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Open Access Research

Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli

Aymerick Eudes12, Darmawi Juminaga13, Edward E K Baidoo1, F William Collins4, Jay D Keasling1235 and Dominique Loqué12*

  • * Corresponding author: Dominique Loqué dloque@lbl.gov

  • † Equal contributors

Author Affiliations

1 Joint BioEnergy Institute, Emeryville, CA 94608, USA

2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

3 California Institute for Quantitative Biosciences and the Synthetic Biology Institute at UC Berkeley, Berkeley, CA 94720, USA

4 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food, Ottawa, ON K1A 0C5, Canada

5 Department of Bioengineering, Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA 94720, USA

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Microbial Cell Factories 2013, 12:62  doi:10.1186/1475-2859-12-62

Published: 28 June 2013

Abstract

Background

Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects. The microbial production of hydroxycinnamoyl anthranilates is an eco-friendly alternative to chemical synthesis or purification from plant sources. We recently demonstrated in yeast (Saccharomyces cerevisiae) that coexpression of 4-coumarate: CoA ligase (4CL) from Arabidopsis thaliana and hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT) from Dianthus caryophyllusenabled the biological production of several cinnamoyl anthranilates upon feeding with anthranilate and various cinnamates. Using engineering strategies to overproduce anthranilate and hydroxycinnamates, we describe here an entire pathway for the microbial synthesis of two Avns from glucose in Escherichia coli.

Results

We first showed that coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 allowed the production of Avn D [N-(4′-hydroxycinnamoyl)-anthranilic acid] and Avn F [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] upon feeding with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) led to the conversion of endogenous tyrosine into p-coumarate and resulted in the production of Avn D from glucose. Second, a 135-fold improvement in Avn D titer was achieved by boosting tyrosine production using two plasmids that express the eleven genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli resulted in the endogenous production of caffeate and biosynthesis of Avn F.

Conclusion

We established a biosynthetic pathway for the microbial production of valuable hydroxycinnamoyl anthranilates from an inexpensive carbon source. The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

Keywords:
Avenanthramide; Tranilast; BAHD; Antioxidant; Anti-inflammatory; Tyrosine; Anthranilate; Hydroxycinnamate; Biological synthesis; Escherichia coli