Abstract
Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.
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References
Aslankoohi, E., Herrera-Malaver, B., Rezaei, M.N., Steensels, J., Courtin, C.M., and Verstrepen, K.J. 2016. Non-conventional yeast strains increase the aroma complexity of bread. PLoS ONE 11, e0165126.
Chi, Z., Chi, Z., Liu, G., Wang, F., Ju, L., and Zhang, T. 2009. Saccharomycopsis fibuligera and its applications in biotechnology. Biotechnol. Adv. 27, 423–431.
Choi, Y.R., Kim, H.J., Lee, J.Y., Kang, H.A., and Kim, H.J. 2013. Chromatographically-purified capsid proteins of red-spotted grouper nervous necrosis virus expressed in Saccharomyces cerevisiae form virus-like particles. Protein Expr. Purif. 89, 162–168.
Choo, J.H., Hong, C.P., Lim, J.Y., Seo, J.A., Kim, Y.S., Lee, D.W., Park, S.G., Lee, G.W., Carroll, E., Lee, Y.W., et al. 2016. Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid. Biotechnol. Biofuels 9, 246.
D’Auria, J.C. 2006. Acyltransferases in plants: a good time to be BAHD. Curr. Opin. Plant Biol. 9, 331–340.
Fujii, T., Nagasawa, N., Iwamatsu, A., Bogaki, T., Tamai, Y., and Hamachi, M. 1994. Molecular cloning, sequence analysis, and expression of the yeast alcohol acetyltransferase gene. Appl. Environ. Microbiol. 60, 2786–2792.
Galaz, S., Morales-Quintana, L., Moya-León, M.A., and Herrera, R. 2013. Structural analysis of the alcohol acyltransferase protein family from Cucumis melo shows that enzyme activity depends on an essential solvent channel. FEBS J. 280, 1344–1357.
Gamero, A., Quintilla, R., Groenewald, M., Alkema, W., Boekhout, T., and Hazelwood, L. 2016. High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation. Food Microbiol. 60, 147–159.
Gautier, R., Douguet, D., Antonny, B., and Drin, G. 2008. HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24, 2101–2102.
Gietz, R.D. and Schiestl, R.H. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34.
Goulet, C., Kamiyoshihara, Y., Lam, N.B., Richard, T., Taylor, M.G., Tieman, D.M., and Klee, H.J. 2015. Divergence in the enzymatic activities of a tomato and Solanum pennellii alcohol acyltransferase impacts fruit volatile ester composition. Mol. Plant 8, 153–162.
Hazelwood, L.A., Daran, J.M., van Maris, A.J.A., Pronk, J.T., and Dickinson, J.R. 2008. The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl. Environ. Microbiol. 74, 2259–2266.
Hu, K., Jin, G.J., Mei, W.C., Li, T., and Tao, Y.S. 2018. Increase of medium-chain fatty acid ethyl ester content in mixed H. uvarum/S. cerevisiae fermentation leads to wine fruity aroma enhancement. Food Chem. 239, 495–501.
Jefferys, B.R., Kelley, L.A., and Sternberg, M.J.E. 2010. Protein folding requires crowd control in a simulated cell. J. Mol. Biol. 397, 1329–1338.
Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., and Sternberg, M.J.E. 2015. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858.
Kruis, A.J., Levisson, M., Mars, A.E., van der Ploeg, M., Garcés Daza, F., Ellena, V., Kengen, S.W.M., van der Oost, J., and Weusthuis, R.A. 2017. Ethyl acetate production by the elusive alcohol acetyltransferase from yeast. Metab. Eng. 41, 92–101.
Lamiable, A., Thévenet, P., Rey, J., Vavrusa, M., Derreumaux, P., and Tufféry, P. 2016. PEP-FOLD3: faster de novo structure prediction for linear peptides in solution and in complex. Nucleic Acids Res. 44, W449–W454.
Lee, S.M., Jung, J.H., Seo, J.A., and Kim, Y.S. 2018. Bioformation of volatile and nonvolatile metabolites by Saccharomycopsis fibuligera KJJ81 cultivated under different conditions-carbon sources and cultivation times. Molecules 23, 2762.
Lilly, M., Bauer, F.F., Lambrechts, M.G., Swiegers, J.H., Cozzolino, D., and Pretorius, I.S. 2006. The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast 23, 641–659.
Lilly, M., Lambrechts, M.G., and Pretorius, I.S. 2000. Effect of increased yeast alcohol acetyltransferase activity on flavour profiles of wine and distillates. Appl. Environ. Microbiol. 66, 744–753.
Lin, J.L. and Wheeldon, I. 2014. Dual N- and C-terminal helices are required for endoplasmic reticulum and lipid droplet association of alcohol acetyltransferases in Saccharomyces cerevisiae. PLoS ONE 9, e104141.
Momoi, M., Tanoue, D., Sun, Y., Takematsu, H., Suzuki, Y., Suzuki, M., Suzuki, A., Fujita, T., and Kozutsumi, Y. 2004. SLI1 (YGR212W) is a major gene conferring resistance to the sphingolipid biosynthesis inhibitor ISP-1, and encodes an ISP-1 N-acetyltransferase in yeast. Biochem. J. 381, 321–328.
Nagasawa, N., Bogaki, T., Iwamatsu, A., Hamachi, M., and Kumagai, C. 1998. Cloning and nucleotide sequence of the alcohol acetyltransferase II gene (ATF2) from Saccharomyces cerevisiae Kyokai No. 7. Biosci. Biotechnol. Biochem. 62, 1852–1857.
Nancolas, B., Bull, I.D., Stenner, R., Dufour, V., and Curnow, P. 2017. Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro. Yeast 34, 239–251.
Nyanga, L.K., Nout, M.J., Smid, E.J., Boekhout, T., and Zwietering, M.H. 2013. Fermentation characteristics of yeasts isolated from traditionally fermented masau (Ziziphus mauritiana) fruits. Int. J. Food Microbiol. 166, 426–432.
Rodriguez, G.M., Tashiro, Y., and Atsumi, S. 2014. Expanding ester biosynthesis in Escherichia coli. Nat. Chem. Biol. 10, 259–265.
Saerens, S.M.G., Delvaux, F.R., Verstrepen, K.J., and Thevelein, J.M. 2010. Production and biological function of volatile esters in Saccharomyces cerevisiae. Microb. Biotechnol. 3, 165–177.
Saerens, S.M.G., Verstrepen, K.J., Van Laere, S.D.M., Voet, A.R.D., Van Dijck, P., Delvaux, F.R., and Thevelein, J.M. 2006. The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. J. Biol. Chem. 281, 4446–4456.
Schneider, J., Rupp, O., Trost, E., Jaenicke, S., Passoth, V., Goesmann, A., Tauch, A., and Brinkrolf, K. 2012. Genome sequence of Wickerhamomyces anomalus DSM 6766 reveals genetic basis of biotechnologically important antimicrobial activities. FEMS Yeast Res. 12, 382–386.
Söding, J. 2005. Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951–960.
Sohn, M.J., Oh, D.B., Kim, E.J., Cheon, S.A., Kwon, O., Kim, J.Y., Lee, S.Y., and Kang, H.A. 2012. HpYPS1 and HpYPS7 encode functional aspartyl proteases localized at the cell surface in the thermotolerant methylotrophic yeast Hansenula polymorpha. Yeast 29, 1–16.
Son, E.Y., Lee, S.M., Kim, M., Seo, J.A., and Kim, Y.S. 2018. Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae. Food Res. Int. 109, 596–605.
Stribny, J., Querol, A., and Perez-Torrado, R. 2016. Differences in enzymatic properties of the Saccharomyces kudriavzevii and Saccharomyces uvarum alcohol acetyltransferases and their impact on aroma-active compounds production. Front. Microbiol. 7, 897.
Su, C., Zhang, K.Z., Cao, X.Z., and Yang, J.G. 2020. Effects of Saccharomycopsis fibuligera and Saccharomyces cerevisiae inoculation on small fermentation starters in Sichuan-style Xiaoqu liquor. Food Res. Int. 137, 109425.
Tashiro, Y., Desai, S.H., and Atsumi, S. 2015a. Two-dimensional isobutyl acetate production pathways to improve carbon yield. Nat. Commun. 6, 7488.
Tashiro, Y., Rodriguez, G.M., and Atsumi, S. 2015b. 2-Keto acids based biosynthesis pathways for renewable fuels and chemicals. J. Ind. Microbiol. Biotechnol. 42, 361–373.
ter Veld, F., Wolff, D., Schorsch, C., Kohler, T., Boles, E., and Poetsch, A. 2013. Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p. Appl. Microbiol. Biotechnol. 97, 8537–8546.
Tiwari, R., Koffel, R., and Schneiter, R. 2007. An acetylation/deacetylation cycle controls the export of sterols and steroids from S. cerevisiae. EMBO J. 26, 5109–5119.
Van Laere, S.D.M., Saerens, S.M.G., Verstrepen, K.J., Van Dijck, P., Thevelein, J.M., and Delvaux, F.R. 2008. Flavour formation in fungi: characterisation of KlAtf, the Kluyveromyces lactis orthologue of the Saccharomyces cerevisiae alcohol acetyltransferases Atf1 and Atf2. Appl. Microbiol. Biotechnol. 78, 783–792.
van Rijswijck, I.M.H., Wolkers-Rooijackers, J.C.M., Abee, T., and Smid, E.J. 2017. Performance of non-conventional yeasts in coculture with brewers’ yeast for steering ethanol and aroma production. Microb. Biotechnol. 10, 1591–1602.
Varela, C. 2016. The impact of non-Saccharomyces yeasts in the production of alcoholic beverages. Appl. Microbiol. Biotechnol. 100, 9861–9874.
Verstrepen, K.J., Van Laere, S.D.M., Vanderhaegen, B.M.P., Derdelinckx, G., Dufour, J.P., Pretorius, I.S., Winderickx, J., Thevelein, J.M., and Delvaux, F.R. 2003. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters. Appl. Environ. Microbiol. 69, 5228–5237.
Verstrepen, K.J., Van Laere, S.D.M., Vercammen, J., Derdelinckx, G., Dufour, J.P., Pretorius, I.S., Winderickx, J., Thevelein, J.M., and Delvaux, F.R. 2004. The Saccharomyces cerevisiae alcohol acetyl transferase Atf1p is localized in lipid particles. Yeast 21, 367–377.
Yoshimoto, H., Fujiwara, D., Momma, T., Ito, C., Sone, H., Kaneko, Y., and Tamai, Y. 1998. Characterization of the ATF1 and Lg-ATF1 genes encoding alcohol acetyltransferases in the bottom fermenting yeast Saccharomyces pastorianus. J. Ferment. Bioeng. 86, 15–20.
Yoshimoto, H., Fujiwara, D., Momma, T., Tanaka, K., Sone, H., Nagasawa, N., and Tamai, Y. 1999. Isolation and characterization of the ATF2 gene encoding alcohol acetyltransferase II in the bottom fermenting yeast Saccharomyces pastorianus. Yeast 15, 409–417.
Yoshioka, K. and Hashimoto, N. 1981. Ester formation by alcohol acetyltransferase from brewer’s yeast. Agri. Biol. Chem. 45, 2183–2190.
Zhu, J., Lin, J.L., Palomec, L., and Wheeldon, I. 2015. Microbial host selection affects intracellular localization and activity of alcohol-O-acetyltransferases. Microb. Cell Fact. 14, 35.
Acknowledgements
This research was supported by the Korean Ministry of Agriculture, Food, and Rural Affairs, Grant No. 918010042HD030 (Strategic Initiative for Microbiomes in Agriculture and Food). This research was supported by the Chung-Ang University Research Scholarship Grants in 2020 (to K.S. Kim). We are grateful to Prof. Ian Wheeldon (University of California Riverside) for providing the pERGmDsRed plasmid and to Azin Rashed for her technical assistance with vector construction.
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Moon, H.Y., Kim, H.J., Kim, K.S. et al. Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation. J Microbiol. 59, 598–608 (2021). https://doi.org/10.1007/s12275-021-1159-8
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DOI: https://doi.org/10.1007/s12275-021-1159-8