Die zentrale Rolle der Methodenentwicklung in der gerichteten Evolution selektiver Enzyme

Qu, Ge; Li, Aitao; Acevedo‐Rocha, Carlos G.; Sun, Zhoutong; Reetz, Manfred T.

doi:10.1002/ange.201901491

Cited by 43 publications

(7 citation statements)

References 275 publications

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“…This resulted to a relatively poor detection capability of the biosensor when compared with Gdh1, manifesting as a narrow linear range. Screening the highly active Cho1 mutants by directed evolution offers an available approach to problem solving [41]. It is still unclear why displaying Cho1 on the surface of yeast resulted to slow growth of the C1 strain although this may have been responsible for its low activity (Supplementary Figure S5).…”

Section: Discussionmentioning

confidence: 99%

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

et al. 2020

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High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L−1. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20–600 mg·dL−1 (1.4–33.3 mmol·L−1) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.

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Section: Discussionmentioning

confidence: 99%

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

et al. 2020

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“…This library contained 3.5 x 10 6 unique variants (an order of magnitude smaller than the previous one) with a 3-fold oversampling factor being needed to cover 95% the library ( Figure S5). The oversampling factor was calculated with the CASTER 2.0 program, based on the combinatorial active site saturation test (CAST) 25 . The CAST library was created using the Assembly of Designed Oligos method 26 , and its quality was checked using the Quick Quality Control (QQC), which consists of sequencing the pooled plasmids in a single reaction.…”

Section: Psefdh Library Design and Constructionmentioning

confidence: 99%

In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

Ramirez

Calvó-Tusell

Stoffel

et al. 2020

Preprint

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CGAR) 2 A b s t r a c t Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes.Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO 2 ). However, formate dehydrogenases (FDHs) are usual specific to NAD + , such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP + , but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP + , specificity towards NADP + , and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP + , we generate a library of >10 6 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate.By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.3 I n t r o d u c t i o n Co-factor regeneration is vital for the continuous operation of biocatalytic processes taking place either within a living cell or in a cell free system 1 . A considerable amount of research has therefore been invested in developing and optimizing enzymatic systems for the in situ regeneration of key cofactors such as ATP, NADH, and NADPH 2,3 . Formate has been long considered as a suitable reducing agent for the regeneration of NADH both in vivo and in vitro 2,4 . This is due to several properties: (i) abundance of formate dehydrogenases (FDHs) that can efficiently transfer reducing power from formate to NAD + 5 ; (ii) formate oxidation is practically irreversible, increasing the efficiency of NADH regeneration; (iii) formate, a small molecule, can easily cross membranes, thus being accessible within cellular compartments; and (iv) the byproduct of formate oxidation, CO 2 , is non-toxic and can be easily expelled from the system.Many biocatalytic processes rely on NADPH rather than NADH 6, 7 . Therefore, in the last 20 years multiple studies aimed at identifying FDHs that can naturally accept NADP + or engineering NAD-dependent FDHs to accept the phosphorylated cofactor [8][9][10][11][12][13][14][15][16] . Whi...

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“…Directed evolution constitutes a powerful tool for optimizing protein properties including activity, substrate scope, selectivity, stability, allostery or binding affinity. By applying iterative rounds of gene mutagenesis, expression and screening (or selection), proteins have been engineered for developing more efficient industrial biocatalytic processes [1][2][3][4] .…”

Section: Introductionmentioning

confidence: 99%

“…Directed evolution has also provided important insights into the relationship between protein sequence and function [4][5][6] , yet understanding the intricacies of non-additive epistatic effects remains a challenge 7 . Epistasis means that the phenotypic consequences of a mutation depend on the genetic background [8][9][10][11] .…”

Section: Introductionmentioning

confidence: 99%

“…Tuning the enzyme conformational dynamics has been recently identified as key for novel activity 21,24,[29][30][31] . 4 Understanding the connection between conformational dynamics and epistasis has been limited to a few model enzymes (mainly beta-lactamases) and a single protein trait (usually activity) as a measure of fitness (this term originally refers to the reproductive success of organisms but it can be applied to protein activity, selectivity or stability) [32][33][34][35] . This contrasts with directed evolution where often two or more traits (e.g., activity and selectivity or stability) are sought for practical purposes 4,36 .…”

Section: Introductionmentioning

confidence: 99%

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

Acevedo‐Rocha

D’Amore

et al. 2020

Preprint

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Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. Yet such efforts are rare and focus only on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report the first multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio-and stereoselective hydroxylation of a steroid. We developed a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative sign 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 beta-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 lessons for engineering P450s.

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Die zentrale Rolle der Methodenentwicklung in der gerichteten Evolution selektiver Enzyme

Cited by 43 publications

References 275 publications

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

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

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Die zentrale Rolle der Methodenentwicklung in der gerichteten Evolution selektiver Enzyme

Cited by 43 publications

References 275 publications

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP+

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

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In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP⁺