Dataset Information

Auxotrophic Selection Strategy for Improved Production of Coenzyme B₁₂ in Escherichia coli.

ABSTRACT: The production of coenzyme B₁₂ using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B₁₂ in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B₁₂ using a previously engineered coenzyme B₁₂-producing E. coli strain. To select a high producer, the coenzyme B₁₂-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B₁₂-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B₁₂ validated the coenzyme B₁₂-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B₁₂ production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B₁₂-producing strains.

SUBMITTER: Noh MH

PROVIDER: S-EPMC7033360 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Auxotrophic Selection Strategy for Improved Production of Coenzyme B12 in Escherichia coli.

Noh Myung Hyun MH Lim Hyun Gyu HG Moon Daeyeong D Park Sunghoon S Jung Gyoo Yeol GY

iScience 20200207 3

The production of coenzyme B12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B12 using a previously engineered coenzyme B12-producing E. coli strain. To select a high producer, the coenzyme B12</sub ...[more]

PMID: 32086013

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Project description:BackgroundMulti-monocistronic and multi-variate vectors were designed, built, and tested for the improved production of cyanidin 3-O-glucoside (C3G) in Escherichia coli BL21 (DE3). The synthetic bio-parts were designed in such a way that multiple genes can be assembled using the bio-brick system, and expressed under different promoters in a single vector. The vectors harbor compatible cloning sites, so that the genes can be shuffled from one vector to another in a single step, and assembled into a single vector. The two required genes: anthocyanidin synthase (PhANS) from Petunia hybrida, and cyanidin 3-O-glucosyltransferase (At3GT) from Arabidopsis thaliana, were individually cloned under PT7, Ptrc, and PlacUV5 promoters. Both PhANS and At3GT were shuffled back and forth, so as to generate a combinatorial system for C3G production. The constructed systems were further coupled with the genes for UDP-D-glucose synthesis, all cloned in a multi-monocistronic fashion under PT7. Finally, the production of C3G was checked and confirmed using the modified M9 media, and analyzed through various chromatography and spectrometric analyses.ResultsThe engineered strains endowed with newly generated vectors and the genes for C3G biosynthesis and UDP-D-glucose synthesis were fed with 2 mM (+)-catechin and D-glucose for the production of cyanidin, and its subsequent conversion to C3G. One of the engineered strains harboring At3GT and PhANS under Ptrc promoter and UDP-D-glucose biosynthesis genes under PT7 promoter led to the production of ~?439 mg/L of C3G within 36 h of incubation, when the system was exogenously fed with 5% (w/v) D-glucose. This system did not require exogenous supplementation of UDP-D-glucose.ConclusionA synthetic vector system using different promoters has been developed and used for the synthesis of C3G in E. coli BL21 (DE3) by directing the metabolic flux towards the UDP-D-glucose. This system has the potential of generating better strains for the synthesis of valuable natural products.

Dataset Information

Auxotrophic Selection Strategy for Improved Production of Coenzyme B₁₂ in Escherichia coli.

Publications

Auxotrophic Selection Strategy for Improved Production of Coenzyme B<sub>12</sub> in Escherichia coli.

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