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Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli.

Eudes A, Juminaga D, Baidoo EE, Collins FW, Keasling JD, Loqué D - Microb. Cell Fact. (2013)

Bottom Line: Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects.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.The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.

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.

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Production of Avn D and Avn F in engineered E. coli coexpressing HCBT and Nt4CL1. Detection of Avn D (A) and Avn F (B) from the culture medium of the W3110 trpD9923 strain harboring the pAvn vector and fed with p-coumarate and caffeate (300 μM), respectively. ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of engineered E. coli strains (upper panels) and of authentic standard solutions (lower panels).
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Figure 2: Production of Avn D and Avn F in engineered E. coli coexpressing HCBT and Nt4CL1. Detection of Avn D (A) and Avn F (B) from the culture medium of the W3110 trpD9923 strain harboring the pAvn vector and fed with p-coumarate and caffeate (300 μM), respectively. ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of engineered E. coli strains (upper panels) and of authentic standard solutions (lower panels).

Mentions: In this study, we primarily focused on the biological synthesis of Avn D, which features a basal core structure of hydroxycinnamoyl anthranilates. For this purpose, a previously characterized E. coli anthranilate-accumulating strain was selected as a chassis [41,42]. In that strain, coexpression of Nt4CL1 and HCBT led to the production of Avn D and Avn F when the culture medium was supplemented only with p-coumarate and caffeate, respectively. This validated the functional expression and activity of both plant enzymes in our chassis. The production system was then affranchised from precursor feeding by additional expression of RgTAL, which converts tyrosine into p-coumarate [43,44] (Figure 2). Avn D biosynthesis was further enhanced by expressing a two-plasmid-based modular biosynthetic pathway for tyrosine overproduction from glucose [45]. Finally, Avn F was also biologically produced de novo upon expression of either Sam5 or HpaBC, which are two hydroxylases that convert p-coumarate into caffeate [46,47].


Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli.

Eudes A, Juminaga D, Baidoo EE, Collins FW, Keasling JD, Loqué D - Microb. Cell Fact. (2013)

Production of Avn D and Avn F in engineered E. coli coexpressing HCBT and Nt4CL1. Detection of Avn D (A) and Avn F (B) from the culture medium of the W3110 trpD9923 strain harboring the pAvn vector and fed with p-coumarate and caffeate (300 μM), respectively. ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of engineered E. coli strains (upper panels) and of authentic standard solutions (lower panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716870&req=5

Figure 2: Production of Avn D and Avn F in engineered E. coli coexpressing HCBT and Nt4CL1. Detection of Avn D (A) and Avn F (B) from the culture medium of the W3110 trpD9923 strain harboring the pAvn vector and fed with p-coumarate and caffeate (300 μM), respectively. ESI-MS spectra were obtained after LC-TOF MS analysis of the culture medium of engineered E. coli strains (upper panels) and of authentic standard solutions (lower panels).
Mentions: In this study, we primarily focused on the biological synthesis of Avn D, which features a basal core structure of hydroxycinnamoyl anthranilates. For this purpose, a previously characterized E. coli anthranilate-accumulating strain was selected as a chassis [41,42]. In that strain, coexpression of Nt4CL1 and HCBT led to the production of Avn D and Avn F when the culture medium was supplemented only with p-coumarate and caffeate, respectively. This validated the functional expression and activity of both plant enzymes in our chassis. The production system was then affranchised from precursor feeding by additional expression of RgTAL, which converts tyrosine into p-coumarate [43,44] (Figure 2). Avn D biosynthesis was further enhanced by expressing a two-plasmid-based modular biosynthetic pathway for tyrosine overproduction from glucose [45]. Finally, Avn F was also biologically produced de novo upon expression of either Sam5 or HpaBC, which are two hydroxylases that convert p-coumarate into caffeate [46,47].

Bottom Line: Oats contain hydroxycinnamoyl anthranilates, also named avenanthramides (Avn), which have beneficial health properties because of their antioxidant, anti-inflammatory, and antiproliferative effects.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.The proposed pathway will serve as a platform for further engineering toward economical and sustainable bioproduction of these pharmaceuticals and other related aromatic compounds.

View Article: PubMed Central - HTML - PubMed

Affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.

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.

Show MeSH
Related in: MedlinePlus