Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli
Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli
Journal of Microbiology and Biotechnology. 2015. Sep, 25(9): 1442-1448
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : March 04, 2015
  • Accepted : May 11, 2015
  • Published : September 28, 2015
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About the Authors
Hyejin Lee
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea
Bong Gyu Kim
Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea
Mihyang Kim
Department of Systems Biotechnology, Chung-Ang University, Anseong 456-756, Republic of Korea
Joong-Hoon Ahn
Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea

The flavonoid apigenin and its O -methyl derivative, genkwanin, have various biological activities and can be sourced from some vegetables and fruits. Microorganisms are an alternative for the synthesis of flavonoids. Here, to synthesize genkwanin from tyrosine, we first synthesized apigenin from p -coumaric acid using four genes ( 4CL , CHS , CHI , and FNS ) in Escherichia coli . After optimization of different combinations of constructs, the yield of apigenin was increased from 13 mg/l to 30 mg/l. By introducing two additional genes ( TAL and POMT7 ) into an apigenin-producing E. coli strain, we were able to synthesize 7- O -methyl apigenin (genkwanin) from tyrosine. In addition, the tyrosine content in E. coli was modulated by overexpressing aroG and tyrA . The engineered E. coli strain synthesized approximately 41 mg/l genkwanin.
Plants produce diverse secondary metabolites such as alkaloids, isoprenoids, and polyphenols [5] . Some of these chemicals have been used as medicines and/or for nutrition [18] . Many natural compounds originating from plants are considered as starting materials for the development of new medicines [1] . Microbial production of plant secondary metabolites has become an attractive topic. Biological pathways for the synthesis of several plant metabolites have been reconstructed in microorganisms, and hosts such as Escherichia coli and Saccharomyces cerevisiae have been engineered to supply precursors for the synthesis of final products [31] .
Flavonoids form a class of phenolic compounds found in plants and can be classified into several groups, including flavanones, flavones, flavonols, anthocyanins, and isoflavones [34] . Among these groups, flavanones are starting compounds for the synthesis of other flavonoid groups. The flavanone naringenin can be synthesized from tyrosine by several enzymes, including tyrosine ammonia lyase (TAL), 4-coumaroyl coenzyme A ligase (4-CL), chalcone synthase (CHS), and chalcone isomerase (CHI) [36] ( Fig. 1 ). Genes for flavanone biosynthesis have been cloned and characterized in various plants [37] , making it possible to assemble these plants genes and reconstruct the flavonoid biosynthesis pathway in microorganisms for the synthesis of a target flavonoid [35] . The typical flavone apigenin is synthesized from naringenin (a flavanone) by flavone synthase (FNS). FNS has stereospecificity and uses only (S)-naringenin as a substrate [22] . Although apigenin itself has several biological activities, including anti-inflammatory [6] , antidepressant [28] , and anticancer activities [7] , the regioselective O -methylation of apigenin (to generate genkwanin) confers new biological activities, including antibacterial [4 , 23] , antiplasmodial [13] , radical scavenging [32] , chemopreventive [8] , and inhibiting 17β-hydroxysteroid dehydrogenase type 1 [3] activities. Although genkwanin has been shown to inhibit the development of cotton-pelletinduced granuloma in rat, the molecular mechanisms of this anti-inflammatory activity remain obscure [29] . Apigenin is found in various fruits and vegetables but the most common sources are parsley and celery [24] . Genkwanin has been identified only in Daphne genkwa [2] . Therefore, in order to explore novel biological functions of genkwanin, alternative approaches for genkwanin acquisition are necessary. Chemical synthesis is one alternative method to obtain genkwanin [21] . Conversion of apigenin into genkwanin using E. coli harboring O -methyltransferase was also successful [9] . By constructing flavonoid biosynthesis pathways in a microbe such as E. coli , diverse flavonoids have been synthesized from glucose [12 , 25 , 30] . However, most previous reports of flavonoid biosynthesis have focused on flavanones and their derivatives. Here, we synthesized the bioactive flavone derivative genkwanin from glucose using E. coli . By introducing six genes involved in genkwanin biosynthesis into an engineered E. coli strain, approximately 41 mg/l genkwanin was synthesized.
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Biosynthesis pathway of 7-O-methylapigenin (genkwanin) from tyrosine. TAL, tyrosine ammonium lyase; 4CL, 4-coumarate CoA ligase; CHS, chalcone synthase; FNS, flavone synthase; POMT7, apigenin 7-O-methyltransferase.
Materials and Methods
- Constructs
The TAL gene from Saccharothrix espanaensis was cloned as previously described [12] . The 4CL ( Os4CL ) and CHS ( PeCHS ) genes were also cloned as previously described [11 , 15] . Both Os4CL and PeCHS were subcloned into the Eco RI/ Not I sites and Nde I/ Kpn I sites of the pCDFDuet vector (Novagene), respectively, and the resulting construct, in which both Os4CL and PeCHS are controlled by an independent T7 promoter, was named pCpPeCHS-pOs4CL. In this construct, both genes are controlled by different T7 promoters. The construct in which both PeCHS and Os4CL are controlled by one T7 promoter was named pCpPeCHS-Os4CL. CHI from Medicago truncatula (GenBank No. XM_003592713) was cloned and inserted into the Nde I/ Kpn I sites of pC-pPeCHS-pOs4CL and pC-pPeCHS-Os4CL. The resulting constructs were named pC-pPeCHS-pOs4CL-pMtCHI and pCpPeCHS-Os4CL-pMtCHI. Flavone synthase (FNS; AY230247) was cloned from parsley [22] and apigenin 7 - O -methyltransferase (POMT7; TC29789) was previously cloned [9] . FNS was subcloned into the Nde I/ Kpn I site of the pET-Duet vector (pE-FNS), and POMT7 was then subcloned into the Sal I/ Not I sites of pPET-Duet containing FNS (pE-POMT7-FNS). Plasmids pA-SeTAL, pA-aroGSeTAL-TyrA, and pA-aroG fbr -TAL-tyrA fbr were constructed previously [11] .
- Production of Apigenin and Genkwanin
To measure the production of apigenin from p -coumaric acid, the E. coli transformant was grown in Luria-Bertani (LB) broth containing 50 μg/ml of chloramphenicol and spectinomycin at 37℃ for 18 h. The culture was inoculated to a fresh LB medium containing 50 μg/ml of chloramphenicol and spectinomycin and incubated with shaking at 37℃ until an OD 600 of 0.8 was attained. IPTG was added to the culture at a final concentration of 1 mM and the culture was allowed to incubate with shaking at 18℃ for 18 h. The cells were harvested and resuspended with M9 medium containing 2% glucose, 0.2% yeast, 50 μg/ml of chloramphenicol, 50 μg/ml spectinomycin, 1 mM IPTG, and 300 μM of p -coumaric acid. The resulting culture was incubated at 30℃ for 24 h with shaking at 180 rpm. The culture was analyzed by high-performance liquid chromatography (HPLC) as described previously [9] .
E. coli harboring pE-POMT7-FNS was used for the production of genkwanin from naringenin. After induction of the proteins as described above, cells were collected and resuspended in fresh LB containing 50 μg/ml ampicillin. Naringenin (100 μM) and IPTG (1 mM) were added to the culture, and the culture was incubated at 30℃ with shaking at 180 rpm for 15 h. The culture was analyzed by HPLC.
To synthesize genkwanin from glucose, an overnight culture of E. coli transformant was inoculated into fresh LB medium and grown until OD 600 = 1.0. Cells were harvested and resupended in M9 medium containing 2% glucose, 2% yeast extract, 1 mM IPTG, and 50 μg/ml antibiotics. Cells were grown at 30℃ for 24 h with shaking. The reaction products were analyzed by HPLC using an Ultimate 3000 HPLC (Thermo Scientific, USA). The separation condition was as described previously [10] .
ESI-MS analyses were performed, on a LCQ fleet instrument (Thermo Scientific, Waltham, MA, USA) coupled to an Ultimate 3000 HPLC system, in the negative-ion mode within the m / z range 100–500 and processed with Xcalibur software (Thermo Scientific). The operating parameters were as follows: spray voltage 4.5 kV, sheath gas 15 arbitrary units, auxiliary gas 10 arbitrary units, heated capillary temperature 275℃, capillary voltage -15 V, tube lens -110 V. Tandem (MS2) or triple (MS3) mass spectrometry analysis was conducted with scan-typeturbo data-dependent scanning (DDS), and the fragment spectra were produced using 35% of normalized collision energies.
Results and Discussion
- Optimization of Apigenin Production
Genkwanin is synthesized from apigenin by 7- O -methylation of POMT7. The yield of apigenin is therefore critical to the subsequent yield of genkwanin. Apigenin is synthesized from p -coumaric acid by four enzymes (4CL, CHS, CHI, and FNS; Fig. 1 ). In E. coli , conversion of naringenin chalcone to naringenin occurs spontaneously and, therefore, the CHI that catalyzes this step is not required. We introduced three genes ( Os4CL , PeCHS , and FNS ) into E. coli (Strain B-AP1 in Table 1 ) and tested if BAP1 synthesized apigenin from p -coumaric acid. Analysis of the culture filtrate by HPLC and mass spectrometry showed that apigenin was synthesized (data not shown).
Plasmids and strains used in the present study.
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Plasmids and strains used in the present study.
It is known that FNS uses ( S )-naringenin as a substrate [22] and that the reaction product of CHI from naringenin chalcone is ( S )-naringenin. Therefore, the final yield of apigenin might be higher when CHI was used in the biosynthetic pathway of naringenin. We made two E. coli transformants (B-AP1 and B-AP2). The B-AP1 contained three genes ( CHS , 4CL , and FNS ), in which naringenin chalcone is spontaneously converted into naringenin, with both ( R )- and ( S )-naringenins being generated. The other strain, B-AP2, harbored CHI as well as the three genes ( CHS , 4CL , and FNS ). In B-AP2, it would be expected that naringenin chalcone is converted into ( S )-naringenin, which would serve as a substrate for FNS. We tested the production of apigenin using B-AP1 and B-AP2 ( Fig. 2 A). B-AP2 produced more apigenin (23 mg/l) than B-AP1 (13 mg/l). This indicates that CHI converted naringenin chalcone into ( S )-naringenin, which could then be used as a substrate by FNS.
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(A) Production of apigenin by different Escherichia coli strains and (B) effect of tyrosine on the production of genkwanin.
4CL and CHS catalyze the first two steps of apigenin biosynthesis. Therefore, coordinated expression of 4CL and CHS in E. coli would be critical to the final yield of apigenin. We made a construct in which 4CL and CHS were cloned in an operon ( i.e. , one promoter controls the expression of both 4CL and CHS ; pC-pPeCHS-Os4CL in Table 1 ), which was named B-AP3. The production of apigenin in B-AP3 was compared with that of B-AP2 ( 4CL and CHS are controlled by independent T7 promoter; pC-pPeCHSpOs4CL). As shown in Fig. 2 A, the yield of apigenin was greater in B-AP3 (30 mg/l) than in B-AP2 (23 mg/l).
Production of Genkwanin from Glucose in E. coli
We showed that the four genes ( Os4CL , PeCHS , MtCHI , and FNS ) worked properly to synthesize apigenin from p -coumaric acid. In order to synthesize genkwain from glucose, two additional genes ( TAL and apigenin 7- O -methyltransferase ( POMT7 )) were needed. TAL uses tyrosine to make p -coumaric acid, and POMT7 catalyzes the conversion of apigenin to genkwanin. The six genes of the genkwanin biosynthetic pathway were introduced into E. coli (B-AP4) and the production of genkwanin was examined. As shown in Fig. 3 B, HPLC spectra generated from E. coli transformants harboring the six genes showed several peaks. One of these peaks (at 12.5 min) had the same retention time as a genkwanin standard. The molecular mass of the peak at 12.5 min was 284 Da ( Fig. 3 E), which corresponded to that of a standard genkwanin ( Fig. 3 D). Besides the molecular ion peak of [M-H]-, the fragmentation patterns at m / z 283 and the MS2 ( m / z 268) and MS3 ( m / z 240) of P5 ( Fig. 3 E) were indistinguishable to those of the standard genkwanin ( Fig. 3 D). In addition, P5 had a similar UV-spectrum with standard genkwanin ( Fig. 3 C). These results suggested that genkwanin was synthesized from glucose in the E. coli transformant.
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HPLC and MS spectra of reaction products from B-AP4. (A) HPLC profile of standard genkwanin; (B) HPLC profile of reaction products. P1 was identified to be p-coumaric acid by comparing with authentic p-coumaric acid. P2 and P3 were likely to be bis-noryangonin (BNY) and naringenin chalcone, respectively. P4 was identified to be apigenin by comparing with authentic apigenin. P5 was identified to be genkwanin. (C) UV spectra of authentic genkwanin and reaction products. (D) MS1, MS2, and MS3 spectra of standard genkwanin (G). (E) MS1, MS2, and MS3 spectra of reaction product (P5).
The content of tyrosine in E. coli is critical, because p -coumaric acid is synthesized from tyrosine. The aroG (3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase) that condenses phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to form DAHP, and tyrA (chorismate mutase/prephenate dehydrogenase) that converts prephenate into 4-hydroxy-phenylpyruvate are the rate-limiting steps of tyrosine biosynthesis [19] . Therefore, overexpression of these two genes in E. coli increases the tyrosine content. In addition, cells carrying feedback inhibition resistance versions of these two genes ( aroG fbr and tyrA fbr ) were able to produce more tyrosine than with wild-type aroG and tyrA [20] . Three E. coli transformants (B-AP4 ~ B-AP6 in Table 1 ) were made and the production of apigenin was examined. As expected, B-AP6 (23 mg/l) produced more genkwanin than E. coli harboring either B-AP4 (7 mg/l) or B-AP5 (10 mg/l) ( Fig. 2 B). HPLC profiles of the reaction product from E. coli harboring pA-aroG fbr -TAL-tyrA fbr still contained apigenin (data not shown). However, lower amounts of apigenin were observed in BAP4 and B-AP5. These results revealed that introducing the feedback-insensitive aroG and the tyrA genes increases the production of flavones. Therefore, introducing the P7OMT gene in the E. coli harboring pA-aroG fbr -TAL-tyrA fbr would likely increase genkwanin production.
For the production of genkwanin from glucose, POMT7 was transformed into B-AP6 and the resulting transfomant was named B-GK. Using B-GK, the production of genkwanin from glucose was examined. Initial cell density was first determined. The cell density was adjusted to OD 600 = 0.5, 1.0, 1.5, and 2.0. The production of genkwanin was analyzed after 24 h incubation at 30℃. The cell density of OD 600 = 1.0 showed the highest yield among the tested cell densities, followed by OD 600 = 0.5, 1.5, and 2.0. Using B-GK at OD 600 = 1.0, the effect of temperature on the production of genkwanin was evaluated at 25℃, 30℃, and 37℃. The incubation temperature at 30℃ gave a higher yield than 25℃ or 37℃. Using the optimized cell density and reaction temperature, the production of genkwanin from glucose using the strain B-GK was monitored for 36 h. Both apigenin and genkwanin were produced rapidly. However, as observed above, apigenin was synthesized more rapidly than genkwanin. The highest production was observed at 30 h, at which time approximately 41 mg/l genkwanin was produced, while approximately 55 mg/l apigenin was remaining ( Fig. 4 ). At 36 h, the yields of both apigenin and genkwanin had declined. At the time, E. coli growth also started declining, which indicated that cells began to die. It seemed that some E. coli cell debris such as lipids and fatty acids inhibited the extraction of apigenin and genkwanin from the culture, which lowered the final yield after 36 h.
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Production of genkwanin by Escherichia coli strain B-GK. We monitored cell growth (filled square), apigenin production (filled circle), and genkwanin production (empty circle), periodically.
Apigenin has been synthesized from p -coumaric acid in Saccharomyces cerevisiae with a final yield of approximately 3.2 mg/l [17] . Apigenin was also synthesized from glucose in E. coli with a yield of 13 mg/l [25] . If it is assumed that there was no degradation during the biosynthesis of genkwanin, approximately 100 mg/l of apigenin was synthesized from glucose in the current study. The final apigenin yield would likely have been higher if apigenin was synthesized from p -coumaric acid. These differences in final yield could be a result of the host organism ( E. coli and yeast) and/or different sources of flavone biosynthesis genes.
Apigenin was biotransformed into genkwanin by Kim et al. [9] with a final yield of approximately 17 mg/l, and some apigenin was not converted into genkwanin. The synthesis of genkwanin from glucose reported here was higher than that from apigenin. Therefore, the current approach could be applicable to the synthesis of apigenin derivatives from cheap or available precursors.
Until recently, flavonoid synthesis from glucose using E. coli was targeted to the synthesis of naringenin. Although naringenin contains several biological activities [14 , 16 , 26 , 38] , flavone and flavonol derivatives have novel activities that are not found in naringenin. Findings from the current study and others [27 , 33 , 38] show that flavone and flavonol derivatives can be synthesized using E. coli or yeast. Although it is still challenging, it should now be possible to synthesize a particular bioactive flavonoid using E. coli .
This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ01114801), Rural Development Administration, Republic of Korea, by the Basic Science Research Program (NRF-2012R1A1A2041132), and by the Priority Research Centers Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2009-0093824)
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