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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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Related in: MedlinePlus

In-vivo enzyme activity of RgTAL towards L-dopa. Production of caffeate detected in the culture medium of an E. coli strain expressing RgTAL and fed with L-dopa. Error bars indicate mean values ± SD from three independent clones. Nd, not detected.
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Figure 5: In-vivo enzyme activity of RgTAL towards L-dopa. Production of caffeate detected in the culture medium of an E. coli strain expressing RgTAL and fed with L-dopa. Error bars indicate mean values ± SD from three independent clones. Nd, not detected.

Mentions: The enzyme complex consisting of a 4-hydroxyphenylacetate 3-hydroxylase (HpaB) and a flavin:NADH reductase (HpaC) from E. coli was tested for the biological production of caffeate and Avn F. The operon hpaBC is involved in 4-hydroxyphenylacetate degradation and several studies showed that the HpaBC enzyme complex can accept a broad range of substrates including tyrosine and p-coumarate [46,52,53]. We constructed a pAvnDF2 plasmid by placing the hpaBC operon under the control of the trc promoter into pAvnD plasmid (Figure 3B). Transformation of pAvnDF2 into E. coli W3110 trpD9923 resulted in the production of small amount of caffeate in the culture medium, but only Avn D could be detected (Table 2). By contrast, co-transformation of pAvnDF2 with pS0 and pY enhanced caffeate production (~115-fold) and led to the biosynthesis of Avn F (~540 nM) in addition to Avn D (Table 2). Unlike the results of the biosynthesis of Avn F using Sam5, the expression of HpaBC maintained higher Avn D titers and did not produce any 3,4,5-trihydroxycinnamate nor entirely deplete p-coumarate content. This suggests that HpaBC is less efficient than Sam5 at converting p-coumarate into caffeate in our system, yet nevertheless Avn F titers using HpaBC were ~5-fold higher compared to those achieved using Sam5 (Table 2). Alternatively, the higher caffeate content and lower AvnF titers obtained using Sam5 could reflect a negative effect of 3,4,5-trihydroxycinnamate on 4CL1 activity. Moreover, we observed a reduction in tyrosine titers compared to those measured from the culturesof E. coli W3110 trpD9923 harboring pAvnD or pAvnDF1. This was probably due to HpaBC activity, which can also convert tyrosine into L-dopa. Conclusively, we found that L-dopa concentration was ~4.4 mM in the culture medium of the pS0/pY/pAvnDF2 strain. Furthermore, based on previous studies showing that some tyrosine ammonia-lyases convert L-dopa into caffeate [46,54], an E. coli strain that expresses RgTAL alone was created and grown in the presence of L-dopa. Interestingly, analysis of the culture medium of the RgTAL strain revealed the presence of caffeate, which was absent in the medium of an empty vector control strain (Figure 5). These results demonstrate that RgTAL exhibits some L-dopa ammonia-lyase activity and suggest that part of the caffeate produced in the strains harboring pAvnDF2 could be derived from L-dopa.


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)

In-vivo enzyme activity of RgTAL towards L-dopa. Production of caffeate detected in the culture medium of an E. coli strain expressing RgTAL and fed with L-dopa. Error bars indicate mean values ± SD from three independent clones. Nd, not detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In-vivo enzyme activity of RgTAL towards L-dopa. Production of caffeate detected in the culture medium of an E. coli strain expressing RgTAL and fed with L-dopa. Error bars indicate mean values ± SD from three independent clones. Nd, not detected.
Mentions: The enzyme complex consisting of a 4-hydroxyphenylacetate 3-hydroxylase (HpaB) and a flavin:NADH reductase (HpaC) from E. coli was tested for the biological production of caffeate and Avn F. The operon hpaBC is involved in 4-hydroxyphenylacetate degradation and several studies showed that the HpaBC enzyme complex can accept a broad range of substrates including tyrosine and p-coumarate [46,52,53]. We constructed a pAvnDF2 plasmid by placing the hpaBC operon under the control of the trc promoter into pAvnD plasmid (Figure 3B). Transformation of pAvnDF2 into E. coli W3110 trpD9923 resulted in the production of small amount of caffeate in the culture medium, but only Avn D could be detected (Table 2). By contrast, co-transformation of pAvnDF2 with pS0 and pY enhanced caffeate production (~115-fold) and led to the biosynthesis of Avn F (~540 nM) in addition to Avn D (Table 2). Unlike the results of the biosynthesis of Avn F using Sam5, the expression of HpaBC maintained higher Avn D titers and did not produce any 3,4,5-trihydroxycinnamate nor entirely deplete p-coumarate content. This suggests that HpaBC is less efficient than Sam5 at converting p-coumarate into caffeate in our system, yet nevertheless Avn F titers using HpaBC were ~5-fold higher compared to those achieved using Sam5 (Table 2). Alternatively, the higher caffeate content and lower AvnF titers obtained using Sam5 could reflect a negative effect of 3,4,5-trihydroxycinnamate on 4CL1 activity. Moreover, we observed a reduction in tyrosine titers compared to those measured from the culturesof E. coli W3110 trpD9923 harboring pAvnD or pAvnDF1. This was probably due to HpaBC activity, which can also convert tyrosine into L-dopa. Conclusively, we found that L-dopa concentration was ~4.4 mM in the culture medium of the pS0/pY/pAvnDF2 strain. Furthermore, based on previous studies showing that some tyrosine ammonia-lyases convert L-dopa into caffeate [46,54], an E. coli strain that expresses RgTAL alone was created and grown in the presence of L-dopa. Interestingly, analysis of the culture medium of the RgTAL strain revealed the presence of caffeate, which was absent in the medium of an empty vector control strain (Figure 5). These results demonstrate that RgTAL exhibits some L-dopa ammonia-lyase activity and suggest that part of the caffeate produced in the strains harboring pAvnDF2 could be derived from L-dopa.

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