inference was only possible because we measured the isotope fractionation due to the ancestral rubisco (ε Rubisco ) and compared it to ANC strain biomass (ε p ), in contrast with the study of (39), which only measured ε p . While our ANC ε p values (≈18-24‰) fell within the range of ε p values derived from the carbon isotope record (22), they exceeded the measured ε Rubisco (Figure 3).…”

The mutation effect reaction norm (mu‐rn) highlights environmentally dependent mutation effects and epistatic interactions

“…In addition, recent studies that support the role of contingency in evolution might be explained by the specificity of mutation effects, adaptive landscape topography or environmental context (Kędzior et al. 2021; Xie et al. 2021).…”

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

“…The resurrection strategy leveraged here importantly provides a means to directly test paradigms of early protein and metabolic evolution, including those associated with the emergence, robustness, and malleability of protein functionality (6, 7, 40-42), the development of protein-protein interactions and complexes (9-11), and the role of evolutionary contingency (43-45). Nevertheless, the experimental application of ancestral sequence resurrection toward the systems-level investigation of earliest evolved, biogeochemically crucial metabolic pathways remains in its infancy, despite the potential for provocative connections between molecular-scale innovations, planetary-scale evolution, and independent, geological records of life (46, 47).…”