A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

Li, Mingji; Wang, Junshu; Geng, Yue; Li, Yikui; Wang, Qian; Liang, Quanfeng; Qi, Qingsheng

doi:10.1186/1475-2859-11-19

Cited by 77 publications

(69 citation statements)

References 34 publications

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“…Then 1 mL water was added to the mixture and mixed thoroughly for 20 s. After phase separation, the heavier chloroform phase was transferred to another new vial for GC analysis. The PHB content was defined as the percentage ratio of the PHB concentration to biomass [27, 49]. …”

Section: Methodsmentioning

confidence: 99%

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Chang¹,

Su²,

Qi³

et al. 2016

Microb Cell Fact

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BackgroundClustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.ResultsBy knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo.ConclusionBy regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0594-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Chang¹,

Su²,

Qi³

et al. 2016

Microb Cell Fact

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“…Li et al integrated the phaCAB operon with a 5CPtacs promoter cluster into E. coli chromosome, to create a system of repetitive promoters for high and stable overexpression; the resulting engineered bacteria accumulate 23.7% PHB of the cell dry weight in batch cultivation [43]. Lee et al produced enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) from glucose, with a yield of 49.5% (85.6% of the maximum theoretical yield), by integration of the PHA biosynthesis and depolymerase genes into the chromosome of E. coli [44].…”

Section: Resultsmentioning

confidence: 99%

PHB Production in Biofermentors Assisted through Biosensor Applications

Poltronieri¹,

Mezzolla²,

D’Urso³

2016

Proceedings of the 3rd International Electronic Conference on Sensors and Applications, 15–30 November 2016; Availabl

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Poly-hydroxy-alcanoates (PHAs) are biodegradable and biocompatible polymers synthesized and accumulated in intracellular compartments in several bacterial species. Polyhydroxyalcanoates (PHAs) are synthesized by numerous prokaryotes, such as Cupriavidus necator (Ralstonia eutropha), Pseudomonas spp., Comamonas spp., in response to stress conditions, i.e., under high carbon and low nitrogen (24:1 ratio). PHA can be synthesized using recombinant microorganisms (provided with the operon phbA/phbB/phbC), escaping the constrains of nutrient request, except addition of high amount of sugar (glucose, lactose, fructose). Recombinant E. coli systems were studied to produce PHB using metabolic engineering. In biofermentors, the critical points are the excess of fermentable sugars and the ratio of nutrients versus cell optical density. In order to allow production in biofermentors in automated system, sensors are envisaged to evaluate critical parameters such as sugar consumption, bacteria concentration and level of synthesis of PHA. The need of fermentors and operation control has compelled for application of three biosensing units, one linked to a Nanodrop to evaluate OD, one linked to an enzymatic reaction chamber to measure sugars consumed by enzyme linked sugar biosensing tools, and one for sampling the bacteria, Nile Blue staining, and fluorescence intensity reads. These detectors will make possible to exploit the full potential of bioreactors optimizing the time of use and maximizing the number of bacteria synthesizing PHA.

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“…We used p1TGL to measure b-galactosidase activity and GFP. p1TGL was constructed from p1TG (Li et al, 2012) by inserting the lacZ gene at the XhoI and SacII sites. The lacZ gene was amplified with primers lacz-up and lacz-down (Table 2).…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli toxin gene hipA affects biofilm formation and DNA release

Zhao

Wang

et al. 2013

Microbiology

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Toxin-antitoxin (TA) systems in Escherichia coli may play a role in biofilm formation, but the mechanism involved remains debatable. It is not known whether the TA systems are responsible for extracellular DNA (eDNA) in biofilms. In this study, we investigated the function of the hipBA TA system in biofilm formation by Escherichia coli strain BW25113. First, the deletion of the HipBA TA system in E. coli BW25113 significantly reduced the biofilm biomass without antibiotic stress. Second, treatment of the BW25113 biofilm with DNase I caused a major reduction in biofilm formation, whereas similar treatment of the hipA mutant biofilm had only a minor effect. Third, the inactivation of HipA reduced the level of eDNA present in biofilm formation, and addition of BW25113 genomic DNA stimulated biofilm formation for both the wild-type and hipA mutant. Fourth, the wild-type cells underwent significantly more cell lysis than the hipA mutant. These results suggest that hipA plays a significant role during biofilm development and that eDNA is an important structural component of E. coli BW25113 biofilms. Thus, the TA system may enhance biofilm formation through DNA release.

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A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli

Cited by 77 publications

References 34 publications

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

PHB Production in Biofermentors Assisted through Biosensor Applications

Escherichia coli toxin gene hipA affects biofilm formation and DNA release

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