Modulation of volatile thiol and ester aromas by modified wine yeast

W.L.P. Bredie and M.A. Petersen (Editors) Flavour Science: Recent Advances and Trends 9 2006 Elsevier B.V. All rights reserved.

113

Modulation of volatile thiol and ester aromas by modified wine yeast Jan H. Swiegers a, Robyn Willmott a, Alana Hill-Ling a, Dimitra L. Capone a, Kevin H. Pardon a, Gordon M. Elsey a, Kate S. Howell a, Miguel A. de Barros Lopes b, Mark A. Sefton a, Mariska Lilly c and Isak S. Pretorius a

aThe Australian Wine Research Institute, PO Box 197, Glen Osmond, Adelaide, Australia," bUniversity of South Australia, North Terrace, Adelaide, Australia," Clnstitute for Wine Biotechnology, Stellenbosch University, Stellenbosch, South Africa

ABSTRACT The volatile thiols, in particular 4-mercapto-4-methylpentan-2-one (4MMP), 3mercaptohexan-l-ol (3MH) and 3-mercaptohexyl acetate (3MHA) are potent aroma shown to contribute strongly to the varietal aroma of Sauvignon Blanc wines. The thiols 4MMP and 3MH exist as non-volatile, aroma-inactive cysteine bound conjugates in the grape must and during fermentation the thiol is cleaved from the precursor. However, no cysteine conjugate for 3MHA has been identified. In this work we showed that 3MHA is formed from 3MH by the wine yeast Saccharomyces cerevisiae during fermentation. Furthermore, the alcohol acetyltransferase, Atflp, the enzyme involved in the formation of the ester ethyl acetate, was shown to be the main enzyme responsible for the formation of 3MHA. Both a laboratory yeast and a commercial wine yeast overexpressing the ATF1 gene produced significantly more 3MHA than the wild-type. Although an atflA laboratory yeast strain showed reduced 3MHA formation, it was not abolished, indicating that other enzymes are also responsible for its formation. Therefore, overexpression of the ATF1 gene in a wine yeast presents the possibility of modulating both the thiol and ester aromas in wine. 1. I N T R O D U C T I O N Sauvignon Blanc wine has characteristic aromas described as box tree, cat urine, broom, grapefruit, blackcurrant and passion fruit [1,2]. These aromas are attributed to three

114 potent volatile thiol compounds: i) 4-mercapto-4-methylpentan-2-one (4MMP), ii) 3mercaptohexyl acetate (3MHA) and iii) 3-mercaptohexan-l-ol (3MH), all having an extremely low perception threshold. These thiols have also been identified in wine varieties such as Colombard, Muscat d'Alsace, Petit Manseng, Gewfirztraminer, Riesling, Cabernet Sauvignon, Merlot and Semillon [1,3]. The volatile thiols 4MMP and 3MH are almost non-existent in the grapes but are released during fermentation from their non-volatile cysteine bound precursor. Although 3MHA is found in significant quantities in wine, no cysteine bound precursors could be identified [4]. Therefore, it appears that 3MHA is formed during fermentation and through the esterification of 3 MH. A large proportion of the characteristic fruity odours of wine are primarily derived from the synthesis of esters by the wine yeast, Saccharomyces cerevisiae. The ATF1- and ATF2-encoded alcohol acetyltransferases of this yeast are responsible for the synthesis of ethyl acetate and isopentyl acetate esters, while the EHTl-encoded ethanol hexanoyl transferase is responsible for synthesising ethyl caproate. However, esters such as these can be degraded by the IAH1 encoded esterase [5-10]. The objective of this study was to investigate the possible role of alcohol acetyltransferases and esterases in the formation o f 3 MHA. 2. M A T E R I A L S AND M E T H O D S

Strains used were commercial wine yeast VIN 13 (Anchor Yeast, South Africa), VIN13ATF 1-s (overexpressing A TF1), VIN 13-ATF2-s (overexpressing A TF2), VIN 13-EHT 1s (overexpressing EHT1), VINI3-IAHI-s (overexpressing IAHI) [6,1 l] and BY4742 wild type and BY4742 atflA (EUROSCARF). Plasmid pATFls [6] was linearised with Apal and transformed into strain BY4742 using the lithium acetate procedure and confirmed by PCR. The growth medium for fermentations consisted of 8% glucose, 0.67% YNB to which 1 mg/1 of 3MH was added after autoclaving. Fermentations were carried out in 250 ml Erlenmeyer flasks equipped with an air lock. The samples were spiked with polydeuterated internal standards for stable isotope dilution analysis (SIDA). The volatile thiols were then analysed by headspace solid phase microextraction coupled with gas chromatography - mass spectrometry (HS-SPME GC-MS). 3. RESULTS The commercial wine yeast, VIN13, was previously used to construct strains overexpressing various genes (ATF1, ATF2, EHT1 and IAH1) involved in ester metabolism [6,11]. These strains were used to investigate the involvement of ester metabolism in the formation of 3MHA. A model medium was spiked with 1 mg/1 of 3MH. Strains were grown overnight in YPD and the 150 ml model medium was inoculated at OD 600 of 0.05. After 2 days of fermentation at 30 ~ samples were taken and the amounts of 3MHA formed were measured using HS-SPME GC-MS (Figure 1). There was a significant increase in the concentration of 3MHA in the ferments

115 conducted with VIN13-ATFI-s and a reduction in the concentration of 3MHA in ferments conducted with VIN13-IAHI-s. It was also shown that this was not due to chemical conversion as 3MH added to sterile fermented model media and left for 2 days at 30 ~ did not result in the formation of 3MHA (data not shown). 800

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Figure 1. Conversion of 3MH to 3MHA by wild type (WT) wine yeast and wine yeast overexpressing ester metabolism related genes (ATF1, ATF2, EHT1 and IAH1). A laboratory yeast strain with deleted A TF1 was investigated, as a deletion for the wine yeast is not available and difficult to construct. Fermentations and analyses were conducted in the same way as described above. After 2 days fermentation at 30 ~ the laboratory strain formed about 12 ~tg/1 of 3MHA. Deletion of the ATF1 gene did not result in the abolishment of 3MHA formation; however, 3MHA production was reduced. As a control, the ATF1 gene was also overexpressed in the laboratory strain and large amounts of 3MHA were formed (Figure 2). a

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Figure 2. The bioconversion ability of laboratory yeast (BY4742) with modified ATF1 expression. (a) A laboratory yeast with a deleted ATF1 (atfl). (b) A laboratory yeast overexpressing A TF1.

116 Previously, the ability of wine yeast to release the thiol, 4MMP, was investigated for the same reason [12]. Fermentations and analysis were conducted in the same way as described above. After 2 days fermentation at 30 ~ analyses was done and a large variation in the ability of wine yeast to form 3 M H A was observed (data not shown). 4. D I S C U S S I O N A N D C O N C L U S I O N In this work we have implicated the A TFl-encoded alcohol acetyltransferase in the formation of 3MHA. The results indicated, firstly, that wine yeast are able to bioconvert 3MH to 3MHA. Secondly, in the collection of enzymes we investigated Atflp appears to have a major role in the conversion of 3MH to 3 M H A as overexpression of ATF1 resulted in significant increase in the amount of 3 M H A formed. Thirdly, our hypothesis was supported by the fact that the esterase lahlp, degrades 3MHA, as overexpression of IAH1 resulted in reduced 3 M H A concentration. Atf2p and Eht 1p did not appear to have a role in the formation of 3MHA. Furthermore, different commercial wine yeast have a large degree of variation in their ability to convert 3MH to 3MHA. This is valuable information for winemakers, helping them to make informed choices on yeast strain selection as a tool to modulate wine flavour. Future work will entail the monitoring of expression of A TF1 during fermentation in order to determine how winemakers can manipulate fermentation conditions to optimise 3 M H A formation. References

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