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| Status |
Public on May 26, 2024 |
| Title |
Genome-scale CRISPRi screen identifies pcnB repression conferring improved physiology for overproduction of free fatty acids in Escherichia coli II |
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Experiment type |
Other
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| Summary |
Microbial physiology plays a pivotal role in construction of a superior microbial cell factory for efficient production of desired products. Here we identified pcnB repression through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS), which confers improved physiology for free fatty acids (FFAs) overproduction in Escherichia coli. The repression of pcnB could improve the stability and abundance of the transcripts involved in proton-consuming system, conferring a global improvement on cell membrane, redox state, and energy level. These physiological advantages facilitated further identification of acrD repression enhancing FFAs efflux. The engineered strain pcnBi-acrDi-fadR+ achieved 35.1āgālā1 FFAs production in fed-batch fermentation, which is the maximum titer in E. coli reported to date. This study underscores the significance of hidden genetic determinants in microbial biosynthesis and sheds light on the role of microbial physiologies in boosting microbial biosynthesis.
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| Overall design |
In this study, we carried out genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS) to identify genetic determinants for FFAs overproduction in Escherichia coli. Two rounds of screening were carried out. In the first round, the transformed cell library (named Tra1) carrying the pCF plasmid and the genome-scale sgRNA expression plasmid library was induced by IPTG and then cultivated for 40 h to sample the cell library before sorting (BS) (named Cul2). Then, BS was dyed with Nile red and sorted by FACS, obtaining the cell library after sorting (AS) (named Sor3). In the second round, the cultured cell library before sorting (BS) (named B-cul2) carrying the pCF-pcnBi plasmid and the genome-scale sgRNA expression plasmid library was sorted by FACS, obtaining the cell library after sorting (AS) (named B-sor3-P5). The Tra1, Cul2, Sor3, B-cul2, and B-sor3-P5 were applied for NGS to get the reads of sgRNA sequence.
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| Contributor(s) |
Cao Y, Fang L, Hao X, Fan J |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
May 19, 2024 |
| Last update date |
May 26, 2024 |
| Contact name |
Lixia Fang |
| E-mail(s) |
lxfang@tju.edu.cn
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| Organization name |
Tianjin University
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| Street address |
135 Yaguan Road, Jinnan District
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| City |
Tianjin |
| ZIP/Postal code |
300350 |
| Country |
China |
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| Platforms (1) |
| GPL26592 |
Illumina NovaSeq 6000 (Escherichia coli str. K-12 substr. MG1655) |
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| Samples (5)
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| GSM8279336 |
plasmid DNA fragments, amplified, the transformed library in the first round |
| GSM8279337 |
plasmid DNA fragments, amplified, library before sorting in the first round |
| GSM8279338 |
plasmid DNA fragments, amplified, library after sorting in the first round |
| GSM8279339 |
plasmid DNA fragments, amplified, library before sorting in the second round |
| GSM8279340 |
plasmid DNA fragments, amplified, library after sorting in the second round |
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| Relations |
| BioProject |
PRJNA1113322 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE267827_NGS-first__Tra1_Cul2_Sor3_.xlsx |
1.6 Mb |
(ftp)(http) |
XLSX |
| GSE267827_NGS-second__B-cul2_B-sor3-P5_.xlsx |
1.6 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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