Bibliographic Details
| Title: |
Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli |
| Authors: |
Jinho Kim, Taek Soon Lee |
| Source: |
Frontiers in Bioengineering and Biotechnology, Vol 11 (2023) |
| Publisher Information: |
Frontiers Media S.A., 2023. |
| Publication Year: |
2023 |
| Collection: |
LCC:Biotechnology |
| Subject Terms: |
CRISPR interference, isoprenol, multiplexed CRISPRi arrays, mevalonate pathway, IPP-bypass pathway, fed-batch cultivation, Biotechnology, TP248.13-248.65 |
| More Details: |
Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction. |
| Document Type: |
article |
| File Description: |
electronic resource |
| Language: |
English |
| ISSN: |
2296-4185 |
| Relation: |
https://www.frontiersin.org/articles/10.3389/fbioe.2023.1296132/full; https://doaj.org/toc/2296-4185 |
| DOI: |
10.3389/fbioe.2023.1296132 |
| Access URL: |
https://doaj.org/article/9f8e6a551be849b8a72d544745a73d44 |
| Accession Number: |
edsdoj.9f8e6a551be849b8a72d544745a73d44 |
| Database: |
Directory of Open Access Journals |
