Metabolic Engineering in Fragaria x ananassa for the Production of

Chapter 19

Downloaded by UNIV OF ARIZONA on December 18, 2014 | http://pubs.acs.org Publication Date (Web): March 18, 2010 | doi: 10.1021/bk-2010-1035.ch019

Metabolic Engineering in Fragaria x ananassa for the Production of Epiafzelechin and Phenylpropenoids W. Schwab,* T. Hoffmann, and M. Griesser Biomolecular Food Technology, Technical University Munich, Hochfeldweg 1, 85354 Freising, Germany *[email protected]

To confirm the in vivo function of a recently cloned strawberry UDP-glucose:anthocyanidin glucosyltransferase (FaGT1) gene, we downregulated its expression in strawberry fruit with a transient RNA interference (RNAi) method. In about one third of the injected fruits this led to a significant downregulation of FaGT1 transcript levels consistent with reduced concentrations of anthocyanin pigments. In contrast, significant levels of epiafzelechin - formed by anthocyanidin reductase (ANR) from pelargonidin - were identified in FaGT1 silenced fruits. Thus, the redirection of the metabolic flux towards the flavan-3-ol through downregulation of FaGT1 offers a new method to increase the levels of this bioactive metabolite in fruit crops. In addition a dormant biosynthetic pathway of strawberry volatiles was uncovered by using the transient RNAi system. Silencing of the flavonoid pathway by downregulation of the chalcone synthase gene (FaCHS) provided phenylpropenoids for the biosynthesis of chavicol and eugenol in the fruits. These studies serve as foundation for metabolic engineering of strawberry flavor.

Strawberry (Fragaria x ananassa) is one of the most popular fruit crops worldwide and is grown in all temperate regions of the world. Much of the popularity of this fruit is due to the attractive flavor and the deep red color. In © 2010 American Chemical Society In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


addition to traditional nutrients such as carbohydrates, vitamins and minerals, strawberries are also rich in phenolic compounds such as flavonoids e.g. epiafzelechin, which is the focus of intense study due to its proliferative effects on osteoblastic cells and selective inhibitory activities against cyclooxygenase-1 (COX-1) over COX-2 (1, 2). The majority of flavonoids in strawberries are anthocyanins, the compounds responsible for the blue, red and purple hues of berries, grapes and other fruits. The genetic and biochemical information about the last steps in anthocyanin biosynthesis in strawberry fruit is still limited although the chemical composition of the anthocyanins has been studied in detail (3). Stable products of the anthocyanin pathway are formed when a glycosyltransferase attaches a sugar molecule to the hydroxyl group at position 3 on the anthocyanidin aglycones. Anthocyanin concentration and flavonoid 3-O-glucosyltransferase activity increase in parallel during fruit ripening (4). Recently, anthocyanidin glucosyltransferase genes have been isolated from strawberry fruits and the recombinant proteins have been characterized (3, 5). Anthocyanidin 3-O-glucosyltransferases have also been isolated from grapes, maize and flowers of many ornamental plants in which anthocyanins are the major determinants of flower color (6). The genome of the model plant Arabidopsis thaliana contains over 100 putative glycosyltransferase sequences, and most of the respective proteins have been expressed heterologously. Out of 91 tested, 29 glycosyltransferases have been reported to accept the flavonol quercetin but only one enzyme from Arabidopsis glycosylates anthocyanidins (7). Phenylpropanoid derivatives like eugenol are assumed to be key flavor components of wild strawberry. It is now known that acetates of hydroxycinnamyl alcohols are the precursors of phenylpropanoid-derived flavor compounds, and the corresponding eugenol synthase (EGS) and isoeugenol synthase (IGS) genes have been functionally characterized in both basil (Ocimum basilicum) and petunia (Petunia hybrid acv. Mitchell) (8, 9). Since hydroxycinnamoyl derivatives accumulate after down-regulation of the chalcone synthase gene (FaCHS) in strawberry fruit, which is the branching point between the flavonoid and the phenylpropanoid pathway (10) we proposed that the anthocyanin pathway can be diverted for the production of phenylpropenes. In the present study we down-regulated an anthocyanindin 3-Oglucosyltransferase gene in strawberry fruit with a recently developed RNAi method to confirm its function in planta and successfully redirected the flavonoid pathway to the phenylpropene pathway by the simultaneous silencing of FaCHS and overexpression of either EGS or IGS.

Materials and Methods Chemicals Solvents and reference compounds were obtained from Sigma, Aldrich, Fluka, Riedel de Haën (all Taufkirchen, Germany), Merck (Darmstadt, Germany) 294 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

or Roth (Karlsruhe, Germany). Anthocyanidins were purchased from Polyphenols Laboratories (Sandnes, Norway).


Construction of pBI-FaGT1i A blunt-end PCR product of the coding sequence of FaGT1 was produced using a high-fidelity polymerase (Finnzymes, Espoo, Finland). The same primers were used to subclone the expression vector. The amplicon was digested with SpeI yielding a fragment of ca. 500 bp which was used for ligation into the binary vector pBI121 that contained XbaI/NheI and SpeI/SacI (Ecl136II) restriction sites separated by an intron from strawberry (10) The procedure yielded the intronhairpin construct pBI-FaGT1i.

Vector Construction of pBI-EGS and pBI-IGS The O. basilicum EGS (accession DQ372812) and P. hybrida IGS (accession DQ372813) coding region were cloned into the binary vector pBI121 replacing GUS (10).

Plant Transfection Transient transfection of strawberry fruit with A. tumefaciens strain AGL0 suspensions containing pBI-EGS, pBI-IGS, pBI-CHSi or pBI-FaGT1i was performed according to a published procedure (10). Strawberry (Fragaria x ananassa) cv. Elsanta plants were grown under standard conditions at 25°C and a 16 h photoperiod. Control experiments were carried out by injecting strawberry fruits with Agrobacterium tumefaciens AGL0 cells carrying a pBI-Intron control construct (10).

Extraction of Volatiles Two g freeze-dried strawberry fruit powder was homogenized with 20 mL of water and centrifuged (5000 g, 10 min). Phenol (10 µg) was added as internal standard. The supernatant was loaded onto an Amberlite XAD-2 polymeric adsorbent (20-60 mesh; 100 g; Aldrich) column. The column was rinsed with 50 mL water and volatiles were eluted with 70 mL diethyl ether. The extract was dried over anhydrous sodium sulphate, concentrated using a Vigreux column, reduced to 50 µl under a stream of N2 and were analyzed by GC-MS.

295 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Figure 1. Metabolite levels in strawberry fruits injected with Agrobacterium tumefaciens harboring pBI-FaGT1i for the down-regulation of FaGT1 and in control fruits injected with pBI-Intron. FW fresh weight.

Figure 2. Section of the flavonoid/anthocyanin pathway illustrating the accumulation of epiafzelechin, epicatechin and catechin due to the reduced activity of FaGT1. LAR leucoanthocyanidin reductase, ANS anthocyanidin synthase, ANR anthocyanidin reductase, FaGT 1 Fragaria x ananassa glucosyltransferase

296 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Metabolite Analysis Freeze-dried strawberry powder (50 mg) was extracted with 250 µL methanol containing 0.2 mg/mL of the internal standars 4-methylumbelliferylβ-D-glucuronide. Methanol was removed and the extract was re-dissolved in 35 µL water for analysis by LC-MS. Metabolite quantification was performed using the QuantAnalysis 1.5 software (Bruker Daltonics, Bremen, Germany) normalizing all results against the internal standard. Each analysis was performed in triplicate. Levels of metabolites determined in the RNAi experiment were displayed as box and whisker plots using the software SigmaPlot 8.0 (Systat Software, Erkrath, Germany). Statistical significance levels were calculated with the Wilcoxon-Mann-Whitney-U-Test (11) using the software package R (www.r-project.org).

Results Accumulation of Epiafzelechin The full-length open reading frame of a UDP-glucose:anthocyanidin glucosyltransferase (FaGT1) was cloned from Fragaria x ananassa cv. Elsanta and heterologously expressed in Escherichia coli (5). The recombinant protein (FaGT1) glucosylated anthocyanidins (pelargonidin, cyanidin) and flavonols (kaempferol, quercetin) in vitro. To elucidate the substrates and function of the glucosyltransferase in strawberry fruit, the expression of FaGT1 was down-regulated by RNA interference (RNAi) using a recently developed transient method (10). One third of the fruits injected with the RNAi-inducing vector pBI-FaGT1i were phenotypically different from fruits injected with the control construct pBI-Intron. The color of the RNAi fruits was generally less intense compared to the bright red color of the the control fruits. A significant down-regulation of FaGT1 transcript expression was determined in the pBI-FaGT1i fruits in comparison to the transcript levels in control fruits. Levels of anthocyanidins, flavonols and phenylpropanoid glucose esters were determined by LC-MS. To identify metabolites with significantly altered concentrations statistical methods such as the Wilcoxon-Mann-Whitney-U-test were applied. In fruits injected with pBI-FaGT1i the level of pelargonidin 3-O-glucoside malonate and pelargonidin 3-O-glucoside decreased significantly (P

Metabolic Engineering in Fragaria x ananassa for the Production of

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