CRISPR-based metabolic pathway engineering

De, Zhao; Zhu, Xinna; Zhou, Hang; Sun, Naxin; Wang, Ting; Bi, Chen

doi:10.1016/j.ymben.2020.10.004

Cited by 36 publications

(24 citation statements)

References 182 publications

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“…Impressive “TYR” (Titer, Yield and Rate) data have been achieved by modulating the metabolic pathway, improving the pathway enzymatic activities, and increasing co-factor availabilities [ 31 , 34 ]. Recently, with the discovery and development of the CRISPR-Cas system, many studies have coupled the system with λRed recombineering to modify the host genome, redirect flux towards the desired products and minimize regulatory inhibitions [ 4 , 37 , 38 ]. In this study, we have further simplified the CRISPR-Cas9 mediated gene deletion protocol by using asymmetric homology arms as donor DNA.…”

Section: Discussionmentioning

confidence: 99%

“…The ability to easily and efficiently modify the genome of BL21 is highly desirable to further improve strain performance for industrial applications. With the breakthrough in genome editing technology, clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) system have become a common genome editing tool applied from microbes to mammals [ 4 ]. CRISPR-Cas9, a class 2 type II system, is well-characterized and the most widely applied because of its simple design [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

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Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

et al. 2022

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Background The recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA with homology arms for precise genome editing. However, generation of homology arms is a time-consuming, costly and inefficient process that is often overlooked. Results In this study, we first optimized a CRISPR-Cas genome engineering protocol in the Escherichia coli (E. coli) BL21 strain and successfully deleted 10 kb of DNA from the genome in one round of editing. To further simplify the protocol, asymmetric homology arms were produced by PCR in a single step with two primers and then purified using a desalting column. Unlike conventional homology arms that are prepared through overlapping PCR, cloning into a plasmid or annealing synthetic DNA fragments, our method significantly both shortened the time taken and reduced the cost of homology arm preparation. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across the BL21 genome. Noteworthy, gRNA design is important for the CRISPR-Cas system and a general heuristic gRNA design has been proposed in this study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene yield by ~ threefold. Conclusions Our work has optimized the homology arms design for gene deletion in BL21. The protocol efficiently edited BL21 to improve lycopene production. The same workflow is applicable to any E. coli strain in which genome engineering would be useful to further increase metabolite production.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

et al. 2022

View full text Add to dashboard Cite

show abstract

“…Impressive "TYR" (Titer, Yield and Rate) data have been achieved by modulating the metabolic pathway, improving the pathway enzymatic activities, and increasing co-factor availabilities [31,34]. Recently, with the discovery and development of CRISPR-Cas system, many studies have coupled the system with λRed recombineering to modify the host genome, redirect flux towards desired products and minimize regulatory inhibitions [4,37,38]. In this study, we have further simplified the CRISPR-Cas9 mediated gene deletion protocol by using asymmetric homolog arm as donor DNA.…”

Section: Discussionmentioning

confidence: 99%

“…To modify the genome of BL21 strain efficiently and easily is highly desirable to further improve the strain performance for industrial applications. With the breakthrough in genome editing technology, clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) system has become a common genome editing tool to be applied from microbes to mammals [4]. CRISPR-Cas9, a class 2 type II system is well-characterized and the most widely applied because of its simple design [1].…”

Section: Introductionmentioning

confidence: 99%

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

Shukal

Lim

Zhang

et al. 2021

Preprint

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BackgroundThe recent CRISPR-Cas coupled with λ recombinase mediated genome recombineering has become a common laboratory practice to modify bacterial genomes. It requires supplying a template DNA or homolog arms for precise genome editing. However, it is often overlooked the process to generate the homolog arms which is a time-consuming, costly and inefficient step.ResultsIn this study, we first optimized CRISPR-Cas protocol in BL21 strain and successfully deleted 10 kb gene from the genome in one round of editing. To further simplify the protocol, asymmetric homolog arms as PCR fragments was used. It can be obtained by one-step PCR reaction with two primers and purified with desalting columns. Unlike conventional homolog arms that are prepared through overlapping PCR, cloning to plasmid or annealing synthetic DNA fragments, our method significantly shortened the time taken and reduced the cost to prepare the homolog arms. To test the robustness of the optimized workflow, we successfully deleted 26 / 27 genes across BL21 genome. Noteworthy, gRNA design is important for CRISPR-Cas system and a general heuristic gRNA design was proposed in the study. To apply our established protocol, we targeted 16 genes and iteratively deleted 7 genes from BL21 genome. The resulting strain increased lycopene production from ~15,000 ppm to > 40,000 ppm.ConclusionsOur work has optimized the homolog arms design for gene deletion in BL21 strains. The protocol efficiently edited BL21 strain to improve lycopene production. The same workflow is applicable to all E. coli strain which would be useful for genome rewiring to further increase metabolite production in microbial cell factories.

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“…In recent years, CRISPRi has emerged as a powerful tool for the targetable reduction of gene expression in E. coli (for reviews, see [ 20 , 21 , 22 ]). CRISPRi uses the exogeneous catalytically inactive protein dCas9 from S. pyogenes in conjunction with single‐guide RNAs (sgRNA) to interfere with transcription of the target gene through steric hindrance [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

Ziegler

Hägele

Gäbele

2021

Engineering in Life Sciences

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CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen‐limited phase. Targeting the promoter region of pdhR enabled pyruvate production during the growth phase of cultivations, and a continued low‐level accumulation during the nitrogen‐limited production phase. Combinatorial targeting of the promoter regions of both aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.

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CRISPR-based metabolic pathway engineering

Cited by 36 publications

References 182 publications

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

Metabolic engineering of Escherichia coli BL21 strain using simplified CRISPR-Cas9 and asymmetric homology arms recombineering

Metabolic Engineering of Escherichia Coli BL21 Strain Using Simplified CRISPR-Cas9 and Asymmetric Homolog Arms Recombineering

CRISPRi enables fast growth followed by stable aerobic pyruvate formation in Escherichia coli without auxotrophy

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