In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection.
Metab Eng
; 59: 15-23, 2020 05.
Article
em En
| MEDLINE
| ID: mdl-31926305
ABSTRACT
Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Compostos Organofosforados
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Piperidonas
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Engenharia de Proteínas
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Evolução Molecular Direcionada
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Isomerases de Aminoácido
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Antimetabólitos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Metab Eng
Assunto da revista:
ENGENHARIA BIOMEDICA
/
METABOLISMO
Ano de publicação:
2020
Tipo de documento:
Article
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