CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion.
Metab Eng
; 75: 192-204, 2023 01.
Article
em En
| MEDLINE
| ID: mdl-36572334
ABSTRACT
Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Microfluídica
/
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Idioma:
En
Revista:
Metab Eng
Assunto da revista:
ENGENHARIA BIOMEDICA
/
METABOLISMO
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China