Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes

Enzyme and Microbial Technology 35 (2004) 293–299

Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes Gérald Bourel a , Jean-Marc Nicaud b , Bethuel Nthangeni b,c , Patricia Santiago-Gomez a , Jean-Marc Belin a , Florence Husson a,∗ a

c

Laboratoire de Microbiologie, UMR UB/INRA 1232, ENSBANA, Université de Bourgogne, Campus Universitaire Montmuzard, 1 Esplanade Erasme, 21 000 Dijon, France b Laboratoire de Microbiologie et Génétique Moléculaire, INRA-CNRS-INAPG UMR 2585, BP 01, 78 850 Thiverval-Grignon, France Department of Microbial, Biochemical and Food Biotechnology, Faculty of Natural and Agricultural Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa Received 19 May 2003; accepted 15 December 2003

Abstract Fatty acid hydroperoxide lyase (HPO lyase) is a cytochrome P450 acting on fatty acid hydroperoxides in many organisms. The expression of green bell pepper HPO lyase in the yeast Yarrowia lipolytica is described for the first time. HPO lyase activity from yeast extract and whole yeast cells is measured and aldehydes production from yeast extract and whole yeast cells is compared. 1200 U/L reaction medium were obtained after 96 h of culture on olive oil rich medium. The quantity of C6-aldehydes produced is the highest (350 mg/L reaction medium) when hydroperoxides are added directly in the culture medium supplemented with olive oil. © 2004 Elsevier Inc. All rights reserved. Keywords: Cloning; Hydroperoxide lyase; Volatile compounds; Bioconversion; Whole cells

1. Introduction Fatty acid hydroperoxide lyase (HPO lyase) is an enzyme which is widely distributed in plants and is involved in the biosynthesis of volatile aldehydes and ␻-oxo-acids, by cleaving a C–C bond of hydroperoxide fatty acids between the carbon of the hydroperoxide group and the neighboring double bond [1]. C6 or C9-aldehydes and the corresponding alcohols, formed by the action of HPO lyase on 13- and 9-hydroperoxides of linoleic or linolenic acid, are important constituents of the characteristic flavors of fruits, vegetables and green leaves [2]. These flavoring molecules are also high value molecules that are widely used in the aroma industry. HPO lyases from green bell pepper have been well characterised and the gene encoding HPL from pepper fruit has been cloned and sequenced [4]. Moreover, in our previous work, we showed that the chloroplast fraction and the purified HPO lyase from green bell pepper were of particular interest in the production of green note molecules, such as hexanal and trans-2-hexenal [3]. However, the quantities of ∗ Corresponding author. Tel.: +33 3 80 39 66 80; fax: +33 3 80 39 66 41. E-mail address: [email protected] (F. Husson).

0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2003.12.014

recovered enzyme were still low and the cost of biocatalyst production too high for industrial process. Until now recombinant expression of common biocatalyst is the preferred way to obtain high quantities of stable and efficient enzymes. Concerning hydroperoxide lyase, several authors have cloned mainly the 13-positional isomers of linoleic acid specific hydroperoxide lyase in bacterial expression system. Bell pepper HPO lyase has already been expressed in Escherichia coli and its characterization led to the suggestion that this enzyme is a novel type of P450 enzymes, termed CYP74 [4]. However, HPO lyase is distinctive among P450s in that it has an extremely high turnover rate, it activates the hydroperoxide moiety, instead of O2 , and it needs no cofactor, such as reducing equivalent, which is usually essential to classic P450 monooxygenases. Noordermeer et al. in 2000 cloned and expressed in E. coli 13-hydroperoxide lyase from alfalfa [5] and the same authors in 2002 described a biocatalytic process for the production of C6-aldehydes with the recombinant enzyme [6]. Matsui et al. [7] in 2000 cloned the fatty acid hydroperoxide lyase of tomato fruits in E. coli. Tijet et al. [8] cloned a hydroperoxide lyase from melon fruit with dual activity on both 9- and 13-fatty acid hydroperoxides. In 2001, Psylinakis et al. [9] expressed hydroperoxide lyase of immature bell pepper fruits in E.

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coli for spectroscopic characterisation. Expression systems for recombinant guava 13-HPO lyase for the production of “green notes” compounds have been proposed [10,11]. Saccharomyces cerevisiae cells containing the recombinant plasmid containing the HPO lyase gene were used directly to produce cis-3-hexenal [11]. The amount of hexanal formed by the recombinant HPL expressed in E. coli was 140 ␮g per 10 ␮l of E. coli cell lysate [10]. There is currently a strong interest in the development of new hosts for the secretion of heterologous proteins. Yeasts are attractive hosts for production of foreign proteins because they combine the advantages of prokaryotic and higher eukaryotic systems. S. cerevisiae has been used extensively for the production of many heterologous proteins since genetic information, host-vector systems, and recombinant DNA techniques for this organism are well-established [12]. Several non-conventional yeasts such as Pichia pastoris [13], Kluyveromyces lactis [14] and Hansenula polymorpha [15] have been explored as new hosts for foreign gene expression. The ascomycetous yeast Yarrowia lipolytica is one of the more intensively studied ‘non-conventional’ species because it is not only of interest for fundamental research but also for biotechnological applications. This yeast has been used to produce citric acid, isopropylmalic acid, erythritol, and mannitol [16–18]. Recently, Y. lipolytica has received special attention as a potential host for the production of heterologous proteins due to its ability to secrete high levels of large proteins such as alkaline extracellular protease and RNAse [19–21]. This yeast was used in industrial applications such as the production of single-cell proteins, peach flavor and citric acid, thus permitting the accumulation of extensive data on its behavior in fermenters [22]. More recently, protein expression and secretion in the yeast Y. lipolytica has been studied [23] for the production of the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Moreover, these authors compare in their study strain containing single copy for the production of lipase in shake flasks (1000 U/mL) and strain containing multiple copy for lipase production in shake flasks (2000 U/mL) in batch (11,500 U/mL) and in fed-batch (90,500 U/mL) which represent several grams per liter. Moreover, Nthangeni et al. [24], have recently demonstrated that activity of cytochrome P450 1A1 (CYP450) was amplified by a factor 50 with a multicopy integrant of Y. lipolytica. Y. lipolytica has been identified as a highly attractive non-conventional yeast that serves as a host for heterologous protein production [25]. Y. lipolytica utilizes very efficiently long- and short-chain triglycerides, such as olive oil and tributyrin, fatty acids and the corresponding n-alkanes, from decane (C10) to octadecane (C18) and longer chains [26]. It has then developed at least two fatty acid carrier systems, one being specific for C12 or C14 fatty acids and the other for C16 or C18 saturated or unsaturated fatty acids [27]. Moreover, HPL gene has been placed under the control of the POX2 promoter which is induced by fatty acids.

For that purpose, Y. lipolytica has been selected in this work for the cloning and expression of hydroperoxide lyase from green bell pepper fruits (HPO lyase). In the present work, we compared HPL activities obtained from the cell extract and whole cells of Y. lipolytica strain expressing HPO lyase from green bell pepper fruit. Moreover, the production of hexanal and trans-2-hexenal was also investigated with the cell extract, whole cells and growing cells as sources of HPL activity. 2. Materials and methods 2.1. Strains and vector E. coli JM109 [28] was used for cloning and plasmid preparation. The Y. lipolytica strain used was PO1d (MatA leu2-270 ura3-302 xpr2-322; strain CLIB139 from the Collection de Levures d’Intérˆet Biotechnologique (Thiverval-Grignon, France, http://www.inra.fr/Internet/ Produits/clib). The plasmid pYEG1 used in this study is a JMP7 derivative which contains the POX2 promoter (promoter of the Y. lipolytica acyl-CoA oxidase 2 gene inducible by fatty acid) -LIP2 terminator (terminator of the Y. lipolytica extracellular lipase gene) expression cassette [29]. The expression cassette is flanked by two ␨ regions for targeting to multiple sites in the genome of PO1d [29]. 2.2. Basic DNA techniques and transformation methods Plasmid DNAs from E. coli clones were isolated by using the QIAprep Miniprep (Qiagen kit). Total Y. lipolytica chromosomal DNA was isolated as previously described [30]. Recombinant DNA techniques were carried out by standard methods [31]. Y. lipolytica transformation by the expression cassette was done as previously described [32]. 2.3. Subcloning and expression of the bell pepper HPO lyase cDNA in Y. lipolytica Cloned bell pepper HPO lyase cDNA was a generous gift from Matsui et al. [4]. The bell pepper HPO lyase gene was first modified by PCR to include flanking AvrII and SfiI restriction sites. PCR was carried out using the HotStarTaq DNA polymerase (Qiagen), according to the following temperature and time probes: 95 ◦ C for 15 min (1 cycle); 92 ◦ C for 1 min, 68 ◦ C for 1 min, 72 ◦ C for 1 min 15 s (25 cycles); 72 ◦ C for 10 min (1 cycle). The synthetic oligonucleotides HPL1 (5 -CCTATCCCTAGGTTAAAATGATACCTATAATGAGCTCTGCTCCTCTATCAACTGC-3 ) with AvrII restriction site and HPL2 (5 -GGGGACAGGCCCGCGGGGCCTACTCAGCAGGCTTTTTTCACAGATGTGAGTG-3 ) with SfiI restriction site, designed over the exact gene sequence (Genbank accession number CA51674), were used. The AvrII-SfiI restriction fragment of the amplified HPO lyase gene was subcloned in

G. Bourel et al. / Enzyme and Microbial Technology 35 (2004) 293–299

295 COOH

COOH

Linoleic acid

Linolenic acid Lipoxygenase

OOH

OOH COOH

COOH

13-hydroperoxy-octadecatrienoic acid 13-HPOT

13-hydroperoxy-octadecadienoic acid 13-HPOD

Hydroperoxide lyase

O

Hexanal

w-oxo-acid

O

trans-2-hexenal

Fig. 1. Formation of hexanal and trans-2-hexenal via the lipoxygenase and hydroperoxide lyase degradation of the polyunsaturated fatty acids: linoleic acid and linolenic acid.

the AvrII-SfiI restriction fragment of the pYEG1 expression cassette giving pYEG-HPL. Digestion of the plasmid pYEG-HPL with NotI yields two fragments: the first fragment represents the pHSS6 moiety of pYEG1 [29], and the second represents the targeting cassette, consisting of the URA3 marker and the expression cassette (␨ region – POX2 promoter – HPO lyase gene – LIP2 terminator – ␨ region). This targeting expression cassette was used to transform Y. lipolytica PO1d to give Y. lipolytica PO1d-HPL strain. 2.4. Bacterial and yeast culture media E. coli cells harbouring the pYEG1 or pYEG-HPL were grown in Luria Bertani Broth [33] containing kanamycin (50 ␮g/mL). The media used for yeast culture were: rich medium (YPD) for cultivation of PO1d, enriched minimal medium (YNBcas) for selection of recombinant strain bearing the targeting expression cassette, and rich glucose olive oil (YOG) for expression of the HPO lyase gene in the recombinant strain. The composition of the media used were as follows (per liter): YPD (10 g of yeast extract, 10 g of tryptone, 20 g of glucose), YNBcas (1.7 g of yeast nitrogen base without amino acid and ammonium sulfate, 4 g of ammonium chloride, 20 g of glucose, 5 g of casamino acids), YOG (5 g of yeast extract, 10 g of tryptone, 10 g of glucose, 20 g of olive oil). YOG medium for expression of HPO lyase gene was inoculated at 2.6 × 107 cells/mL (1 g cells/L). 2.5. Preparation of hydroperoxide of linoleic acid and assays of hydroperoxide lyase activity Preparation of hydroperoxide and assays of hydroperoxide lyase activity were done as previously described [34]. The substrate olein (Nouracid LE 80) (31%, w/v) (a source of 48% ␣-linolenic acid and 18% linoleic acid) (Akzo-Nobel, France) was suspended in oxygenated borate buffer (0.1 M, pH 9.5) containing 1% (v/v) Tween 80 and soybean flour (21%, w/v) as the lipoxygenase source giv-

ing rise to 13-hydroperoxides of linolenic acid and linoleic acids (13-HPOT and 13-HPOD), respectively. Upon action of hydroperoxide lyase activity the 13-HPOT and 13-HPOD resulted in the formation of trans-2-hexenal and hexanal, respectively (Fig. 1). For aldehydes extraction and analysis, 4 mL of the reaction mixture was removed and centrifuged (20,000 × g, 5 min, 4 ◦ C). The internal standard (isoamyl acetate) was added to the supernatant and the mixture was extracted with 4 mL of diethyl ether. The ether phase was then analyzed in an HP6890 gas chromatograph with an HP-Innowax capillary column using N2 as a carrier gas at a linear flow rate of 4.3 mL min−1 . The split injector and the flame ionization detector temperature were set to 250 and 300 ◦ C, respectively. The oven temperature was programmed to increase from 35 to 160 ◦ C at a rate of 5 ◦ C min−1 and then at a rate of 2 ◦ C min−1 to 200 ◦ C. 2.6. Production of hexanal and trans-2-hexenal using yeast cell-free extracts and resting yeast cells Y. lipolytica PO1d-HPL strain was grown in 50 mL YOG medium in 250 mL baffled Erlenmeyer flasks at 150 rpm and 27 ◦ C. Cells were harvested by centrifugation (2500 × g, 5 min, 4 ◦ C) and washed three times with Tris–HCl buffer (100 mM, pH 8.5). Cell-free extracts were prepared by grinding washed cells using a mortar and a pestle in liquid N2 . The cell lysate was then transferred into a 50 mL conical tube on ice containing Tris–HCl buffer (100 mM, pH 8.5) Triton-X100 (2% w/v) at a ratio of 1 g of cell lysate per 1 mL of buffer, and then stirred gently for 1 h at 4 ◦ C. The solution was centrifuged (10,000 × g, 20 min, 4 ◦ C) and the resulting supernatant, containing solubilized HPO lyase was used for hydroperoxide lyase assay and for the production of C6-aldehydes. The production of C6-aldehydes using yeast cell-free extracts was performed in an enclosed glass vial (10 mL) fitted with a rubber stopper. A typical reaction mixture (3 mL), consisted of MES-KOH buffer (200 mM, pH 5.5, 25 ◦ C) to which hydroperoxide of linoleic acid was

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added and emulsified by sonication to obtain the desired concentration. The cell-free extract containing two units of HPO lyase was added to the mixture followed by stirring. The vial was placed in a water bath at 25 ◦ C and the reaction was allowed to proceed for 1 h after which aldehydes were extracted. The production of hexanal and trans-2-hexenal using resting cells was performed as already described, but the cell-free extract was replaced by whole resting cells. The production of aldehydes by growing cells was performed by adding HPO directly into the culture medium at 14 h of the culture period, the time corresponding to the point at which all the glucose in the medium is exhaustively consumed and the POX2 promoter is induced.

period (120 h) (Fig. 2). The reaction conditions were as described for cellular extract activities. Maximal HPL activity of 1000 U/L reaction medium was obtained with resting cells at 86 h. It is interesting to note that at this time point, cellular extract and whole cells show similar HPL activity. Moreover, Nicaud et al. [23], have shown that enzymatic activity could be increased 100-fold when both gene amplification (construction of a multi-copy transformant) and growth to high cell density (growth in fed-batch) were combined. The use of whole cells is interesting in that it avoided the lysis of the yeast cells, it decreased the cost of the final process and could then be more easily transposable to an industrial process. 3.4. In vivo aldehydes production

3. Results 3.1. Functional expression of the HPL-gene in Y. lipolytica The full-length cDNA was cloned into the expression plasmid pYEG1, allowing the expression of a native, functional enzyme. Analysis of the proteins from the transformed Y. lipolytica cells by SDS–PAGE showed one supplementary band at 55 kDa compared to the proteins from the wild strain of Y. lipolytica (W29) (data not shown).

For the in vivo aldehydes production, strain PO1d-HPL was grown in YOG After 14 h growth (cell densities: 3 × 1011 cells/L), different concentrations of HPO (0, 4, 10, 50 and 75 mM) were added directly to the medium and final hexanal and trans-2-hexenal concentrations were determined (Fig. 3). The production of hexanal in the culture medium containing HPO increased rapidly with maximal concentrations obtained at about 20 h of the culture (Fig. 3A). With 75 mM of HPO, maximum hexanal production (270 mg/L) was obtained at 20 h of culture (6 h

3.2. HPL activity using cellular extracts

3.3. HPL activity using resting cells

350 300 hexanal (mg/L)

HPL activity was measured in cellular extracts obtained from Y. lipolytica PO1d-HPL cultures withdrawn after various times of growth in the medium supplemented with olive oil (times 24, 48, 72, 96 and 120 h after inoculation, Fig. 2). The highest HPL activity (1200 U/L reaction medium) was obtained at 96 h, during the stationary phase.

250 200 150 100 50

HPL activity was also measured with whole cells harvested at different times of culture and during the same

(A)

0 0

20

0

20

40

60

80

1400

50

1200 1000 800 600 400

T-2-hexenal (mg/L)

18 16 14 12 10 8 6 4 2 0

HPL activity (U/L)

biomass (g/L)

60

6

8 14 24 30 48 54 78 86 92 96 110 116 134

20

0

0 3

30

10

200 0

40

(B)

40 60 culture time (hours)

80

culture time (hours)

Fig. 2. Growth of Y. lipolytica on olive oil supplemented medium (YOG) inoculated at 2.6 × 107 cells/mL (䊏) and HPL activity obtained with cell extracts () and with whole cells (䉱). Experiments made in triplicate, only one is represented here.

Fig. 3. Production of hexanal (A) and trans-2-hexenal (B) by Y. lipolytica cells cultured in YOG medium containing different concentrations of HPO (HPO were added in the medium at 14 h of culture: (䊉) 0 mM, (䊏) 4 mM, (䉱) 10 mM, (×) 50 mM, (䉬) 75 mM. Experiments made in triplicate, only one is represented here.

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after the addition of HPO); followed by a decrease in hexanal concentration down to 50 mg/L at 70 h of culture. The maximum hexanal concentration (312 mg/L) in the culture medium containing 50 mM HPO was obtained at 40 h of culture. With 10 mM of HPO, the hexanal produced at time 20 h (50 mg/L) decreased slowly during time. There was, however, no detectable hexanal production observed in the culture medium with 4 mM of HPO. These results showed that 13-HPO was rapidly converted into hexanal during the first 6 h followed by a decreased in hexanal concentration. This decrease could be attributed to the transformation of hexanal into hexanol by fatty aldehyde dehydrogenase (FALDH) enzymes. Indeed, survey of Y. lipolytica sequences revealed four putative FALDH genes in Y. lipolytica (Nicaud et al., unpublished) which might be induced by the production of hexanal resulting in its degradation into the alcohol. Similarly, in vivo production of trans-2-hexenal was obtained reflecting bioconversion of HPOT. However, the production of trans-2-hexenal in the culture medium containing all the tested concentrations of HPO increased relatively slowly as compared to the accumulation of hexanal, with maximal concentrations obtained at about 40 h of the culture (Fig. 3A). Increased incubation resulted in the decline of trans-2-hexenal, probably as a result of enzymatic degradation. Maximal production of trans-2-hexenal (50 mg/L) was observed at 40 h with 50 mM HPO added in the culture medium (Fig. 3B). The maximum molar conversion yield of 6.22% was obtained at 40 h of culture with 75 mM HPO added in the culture medium for hexanal and 1% for trans-2-hexenal with 50 mM HPO. 3.5. Bioconversion activity using yeast extract, resting cells and growing cells Production of hexanal and trans-2-hexenal were assayed with enzymatic extracts from cellular extract, whole cells and growing cells obtained at 96 h of culture on olive oil medium (Table 1). In all the cases, several concentrations of HPO ranging from 10 to 100 mM and several quantities of enzyme were tested. The best enzyme/substrate ratio was 1 unit of enzyme for 100 mM HPO. Hexanal was the major aldehyde produced from cellular extract, 300 mg/L. Trans-2-hexenal was also produced but in a quite lower quantity, 25 times less than hexanal (12 mg/L). The bioconversion of HPO with the resting cells for 1 h resulted in the production of 220 mg hexanal/L while no

Table 1 Production of C6-aldehydes with cellular extract, whole cells and growing cells of Y. lipolytica expressing HPL gene of green bell pepper fruit

Hexanal (mg/L) Trans-2-hexenal (mg/L)

Cellular extract

Whole cells

Growing cells

300 12

220 –

300 50

297

trans-2-hexenal was observed. The total aldehyde quantity (312 mg/L) obtained with cellular extract of Y. lipolytica during 1 h of bioconversion was quite similar to that produced by growing cells on YOG-HPO medium at 40 h (350 mg/L). Bioconversion activity with whole cells during 1 h resulted in the lowest production levels (220 mg/L).

4. Discussion We have been able to produce HPL activity of 1200 U/L reaction medium with a cellular extract of Y. lipolytica grown on olive oil and a mixture of 13-HPOD and 13-HPOT. We also obtained 1000 U/L of HPL activity with whole cells of Y. lipolytica. This value is comparatively lower than that obtained by Noordermeer et al. [6] who obtained 3000 and 8000 U/L for 13 HPOD and 13-HPOT with E. coli as expression host. Delcarte et al. [35], found 7900 U/L of HPL activity using 13-HPOT from extracts of E. coli cells grown on TB medium supplemented 0.5 mM IPTG [35]. Nevertheless, our results are quite promising for the production of “green note” aldehydes with resting yeast cells avoiding laborious and time-consuming methods involved in cellular extractions. We have investigated hexanal and trans-2-hexenal production with cell extracts, resting cells and growing cells of Y. lipolytica-HPL strain. The results presented here show that the quantities of C6-aldehydes produced were almost the same when HPO was added directly into the culture medium (350 mg/L) or when cell extracts were used (312 mg/L). These values were comparatively higher than the value obtained with the resting cells (220 mg/L). In our previous work [3] we showed that a maximum of 7500 mg aldehydes/g of protein could be produced by the purified enzyme and 392 and 88 mg of aldehydes were produced, respectively, by the chloroplast fraction and the crude extract of green bell pepper fruit. If we compare these results with the results obtained in this study; 350 mg C6 aldehydes/L or/1200 U or/8.57 mg proteins can be converted to 41 g C6 aldehydes/g protein, we can conclude that Y. lipolytica is a good host for the expression of HPL enzyme and for the production of C6-aldehydes. Moreover, it has been shown previously that fatty acid hydroperoxides can be particularly toxic to cells such as S. cerevisiae [36]. These authors have shown that linoleic acid hydroperoxide is toxic to wild-type S. cerevisiae at concentrations as low as 0.2 mM relative to other peroxides. An important role for oxygen, glutathione and the transcriptional activator yAP-1 in the cellular response to linoleic acid hydroperoxide was shown. Moreover, total glutathione peroxidase activity increased following treatment with linoleic acid hydroperoxide, indicating a possible detoxification role for this enzyme in yeast. In our work, we did not observe toxicity towards Y. lipolytica by HPO at all the concentrations tested. The cellular responses allowing the tolerance to high linoleic and linolenic acid hy-

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droperoxide in Y. lipolytica remain to be investigated in the future. In conclusion, we have demonstrated that Y. lipolytica could be a useful host for the expression of HPL. We demonstrate that, with such yeast, we established a simple process that could yield high quantities of C6-aldehydes. There exists a scope for the optimization of the production of HPL activity, given the fact that Y. lipolytica mutants deficient of FALDH activities that otherwise degrade the produced C6-aldehydes could be constructed. Furthermore, POX2 promoter expression systems constructed for multiple copy integration into the Y. lipolytica genome are available [23]. These could be used to introduce multiple HPL genes into the Y. lipolytica genome for fed-batch cultivation and production of the HPL activity.

Acknowledgments The authors are thankful to Dr. Kenji Matsui for providing the cDNA of green bell pepper and to Aline Mina-Passi for her technical help. This work was supported by the Institut National de la Recherche Scientifique, the Centre National de la Recherche Scientifique and by the Région bourgogne.

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