H2020Индивидуална стипендия2016–2018

TIMEnzyme · Implementation of Enzymatic Activity in a Naïve, de novo Designed Protein Scaffold by Rational Design and Laboratory Evolution

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2016-03-01 → 2018-02-28
Финансиране от ЕС: 175 420 €
Участници: 1
Схема: MSCA-IF-EF-ST

Линиите свързват координатора с партньорите.

Накратко на български

Изкуствено създадени протеини се модифицират, за да действат като ензими, които например изграждат или разкъсват въглеродни връзки. Това помага за замяната на традиционните химически процеси в индустрията с по-екологични методи за синтез на ценни вещества.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Implementation of Enzymatic Activity in a Naïve, de novo Designed Protein Scaffold by Rational Design and Laboratory Evolution

The chemical and pharmaceutical industry is under increasing pressure to replace traditional chemical catalysis with environmentally benign approaches for the synthesis of high-value compounds. Biocatalytic transformations, that is using enzymes as catalysts, generally offer sustainability in combination with high selectivity and catalytic activity. However, most natural enzymes do not meet the requirements of large-scale industrial processes and/or do not catalyze the relevant non-natural reactions with sufficient efficiency. A powerful strategy to tackle this problem is the development of de novo enzymes using a combined approach of computational design and directed evolution. The TIMEnzyme project is centered around recently produced de novo protein scaffolds, which have no sequence homology with natural proteins, and may thus provide an unbiased starting point for the design of novel enzymes and their subsequent optimization by laboratory evolution. The overall goal was to functionalize these protein scaffolds and test their evolvability towards a synthetically valuable activity. We chose to design de novo aldolases catalyzing the stereoselective cleavage or formation of carbon-carbon bonds (Fig. 1). While the work on this project is still in progress at the time this report has been filed, important milestones were achieved and published during the time period of the fellowship, as described in further detail below. This study has been performed in close collaboration with the laboratory of Prof. David Baker (University of Washington, Seattle, USA).

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

The proposed research project aims at implementing enzymatic activity in a de novo designed, unbiased protein scaffold. First, simplified active site arrangements deduced from two previously evolved model enzymes (Kemp eliminase and retro-aldolase) will be implanted in the scaffold. This will allow evaluating the extent to which a fully computationally designed and naïve protein can be functionalized and evolved. A recently established fluorescence-based microfluidics setup will be utilized to screen large DNA libraries of several million clones per round of laboratory evolution. The artificial protein scaffold, kindly provided by Prof. David Baker (University of Washington), has been designed to adopt a minimalist TIM barrel fold, which is the most abundant and diversely evolved protein fold found for natural enzymes. Here, the substrate binding pocket is usually formed by an extended loop region on one side of the scaffold, which is not yet present in the naïve, designed variant. Thus, in the second step, I will generate a randomized library of loop fragments, insert into the scaffold and screen for improved activity. This approach can be extended from the retro-aldolase model reaction towards synthetic aldolases that catalyze the stereospecific formation of a new carbon-carbon bond. Ultimately, the aim is to evaluate whether loop libraries are an appropriate tool to broaden the substrate scope of these enzymes. Furthermore, the proposal contains a second, independent approach to equip the artificial scaffold with novel enzymatic functionality. Here, I plan to design a covalent dimer of two artificial TIM barrels carrying a cofactor-dependent active site in the dimeric interface. This work will not only generate fundamental insight into the evolution of catalytic activity, it also has great potential to contribute to the development of general strategies for creating enzymes with novel functionality, and thus, prospective applications in industry or medicine.

Оригинален текст от CORDIS (на английски).

Участници

EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH · ZuerichКоординаторШвейцария