Abstract
BackgroundOats 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.ResultsWe 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.ConclusionWe 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.
Highlights
Oats contain hydroxycinnamoyl anthranilates, named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects
We primarily focused on the biological synthesis of Avn D, which features a basal core structure of hydroxycinnamoyl anthranilates
Expression of Nt4CL1 and HCBT in E. coli strain W3110 trpD9923 E. coli W3110 trpD9923 strain is a tryptophan auxotroph that over-accumulates anthranilate due to a nonsense mutation in the trpD gene, which abolishes anthranilate phosphoribosyltransferase activity but does not affect anthranilate synthase activity [41,42]
Summary
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. Hydroxycinnamoyl anthranilates are part of the large cinnamoyl anthranilates family, a class of molecules with beneficial health properties. Avenanthramides (Avns) are natural hydroxycinnamoyl anthranilates found in oats (Avena sativa L.) at low concentrations Tranilast has anti-inflammatory and antiproliferative effects and is currently evaluated clinically for the treatment of multiple sclerosis and various arthritic conditions [22,23,24]. The antitumor potential of tranilast has been evidenced in several clinical trials [25], and the design of analogues that exhibit higher anti-fibrotic activity has been extensively investigated [26,27,28,29,30]
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