Selective Enrichment of Slow-Growing Bacteria in a Metabolism-Wide CRISPRi Library with a TIMER Protein

Beuter, Dominik; Gomes-Filho, José Vicente; Randau, Lennart; Díaz-Pascual, Francisco; Drescher, Knut; Link, Hannes

doi:10.1021/acssynbio.8b00379

Cited by 18 publications

(9 citation statements)

References 31 publications

(60 reference statements)

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“…The CRISPRi library has a unique advantage over gene knockouts for engineering bioproduction in that the level of essential genes can be titrated, allowing perturbation of the core metabolic network 55 . Cultures where a 'metabolic switch' shifts metabolism away from growth can be more productive 14,38,56,57 .…”

Section: Discussionmentioning

confidence: 99%

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

et al. 2020

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Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactatesecreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.

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Section: Discussionmentioning

confidence: 99%

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

et al. 2020

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“…All data are averaged through three independent experiments metabolism of industrial strains. Recent advances in the methods for selection of slow-growing microbes using CRISPRi technology [70] and laboratory evolution, have been applied to rebalance cellular metabolism [71] and could be solutions for development of industrial strains optimized for metabolite production.…”

Section: Application Of the Mutants For Metabolite Production (Egfp mentioning

confidence: 99%

Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli

Jung

Lim

et al. 2019

Microb Cell Fact

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Background: Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate. Results: As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13 C metabolic flux analysis (13 C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants. Conclusions: The elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.

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“…The CRISPRi library has a unique advantage over gene knockout libraries for engineering bioproduction in that the level of essential genes can be titrated, allowing perturbation of the core metabolic network 66 . Cultures where a 'metabolic switch' shifts metabolism away from growth can be more productive 53,67,68 .…”

Section: Discussionmentioning

confidence: 99%

Pooled CRISPRi screening of the cyanobacteriumSynechocystissp. PCC 6803 for enhanced growth, tolerance, and chemical production

Yao

Shabestary²,

Björk³

et al. 2019

Preprint

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We developed an inducible CRISPRi gene repression library in the cyanobacterium Synechocystis sp. PCC 6803, where all annotated genes are targeted for repression. We used the library to estimate gene fitness in multiple conditions. The library revealed several mutants with increased specific growth rates (up to 17%), and transcriptomics of these mutants revealed common upregulation of genes within photosynthetic electron flow. We challenged the library with L-lactate stress to find more tolerant mutants. Repression of the peroxiredoxin Bcp2 increased growth rate by 49% in the presence of 0.1 M L-lactate. Finally, the library was transformed into a L-lactate-secreting strain, and droplet microfluidics sorting of top producers enriched sgRNAs targeting nutrient assimilation, redox modulation, and cyclic-electron flow.Several clones showed increased productivity in batch cultivations (up to 75%). In some cases, tolerance or productivity was enhanced by partial repression of essential genes, which are difficult to access by transposon insertion.

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Selective Enrichment of Slow-Growing Bacteria in a Metabolism-Wide CRISPRi Library with a TIMER Protein

Cited by 18 publications

References 31 publications

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes

Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli

Pooled CRISPRi screening of the cyanobacteriumSynechocystissp. PCC 6803 for enhanced growth, tolerance, and chemical production

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