Ancestral sequence reconstruction for protein engineers

Spence, Matthew A.; Kaczmarski, Joe A.; Saunders, Jake; Jackson, C.J.

doi:10.1016/j.sbi.2021.04.001

Cited by 110 publications

(107 citation statements)

References 55 publications

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Section: Discussionmentioning

confidence: 99%

“…Attempts to explore protein sequence-function space via rational design can be hindered by local adaptive valleys. However, evolutionarily guided sequence exploration provides an effective strategy to efficiently traverse functional sequence space already “vetted” by natural selection and can integrate the effects of epistatic interactions that are challenging to predict a priori (8, 37, 38). A historical, synthetic biology approach focused within the lineage of the utilized model host system is perhaps more likely to yield a functional phenotype because introduced changes are constrained to the evolutionary “vernacular” of native proteins and their interaction networks (39).…”

Section: Discussionmentioning

confidence: 99%

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Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Garcia

Harris

Rivier

et al. 2022

Preprint

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Understanding the coevolutionary dynamics between proteins, cellular networks, and environmental pressures remains an outstanding challenge in biology. The effects of molecular constraints on the development of metabolic pathways that shape the biosphere remain underexplored. Extant genes, relics of a >3-billion-year history of life, offer the means to experimentally reconstruct protein evolutionary trajectories. Surveying life s extinct genetic repertoire may unlock adaptive states to environmental conditions unobserved today. However, the lack of suitable experimental systems poses a hurdle for such strategies, particularly for the reconstruction of biogeochemically critical ancient proteins and their supporting networks. To fill this gap, here we developed an evolutionarily guided, systems biology and biochemistry approach for the resurrection and functional characterization of ancient nitrogen fixation machinery in Azotobacter vinelandii. We report the recovery of active, ancestral nitrogenase enzymes and observe the conservation of core, catalytic features that are robust to numerous residue-level changes and modular incorporation of ancestral protein components. These results highlight the preservation of protein features vital for primary function over long timescales. An ancient-modern hybrid experimental strategy enables the reconstruction and historical characterization of essential biosystems, providing an evolutionarily vetted toolbox for the study, resurrection, and engineering of life s key metabolic innovations.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Garcia

Harris

Rivier

et al. 2022

Preprint

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“…1) 39 Thus, in prokaryotic expression systems the 8 kDa and 50 kDa subunits have to be expressed separately and assemble into a heterodimeric complex. 33,34 There are many experimental and computational methods that have been developed to improve enzyme function and stability, such as bioinformatics-based approaches like consensus design 40,41 or ancestral sequence reconstruction, 42,43 or forcefieldbased approaches like Rosetta 44 or FoldX. 45 However, both approaches have limitations.…”

Section: Introductionmentioning

confidence: 99%

Computational design and experimental characterisation of a stable human heparanase variant

et al. 2022

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“…Consensus guided mutagenesis (CGM) is a semi-rational approach, where amino acid sequences are inferred from the alignment of a group of homologous enzymes. Often, the recreated sequence shows an increased thermal stability (Spence et al, 2021;Thornton, 2004), a property required by the ideal catalysts to be used in oil degumming. However obtaining thermostable enzymes that retain at the same time the catalytic e ciency of their mesophilic counterparts represents an additional challenge (Furukawa et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Sustainable refining of vegetable oil made easy with a designer phospholipase C enzyme

Val

Marchisio

Nardo

et al. 2022

Preprint

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Enzymes are extraordinary catalysts and a preferred tool for green chemistry, but they have not evolved to tolerate the harsh conditions required in large scale chemical processes, making in most cases the replacement of inorganic catalysts extremely expensive. However, continuous knowledge generated by protein engineers help to realize the promise of using biocatalysts to address critical environmental and industrial challenges. The increasing demand for food and biofuels pressures the vegetable oil industry to continuously improve the manufacturing efficiency and at the same time to mitigate the environmental pollution caused by the refineries. Enzymatic degumming is a key enabling technology to obtain cleaner and more efficient methods, but advanced enzymes are needed for a widespread adoption. Here we present an enzymatic degumming process based on chPLC, a synthetic phospholipase C with remarkable thermal stability and catalytic properties. The process developed does not require modifications in current soybean oil factories, avoiding upfront investments, and is four times faster than the enzymatic processes available in the market. A techno-economic analysis predicts a ~ 60% cost reduction for a base-case scenario, establishing the largely expected conditions to promote the global adoption of enzymatic oil degumming by refineries of all sizes. To the best of our knowledge, this is the first report showing the application of a synthetic enzyme designed to address an industrial need with impact in both, the global economy and the environment, in the billion-dollar range.

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Ancestral sequence reconstruction for protein engineers

Cited by 110 publications

References 55 publications

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Computational design and experimental characterisation of a stable human heparanase variant

Sustainable refining of vegetable oil made easy with a designer phospholipase C enzyme

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