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 3 publications

(1 citation statement)

References 66 publications

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“…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.

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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.

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Section: Introductionmentioning

confidence: 99%

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Xiao,

Tan,

et al. 2024

Front. Bioeng. Biotechnol.

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

Vercauteren,

Fiesack,

Maroc

et al. 2024

FEMS Microbiology Reviews

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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.

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Application of functional genomics for domestication of novel non-model microbes

Bales,

Vergara,

Eckert

2024

Journal of Industrial Microbiology and Biotechnology

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With the expansion of domesticated microbes producing biomaterials and chemicals to support a growing circular bioeconomy, the variety of waste and sustainable substrates that can support microbial growth and production will also continue to expand. The diversity of these microbes also requires a range of compatible genetic tools to engineer improved robustness and economic viability. As we still do not fully understand the function of many genes in even highly studied model microbes, engineering improved microbial performance requires introducing genome-scale genetic modifications followed by screening or selecting mutants that enhance growth under prohibitive conditions encountered during production. These approaches include adaptive laboratory evolution, random or directed mutagenesis, transposon-mediated gene disruption, or CRISPR interference (CRISPRi). Although any of these approaches may be applicable for identifying engineering targets, here we focus on using CRISPRi to reduce the time required to engineer more robust microbes for industrial applications. One-Sentence Summary The development of genome scale CRISPR-based libraries in new microbes enables discovery of genetic factors linked to desired traits for engineering more robust microbial systems.

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

Cited by 3 publications

References 66 publications

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production

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

Application of functional genomics for domestication of novel non-model microbes

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