Dataset Information

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities.

ABSTRACT: The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of Corynebacterium glutamicum to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based expression of shikimate pathway genes revealed that 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, encoded by aroG, and DHQ synthase, encoded by aroB, are key enzymes for shikimic acid production in C. glutamicum. We constructed a C. glutamicum strain with aroG, aroB and aroE3 integrated. This strain produced 13.1 g/L of shikimic acid from 50 g/L of glucose, a yield of 0.26 g-shikimic acid/g-glucose, and retained both its phosphotransferase system and its pyruvate kinase activity. We also endowed ?-glucosidase secreting ability to this strain. When cellobiose was used as a carbon source, the strain produced shikimic acid at 13.8 g/L with the yield of 0.25 g-shikimic acid/g-glucose (1 g of cellobiose corresponds to 1.1 g of glucose). These results demonstrate the feasibility of producing shikimic acid and its derivatives using an engineered C. glutamicum strain from cellobiose as well as glucose.

SUBMITTER: Sato N

PROVIDER: S-EPMC7511668 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Metabolic Engineering of Shikimic Acid-Producing <i>Corynebacterium glutamicum</i> From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities.

Sato Naoki N Kishida Mayumi M Nakano Mariko M Hirata Yuuki Y Tanaka Tsutomu T

Frontiers in bioengineering and biotechnology 20200910

The production of aromatic compounds by microbial production is a promising and sustainable approach for producing biomolecules for various applications. We describe the metabolic engineering of <i>Corynebacterium glutamicum</i> to increase its production of shikimic acid. Shikimic acid and its precursor-consuming pathways were blocked by the deletion of the shikimate kinase, 3-dehydroshikimate dehydratase, shikimate dehydratase, and dihydroxyacetone phosphate phosphatase genes. Plasmid-based ex ...[more]

PMID: 33015020

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Project description:To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63(up), and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the ?-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63(up) mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63(up) fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

Dataset Information

Metabolic Engineering of Shikimic Acid-Producing Corynebacterium glutamicum From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities.

Publications

Metabolic Engineering of Shikimic Acid-Producing <i>Corynebacterium glutamicum</i> From Glucose and Cellobiose Retaining Its Phosphotransferase System Function and Pyruvate Kinase Activities.

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