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

Peng, Qiqun; Bao, Weiwei; Geng, Binan; Yang, Shihui

doi:10.1016/j.synbio.2024.02.002

Cited by 4 publications

(1 citation statement)

References 66 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Zymomonas mobilis is a facultative anaerobic bacterium that is generally regarded as safe (GRAS) and has excellent industrial characteristics, such as high sugar uptake and conversion efficiency, few byproducts, broad range of pH (pH 3.5-7.5) and temperature (24 °C-40 °C), phage resistance, mature genome-editing tools, and high-quality genome-scale metabolic model, which has made it an important chassis for constructing microbial cell factories for diverse biochemicals, such as lignocellulosic ethanol, PHB, lactate, acetoin, and isobutanol (Xia et al, 2019;Qiu et al, 2020;Li et al, 2022;Bao et al, 2023;Hu et al, 2023;Peng et al, 2024).…”

Section: Introductionmentioning

confidence: 99%

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Xiao,

Tan,

et al. 2024

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by β-farnesene synthase for β-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for β-farnesene production, five heterologous β-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest β-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap–AaBFS) doubled its β-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet–AaBFS), which can be further improved by overexpressing the Pgap–AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on β-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the β-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing dxs, ispG, and ispH. Finally, the β-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant β-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

show abstract

Section: Introductionmentioning

confidence: 99%

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Xiao,

Tan,

et al. 2024

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

Illuminating anions in biology with genetically encoded fluorescent biosensors

Cook,

Phelps,

Tutol

et al. 2025

Current Opinion in Chemical Biology

View full text Add to dashboard Cite

The rise and future of CRISPR-based approaches for high-throughput genomics

Vercauteren,

Fiesack,

Maroc

et al. 2024

FEMS Microbiology Reviews

View full text Add to dashboard Cite

Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized the field of genome editing. To circumvent the permanent modifications made by traditional CRISPR techniques and facilitate the study of both essential and non-essential genes, CRISPR interference (CRISPRi) was developed. This gene silencing technique employs a deactivated Cas effector protein and a guide RNA to block transcription initiation or elongation. Continuous improvements and a better understanding of the mechanism of CRISPRi have expanded its scope, facilitating genome-wide high-throughput screens to investigate the genetic basis of phenotypes. Additionally, emerging CRISPR-based alternatives have further expanded the possibilities for genetic screening. This review delves into the mechanism of CRISPRi, compares it with other high-throughput gene-perturbation techniques and highlights its superior capacities for studying complex microbial traits. We also explore the evolution of CRISPRi, emphasizing enhancements that have increased its capabilities, including multiplexing, inducibility, titratability, predictable knockdown efficacy, and adaptability to non-model microorganisms. Beyond CRISPRi, we discuss CRISPR activation, RNA-targeting CRISPR systems and single-nucleotide resolution perturbation techniques for their potential in genome-wide high-throughput screens in microorganisms. Collectively, this review gives a comprehensive overview of the general workflow of a genome-wide CRISPRi screen, with an extensive discussion of strengths and weaknesses, future directions and potential alternatives.

show abstract

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

Cited by 4 publications

References 66 publications

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Illuminating anions in biology with genetically encoded fluorescent biosensors

The rise and future of CRISPR-based approaches for high-throughput genomics

Contact Info

Product

Resources

About