Directed -in vitro- evolution of Precambrian and extant Rubiscos

Abstract

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere’s carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

“…Indeed, through ancestral sequence reconstruction and resurrection (i.e., the functional expression of inferred ancestral nodes in modern microbes), “ paleoenzymologists ” are bringing back protein sequences from long-extinct organisms in an attempt to scrutinize the events of natural molecular evolution. From a strict protein engineering point of view, resurrected enzymes may exhibit a range of appealing biochemical traits, such as stability, promiscuity, and heterologous expression, all of which characteristics may be interesting to instill in modern enzymes ( 5 – 12 ). Under the assumption that ancient microorganisms could not afford a broad repertoire of specialist enzymes (due to the high associated metabolic cost), the majority of the tasks within ancestral microorganisms were performed by just a few generalist biocatalysts, which in turn had to adapt to the harsh environments of ancient earth (particularly those in the Precambrian period) ( 13 – 16 ).…”

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

“…Here, we explored the indicators of nitrogenase metal usage history by a combined method relying on ancestral sequence reconstruction, an evolutionary approach by which inferred, historical protein sequence information can be linked to functional inferences of molecular properties evidenced by computational analyses or laboratory experiments (Aadland, Pugh, & Kolaczkowski, ; Benner, Sassi, & Gaucher, ; Thornton, ). These paleogenetic approaches have been increasingly applied in biogeochemically relevant molecular studies to offer insights into the coevolution of life and Earth (Garcia & Kacar, ; Gomez‐Fernandez et al, ; Kacar, Hanson‐Smith, Adam, & Boekelheide, ). We reconstructed the phylogenetic history of Mo‐, V‐, and Fe‐nitrogenases in order to resurrect ancestral nitrogenases in silico, as well as to map the taxonomic distribution of cofactor biosynthetic components considered necessary for cofactor assembly (Boyd, Anbar, et al, ; Curatti et al, ; Dos Santos et al, ; Hu et al, ; Shah et al, , ; Tal et al, ).…”

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

“…Second, despite being so overrepresented in kinetic studies, rubisco's k cat values span the smallest dynamic range across all catalytically characterized enzymes (Flamholz et al, 2019). Third, multiple efforts to improve the rate of rubisco carboxylation have made limited progress (Mueller-Cajar et al, 2007;Gomez-Fernandez et al, 2018;. Cases where some increase in the net carboxylation capacity of rubisco was achieved (via directed evolution) include only form-I and form-III isoforms (Wilson et al, 2016;Zhou & Whitney, 2019).…”