Substantial Improvement of an Epimerase for the Synthesis of D-Allulose by Biosensor-Based High-Throughput Microdroplet Screening.
Angew Chem Int Ed Engl
; 62(10): e202216721, 2023 03 01.
Article
em En
| MEDLINE
| ID: mdl-36658306
ABSTRACT
Biosynthesis of D-allulose has been achieved using ketose 3-epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D-allulose-responsive transcriptional regulator for real-time monitoring of D-allulose. An ultrahigh-throughput droplet-based microfluidic screening platform was further constructed by coupling with this D-allulose-detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D-allulose 3-epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure-guided rational design and directed evolution, in which a mutant M3-2 was identified with 17-fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor-based microdroplet screening platform.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Racemases e Epimerases
/
Frutose
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article