Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

Ting, Wan-Wen; Tan, Shih‐I; Ng, I‐Son

doi:10.1186/s40643-020-00342-6

Cited by 23 publications

(11 citation statements)

References 42 publications

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“…To further improve biosafety, an auxotrophic strain E. coli WM3064 which has deficiency of dap A gene was used as the final chassis. The pHK‐L5‐Km plasmid with T7RNA polymerase, 18 was transformed into E. coli WM3064 using the heat‐shock method. Subsequently, pSU‐NOSAD was transformed into the WM3064 competent cells that harbored T7RNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

A systematic approach to reduce intraocular pressure for the treatment of glaucoma

Hsu

Huang

et al. 2022

Biotechnology Progress

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Glaucoma is the leading cause of irreversible blindness due to increased intraocular pressure (IOP) in the eye. We have developed a novel treatment option for glaucoma based on a real-time IOP-dependent nitric oxide synthase (NOS) and packed in a therapeutic contact lens to reduce the IOP. First, 1.6 nmole nitric oxide was produced from the genetic chassis, which was optimized for isopropyl β-d-1-thiogalactopyranoside (IPTG) induction in a T7 expression system. For biosafety concerns to human being, the csgAD genes responsible for curli biofilm formation in Escherichia coli were co-expressed with NOS in the designated NOSAD strain to strengthen the adherence of cells to the contact lens, thereby preventing the contamination into the eyes. Moreover, NOSAD is a diaminopimelic acid (DAP) auxotrophic strain, which cannot survive without supplementation of DAP and reached the critical consideration of biosafety to the environment. We also demonstrated that the nitric oxide diffusion was 3.6-times enhanced from penetration into the aqueous humor of porcine eyes. The deformation ratio of the contact lens was correlated to the change of IOP by using a digital image correlation (DIC) system in a porcine eye model. The novel systematic approach provides an alternative treatment for glaucoma patients in the future.

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Section: Methodsmentioning

confidence: 99%

A systematic approach to reduce intraocular pressure for the treatment of glaucoma

Hsu

Huang

et al. 2022

Biotechnology Progress

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“…T7RNAP and the cognate T7 promoter have been broadly employed as an efficient system for high-level recombinant expression in E. coli DE3 strain. ,, The high orthogonality of T7RNAP and T7 promoter rendered an energy competition between the T7 system and the housekeeping proteins, thus triggering metabolic burden and hampering cell growth . Hence, fine-tuning of T7RNAP expression was inevitably required to optimize production by varying the genetic elements, including promoter strengthens (e.g.,E.…”

Section: Introductionmentioning

confidence: 99%

Reprogramming T7RNA Polymerase in Escherichia coli Nissle 1917 under Specific Lac Operon for Efficient p-Coumaric Acid Production

Effendi

2022

ACS Synth. Biol.

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Lac operon is the standard regulator used to control the orthogonality of T7RNA polymerase (T7RNAP) and T7 promoter inEscherichia coli BL21(DE3) strain for protein expression. However,E. coliNissle 1917 (EcN), the unique probiotic strain, has seldom been precisely adapted to the T7 system. Herein, we applied bioinformatics analysis on Lac operon from different strains, and it was observed that a weak promoter for LacI repressor existed in EcN. Furthermore, X-gal assay revealed a strong expression of lacZ in EcN. We demonstrated that Lac operon significantly affected the protein expression in the two T7-derived EcN, in which T7RNAP was integrated at lambda (ET7L) and HK022 (ET7H), respectively. Different combinations of replication origin, chaperonin GroELS, inducer, and medium were explored to fine-tune the best strain with tyrosine ammonia-lyase (TAL) for p-coumaric acid (pCA) production, which is one of the essential bioactive compounds for human health. Finally, the highest pCA conversion of 78.8% was achieved using RRtL (plasmid form) under the optimum condition, and a 51.5% conversion was obtained with L::Rt strain which has integrated T7-RtTAL at HK022 of ET7L in the simulated gut environment. The appropriate reprogramming of T7RNAP expedites EcN as an effective and promising cell factory for live bacterial therapeutics in the future.

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“…The ideal production of recombinant proteins by BL21 (DE3) includes two periods, where biomass is accumulated rapidly during non-inducing period, while protein production is achieved during the inducing period. Moreover, the expression level of T7 RNAP is important for the production of recombinant proteins [ 6 ]. For easy-to-express proteins, production of proteins in BL21 (DE3) is often limited because a large portion of feedstock will be used to produce biomass [ 7 , 8 ].…”

Section: Introductionmentioning

confidence: 99%

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

Liu

et al. 2021

Microb Cell Fact

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Escherichia coli is the most widely used bacterium in prokaryotic expression system for the production of recombinant proteins. In BL21 (DE3), the gene encoding the T7 RNA polymerase (T7 RNAP) is under control of the strong lacUV5 promoter (PlacUV5), which is leakier and more active than wild-type lac promoter (PlacWT) under certain growth conditions. These characteristics are not advantageous for the production of those recombinant proteins with toxic or growth-burdened. On the one hand, leakage expression of T7 RNAP leads to rapid production of target proteins under non-inducing period, which sucks resources away from cellular growth. Moreover, in non-inducing or inducing period, high expression of T7 RNAP production leads to the high-production of hard-to-express proteins, which may all lead to loss of the expression plasmid or the occurrence of mutations in the expressed gene. Therefore, more BL21 (DE3)-derived variant strains with rigorous expression and different expression level of T7 RNAP should be developed. Hence, we replaced PlacUV5 with other inducible promoters respectively, including arabinose promoter (ParaBAD), rhamnose promoter (PrhaBAD), tetracycline promoter (Ptet), in order to optimize the production of recombinant protein by regulating the transcription level and the leakage level of T7 RNAP. Compared with BL21 (DE3), the constructed engineered strains had higher sensitivity to inducers, among which rhamnose and tetracycline promoters had the lowest leakage ability. In the production of glucose dehydrogenase (GDH), a protein that causes host autolysis, the engineered strain BL21 (DE3::ara) exhibited higher biomass, cell survival rate and foreign protein expression level than that of BL21 (DE3). In addition, these engineered strains had been successfully applied to improve the production of membrane proteins, including E. coli cytosine transporter protein (CodB), the E. coli membrane protein insertase/foldase (YidC), and the E. coli F-ATPase subunit b (Ecb). The engineered strains constructed in this paper provided more host choices for the production of recombinant proteins.

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Development of chromosome-based T7 RNA polymerase and orthogonal T7 promoter circuit in Escherichia coli W3110 as a cell factory

Cited by 23 publications

References 42 publications

A systematic approach to reduce intraocular pressure for the treatment of glaucoma

A systematic approach to reduce intraocular pressure for the treatment of glaucoma

Reprogramming T7RNA Polymerase in Escherichia coli Nissle 1917 under Specific Lac Operon for Efficient p-Coumaric Acid Production

Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production

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