Abstract
Ethyl-acetate is important for the flavor and aroma of the alcoholic beverages, therefore, there have been extensive efforts toward increasing its production by engineering yeast strains. In this study, we reported a new approach to breed non-genetic modified producing yeast strain with higher ethyl-acetate production for beer brewing. First, we demonstrated the positive effect of higher acetic acid concentration on inducing the expression of acetyl-CoA synthetase (ACS). Then, we applied adaptive laboratory evolution method to evolve strain with higher expression level of ACS. As a result, we obtained several evolved strains with increased ACS expression level as well as ethyl-acetate production. In 3 L scale fermentation, the optimal strain EA60 synthesized more ethyl-acetate than M14 at the same time point. At the end of fermentation, the ethyl-acetate production in EA60 was 21.4% higher than M14, while the other flavor components except for acetic acid were changed in a moderate degree, indicating this strain had a bright prospect in industrial application. Moreover, this study also indicated that ACS1 played a more important role in increasing the acetic acid tolerance of yeast, while ACS2 contributed to the synthesis of cytosol acetyl-CoA, thereby facilitating the production of ethyl-acetate during fermentation.
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References
Armando JW, Boghigian BA, Pfeifer BA (2012) LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Lett Appl Microbiol 54(2):140–148. https://doi.org/10.1111/j.1472-765X.2011.03184.x
Bloem A, Sanchez I, Dequin S, Camarasa C (2016) Metabolic impact of redox cofactor perturbations on the formation of aroma compounds in Saccharomyces cerevisiae. Appl Environ Microbiol 82(1):174–183. https://doi.org/10.1128/aem.02429-15
Chen Y, Siewers V, Nielsen J (2012) Profiling of cytosolic and peroxisomal acetyl-coa metabolism in Saccharomyces cerevisiae. PLoS ONE 7(8):e42475. https://doi.org/10.1371/journal.pone.0042475
Ding J, Holzwarth G, Penner MH, Patton-Vogt J, Bakalinsky AT (2015) Overexpression of acetyl-CoA synthetase in Saccharomyces cerevisiae increases acetic acid tolerance. FEMS Microbiol Lett 362(3):1–7. https://doi.org/10.1093/femsle/fnu042
Dong J, Wang P, Fu X, Dong S, Li X, Xiao D (2019) Increase ethyl acetate production in Saccharomyces cerevisiae by genetic engineering of ethyl acetate metabolic pathway. J Ind Microbiol Biotechnol 46(6):801–808. https://doi.org/10.1007/s10295-019-02142-0
Dzialo MC, Park R, Steensels J, Lievens B, Verstrepen KJ (2017) Physiology, ecology and industrial applications of aroma formation in yeast. FEMS Microbiol Rev 41(1):S95-s128. https://doi.org/10.1093/femsre/fux031
Fujii T, Kobayashi O, Yoshimoto H, Furukawa S, Tamai Y (1997) Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene. Appl Environ Microbiol 63(3):910–915
Lei H, Li H, Mo F, Zheng L, Zhao H, Zhao M (2013a) Effects of Lys and His supplementations on the regulation of nitrogen metabolism in lager yeast. Appl Microbiol Biotechnol 97(20):8913–8921
Lei H, Zheng L, Wang C, Zhao H, Zhao M (2013b) Effects of worts treated with proteases on the assimilation of free amino acids and fermentation performance of lager yeast. Int J Food Microbiol 161(2):76–83
Lian J, Mishra S, Zhao H (2018) Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications. Metab Eng 50:85–108. https://doi.org/10.1016/j.ymben.2018.04.011
Lian J, Si T, Nair NU, Zhao H (2014) Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains. Metab Eng 24:139–149. https://doi.org/10.1016/j.ymben.2014.05.010
Lilly M, Bauer FF, Lambrechts MG, Swiegers JH, Cozzolino D, Pretorius IS (2006) The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast 23(9):641–659. https://doi.org/10.1002/yea.1382
Lin JL, 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(8):e104141. https://doi.org/10.1371/journal.pone.0104141
Liu C, Li Q, Niu C, Zheng F, Li Y, Zhao Y, Yin X (2017) Genome sequence of the lager-brewing yeast Saccharomyces sp. strain m14, used in the high-gravity brewing industry in China. Genome Announc. https://doi.org/10.1128/genomeA.01194-17
Mason AB, Dufour JP (2000) Alcohol acetyltransferases and the significance of ester synthesis in yeast. Yeast 16(14):1287–1298. https://doi.org/10.1002/1097-0061(200010)16:14%3c1287::aid-yea613%3e3.0.co;2-i
Nykänen L, Nykänen I, Suomalainen H (1977) Distribution of esters produced during sugar fermentation between the yeast cell and the medium. J Inst Brew 83(1):32–34
Peddie HA (1990) Ester formation in brewery fermentations. J Inst Brew 96(5):327–331
Pires EJ, Teixeira JA, Branyik T, Vicente AA (2014) Yeast: the soul of beer’s aroma–a review of flavour-active esters and higher alcohols produced by the brewing yeast. Appl Microbiol Biotechnol 98(5):1937–1949. https://doi.org/10.1007/s00253-013-5470-0
Saerens S, Verbelen P, Vanbeneden N, Thevelein J, Delvaux F (2008) Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast. Appl Microbiol Biotechnol 80(6):1039–1051
Shi TT, Li P, Chen SJ, Chen YF, Guo XW, Xiao DG (2017) Reduced production of diacetyl by overexpressing BDH2 gene and ILV5 gene in yeast of the lager brewers with one ILV2 allelic gene deleted. J Ind Microbiol Biotechnol 44(3):397–405. https://doi.org/10.1007/s10295-017-1903-6
van den Berg MA, de Jong-Gubbels P, Kortland CJ, van Dijken JP, Pronk JT, Steensma HY (1996) The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation. J Biol Chem 271(46):28953–28959
Verstrepen KJ, Van Laere SD, Vanderhaegen BM, Derdelinckx G, Dufour JP, Pretorius IS, Winderickx J, Thevelein JM, Delvaux FR (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(9):5228–5237
Xu X, Liu C, Niu C, Wang J, Zheng F, Li Y, Li Q (2018a) Rationally designed perturbation factor drives evolution in Saccharomyces cerevisiae for industrial application. J Ind Microbiol Biotechnol 45(10):869–880. https://doi.org/10.1007/s10295-018-2057-x
Xu X, Wang J, Bao M, Niu C, Liu C, Zheng F, Li Y, Li Q (2018b) Reverse metabolic engineering in lager yeast: impact of the NADH/NAD+ ratio on acetaldehyde production during the brewing process. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-018-9517-0
Yoshimoto H, Fujiwara D, Momma T, Ito C, Sone H, Kaneko Y, 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(1):15–20
Yoshioka K, Hashimoto N (1984) Acetyl-CoA of brewers’ yeast and formation of acetate esters. Agric Biol Chem 48(1):207–209. https://doi.org/10.1080/00021369.1984.10866117
Zhang S, Guo F, Yan W, Dong W, Zhou J, Zhang W, Xin F, Jiang M (2020) Perspectives for the microbial production of ethyl acetate. Appl Microbiol Biotechnol 104(17):7239–7245. https://doi.org/10.1007/s00253-020-10756-z
Acknowledgements
This work was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program of Introducing Talents of Discipline to Universities (No. 111-2-06), the National High Technology Research and Development program 863 (No. 2018YFD0400403) and the National Science Foundation (Nos. 31771963 and 31901626).
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Xu, X., Niu, C., Liu, C. et al. Screening lager yeast with higher ethyl-acetate production by adaptive laboratory evolution in high concentration of acetic acid. World J Microbiol Biotechnol 37, 125 (2021). https://doi.org/10.1007/s11274-021-03082-7
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DOI: https://doi.org/10.1007/s11274-021-03082-7