Unbiased libraries in protein directed evolution

Sayous, Victor; Lubrano, Paul; Li, Yanyan; Acevedo‐Rocha, Carlos G.

doi:10.1016/j.bbapap.2019.140321

Cited by 28 publications

(20 citation statements)

References 88 publications

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“…Such landscapes provide insights on the different routes that evolution can take. Additionally, engineering proteins by single mutational steps 53 is a highly successful strategy in directed evolution 2 , 4 , 5 , 38 , 54 , 55 . To explore the step-wise accessibility in the evolution of parental --- towards III, we constructed a fitness “pathway” landscape 56 based on both activity and selectivity (Fig.…”

Section: Resultsmentioning

confidence: 99%

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Acevedo‐Rocha

D’Amore

et al. 2021

Nat Commun

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Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.

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Section: Resultsmentioning

confidence: 99%

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Acevedo‐Rocha

D’Amore

et al. 2021

Nat Commun

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“…One solution is the development of smart libraries restricted to specific mutational changes targeting protein traits described above, rather than searching over all of the protein sequence space. Multiple tools are available to better identify residues that may alter function (Sebestova et al 2014;Wrenbeck et al 2019) and for making focused libraries (Sayous et al 2020). In a recent study, ML was used to computationally identify residues that could be modified for a new-to-nature chemical transformation by using a much smaller mutagenesis library (Wu et al 2019).…”

Section: Roadblocks and Practical Considerations For Evolution-aided Metabolic Engineeringmentioning

confidence: 99%

Evolution-aided engineering of plant specialized metabolism

et al. 2021

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The evolution of new traits in living organisms occurs via the processes of mutation, recombination, genetic drift, and selection. These processes that have resulted in the immense biological diversity on our planet are also being employed in metabolic engineering to optimize enzymes and pathways, create new-to-nature reactions, and synthesize complex natural products in heterologous systems. In this review, we discuss two evolution-aided strategies for metabolic engineering-directed evolution, which improves upon existing genetic templates using the evolutionary process, and combinatorial pathway reconstruction, which brings together genes evolved in different organisms into a single heterologous host. We discuss the general principles of these strategies, describe the technologies involved and the molecular traits they influence, provide examples of their use, and discuss the roadblocks that need to be addressed for their wider adoption. A better understanding of these strategies can provide an impetus to research on gene function discovery and biochemical evolution, which is foundational for improved metabolic engineering. These evolution-aided approaches thus have a substantial potential for improving our understanding of plant metabolism in general, for enhancing the production of plant metabolites, and in sustainable agriculture.

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“…The genetic improving of enzymes by means of rational (or semirational) design [32][33][34][35][36][37], directed evolution [4,[38][39][40][41][42][43][44][45] or even de novo enzyme design [46][47][48][49] helped with machine-learning technologies [50][51][52][53], a very powerful (although rather complex) approach. The chemical modification of enzymes is also a usual technique to improve enzyme features [54][55][56][57].…”

Section: Enzymatic Biocatalysismentioning

confidence: 99%

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

et al. 2020

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Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called “combilipases” and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes.

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Unbiased libraries in protein directed evolution

Cited by 28 publications

References 88 publications

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Evolution-aided engineering of plant specialized metabolism

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

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