Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production

Li, Songyuan; Jendresen, Christian Bille; Landberg, Jenny Marie; Pedersen, Lasse Ebdrup; Sonnenschein, Nikolaus; Jensen, Sheila Ingemann; Nielsen, Alex Toftgaard

doi:10.1021/acssynbio.9b00143

Cited by 37 publications

(33 citation statements)

References 40 publications

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“…Knockdowns with CRSIPR interference were also used for synthetic growth switches. A CRISPRi library targeting more than 12 000 loci in the E. coli genome, identified targets that could efficiently decouple growth from production (Li et al 2020). Therefore, the authors measured which CRISPRi targets reduce growth and which targets increase production of GFP, and the overlap between these targets were promising targets for an effective growth switch.…”

Section: Non-growing Bacteria In Industrial Biotechnologymentioning

confidence: 99%

Metabolism of non-growing bacteria

Lempp

Lubrano

Bange

et al. 2020

Biological Chemistry

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A main function of bacterial metabolism is to supply biomass building blocks and energy for growth. This seems to imply that metabolism is idle in non-growing bacteria. But how relevant is metabolism for the physiology of non-growing bacteria and how active is their metabolism? Here, we reviewed literature describing metabolism of non-growing bacteria in their natural environment, as well as in biotechnological and medical applications. We found that metabolism does play an important role during dormancy and that especially the demand for ATP determines metabolic activity of non-growing bacteria.

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Section: Non-growing Bacteria In Industrial Biotechnologymentioning

confidence: 99%

Metabolism of non-growing bacteria

Lempp

Lubrano

Bange

et al. 2020

Biological Chemistry

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“…It is difficult to predict exactly how these polar effects might have impacted the results of this study. However, in our previous library screen, repression of eno and yciH did not result in significant inhibition of growth or an increase in GFP production, indicating that the results seen for pyrF and pyrG inhibition are not affected by downstream eno and yciH inhibition, respectively (S. Li et al, 2020).…”

Section: Resultsmentioning

confidence: 85%

“…Partial CRISPRi‐based inhibition of citrate synthase GltA increased the productivity of butanol in the cyanobacteria Synechocystis (Shabestary et al, 2018), and CRISPRi‐based inhibition of pyrF improved the yield of mevalonate almost fivefold in E. coli (S. Li, Jendresen, Grünberger et al, 2016). To screen the E. coli genome for additional promising growth decoupling targets, we established a genome‐wide sgRNA library and screened it to identify several targets that increased the green fluorescent protein (GFP) production and inhibited growth (S. Li et al, 2020). The results from this study indicated an enrichment of promising targets among genes involved in biosynthesis of purines and pyrimidines.…”

Section: Introductionmentioning

confidence: 99%

CRISPR interference of nucleotide biosynthesis improves production of a single‐domain antibody in Escherichia coli

Landberg

Wright

Wulff

et al. 2020

Biotech & Bioengineering

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Growth decoupling can be used to optimize the production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference to increase the production of a single-domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways and found that the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAbproducing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.

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“…PCC 6803 lead to the identification of gltA and pcnB facilitating the productivity of L-lactate and bcp2, the L-lactate tolerance related gene (Yao et al, 2020). A pooled CRISPRi screen facilitated the discovery of growth switches sibB/ibsB, which can be applied to decoupling cell growth and protein production in E. coli (Li et al, 2020).…”

Section: Application Of Crispri In High-throughput Screenmentioning

confidence: 99%

Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects

Zhang

Shao

et al. 2021

Front. Microbiol.

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Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist’s toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.

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Genome-Wide CRISPRi-Based Identification of Targets for Decoupling Growth from Production

Cited by 37 publications

References 40 publications

Metabolism of non-growing bacteria

Metabolism of non-growing bacteria

CRISPR interference of nucleotide biosynthesis improves production of a single‐domain antibody in Escherichia coli

Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects

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