Abstract
During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.
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References
McGovern PE, Zhang JZ, Tang JG, Zhang ZQ, Hall GR, Moreau RA, Nun͂ez A, Butrym ED, Richards MR, Wang CS, Cheng GS, Zhao ZJ, Wang CS (2005) Fermented beverages of pre- and proto-historic China. P Natl Acad Sci USA 101:17593–17598
Zhang WX, Qiao ZW, Shigematsu T, Tang YQ (2005) Analysis of the bacterial community in Zaopei during production of Chinese Luzhou-flavor Liquor. J I Brewing 111:215–222
Chen YF, Li F, Guo J, Liu GX (2013) Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1. J Ind Microbiol Biot 41:563–572
Li XW, Guo DY, Cheng YB, Zhu FY, Deng ZX, Liu TG (2014) Overproduction of fatty acids in engineered Saccharomyces cerevisiae. Biotechnol Bioeng 111:1841–1852
Zhao JS, Zheng J, Zhou RQ, Shi B (2012) Microbial community structure of pit mud in a Chinese strong aromatic liquor fermentation pit. J Inst Brew 118:356–360
Saerens SM, Verstrepen KJ, Van Laere SD (2006) The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzyme with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity. J Biol Chem 281:4446–4456
Peddie HA (1990) Ester formation in brewery fermentations. J Inst Brew 96:327–331
Zhang JW, Zhang CY, Dai LH, Dong J, Liu YL et al (2012) Effects of overexpression of the alcohol acetyltransferase-encoding gene ATF1 and disruption of the esterase-encoding gene IAH1 on the flavour profiles of Chinese yellow rice wine. Int J Food Sci Tech 47:2590–25968
Ichikawa E, Hosokawa N, Hata Y et al (1991) Breeding of a sake yeast with improved ethyl caproate productivity. Agric Biol Chem 55:2153–2154
Lian JZ, Zhao HM (2015) Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites. J Ind Microbiol Biot 42:437–451
Lilly M, Bauer FF, Lambrechts MG, Swiegers JH (2006) The effect of increased yeast alcohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates. Yeast 23:641–659
Fujii T, Kobayshi O, Yoshimoto H et al (1997) Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene. Appl Environ Microb 63:910–915
Shi S, Chen Y, Siewers V, Nielsen J (2014) Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of ACC1. mBio 5(3):e01130–14
Furukawa K, Yamada T, Mizoguchi H (2003) Increased ethyl caproate production by inositol limitation in Saccharomyces cerevisiae. J Biosci Bioeng 5:448–454
Kaadige MR, Lopes JM (2006) Analysis of Opi1p repressor mutants. Curr Genet 49:30–38
Hablacher M, Ivessa AS et al (1993) Acetyl-coA carboxylase from yeast is an essential enzyme and is regulated by factors that control phospholipid metabolism. J Biol Chem 268:10946–10952
Choi JW, Silva NA (2014) Improving polyketide and fatty acid synthesis by engineering of the yeast acetyl-CoA carboxylase. J Biotechnol 187:56–59
Zheng XW, Tabrizi MR et al (2011) Daqu-a traditional Chinese liquor fermentation starter. J Inst Brew 117:82–90
Cordente AG, Curtin CD, Varela C, Pretorius IS (2012) Flavour-active wine yeasts. Appl Microbiol Biotechnol 96:601–618
Saerens SM, Delvaux FR, Verstrepen KJ, Thevelein JM (2010) Production and biological function of volatile esters in Saccharomyces cerevisiae. Microb Biotechnol 3:165–177
Jong BW, Shi SB et al (2014) Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration. J Ind Microbiol Biotechnol 42:477–486
Yang B, Chen HQ (2014) Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli. J Appl Microbiol 117:430–439
Gibson DG (2011) Enzymatic assembly of overlapping DNA fragments. Methods Enzymol 498:350–360
Lilly M, Lambrechts M, Pretorius I (2000) Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Appl Environ Microbiol 66:744–753
Tehlivets O, Scheuringer K, Kohlwein SD (2006) Fatty acid synthesis and elongation in yeast. Biochim Biophys Acta 1771:255–270
Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346
Runguphan W, Keasling JD (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metab Eng 21:103–113
Sumby KM, Grbin PR, Jiranek V (2010) Microbial modulation of aromatic esters in wine: current knowledge and future prospects. Food Chem 121:1–16
Fan W, Qian MC (2005) Headspace solid phase microextraction and gas chromatography-olfactometry dilution analysis of young and aged Chinese “yanghe daqu” liquors. J Agric Food Chem 53:7931–7938
Nielsen J, Larsson C et al (2013) Metabolic engineering of yeast for production of fuels and chemicals. Curr Opin Biotechnol 24:398–404
Wu XH, Zheng XW, Vervoort J (2009) Characterization of Chinese liquor starter, “Daqu”, by flavor type with 1H NMR-based nontargeted analysis. J Agric Food Chem 57:11354–11359
Gao L, Chiou W, Tang H (2007) Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS. J Chromatogr B853:303–313
Park YC, Shaffer CE, Bennett GN (2009) Microbial formation of esters. Appl Microbiol Biotechnol 85:13–25
Chen LW, Zhang JH, Lee J (2014) Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism. Appl Microbiol Biotechnol 98:6739–6750
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This research was financed by the program for the National High Technology Research and Development Program of China (Grant No. 2013AA102108) and the National Natural Science Foundation of China (Grant No. 31271916).
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Chen, Y., Luo, W., Gong, R. et al. Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with OPI1 deletion. J Ind Microbiol Biotechnol 43, 1261–1270 (2016). https://doi.org/10.1007/s10295-016-1795-x
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DOI: https://doi.org/10.1007/s10295-016-1795-x