Dataset Information

Production of Deuterated Cyanidin 3-O-Glucoside from Recombinant Escherichia coli.

ABSTRACT: Anthocyanins are plant secondary metabolites that, despite their chemical instability, have found many applications as natural food colorants. They are also known for their beneficial health effects because of their antioxidant and anticancer properties. More stable versions of these molecules, particularly at neutral pH conditions, are required to study the anthocyanin pharmacokinetic properties and obtain effective therapeutic results. In the present report, a cost-effective technique was developed to prepare the deuterated anthocyanin using recombinant Escherichia coli as a production host and deuterated glycerol and D2O in the culture media. This approach resulted in the formation of endogenous deuterated uridine 5'-diphosphate-glucose that was further incorporated by the recombinant anthocyanin pathway, resulting in the formation of deuterated cyanidin 3-O-glucoside (C3G). The deuterium exchange of O-D and C-D were studied by liquid chromatography (LC)-mass spectrometry and NMR analysis. The labeled C3G, purified by high-performance LC showed a stable nature at pH 7.0 as compared to nondeuterated C3G.

SUBMITTER: Gupta M

PROVIDER: S-EPMC6173498 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Production of Deuterated Cyanidin 3-<i>O</i>-Glucoside from Recombinant <i>Escherichia coli</i>.

Gupta Mamta M Zha Jian J Zhang Xing X Jung Gyoo Yeol GY Linhardt Robert J RJ Koffas Mattheos A G MAG

ACS omega 20180924 9

Anthocyanins are plant secondary metabolites that, despite their chemical instability, have found many applications as natural food colorants. They are also known for their beneficial health effects because of their antioxidant and anticancer properties. More stable versions of these molecules, particularly at neutral pH conditions, are required to study the anthocyanin pharmacokinetic properties and obtain effective therapeutic results. In the present report, a cost-effective technique was deve ...[more]

PMID: 30320269

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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.

Project description:In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Dataset Information

Production of Deuterated Cyanidin 3-O-Glucoside from Recombinant Escherichia coli.

Publications

Production of Deuterated Cyanidin 3-<i>O</i>-Glucoside from Recombinant <i>Escherichia coli</i>.

Similar Datasets

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