CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion
Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved..
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.
| Medienart: |
E-Artikel |
|---|
| Erscheinungsjahr: |
2023 |
|---|---|
| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:75 |
|---|---|
| Enthalten in: |
Metabolic engineering - 75(2023) vom: 15. Jan., Seite 192-204 |
| Sprache: |
Englisch |
|---|
| Beteiligte Personen: |
Yu, Xinyu [VerfasserIn] |
|---|
| Links: |
|---|
| Themen: |
Journal Article |
|---|
| Anmerkungen: |
Date Completed 17.01.2023 Date Revised 02.02.2023 published: Print-Electronic Citation Status MEDLINE |
|---|
| doi: |
10.1016/j.ymben.2022.12.004 |
|---|
| funding: |
|
|---|---|
| Förderinstitution / Projekttitel: |
|
| PPN (Katalog-ID): |
NLM350838240 |
|---|
| LEADER | 01000naa a22002652 4500 | ||
|---|---|---|---|
| 001 | NLM350838240 | ||
| 003 | DE-627 | ||
| 005 | 20231226045909.0 | ||
| 007 | cr uuu---uuuuu | ||
| 008 | 231226s2023 xx |||||o 00| ||eng c | ||
| 024 | 7 | |a 10.1016/j.ymben.2022.12.004 |2 doi | |
| 028 | 5 | 2 | |a pubmed24n1169.xml |
| 035 | |a (DE-627)NLM350838240 | ||
| 035 | |a (NLM)36572334 | ||
| 035 | |a (PII)S1096-7176(22)00153-7 | ||
| 040 | |a DE-627 |b ger |c DE-627 |e rakwb | ||
| 041 | |a eng | ||
| 100 | 1 | |a Yu, Xinyu |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion |
| 264 | 1 | |c 2023 | |
| 336 | |a Text |b txt |2 rdacontent | ||
| 337 | |a ƒaComputermedien |b c |2 rdamedia | ||
| 338 | |a ƒa Online-Ressource |b cr |2 rdacarrier | ||
| 500 | |a Date Completed 17.01.2023 | ||
| 500 | |a Date Revised 02.02.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 7 | |a Recombinant Proteins |2 NLM | |
| 700 | 1 | |a Li, Shuang |e verfasserin |4 aut | |
| 700 | 1 | |a Feng, Huibao |e verfasserin |4 aut | |
| 700 | 1 | |a Liao, Xihao |e verfasserin |4 aut | |
| 700 | 1 | |a Xing, Xin-Hui |e verfasserin |4 aut | |
| 700 | 1 | |a Bai, Zhonghu |e verfasserin |4 aut | |
| 700 | 1 | |a Liu, Xiuxia |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Chong |e verfasserin |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Metabolic engineering |d 1997 |g 75(2023) vom: 15. Jan., Seite 192-204 |w (DE-627)NLM097535249 |x 1096-7184 |7 nnns |
| 773 | 1 | 8 | |g volume:75 |g year:2023 |g day:15 |g month:01 |g pages:192-204 |
| 856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.ymben.2022.12.004 |3 Volltext |
| 912 | |a GBV_USEFLAG_A | ||
| 912 | |a GBV_NLM | ||
| 951 | |a AR | ||
| 952 | |d 75 |j 2023 |b 15 |c 01 |h 192-204 | ||