Microbial production of natural raspberry ketone

1 Introduction

Raspberry ketone [4-(4-hydroxyphenyl)-butan-2-one] is akey flavour molecule with typical raspberry flavour char-acteristics and a low odour threshold. Raspberry ketone isone of the most expensive flavour components used in thefood industry. Up to $20 000/kg may be paid for the natu-ral compound, which is found in raspberries and also inother fruits such as peaches, grapes, apples and variousberries, vegetables (e.g., rhubarb) and in the bark of trees(e.g., yew, maple and pine). Raspberry ketone can be usedin the aroma formulation of, for instance, strawberry, kiwi,cherry and other berries. However, none of the fruits list-ed are currently used to obtain raspberry ketone, as the

low content in these fruits makes the extraction and pu-rification process unprofitable. For instance, if raspberryketone was extracted from raspberry fruit, one would facethe limited availability of this fruit outside the growingseason, and its price. Yields are typically very low: thecontent is usually 1–4 mg/kg raspberries [1].

Chemical synthesis of raspberry ketone can beachieved via the condensation of p-hydroxybenzaldehydewith acetone [2]. However, according to food laws, thisproduct of chemical synthesis cannot be marketed as a“natural” flavour compound [3]. Therefore, we set out toclone plant genes involved in the biosynthesis of raspber-ry ketone, and express the pathway in micro-organisms,to produce natural raspberry flavour compounds.

In the fruit of raspberry (Rubus idaeus), the naturalsynthesis of raspberry ketone initiates with the phen-ylpropanoid pathway [4] (Fig. 1). In the first step,coumaroyl-CoA (which is present in many plant tissues asintermediate of the lignin biosynthetic pathway) is con-densed with one malonyl-CoA to form p-hydroxybenza-lacetone [4-(4-hydroxyphenyl)-but-3-ene-2-one]. The en-zyme catalysing this step is called benzalacetone syn-thase (BAS). In the second step, the double bond in p-hy-

Research Article

Microbial production of natural raspberry ketone

Jules Beekwilder1, Ingrid M. van der Meer1, Ole Sibbesen2, Mans Broekgaarden1, Ingmar Qvist3, Joern D. Mikkelsen2 and Robert D. Hall1

1 Plant Research International, Wageningen, The Netherlands2 Danisco Innovation, Copenhagen, Denmark3 Danisco Flavours, Wellingborough, England

Raspberry ketone is an important compound for the flavour industry. It is frequently used in prod-ucts such as soft drinks, sweets, puddings and ice creams. The compound can be produced by or-ganic synthesis. Demand for “natural” raspberry ketone is growing considerably. However, thisproduct is extremely expensive. Consequently, there is a remaining desire to better understandhow raspberry ketone is synthesized in vivo, and which genes and enzymes are involved. With thisinformation we will then be in a better position to design alternative production strategies such asmicrobial fermentation. This article focuses on the identification and application of genes poten-tially linked to raspberry ketone synthesis. We have isolated candidate genes from both raspberryand other plants, and these have been introduced into bacterial and yeast expression systems.Conditions have been determined that result in significant levels of raspberry ketone, up to 5 mg/L.These results therefore lay a strong foundation for a potentially renewable source of “natural”flavour compounds making use of plant genes.

Keywords: Raspberry ketone · Natural flavour · Fermentation

Correspondence: Dr. Jules Beekwilder, PO box 16, 6700 AA Wageningen,The NetherlandsE-mail: [email protected]: +31-317-418094

Abbreviations: BAR, benzalacetone reductase; BAS, benzalacetone syn-thase; CHS, chalcone synthase; 4CL, 4-coumarate-coenzyme A ligase; STS,stilbene synthase

Received 23 April 2007Revised 13 June 2007Accepted 27 June 2007

BiotechnologyJournal DOI 10.1002/biot.200700076 Biotechnol. J. 2007, 2, 1270–1279

1270 © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1271

Biotechnol. J. 2007, 2, 1270–1279 www.biotechnology-journal.com

droxybenzalacetone is reduced, resulting in raspberry ke-tone. This step is catalysed by benzalacetone reductase(BAR), and requires the presence of NADPH.

There is evidence that, in raspberry, a relationship ex-ists between BAS and chalcone synthase (CHS). BAS ac-tivity during fruit ripening parallels pigment formation [4].The colour in ripe raspberry fruit is predominantly deter-mined by the formation of red anthocyanins, which arederived from the phenyl-propanoid pathway [5]. CHS is akey enzyme in this pathway. CHS is a member of the typeIII polyketide synthase family, and BAS is also likely to bea member of this family. Whereas BAS condenses onemalonyl-CoA with one coumaroyl-CoA to form p-hydrox-ybenzalacetone, CHS condenses three malonyl-CoA unitswith one p-coumaroyl-CoA to form naringenin chalcone.This naringenin chalcone may then be further trans-formed into anthocyanins such as cyanidin sophoroside.Moreover, a CHS homologue from rhubarb has beencloned, which, after expression in Escherichia coli andpurification of the recombinant protein, showed in vitrosynthesis of p-hydroxybenzalacetone, but not of narin-genin [6]. However, such an enzyme has never been iden-tified in raspberry.

The NADPH-dependent reductase BAR has to datehardly been investigated. It is known that p-hydroxyben-zalacetone can be transformed to raspberry ketone by fun-gi or yeasts such as Pichia, Saccharomyces and Beauve-ria [7]. However, no gene has yet been identified that isconnected to this enzyme activity. Also, to our knowl-edge, no such activity has been reported for bacteria.

Attempts to biosynthesise raspberry ketone have pre-viously been described. Kosjek et al. [8] described amethod using a micro-organism that has a secondary al-cohol dehydrogenase (ADH), which can convert the pre-

cursor betuloside into raspberry ketone. However, betulo-side is a compound of limited availability.

We describe a method in which a raspberry CHS is in-corporated into a raspberry ketone production pathway inE. coli. Successful production of raspberry ketone wasachieved.

2 Materials and methods

2.1 Materials

Raspberries were obtained from the PPO-field station inRandwijk (The Netherlands). The p-hydroxybenzalace-tone was from Pfaltz & Bauer (Waterbury, CT, USA), narin-genin chalcone, naringenin and raspberry ketone werefrom Apin Chemicals Ltd. (Abingdon, UK), while all otherchemicals were from Sigma (St Louis, MO, USA). Restric-tion enzymes were from Invitrogen. All DNA manipula-tions were done in E. coli XL-1 blue. Expression was per-formed in E. coli BL21-CodonPlus-RIL, supplied with plas-mid pREP4.

2.2 Cloning of raspberry RiCHS

Total RNA was isolated from ripe Tulameen raspberries.Raspberries (5 g) were homogenized in liquid nitrogen,and incubated for 1 h at 65°C in 50 mL extraction buffer(2% cetyltrimethylammonium bromide, 100 mM Tris pH 8.2, 1.4 M NaCl, 20 mM EDTA, 0.1% 2-mercaptoethanol).This was extracted twice with chloroform, and RNA wasprecipitated with 33 mL 10 M LiCl at 4°C overnight. Pre-cipitated RNA was dissolved in 1 mL TE buffer and ex-tracted with phenol/chloroform. Copy DNA was synthe-sized from 1 µg total RNA using a polyT primer accordingto standard procedures. Primers RiCHSa (5’-CAAGT-GAACCCAGCCATGGTCAAGTTGAAGCTGCCA-CACTG) and RiCHSs (5’-CTTTCTCCACAGACTCGA-GATGGTGACCGTCGATGAAGTC) and Pfu polymerase(Stratagene) were used to amplify the full protein codingregion of a CHS from raspberry (GenBank accessionAF292367; [9]). The fragment, 1136 nucleotides in size,was digested with NcoI and XhoI, and ligated into pRSETA (Invitrogen), yielding plasmid pRSETA-RiCHS.The sequence of the encoded protein was 98% identicalto the aromatic polyketide synthase in AF292367, with 7residues out of 391 differing. It is even more identical(99%, 3 out of 391 residues differ) to a protein called CHS6(AF400567), described in [10].

2.3 Mutagenesis of CHS

The DNA of pRSETA-CHS#1 was mutated by overlap-ex-tension PCR. Plasmid pRSETA-RiCHS was used as a tem-plate. Two PCR reactions were performed in parallel, oneusing oligonucleotides CHSmutA (5’-CGTCACCGAG-

S-CoA

O

HO

HO

O

S-CoA

O

HO

HO

OH

O

OH

O

raspberry ketoneanthocyanins

CHS BAS

OH

OBAR

OH

O

HO

phenylalanine

lignin

4CL

p-coumaric acid

coumaroyl-CoA malonyl-CoA

benzalacetonenaringenin chalcone

OH

Figure 1. Biosynthetic pathway of raspberry anthocyanins and raspberryketone. The biosynthesis of these compounds starts by the coupling of p-coumaric acid to CoA by the 4CL enzyme. Subsequently, the coumaroyl-CoA is converted into either naringenin chalcone, or to p-hydroxybenza-lacetone, depending on the number of malonyl-CoA units added.

TATGGCTTGGCCCACAAG) and RiCHSs, and the otherusing oligonucleotides RiCHSa and CHSmutS (5’-GC-CAAGCCATACTCGGTGACGGTGCTGC) using Pfupolymerase. Resulting fragments were purified from geland 100 ng of each fragment was used as a template forfusion PCR using oligonucleotides RiCHSa and RiCHSs.The resulting fragment was cloned into pRSETA usingXhoI and NcoI restriction enzymes yielding plasmidpRSET-RiCHS-Mut. Sequence analysis of this plasmidconfirmed the presence of the designed mutations.

2.4 Plasmids pAC-4CL-RiCHS, pAC-4CL-RiCHS-Mut andpAC-4CL-PhCHS

RiCHS and RiCHS-Mut DNA was amplified from the con-structs described above using oligonucleotides CHS-DUETS (5’-TATATAGATCTTGTGACCGTCGATGAAG)and CHSDUETA (5’-TATATGGTACCTCAAGTTGAAGCTGCCAC). The amplification products were digested us-ing restriction enzymes KpnI and BglII, and cloned intopACYC-DUET-1 (Novagen). The resulting plasmids weretermed pAC-RiCHS and pAC-RiCHS-Mut. The Petunia xhybrida CHS gene was cleaved from plasmid pFLAP600([11]; kindly provided by Arnaud Bovy) using restrictionenzymes BglII and XhoI, and ligated into pACYC-DUET-1,yielding plasmid pAC-PhCHS.

The tobacco 4-coumarate-coenzyme A ligase (4CL)-2 cDNA was added to all three constructs, and to pACYC-DUET-1 as described in Beekwilder et al. [12], yieldingplasmids pAC-4CL-RiCHS, pAC-4CL-RiCHS-Mut, pAC-4CL-PhCHS and pAC-4CL. Plasmid pAC-4CL-VvSTS hasbeen described elsewhere [12].

RiCHS was amplified from pAC-4CL-CHS by primersCHSNco (5’-TATAGGTACCTCAGTTGAAGCTGCCACACTG) and CHSEco (5’-TATACTCGAGAATTCTCAAGTTGAAGCTGCCACAC). 4CL was amplified from pAC-4CL-CHS using oligonucleotides DUETS (5’-GGATCTCGACGCTCTCCCT) and DUETASEco (5’-TATGAATTCGATTATGCGGCCGTGTACAA). Both amplification products weredigested with EcoRI and NcoI and cloned into plasmidpBAD-HisA (Invitrogen), to yield plasmids pBAD-RiCHSand pBAD-4CL.

2.5 In vitro BAS assay

Plasmids pRSET-A, pRSET-RiCHS and pRSET-RiCHS-Mut were used for transformation of E. coli BL21. Individ-ual transformants were inoculated in LB medium with 1%glucose and 100 mg/L ampicillin and grown overnight at37°C. The overnight cultures were diluted 1:100 in 50 mL2xYT medium with 50 mg/L ampicillin, and further incu-bated at 37°C. At a density (measured at 600 nm) of 0.4,IPTG was added to 1 mM and cultures were grownovernight at 28°C and 250 rpm. The next day, the cultureswere centrifuged and cell pellets were dissolved in 2 mLbuffer A (50 mM Tris pH 8, 300 mM NaCl, 10 mM imida-

zole). After sonication and centrifugation, the super-natant was transferred to a NiTED Protein Purificationcolumn (ActivMotiv), and treated as prescribed by themanufacturer. About 10 µg eluted protein was mixed in200 µL 50 mM Tris pH 7.0, with p-coumaroyl-CoA (10 µM,kindly provided by Stefan Martens) and malonyl-CoA (50 µM) and incubated for 1 h at 30°C, after which the reaction was stopped by addition of 400 µL methanol with 0.75% formic acid, and samples were analysed onHPLC (see below).

2.6 Bioconversion of p-coumaric acid by E. coli

Plasmids were transferred into E. coli BL21 cells and se-lected on the appropriate antibiotics (30 mg/L chloram-phenicol in case of pAC-plasmids; 100 mg/L ampicillin incase of pRSET plasmids or pBAD plasmids) in the pres-ence of 1% glucose. Overnight cultures of individualcolonies were made in LB medium with antibiotics and1% glucose. For expression, the overnight cultures werediluted 1:100 in 50 mL 2xYT medium, and grown at 37°Cuntil a density (measured at 600 nm) of 0.4 was reached.At this point, p-coumaric acid was added to 3 mM andIPTG to 1 mM, and cultures were grown overnight at 28°Cand 250 rpm. The next day (after 20 h), the cultures weretransferred to separation funnels, and 20 mL ethyl acetatewas added under vigorous mixing. The upper (ethyl ac-etate) phases were collected in centrifuge tubes, sonicat-ed for 10 min and centrifuged for 5 min at 1200 × g. Fromthe clear ethyl acetate phases, solvent was aspired undera nitrogen flow. Dried product was dissolved in 1 mL eth-yl acetate, and analysed on HPLC and GC-MS.

2.7 Expression in yeast

For expression in yeast, the RiCHS gene was amplifiedfrom pRSET-RiCHS and pRSET-RiCHS-Mut using oligo-nucleotides ESCCHSfw (5’-ATATGGATCCACCATGGTGACCGTCGATGAAG) and ESCCHSre (5’-TATATACTCGAGTCAAGTTGAAGCTGCCAC), and cloned into vectorpESC-trp (Stratagene) using BamHI and XhoI restrictionenzymes. This yielded plasmids pESC-RiCHS and pESC-RiCHS-Mut. Subsequently, the 4CL fragment was re-cloned from pAC-4CL-CHS into pESC-RiCHS and pESC-RiCHS-Mut using EcoRI and NotI restriction enzymes.The resulting plasmids (pESC-4CL-CHS and pESC-4CL-CHS-Mut) and control vector pESC-Trp were introducedinto the yeast S. cerevisiae YPH499, where they weremaintained by tryptophan auxotrophy complementation.Cultures (50 mL, YPGal-medium) were grown in the pres-ence of galactose, to induce recombinant gene expres-sion. At a culture density of 0.4 (at 600 nm), p-coumaricacid (3 mM) was added, after which the cultures aregrown overnight at 30°C and 250 rpm. After 20 h, the cul-tures were extracted with ethyl acetate as describedabove, and extracts were analysed on HPLC.

BiotechnologyJournal Biotechnol. J. 2007, 2, 1270–1279




2.8 Fermentation

E. coli BL21 containing the pAC-4CL-RiCHS vector wasgrown in an 800-mL fermenter (Braun Biotech, Biostat Q),using 2xYT as growth medium. Temperature was kept at25°C, and pH at 7.5. Samples were taken at various timepoints. Samples were extracted with ethyl acetate andanalysed on a Perkin Elmer Autosystem XL GC fitted withan FID detector and a Varian FactorFour VF-5ms 30 m ×0.25 mm column with helium as carrier. Injector tempera-ture was 270°C, detector temperature 300°C. Oven tem-perature was at 50°C for the first 2 min after injection, andthereafter raised by 15°C/min to 230°C. Ethylvanillin (100 mg/L) was used as an internal standard in the samples. The chromatograms were normalized accordingto the standard, and raspberry ketone was measured using a standard curve generated by using authenticcompounds.

2.9 HPLC and GC-MS analysis

From each extract, 10 µL was injected into an HPLC sys-tem using a Luna 3u C18(2) 150 × 4.6 mm column (Phe-nomenex). The HPLC set-up was composed of a Waters600 controller and a Waters 996 Photo Diode Array detec-tor. The column was used at 40°C, flow rate 1 mL/min. Theproducts were eluted with a linear gradient from 95%buffer A (0.1% formic acid in water) and 5% buffer B (100%acetonitrile) to 50% buffers A and B in 37 min.

Of each sample, 2 µL was analysed by GC-MS using agas chromatograph (5890 series II, Hewlett-Packard)equipped with a 30 m × 0.25 mm 5MS column (Hewlett-Packard) and a mass-selective detector. The GC was pro-grammed at an initial temperature of 45°C, with a ramp of10°C/min to 220°C and final time of 5 min and a constantcolumn flow of 1.0 mL/min. The injection port, interface,and MS source temperatures were 250°C, 290°C, and180°C, respectively. Compounds were detected in the se-lected-ion-monitoring mode (70 eV): m/z 107, 121 and 164.Masses at m/z 107 and 164 are typical for raspberry ke-tone. The peak surface at m/z 107, eluting around 15 min,

was used as a measure for the amount of raspberry ketoneformed. Values were normalized by use of an internal stan-dard [1 µg/mL 4-(4-methoxyphenyl)-2-butanone (Aldrich)]in the ethyl acetate used for extraction, which was de-tected at m/z 121.

3 Results

3.1 CHS and BAS activity in vitro

The first aim of this study was to obtain a CHS-like genefrom raspberry that could drive synthesis of p-hydroxy-benzalacetone. A known CHS gene was amplified fromripe raspberry (Rubus idaeus) fruit cDNA. The cDNA wascloned into the E. coli expression vector pRSETA (yieldingpRSET-RiCHS), in such a way that the CHS protein isfused to an N-terminal His-tag, which allows convenientpurification for enzymatic tests (Fig. 2). Subsequently,mutations were introduced into this plasmid (Fig. 2B),which were aimed at altering the properties of thecoumaroyl-CoA binding site at residues 214 (Leu) and 215(Phe) in RiCHS. The introduced mutations alter theseresidues to conform to those present in rhubarb BAS (Ileand Leu, respectively; Fig. 2B, [13]), and the resultingplasmid was referred to as pRSET-RiCHS-Mut (Fig. 2).Both wild-type and mutant CHS fusion proteins were pro-duced in E. coli, and subsequently purified using their N-terminal His-tag. They were then incubated withcoumaroyl-CoA and malonyl-CoA and products wereanalysed using HPLC. As expected from literature, theRiCHS protein converted the substrates mainly intonaringenin chalcone (not shown; [10]).

However, the mutant enzyme displayed poorly repro-ducible behaviour when it was tested a few times underthe same conditions. It never produced any naringeninchalcone, but in some tests, the formation of p-hydroxy-benzalacetone was observed. In others the mutant en-zyme appeared to be inactive, as was also reported for asimilar Scutellaria CHS mutant [13]. It was therefore con-ceived that the mutant RiCHS enzyme would perform bet-

4CL

RiCHS-MutHis

RiCHS

T7

T7 T7

4CL RiCHS-MutT7 T7

4CL PhCHST7 T7

4CL VvSTST7 T7

4CLT7

RiCHST7

RiCHSHisT7

4CLBAD

RiCHSBAD

RiCHSGal14CL Gal10

RiCHS-MutGal14CL Gal10 pESC-TrpTrppESC-4CL-CHS-Mut

pESC-TrpTrppESC-4CL-CHS

pBAD-HisAAmppBAD-RiCHS

pBAD-HisAAmppBAD-4CL

pACYC-DUET-1CampAC-RiCHS

pACYC-DUET-1CampAC-4CL

pACYC-DUET-1CampAC-4CL-VvSTS

pACYC-DUET-1CampAC-4CL-PhCHS

pACYC-DUET-1CampAC-4CL-RiCHS-Mut

pACYC-DUET-1CampAC-4CL-RiCHS

pRSET-AAmppRSET-RiCHS-Mut

pRSET-AAmppRSET-RiCHS

vectorselectionname 747 783Leu Val Gly Gln Ala Leu Phe Gly Asp Gly Ala Ala

RiCHS CTT GTG GGC CAA GCC TTG TTC GGT GAC GGT GCT GCA RiCHS-Mut CTT GTG GGC CAA GCC ATA CTC GGT GAC GGT GCT GCA

Leu Val Gly Gln Ala Ile Leu Gly Asp Gly Ala Ala

Figure 2. Constructs used in this study. (A) Indicated are the promoters(arrows), terminators (crosses), construct name, antibiotic resistance andvector. (B) The region from nucleotide 747 to 783 in RiCHS and RiCHS-Mut, and the encoded amino acid sequences. Altered residues are indicat-ed in bold.

A B

ter in an in vivo system, where circumstances (the cyto-plasmic environment) would be more constant and freshenzyme is continuously being synthesized.

3.2 E. coli and S. cerevisiae are hardly affected by raspberryketone

The suitability of two microbial hosts for producing rasp-berry ketone was investigated. Firstly, the maximum con-centrations of raspberry ketone and the intermediateproduct p-hydroxybenzalacetone that could be toleratedby cultures without severely affecting microbial growthwere determined. In Table 1, the concentrations of eachcompound are given leading to a reduction of 50% in thedensity of the culture (IC50). Inhibitory concentrations arebetween 0.1 and 1 g/L, indicating that microbial growthis unlikely to be significantly affected when these mi-crobes are used in a production system. Thus, it appearsthat both E. coli and yeast are not very sensitive for eitherintermediate or product of raspberry ketone synthesis.

3.3 Bioconversion of benzalacetone by E. coli

The activity of BAR can easily be recruited from fungi likeSaccharomyces [7]. However, no such reductase activityhas been documented for bacteria. To assess the reduc-tase activity of E. coli cultures, a fresh overnight culture ofE. coli BL21 was diluted 200-fold in 2xYT medium, sup-plemented with 10 µM p-hydroxybenzalacetone, and in-cubated overnight. The next day, raspberry ketone wasanalysed using GC-MS, and a concentration of 4 µM wasdetected. Consequently, the E. coli culture demonstrateda capacity to turn over 40% of p-hydroxybenzalacetoneinto raspberry ketone.

To further characterize the BAR activity of E. coli, afresh overnight culture of E. coli BL21 was divided into anextracellular fraction (centrifuged medium) and intracel-lular fraction (pelleted cells, resuspended in buffer, soni-cated and centrifuged again). In both fractions, hardly anyreductase activity was observed when incubated with 5 mM p-hydroxybenzalacetone. However, when 5 mMNADPH was added, the intracellular fraction was capableof producing 2.4 mM raspberry ketone in 30 min. The ex-

tracellular fraction remained inactive. Thus, it was con-cluded that E. coli BL21 contains intracellular, NADPH-dependent BAR activity.

3.4 Raspberry CHS produces benzalacetone in E. coli in vivo

A microbial production system for raspberry ketone wasdesigned. For the synthesis of raspberry ketone from asimple substrate, the micro-organism would need to (i)convert p-coumaric acid into coumaroyl-CoA, (ii) con-dense coumaroyl-CoA into p-hydroxybenzalacetone, and(iii) convert p-hydroxybenzalacetone into raspberry ke-tone (Fig. 1). From previous work it is known thatcoumaroyl-CoA can be provided in E. coli cells by expres-sion of the 4CL-2 gene from tobacco, and feeding with p-coumaric acid [12]. Moreover, it was also observed that E.coli contains an endogenous BAR activity. We thus aimedto establish the raspberry ketone synthesis pathway byintroducing the RiCHS-Mut protein, which seemed to beable to convert coumaroyl-CoA into p-hydroxybenzalace-tone. Wild-type RiCHS protein would serve as a control totest the performance of the system by monitoring narin-genin production.

To establish the putative production system, bothCHS variants (RiCHS and RiCHS-mut) were recloned intoE. coli vector pACYC-DUET-1, together with the 4CL-2gene. In this system, both genes (4CL-2 and RiCHS) areunder control of the T7 promoter. In a previous publica-tion, we described this system for stilbene synthase (STS)[12]. Both plasmids were expressed in E. coli BL21 andcompared to the empty vector.

To test the performance of the system, fresh culturesof pAC-4CL-RiCHS, pAC-4CL-RiCHS-Mut and pACYC-DUET were incubated overnight with 3 mM p-coumaricacid. Cultures were extracted with ethyl acetate, andHPLC-PDA analyses and GC-MS analyses were per-formed. Using HPLC analysis, a peak co-eluting withnaringenin was detected in the chromatogram at 280 nmof the pAC-4CL-RiCHS culture extract, but not in the oth-er extracts (Fig. 3A). This indicated that p-coumaric acidwas modified by the 4CL enzyme into coumaroyl-CoA,which was in turn accepted by the CHS enzyme as a sub-strate. The expected product from CHS, naringenin chal-cone, spontaneously converts into naringenin at neutralpH [14]. When the chromatogram at 312 nm was inspect-ed, a significant peak was observed at the position of p-hydroxybenzalacetone, with an absorption maximum at323 nm. This peak was not predominantly present in thecandidate BAS strain pAC-4CL-RiCHS-Mut extract, but,surprisingly, in the pAC-4CL-RiCHS extract, where it wasclearly distinguishable from the background (Fig. 3B). Theidentity of the product was confirmed as p-hydroxyben-zalacetone using MS (see below). Thus, we concludedthat the wild-type RiCHS was unexpectedly able to con-vert p-coumaric acid into p-hydroxybenzalacetone invivo.



Table 1. IC50 values of raspberry ketone and p-hydroxybenzalacetone inE. coli and yeasta)

p-Hydroxy-Raspberry ketone

benzalacetone IC50 (g/L)

IC50 (g/L)

E. coli BL21 0.3 ± 0.1 0.9 ± 0.2S. cerevisiae YPH499 0.10 ± 0.03 0.5 ± 0.2

a) Fresh overnight cultures of E. coli BL21 and S. cerevisiae YPH499 were dilut-ed 200-fold in 30 mL of 2xYT medium to which 40 μL ethanol was addedcontaining increasing concentrations of either p-hydroxybenzalacetone orraspberry ketone. After overnight incubation, the density of the cultures wasmeasured at 600 nm.



GC-MS analysis further confirmed the establishmentof a raspberry ketone production pathway in E. coli. Avery clear peak was observed in the GC-MS chro-matogram, recorded at m/z 164 (Fig. 3C), with the sameretention time and mass spectrum as raspberry ketone.This peak was also present in the pAC-4CL-RiCHS-Mutextract, but was much lower. Thus, it appeared that thereductase activity of E. coli had converted p-hydroxyben-zalacetone into raspberry ketone.

In a second experiment, the localization of raspberryketone was tested. A culture of pAC-4CL-RiCHS after ithad been fed with p-coumaric acid for 2 days was sepa-rated into cell pellet and culture medium by centrifuga-tion, and both fractions were extracted. It was observedthat 0.7 mg/L raspberry ketone could be isolated from theculture medium, while only 0.1 mg/L was detected in thebacterial pellet. This indicates that most synthesizedraspberry ketone had been secreted by the bacteria intothe medium.

3.5 Raspberry ketone production in yeast in vivo

The p-hydroxybenzalacetone synthase activity observedfor RiCHS was not restricted to the environment of a liv-ing E. coli cell, but appeared also be achievable using oth-er microbial systems. This was demonstrated when theRiCHS gene was introduced in the yeast S. cerevisiaeYPH499. For expression in yeast, the 4CL gene and theRiCHS gene or the mutant RiCHS-Mut gene were intro-duced into the yeast expression vector pESC-Trp (Fig. 2),which allows expression of both genes when induced bygalactose. The pESC-Trp, pESC-4CL-RiCHS and pESC-4CL-RiCHS plasmids were introduced in the yeast strainYPH499. Cultures (10 mL) were grown in the presence ofgalactose to induce recombinant gene expression, and p-coumaric acid (3 mM) was added, after which the cultureswere grown overnight at 30°C. The cultures were extract-ed with ethyl acetate, and analysed using HPLC. Again,as was the case with E. coli, the pESC-4CL–RiCHS strainshowed production of p-hydroxybenzalacetone, in addi-tion to naringenin chalcone and naringenin (Table 2). Theextract from the yeast cultures was also analysed on anHPLC coupled to a Q-TOF-MS (Fig. 4). Figure 3C showsthat the peak eluting at Rf =27.8 min in the HPLC-MS isdominated by a compound of m/z 163.0761 [M+H], whichis only 1.2 ppm different from the predicted mass of p-hy-droxybenzalacetone (C10H10O2; calculated mass [M+H] =163.0759). Moreover, this compound has the same re-tention time and absorption spectrum (λmax=323 nm) as ap-hydroxybenzalacetone standard, which confirms thepresence of p-hydroxybenzalacetone in the yeast culturemedium.

In the pESC-4CL–RiCHS-Mut culture, no naringeninor naringenin chalcone was observed, and only a low

13.60 14.00 14.40 14.80 15.20 15.60

C

t

Minutes23.00 24.00 25.00 26.00 27.00 28.00 29.00

t

Minutes23.00 24.00 25.00 26.00 27.00 28.00 29.00

312nmB

m/z 164

A 280nm

MinutesMinutes

38.5 39.0 39.5 40.0 40.5 41.0 41.5 42.0 42.5 43.0

A 280nm

MinutesMinutes

38.5 39.0 39.5 40.0 40.5 41.0 41.5 42.0 42.5 43.0

Figure 3. Comparison of culture extracts from BL21-pAC-DUET1 (lower),pAC-4CL-RiCHSmut (middle) and pAC-4CL-RiCHS (upper). (A) HPLCanalysis: area from 38 to 43 min, recorded at 280 nm. The position ofnaringenin is indicated (arrow). (B) HPLC analysis: area from 23 to 30 min, recorded at 312 nm. The position of p-hydroxybenzalacetone is indicated (arrow). (C) GC-MS chromatogram, recorded at m/z 164. Theposition of raspberry ketone is indicated (arrow).

Table 2. Production of p-hydroxybenzalacetone, naringenin and naringeninchalcone in yeasta)

p-Hydroxy- Naringenin Naringenin

construct benzalacetone chalcone (mg/L)

(mg/L) (mg/L)

pESC-Trp 0.000 0.000 0.000pESC-4CL-RiCHS 0.060 0.086 0.296pESC-4CL-RiCHS-Mut 0.004 0.000 0.000

a) Concentrations are the average of two independent measurements.

amount of p-hydroxybenzalacetone (Table 2). GC-MSanalysis was used to detect raspberry ketone in the yeastculture extracts. However, as observed for the productionof resveratrol in yeast [12], most p-coumaric acid wasfound to be converted into hydroxyphenyl-propionic acid,which hampered proper detection of raspberry ketone inthe GC-MS. Nevertheless, it was concluded that theRiCHS was capable of BAS activity, also in a yeast envi-ronment.

3.6 Raspberry ketone is an in vivo product of other enzymesin the CHS family

In the previous paragraphs, the capability of the RiCHSprotein to drive synthesis of p-hydroxybenzalacetone invivo was observed. As a next step, we quantitatively com-pared its production to that of other members from theCHS family. The Petunia CHS gene, involved in produc-tion of flower colour, and the grape STS, involved in theproduction of resveratrol, were introduced into the sameE. coli expression system in combination with the 4CLgene (Fig. 2). Both constructs were compared to pAC-

4CL-RiCHS and the empty vector. RiCHS was the most ef-ficient enzyme, but all enzymes tested were capable ofproducing some raspberry ketone (Table 3). The amountof naringenin produced by raspberry RiCHS (0.44 mg/L)only slightly exceeded the sum of the raspberry ketoneand p-hydroxybenzalacetone produced (0.39 mg/L). Thepetunia CHS produced 0.24 mg/L naringenin, and 0.10 mg/L p-hydroxybenzalacetone and raspberry ketone.The grape VvSTS was clearly more focussed on stilbenesynthesis (5 mg/L resveratrol) than on raspberry ketonesynthesis (0.07 mg/L). Thus, we conclude that the RiCHSmay be somewhat more active as a BAS than the othertwo tested polyketide synthases, but it is certainly notunique for this activity.

3.7 RiCHS is the limiting enzyme in the E. coli raspberryketone biosynthesis pathway

In the system described above, both RiCHS and 4CL areunder control of the same T7 promoter. We wanted to testthe role of the individual enzymes for synthesis of rasp-berry ketone. For this, the 4CL and RiCHS genes wereplaced on separate, compatible plasmids, each undercontrol of either the T7 promoter or the arabinose-in-ducible P(BAD) promoter, forming plasmids pBAD-RiCHSand pBAD-4CL (arabinose-controlled promoter) and pAC-4CL and pAC-RiCHS (IPTG inducible) (Fig. 2). Plasmidswere combined in E. coli BL21, and production of narin-genin and raspberry ketone was monitored using HPLCand GC-MS.

In one experiment, E. coli with pBAD-RiCHS and pAC-4CL was tested. The RiCHS expression was varied(0–0.1% arabinose), while 4CL expression was kept con-stant (1 mM IPTG) (Fig. 5A). Conspicuously, the raspber-ry ketone formation had an optimum at 0.03–0.05% arabi-nose, and was less efficient at higher concentration of ara-binose, while the naringenin production was always high-er at higher concentrations of arabinose. Apparently, BASactivity and CHS activity behaved differently upon in-crease of RiCHS expression.

In a second experiment, E. coli BL21 with pAC-RiCHSand pBAD-4CL was tested. In this the 4CL expression



Figure 4. p-Hydroxybenzalacetone production by yeast. Culture extractsfrom YPH499-pESC-4CL-RiCHS (A) and YPH499-pESC-Trp (B) were com-pared on HPLC-MS. Shown are total mass counts from the chromatogramfrom Rf = 27 to 30 min. In (C), the mass spectrum recorded at 27.8 min isshown. Also indicated is the structure of p-hydroxybenzalacetone, and itscalculated mass ([M+H]).

Table 3. Production of p-hydroxybenzalacetone, raspberry ketone andnaringenin in E. coli

p-Hydroxy- Raspberry Naringenin

Construct benzalacetone ketone (mg/L)

(mg/L) (mg/L)

pAC-DUET-1 0.000 0.000 0.000pAC-4CL-RiCHS-mut 0.005 0.006 0.000pAC-4CL-RiCHS 0.108 0.284 0.440pAC-4CL-PhCHS 0.022 0.074 0.244pAC-4CL-VvSTS 0.015 0.052 5.01a)

a) Here the concentration of resveratrol instead of naringenin is given.



was regulated (0–0.1% arabinose), while RiCHS expres-sion was kept constant (1 mM IPTG) (Fig. 5B). The maxi-mal amounts of raspberry ketone and naringenin pro-duced in this experiment were both relatively low (0.016 mg/L and 0.07 mg/L; 7.5 and 2.5 times less thanthat seen in Fig. 5A, respectively). The quantity of eachcompound seemed to be related, and reached a plateau at0.05% arabinose. These data indicate that the RiCHS isthe limiting factor in the production system.

3.8 Fermentation yields 5 mg/L raspberry ketone

E. coli BL21 containing the pAC-4CL-RiCHS vector wasgrown in a fermenter, and enzyme production was in-duced after 28 h by addition of IPTG and p-coumaric acid.A distinct raspberry ketone peak was present in the 42-hand 46-h samples; after 46 h this corresponded to a con-centration of 5 mg raspberry ketone/L (Fig. 6). p-Hydrox-ybenzalacetone was not observed, indicating that gener-ation of p-hydroxybenzalacetone is rate limiting underthese circumstances. Induction of the cells also resulted

in the accumulation of a large amount of another com-pound (Fig. 6: peak at 9.6 min). This compound was iden-tified as indole by GC-MS analysis.

4 Discussion

The enzymes responsible for raspberry ketone formationare interesting from a biotechnological point of view. Suchenzymes could be recruited for production of raspberryketone by micro-organisms or plants, to achieve a sus-tainable production system for producing this flavouringcompound, to include it in artificial, but “natural” aromas.For these reasons, identification of the enzymes has beenpursued by several groups. The focus has been on the p-hydroxybenzalacetone synthesis, since the reductase ac-tivity can be performed by yeasts, such as Beauveria [7].So far, it has been assumed that raspberry contains a BAS,which is distinguished from CHS. In rhubarb, such a BAShas indeed been found and this enzyme produces exclu-sively p-hydroxybenzalacetone, and not naringenin chal-cone [6].

A biochemical strategy to isolate BAS from raspberrywas unsuccessful. When purifying BAS activity fromraspberry, Borejsza-Wysocki and Hrazdina [15] showedthat CHS and BAS co-purify, and reacted with the sameantisera. However, CHS and BAS activity in the same en-zyme preparation showed a different response to treat-ments with, for instance, 2-mercaptoethanol and ethyleneglycol. Also, CHS activity and BAS activity showed dif-ferent induction patterns upon treatment of raspberry cellcultures with yeast extract, suggesting that these en-zymes are not one and the same molecule. However, CHSand BAS could not be separated by biochemical tech-niques.

A molecular strategy to identify a raspberry BAS hasso far also been unsuccessful. In total, six different rasp-berry CHS-like proteins have been isolated by amplifyingraspberry CHS-like sequences [9, 10]. When tested in anin vitro assay, using purified recombinant enzyme and p-coumaroyl-CoA and malonyl-CoA as substrates, “negligi-ble benzalacetone synthesis was observed” [10], whichwe confirmed in this work. Tested in vivo, when CHS isco-expressed with 4CL, highly significant BAS is exhibit-ed by RiCHS. In E. coli, the production of raspberry ketone(0.28 mg/L), and its precursor p-hydroxybenzalacetone(0.11 mg/L) was only slightly lower than production ofnaringenin (0.44 mg/L). We conclude from this findingthat the natural in vivo environment in these living cells isconducive to p-hydroxybenzalacetone synthesis byRiCHS. Apparently, this in vivo environment is difficult tomimic in vitro. Nevertheless, combining 4CL activity,CHS/BAS activity and BAR activity, provided by an en-dogenous E. coli enzyme, leads to a system where signif-icant amounts of the valuable flavour compound can beproduced.

Arabinose-induced RiCHS expressionconstant 4CL expression

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

concentration arabinose (%)

rasp

ber

ry k

eto

ne

(mg

/L)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

nar

ing

enin

(m

g/L

)

raspberry ketone

naringenin

Arabinose-induced 4CL expressionconstant RiCHS expression

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0.02

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1

concentration arabinose (%)

rasp

ber

ry k

eto

ne

(mg

/L)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.1

nar

ing

enin

(m

g/L

)

raspberry ketone

naringenin

A

B

Figure 5. Influence of variation in expression of RiCHS (upper) or 4CL(lower). Cultures of BL21with pBAD-RiCHS and pAC-4CL (top) or BL21with pBAD-4CL and pAC-RiCHS were grown overnight in the presence of3 mM p-coumaric acid, 0.1 mM IPTG and increasing concentrations ofarabinose. HPLC analysis was used to monitor naringenin concentration(open squares), and GC-MS to monitor raspberry ketone concentration(solid diamonds).

The differences between the in vitro and the in vivoproperties of RiCHS are difficult to interpret. Some cluemay be provided by the experiment shown in Fig. 5. In-creasing the amount of RiCHS in a cell (by inducing itspromoter more strongly) led to an increased CHS activity,while BAS activity was decreased (Fig. 5A). This resultwas further confirmed when an E. coli was tested whereboth pAC-4CL-RiCHS (low copy) and pRSET-RiCHS (highcopy) were used for expression (not shown). In that case,the presence of pRSET-RiCHS (producing much moreRiCHS) led to five times more naringenin, while theamount of raspberry ketone was unaffected. Thus, also inthis case, CHS activity and BAS activity, deriving fromthe same gene, respond differently to circumstances suchas enzyme concentration. The effect of enzyme concen-tration could relate to the fact that CHS enzymes usuallyoccur as a homodimer [16, 17]. The increased concentra-tion of RiCHS in Fig. 5 may have led to an increase in the

number of dimers, and a slight decrease in the number ofmonomers. One could envisage that the active site pock-et of a CHS in a dimer may have properties different froma CHS in a monomer. In the monomer, this could possiblylead to an increased premature release of a p-hydroxy-benzalacetone polyketide. However, many more experi-ments are needed to support this hypothesis.

Raspberry ketone synthesis in raspberry fruit may alsobe mediated by CHS. The in vivo activity in E. coli andyeast of a 4CL-CHS pathway may reflect the situation inraspberry. The concentration of raspberry ketone in rasp-berry fruit is much lower than that of anthocyanins, whichare the major end-product of CHS activity in raspberry.Where raspberry ketone occurs in concentrations be-tween 1 and 4 mg/kg fruit [1], anthocyanins occur at400–500 mg/kg fruit [18]. It is easily conceivable that thisrelatively small amount of raspberry ketone in the fruit isa side product from RiCHS, and that this enzyme medi-ates both CHS and BAS activity.

The produced quantities of both naringenin and rasp-berry ketone do not dramatically differ from other plant-specific phenyl-propanoids produced in microbes by re-combinant expression. In our system, feeding p-coumar-ic acid and expressing tobacco 4CL2 and raspberry CHS,we observed production of 0.3 mg/L (flasks) to 5 mg/L (fer-menter). In another study, three genes were co-expressedin E. coli, including a phenylalanine-ammonia lyase (PAL)from Rhodotorula rubra, 4CL from Streptomyces coelicol-or and CHS from Glycyrrhiza echinata [19]. Using tyrosineas a precursor, formation of naringenin was observed to amaximum concentration of 0.45 mg/L. Similarly, the 4CLand CHS from Arabidopsis thaliana have been expressedin E. coli, together with the tyrosine-ammonia lyase (TAL)from Rhodobacter sphaeroides, which led to a productionof about 20 mg/L naringenin [20].

We observed that, in our system, expression of theRiCHS enzyme was the limiting factor (Fig. 5B). This is inkeeping with turnover numbers reported for CHS(kcat=2.2/min; [21]) and 4CL (kcat is around 1200/min, cal-culated from [22]), which indicate that 4CL is a muchmore efficient enzyme. It is known that the CHS enzymeis part of an enzyme complex, in which the substrate ischannelled from one enzyme to the other [23]. Derivingboth genes from the same organism may enhance thiscomplex formation, mediated through specific surface in-teractions between 4CL and CHS proteins, and therebyimprove channelling of the substrate. This may lead to ahigher metabolic flux and to a higher production of valu-able compounds.

None of the previously published studies on produc-tion of flavonoids in E. coli reported any formation of rasp-berry ketone or p-hydroxybenzalacetone [19, 20]. Our datashow that not only RiCHS but also PhCHS and VvSTS arecapable of BAS activity, which would suggest that alsothe CHS enzymes from Arabidopsis and Glycyrrhiza pro-duce these compounds in E. coli. However, the authors



Figure 6. Fermentation of E. coli poAC-4CL-RiCHS. Cells were induced byaddition of 1 mM IPTG after 28 h of growth, at the same time 3 mM p-coumaric acid and the culture was further grown until 46 h after inocula-tion. An OD600 of 20 was reached after 28 h and this remained constant af-ter induction. Shown are compounds extracted from the culture at differ-ent time points, and analysed on GC using flame-ionisation detection.Compounds raspberry ketone, ethylvanillin and indole are indicated.



may have overlooked this pathway, since most p-hydrox-ybenzalacetone is converted into raspberry ketone, andthis compound is only efficiently detected by GC-MS.

In conclusion, we report on the establishment of a mi-crobial production system for the flavour compound rasp-berry ketone, using enzymes from plant sources. Futureefforts will be focused on expression of this plant-specificpathway in food-grade micro-organisms, such as Lacto-coccus lactis [24].

The authors acknowledge Francel Verstappen and HarryJonker for technical advise. J.B., I.v.d.M. and R.H. ac-knowledge Danisco Flavours for financial support. Part ofthis research was carried out in the frame of the EU 6thFramework project FOOD-CT-2005-007130 “FLORA”.

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