{"title":"Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle","authors":"Sefli Sri Wahyu Effendi, and , I-Son Ng*, ","doi":"10.1021/acssynbio.4c00318","DOIUrl":null,"url":null,"abstract":"<p >Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories’ sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic <i>Escherichia coli</i> Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO<sub>2</sub>-fixing system. Herein, EcN was equipped with a simultaneous CO<sub>2</sub> fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO<sub>2</sub>-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the <i>pfk</i>AB and <i>zwf</i> genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO<sub>2</sub> fixation. The CO<sub>2</sub>-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO<sub>2</sub> release by 77%. CO<sub>2</sub> fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acssynbio.4c00318","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Synthetic Biology","FirstCategoryId":"99","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acssynbio.4c00318","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories’ sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2-fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pfkAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO2 fixation. The CO2-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO2 release by 77%. CO2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.
期刊介绍:
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.