Production of hydroxycinnamoyl anthranilates from glucose in Escherichia coli
- Aymerick Eudes†1, 2,
- Darmawi Juminaga†1, 3,
- Edward E K Baidoo1,
- F William Collins4,
- Jay D Keasling1, 2, 3, 5 and
- Dominique Loqué1, 2Email author
© Eudes et al.; licensee BioMed Central Ltd. 2013
Received: 25 May 2013
Accepted: 18 June 2013
Published: 28 June 2013
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 caryophyllus enabled 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.
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 (Rg TAL) 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.
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.
KeywordsAvenanthramide Tranilast BAHD Antioxidant Anti-inflammatory Tyrosine Anthranilate Hydroxycinnamate Biological synthesis Escherichia coli
Using microbes for biological synthesis of therapeutic drugs or precursors offers an alternative production strategy to commonly employed methods such as direct extraction from source organisms or chemical synthesis. Microbial expression systems have several advantages such as reduced requirements for toxic chemicals and natural resources; consistent quality; scalability; simple extraction; and potential for higher synthesis efficiency [31, 32]. Taking into consideration the expanding number of therapeutic applications for cinnamoyl anthranilates, as well as the fact that these molecules are currently synthesized chemically or extracted from food sources [33, 34], we attempted to design a pathway for their de novo production from glucose using E. coli as a production platform.
HCBT is an acetyltransferase from the BAHD family , which couples p-coumaroyl-CoA with anthranilate via an amide bond to produce Avn D (Figure 1B) ; while 4CL enzymes convert cinnamates into their corresponding CoA thioesters . We previously engineered a yeast strain that coexpresses 4CL and HCBT for the production of several cinnamoyl anthranilates, such as Avn D and Avn F, upon feedings with anthranilate and various cinnamates. This highlighted the potential of using these enzymes for the biological production of cinnamoyl anthranilates . E. coli is a host of choice for the expression of complex pathways and the production of elaborate molecules such as aromatic compounds from cheap carbon sources [39, 40].
Results and discussion
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]. This strain was shown to be suitable for metabolic engineering because expression of genes from the shikimate pathway further increased anthranilate production . We first constructed pAvn plasmid for coexpression of Nt4CL1, which encodes a 4CL that converts p-coumarate and caffeate into their corresponding CoA thioesters , and HCBT. To confirm that HCBT can catalyze the condensation of coumaroyl-CoA with anthranilate and produce Avn D in W3110 trpD9923, the strain was transformed with pAvn and grown in the presence of p-coumarate as a precursor. Cultures of W3110 trpD9923 harboring an empty vector were also grown as a negative control. Only in the case of the strain expressing pAvn, LC-TOF MS analysis of the culture medium revealed a peak (Rt = 11.75 min) that corresponds to Avn D by comparison with an authentic standard solution (Figure 2A). Similarly, the engineered strain produced some Avn F (Rt = 10.56 min) when p-coumarate was substituted by caffeate in the medium (Figure 2B). This result confirms the affinity of HCBT for caffeoyl-CoA. It also demonstrates secretion of Avn outside of the production host, because the Avn D and Avn F content inside E. coli cells represented less than 5% of the amount quantified from the medium (data not shown).
Biosynthesis of Avn D from glucose and titer improvement using a tyrosine overproduction strategy
Production of Avn D and precursors by engineered W3110 trpD9923 E . coli strains
Empty pZS21 + pBbB5a
1646 ± 63
9.3 ± 1.3
pS0 + pY
5878 ± 311
6201 ± 598
1564 ± 58
6.8 ± 0.9
1.0 ± 0.1
0.2 ± 0.1
pS0 + pY + pAvnD
5780 ± 251
5963 ± 219
32.6 ± 3.4
27.3 ± 1.4
Conversion of p-coumarate into caffeate and production of Avn F using Sam5
Production of Avn F and precursors by engineered W3110 trpD9923 E . coli strains
1498 ± 76
6.0 ± 1.0
0.28 ± 0.07
pS0 + pY + pAvnDF1
5802 ± 298
6286 ± 150
65.1 ± 8.3
0.07 ± 0.00
0.11 ± 0.04
1521 ± 44
2.0 ± 0.2
0.10 ± 0.01
0.13 ± 0.03
0.03 ± 0.00
pS0 + pY + pAvnDF2
5644 ± 288
2503 ± 313
11.9 ± 2.2
14.9 ± 1.6
4.1 ± 0.7
0.54 ± 0.16
Conversion of p-coumarate into caffeate and production of Avn F using the HpaBC complex
This work is an example of biological production of valuable aromatic metabolites using a tyrosine-overproducing strategy applied to an anthranilate-accumulating strain. Considering the anthranilate titers achieved with the strain containing only the shikimate and tyrosine modules, the maximum theoretical yield for Avn D in this background would be ~5.8 mM. However, much lower Avn D titers were obtained for the strain harboring pS0, pY and pAvnD, probably due to poor conversion of tyrosine into p-coumarate as previously observed in various studies using heterologous expression of TALs [46, 49, 50], and potentially to the limited intracellular pools of coenzyme A availability . It is particularly noteworthy that, because of its specificity to anthranilate as an acceptor, the BAHD acyltransferase HCBT allowed the exclusive biological synthesis of cinnamoyl anthranilates. For instance, no mass peaks corresponding to other phenylpropenoyl-amino acid amides consisting of a tryptophan, tyrosine or an L-dopa moiety — nor to hydroxycinnamate esters of shikimate or quinate — could be detected in the culture medium of our different E. coli Avn-producing strains.
The discovery that Rg TAL has L-dopa ammonia-lyase (DAL) activity is of interest and provides some opportunities for the design of new enzymes with a higher DAL/TAL activity ratio. In combination with tyrosine hydroxylase complexes such as HpaBC, such engineered DALs could be used to improve the bioproduction of caffeate from tyrosine via L-dopa and without generating p-coumarate as an intermediate, a competitive precursor for the biosynthesis of Avn F. Furthermore, the impact of expressing in our system 4CLs other than Nt4CL1 should be considered; especially in regard to production of Avn F, because Nt4CL1 is known to be less active with caffeate as a substrate compared to p-coumarate [56, 57]. Finally, our rationally designed pathway can serve as a framework for improvement of Avn production using combinatorial approaches that have been shown previously to increase tyrosine production . As an adjunct to the recent development of procedures that use safe methylating agents , this study describes a basis for eco-friendly production of cinnamoyl anthranilates such as Avn D and Avn F and can serve as a scaffold for the synthesis of more elaborate molecules such as tranilast and its analogs.
Chemicals and enzymes
The following chemicals and enzymes were used in this study: p-coumarate, L-tyrosine, anthranilate, L-dopa, isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO, USA), caffeate (MPBiomedicals, Solon, OH, USA), 3,4,5-trihydroxycinnamate (Apin Chemicals Ltd, Abingdon, UK), restriction enzymes (NEB, Ipswich, MA, USA), PhusionHigh-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA), Rapid DNA ligase Kit (Roche Applied Science, Indianapolis, IN, USA). All the enzymes were used in accordance with instructions provided by the manufacturers. N-(4′-hydroxy-(E)-cinnamoyl)-anthranilate (Avn D) and N-(3′,4′-dihydroxy-(E)-cinnamoyl)-anthranilate (Avn F) were prepared as described [1, 38].
Strains, plasmids, media, and growth conditions
Plasmids and strains used in this study
Plasmid or Strain
pSC101; Kanr PLtetO-1
pBBR1; AmprlacI PlacUV5
p15A; CmrlacI PlacUV5
colE1; AmprlacI Ptrc
colE1; KanrlacI PT7
Cinnamoyl anthranilates plasmids
W3110 [F-λ- INV (rrnD-rrnE) 1] tryptophan auxotroph, randomly mutagenized by treatment with ultraviolet radiation
Construction of plasmids
The BglBricks cloning strategy and the BglBricks vectors [62, 63] were used for gene assembly. All the forward primers consist of a BglII restriction site at the 5′-end, followed by the Shine-Dalgarno sequence prior to the start codon. The reverse primers consist of the XhoI and BamHI restriction sites at the 5′-end. For the pAvn construct, the gene sequence encoding HCBT (GenBank: CAB06427) from  was amplified using the primers HCBTfw and HCBTrv listed in Additional file 2: Table S1. The PCR product was digested with BglII / XhoI and ligated into the pBbA5c plasmid  between theBamHI and XhoI restriction sites. The cDNA clone corresponding to Nt4CL1 (GenBank: U50845) from  was amplified using the primers 4CLfw and 4CLrv (Additional file 2: Table S1), digested with BglII / XhoI and ligated into the pBbA5c::HCBT construct previously digested with BamHI / XhoI to yield the pAvn plasmid.
For the construction of pAvnD, a gene sequence encoding Rg TAL (GenBank: AAA33883) was synthesized (Genescript, NJ, USA) and amplified using the primers TALfw and TALrv listed in Additional file 2: Table S1. The PCR product was digested with BglII / XhoI and ligated downstream Nt4CL1 into pAvn previously digested with BamHI / XhoI. The Rg TAL gene sequence was also ligated downstream the T7 promoter into the pBbE7k plasmid  between the BamHI and XhoI sites to obtain the pRgTAL construct.
For the pAvnDF1 construct, a gene sequence encoding Sam5 (GenBank: ABC88666.1) was synthesized (Genescript, NJ, USA) with the BglBricks restriction sites EcoRI and BglII followed by the Shine-Dalgarno sequence at the 5′-end, and with BamHI and XhoI restriction sites at the 3′-end. The sam5 fragment was released by BglII / XhoI digestions and cloned between the BamHI and XhoI sites of the pBbE1a plasmid , downstream the terminator – promoter combination sequence T1-Ptrc, to yield the pSam5 plasmid. The T1-Ptrc-Sam5 fragment was released from pSam5 with BglII / XhoI digestions and ligated downstream Rg TAL into pAvnD previously digested with BamHI and XhoI.
For the pAvnDF2 construct, the hpaBC operon, which encodes HpaB (GenBank: CAQ34705) and HpaC (GenBank: CAQ34704) was amplified from E. coli BL21 (DE3) genomic DNA using primers hpaBCfw and hpaBCrv (Additional file 2: Table S1). The PCR product was ligated into the pCR-4Blunt-TOPO vector (Life technologies, Foster City, CA, USA) and a sequenced-verified clone was cured by site-directed mutagenesis to remove an internal BglII restriction site (nucleotides 83–88) using the primers SDM-BglIIfw and SDM-BglIIrv (Additional file 2: Table S1). The cured hpaBC operon was cloned into the pBbE1a plasmid downstream the T1-Ptrc sequence. The T1-Ptrc-hpaBC fragment was released with BglII / XhoI digestions and ligated downstream Rg TAL into pAvnD previously digested with BamHI and XhoI. For the construction of the pY plasmid, the tyrosine operon in the pY1 plasmid  was released with BglII / XhoI digestions and cloned into the pBbB5a plasmid between the BamHI and XhoI restriction sites.
LC-MS analysis of cinnamoyl anthranilates and precursors
All metabolites were quantified using HPLC–electrospray ionization (ESI)–time-of-flight (TOF) MS. An aliquot of the culture medium was cleared by centrifugation (21,000xg, 5 min, 4°C), mixed with an equal volume of cold methanol–water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cut off regenerated cellulose membrane; Millipore, Billerica, MA) prior to analysis. For the quantification of intracellular Avn, a cell pellet from 5 ml of culture was washed three times with water, suspended in cold methanol–water (1:1, v/v), sonicated twice for 30 s and centrifuged (21,000xg, 5 min, 4°C). The supernatant was collected and filtered before analysis. The separation of metabolites was conducted on the fermentation-monitoring HPX-87H column with 8% cross-linkage (150-mm length, 7.8-mm inside diameter, and 9-μm particle size; Bio-Rad, Richmond, CA) using an Agilent Technologies 1100 Series HPLC system. A sample injection volume of 10 μl was used throughout. The sample tray and column compartment were set to 4 and 50°C, respectively. Metabolites were eluted isocratically with a mobile-phase composition of 0.1% formic acid in water at a flow rate of 0.5 ml/min. The HPLC system was coupled to an Agilent Technologies 6210 series time-of-flight mass spectrometer (for LC-TOF MS) via a MassHunter workstation (Agilent Technologies, CA). Drying and nebulizing gases were set to 13 liters/min and 30 lb/in2, respectively, and a drying-gas temperature of 330°C was used throughout. ESI was conducted in the negative ion mode and a capillary voltage of −3,500 V was utilized. All other MS conditions were described previously . Metabolites were quantified via seven-point calibration curves of authentic standard compounds for which the R2 coefficients were ≥0.99.
Tyrosine ammonia lyase
Authors are thankful to Dr. Carsten Rautengarten for providing the Nt4CL1 cDNA clone and Sabin Russell for language editing of the manuscript. This work was part of the DOE Joint BioEnergy Institute (http://www.jbei.org) supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.
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