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Biosensor-assisted CRISPRi high-throughput screening to identify genetic targets in Zymomonas mobilis for high d-lactate production

Authors :
Qiqun Peng
Weiwei Bao
Binan Geng
Shihui Yang
Source :
Synthetic and Systems Biotechnology, Vol 9, Iss 2, Pp 242-249 (2024)
Publication Year :
2024
Publisher :
KeAi Communications Co., Ltd., 2024.

Abstract

Lactate is an important monomer for the synthesis of poly-lactate (PLA), which is a substitute for the petrochemical plastics. To achieve the goal of high lactate titer, rate, and yield for commercial production, efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance. In this study, an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence, which could be the mutant strains with increased d-lactate production. Then, LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference (CRISPRi) library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome. Specifically, two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting (FACS), respectively. Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production, further knockout of ZMO1323 and ZMO1530 resulted in a 15% and 21% increase of d-lactate production, respectively. This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z. mobilis, but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

Details

Language :
English
ISSN :
2405805X
Volume :
9
Issue :
2
Database :
Directory of Open Access Journals
Journal :
Synthetic and Systems Biotechnology
Publication Type :
Academic Journal
Accession number :
edsdoj.57565f89c60d416785f9a15c92dbd65e
Document Type :
article
Full Text :
https://doi.org/10.1016/j.synbio.2024.02.002