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Biosynthesis of flavone C-glucosides in engineered Escherichia coli

Abstract

Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T7 promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 μM) and 11% (9 mg/L; 22 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-β-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 μM) and 14 mg/L (17%, 34 μM) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 μM) conversion of chrysin to chrysin 6-C-β-D-glucoside at 48 h upon supplementation of 200 μM of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 μM) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and β-glucosidase enzyme, than O-glucosylated derivative of chrysin.

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Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant # PJ01111901), Rural Development Administration, Republic of Korea.

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Correspondence to Jae Kyung Sohng.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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The authors declare that they have no conflict of interest.

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Shrestha, A., Pandey, R.P., Dhakal, D. et al. Biosynthesis of flavone C-glucosides in engineered Escherichia coli. Appl Microbiol Biotechnol 102, 1251–1267 (2018). https://doi.org/10.1007/s00253-017-8694-6

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  • DOI: https://doi.org/10.1007/s00253-017-8694-6

Keywords

  • C-glycosyltransferase
  • Flavone
  • Chrysin
  • Luteolin
  • C-glucosylation