CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in <i>E. coli</i>

Cress, Brady F.; Toparlak, Ö. Duhan; Guleria, Sanjay; Lebovich, Matthew; Stieglitz, Jessica T; Englaender, Jacob A.; Jones, J. Andrew; Linhardt, Robert J.; Koffas, Mattheos

doi:10.1021/acssynbio.5b00012

Cited by 152 publications

(150 citation statements)

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“…One possible solution is the deletion or downregulation of the genes in competitive pathways. Previous work has indicated that the TCA cycle is likely to be a competitive pathway (35,38,39), and this work showed that deletion of the sucA gene in the TCA cycle does improve mevalonate yield. An alternative solution applied here is the enhancement of the downstream pathway for the desired product, and although it could not eliminate the production of by-products, it did increase the carbon flux to the desired product.…”

Section: Discussionmentioning

confidence: 75%

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

Wang

Niyompanich

Tai

et al. 2016

Appl Environ Microbiol

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Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmidbased fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into ␤-methyl-␦-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA. The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ϳ0.43 g/liter/h) and yields (67.8 to ϳ71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fedbatch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (ϳ1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask). IMPORTANCEMetabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals. Mevalonate pathways, important in the production of isoprenoids, are broadly present in eukaryotes, archaea, and some prokaryotes (1-3). Besides a precursor for the production of isoprenoids (2, 3), mevalonate has been used in cosmetics and as a building block for the production of sustainable polymers (4). Recently, it has been demonstrated that mevalonate could be transformed, by simple chemical steps, into a branched lactone ␤-methyl-␦-valerolactone (␤M␦VL), and its copolymerization with lactides results in a new class of polyesters with tunable mechanical properties (5). These potential applications motivate the development of ...

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Section: Discussionmentioning

confidence: 75%

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

Wang

Niyompanich

Tai

et al. 2016

Appl Environ Microbiol

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“…Similar to lycopene, C3G in the colorimetric assay can be used as a screening molecule for desirable phenotypes in applications such as combinatorial engineering (37,38). The calibration curve generated could be utilized for rapid C3G concentration quantification without the need for complex and timeconsuming analytical tools.…”

Section: Resultsmentioning

confidence: 99%

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

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bAnthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (؉)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

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“…1e). Several examples, such as the type III CRISPR-Csy4 [25,76] or natural CRISPR array [1,14] have been shown to efficiently generate multiple gRNAs from a single transcript (Fig. 1e).…”

Section: The Grna Characteristics and Extensionsmentioning

confidence: 99%

“…Method: CRISPR/Cas9-coupled recombineering mediated with the recombinase RecT Multiplexed repression with proof-ofconcept in naringenin production [14] CRISPRi Escherichia coli…”

Section: The Grna Characteristics and Extensionsmentioning

confidence: 99%

“…As mentioned above, besides its efficient endonuclease activity, CRISPR can enable gene expression modulation through the deactivated form of the Cas9 protein, dCas9 [59,79]. Once bound to, or in the vicinity of the transcriptional start site (TSS), the gRNA:dCas9 complex can significantly alter the transcriptional expression by physically interfering with RNA polymerase binding [14,43,79]. Wu et al recently exploited this strategy in E. coli where they did a selective knockdown of gene expression of enzymes that could divert the carbon flux away from the production of 1,4-Butanediol (BDO) [99].…”

Section: Dcas9-transcriptional Regulationmentioning

confidence: 99%

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Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli

Cited by 152 publications

References 61 publications

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

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