Dataset Information

Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

ABSTRACT: The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene repressing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L, which was 7.4-fold higher than the control strain. The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for producing other important products that require malonyl-CoA as a precursor.

SUBMITTER: Wu J

PROVIDER: S-EPMC4555050 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

Wu Junjun J Du Guocheng G Chen Jian J Zhou Jingwen J

Scientific reports 20150901

The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed for fine-tuning central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target gene ...[more]

PMID: 26323217

Similar Datasets

Project description:Beta (?)-carotene (C40H56; a provitamin) is a particularly important carotenoid for human health. Many studies have focused on engineering Escherichia coli as an efficient heterologous producer of ?-carotene. Moreover, several strains with potential for use in the industrial production of this provitamin have already been constructed via different metabolic engineering strategies. In this study, we aimed to improve the ?-carotene-producing capacity of our previously engineered E. coli strain ZF43?gdhA through further gene deletion and metabolic pathway manipulations. Deletion of the zwf gene increased the resultant strain's ?-carotene production and content by 5.1 and 32.5%, respectively, relative to the values of strain ZF43?gdhA, but decreased the biomass by 26.2%. Deletion of the ptsHIcrr operon further increased the ?-carotene production titer from 122.0 to 197.4 mg/L, but the provitamin content was decreased. Subsequently, comparative transcriptomic analysis was used to explore the dynamic transcriptional responses of the strains to the blockade of the pentose phosphate pathway and inactivation of the phosphotransferase system. Lastly, based on the analyses of comparative transcriptome and reduction cofactor, several strategies to increase the NADPH supply were evaluated for enhancement of the ?-carotene content. The combination of yjgB gene deletion and nadK overexpression led to increased ?-carotene production and content. The best strain, ECW4/p5C-nadK, produced 266.4 mg/L of ?-carotene in flask culture and 2,579.1 mg/L in a 5-L bioreactor. The latter value is the highest reported from production via the methylerythritol phosphate pathway in E. coli. Although the strategies applied is routine in this study, the combinations reported were first implemented, are simple but efficient and will be helpful for the production of many other natural products, especially isoprenoids. Importantly, we demonstrated that the use of the methylerythritol phosphate pathway alone for efficient ?-carotene biosynthesis could be achieved via appropriate modifications of the cell metabolic functions.

Dataset Information

Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

Publications

Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure