Enhancing Medium-Chain Fatty Acid Ethyl Ester Production During

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Chemistry and Biology of Aroma and Taste

Enhancing Medium Chain Fatty Acid Ethyl Ester Production During Beer Fermentation Through EEB1 and/ or ETR1 Overexpression in Saccharomyces pastorianus Hua Yin, Ling-Pu Liu, Mei Yang, Xiao-Tong Ding, Shiru Jia, Jian-Jun Dong, and Cheng Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00128 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Journal of Agricultural and Food Chemistry

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Enhancing Medium Chain Fatty Acid Ethyl Ester Production

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During Beer Fermentation Through EEB1 and/or ETR1

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Overexpression in Saccharomyces pastorianus

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Hua Yin1, Ling-Pu Liu2,3, Mei Yang1, Xiao-Tong Ding2,3, Shi-Ru Jia2,3, Jian-Jun

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Dong1,2, Cheng Zhong2,3,*

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1. State Key Laboratory of Biological Fermentation Engineering of Beer, Tsingtao

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Brewery Co Ltd, R&D Ctr, Qingdao 266101, Shandong, P. R. China

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2. State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science &

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Technology, Tianjin 300457, P. R. China

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3. Key Laboratory of Industrial Fermentation Microbiology, (Ministry of Education),

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Tianjin University of Science & Technology, Tianjin 300457, P. R. China

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*Corresponding author: Tel.: +86-22-60601268; Fax: +86-022-60602298

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E-mail: [email protected]; [email protected] (Cheng Zhong)

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ACS Paragon Plus Environment


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Abstract

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Esters are important flavor compounds in alcoholic beverages. Although they are

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present in trace levels, esters play a key role in flavor formation, especially fruity

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flavors, in beverages. Low ester contents result in bland beer and unpleasant flavor. In

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this study, three recombinant strains, ethanol O-acyltransferase-encoding EEB1

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overexpression strain (31194::EEB1), 2-enoyl thioester reductase-encoding ETR1

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overexpression strain (31194::ETR1), and EEB1/ETR1 co-overexpression strain

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(31194::EEB1::ETR1), were constructed. Ethyl hexanoate productions by 31194::EEB1

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and 31194::EEB1::ETR1 were all 106% higher than that by the parental strain. Further,

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ethyl octanoate production by 31194::EEB1 and 31194::EEB1::ETR1 were enhanced by

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47% and 41%, respectively, compared to that by the parental strain 31194. However,

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no difference was observed between 31194::ETR1 and the parental strain in terms of

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ethyl hexanoate and ethyl octanoate production. This indicates that while the EEB1

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overexpression in Saccharomyces pastorianus enhanced ethyl hexanoate and ethyl

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octanoate production, ETR1 expression level did not affect the extracellular

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concentration of these esters.

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Keywords: Beer, Saccharomyces pastorianus, Ethanol O-acyltransferases, Medium

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chain fatty acid ethyl esters

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1. Introduction

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Preferable beer flavors can be obtained with the right balance between its volatile

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and non-volatile constituents such as higher alcohols, aldehydes, organic acids, esters,

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ketones, phenols, and sulfides1, 2. While some parameters such as yeast activity, wort

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composition, and operative conditions (temperature management, etc) have an impact

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on beer flavor. For example, in wine must fermentation, the emission of large

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amounts of carbon dioxide and water vapor leads to a nonstop stripping-off aroma

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active compounds3.

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fermentation4. Higher alcohols, also known as fusel alcohols, and aromatic esters are

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two of the most abundant and important aromatic compounds found in beer5. Medium

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chain fatty acid (MCFA) ethyl esters are thought to contribute to fatty odor and

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bitterness in Japanese sake6. Although esters are present only in trace amounts, even

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slight changes in their concentrations can affect the final sensorial quality of

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fermented beverages7.

A part of flavor-active compounds are produced by yeast during

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Fermented beverages primarily contain two types of aromatic esters. The first

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type is acetate esters. Alcohol acetyl transferase (AAtase; EC2.3.1.84) catalyzes the

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synthesis of acetate esters, such as isoamyl acetate (banana-like flavor), ethyl acetate

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(solvent-like flavor), and phenyl ethyl acetate (rose-like flavor), from acetyl-CoA and

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the corresponding alcohols. Previous studies have shown that there are three types of

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AATase, namely AATaseI, Lg-AATase, and AATaseII, which are encoded by ATF1,

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Lg-ATF1, and ATF2 respectively8-10. Overexpression of ATF1 gene in Kluyveromyces

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marxianus during mezcal production resulted in enhanced levels of primary esters, 3



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including ethyl acetate, ethyl hexanoate, ethyl octanoate, 2-phenylethyl acetate,

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isoamyl decanoate, ethyl tetradecanoate, ethyl hexadecanoate, and ethyl linoleate.

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SLI1 encodes for an N-acetyltransferase with possible activity towards ester

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biosynthesis11. Disruption of ATF1 and SLI1 also reduced ethyl acetate production (by

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24% and 28.4%, respectively), but did not have a significant effect on the other esters

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in K.marxianus12. Further, during beer preparation, significantly higher levels of

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isoamyl acetate and ethyl acetate were obtained with yeast strains that

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overexpressATF1 and ATF2 than with wild-type yeast strains13, 14.

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The second type is ethyl esters, which mainly include ethyl hexanoate (apple-like

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flavor), ethyl octanoate (apple-like flavor), and ethyl decanoate (flower-like flavor)4,

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15.

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by ethanol O-acyltransferases, which are primarily encoded by EHT1 and EEB1 that

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belong to a three-member S. cerevisiae gene family (YBR177c/EHT1, YBR177c/EEB1,

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and YMR210w). These enzymes catalyze the condensation reaction between an

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acyl-CoA component and ethanol to form MCFA ethyl esters16. Lower concentrations

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of ethyl butanoate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate were

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obtained during fermentation using S. cerevisiae deletion strain BY4741eeb1 than

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during fermentation using wild-type strain16. In contrast to these results, deletion of

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EHT1 did not affect ethyl butanoate and ethyl decanoate production and resulted in

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only minor decreases in levels of ethyl hexanoate (36%) and ethyl octanoate (20%)16.

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Furthermore, ethyl ester (ethyl butanoate, ethyl hexanoate, and ethyl decanoate)

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concentrations obtained using double deletion strain BY4741eeb1eht1 were similar

Previous studies have demonstrated that ethyl ester production in yeast is catalyzed

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to those obtained with single deletion strain BY4741 eeb1. This suggests that EEB1

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plays a more dominant role in MCFA ethyl ester synthesis than EHT116.

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During the formation of MCFA ethyl esters, ethanol O-acyltransferases catalyze

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the condensation reaction between an acyl-CoA component and ethanol17. As

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acyl-CoA is a substrate of this reaction, its concentration is an important factor for the

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formation of MCFA ethyl esters. In order to enhance the formation of acyl-CoA,

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ETR1, which encodes intramitochondrial fatty acid synthesis (FAS) type II 2-enoyl

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thioester reductase in yeast, was selected as the target gene. 2-enoyl thioester

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reductase Etr1p is a key enzyme for fatty acid elongation, and catalyzes the last

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reaction of each fatty acid elongation cycle in the endoplasmic reticulum18.

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In the current study, recombinant Saccharomyces pastorianus strains that

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overexpress EEB1 and/or ETR1 were constructed to investigate the effect of two

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genes at the MCFA ethyl ester production during beer fermentation. Levels of ethyl

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hexanoate and ethyl octanoate produced by these strains after fermentation for 9 d

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were measured via headspace-gas chromatography (HS-GC). Further, the

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recombinant strains were characterized in terms of mRNA levels, ethyl hexanoate and

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ethyl octanoate production capacity, and other fermentation performance parameters.

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2. Materials and methods

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2.1 Strains, plasmids, and culture conditions

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All plasmids and strains used in this study are listed in Table 1. The parent strain,

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Saccharomyces pastorianus TCCC 31194, was an industrial lager brewing strain, and

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kindly provided by the State Key Laboratory of Biological Fermentation Engineering 5



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of Beer (Tsingtao Brewery Co., Ltd. Shandong Province, China). Escherichia coli

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DH5α strain conserved in our laboratory was used. pMD18-T Vector was purchased

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from Takara Biotechnology (Dalian) Co., Ltd., and plasmids pUG6 and pYPGE15

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were obtained from our laboratory.

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Escherichia coli was grown at 37℃ in a Luria-Bertani (LB) medium (composed

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of 10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract) supplemented with

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ampicillin (100 μg/mL) or kanamycin (100 μg/mL) to select positive E. coli

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transformants. Yeast extract peptone dextrose (YEPD) medium (composed of 10 g/L

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yeast extract, 20 g/L peptone, and 20 g/L glucose) was used to cultivate the yeast

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strains. YEPD plates were supplemented with 80 μg/mL filter-sterilized G418

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antibiotic (Promega, USA) to select for yeast transformants harboring KanMX gene.

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All the solid media used in this study were prepared with 20 g/L agar.

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2.2 Construction of pEEB1, pETR1, and pEEB1_ETR1 plasmids and

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transformation

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Genomic DNA (template DNA) of S. pastorianus was isolated using Dr.

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GenTLE® (from Yeast) High Recovery (Takara Bio, Shiga, Japan). The polymerase

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chain reaction (PCR) primers used in this study are listed in Table 2.

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The plasmids (pEEB1, pETR1 and pEEB1_ETR1) had a G418 selection marker

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derived from plasmid pUG6. The EEB1 gene (PCR product 1) of yeast was amplified

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with EEB1-F and EEB1-R primers, while the CYC1 terminator region (PCR product 2)

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of plasmid pYPGE15 was amplified with CYC1(ET)-F and CYC1(TK)-R primers.

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The loxP-KanMX-loxP fragment (PCR product 3) of plasmid pUG6 was amplified 6


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with

KanMX(TK)-F

and

KanMX(S)-R

primers.

Subsequently,

ECK

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(EEB1+CYC1+KanMX) fragment (PCR product ECK) was obtained via fusion PCR

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of PCR products 1, 2, and 3 using EEB1-F and KanMX(S)-R primers with BamH I

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and Sma I restriction sites. This fragment was cloned into a multi-cloning site of

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pYPGE15, forming a plasmid containing the EEB1 gene, which is controlled by the

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PGK1 promoter and CYC1 terminator, and G418 resistance gene KanMX. This

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plasmid was named pEEB1. The process followed for the construction of pETR1,

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containing the ETR1 gene, which is controlled by the PGK1 promoter and CYC1

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terminator, and G418 resistance gene KanMX is similar to that of pEEB1.

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Plasmid pEEB1_ETR1 was constructed for overexpression of EEB1 and ETR1

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genes. The PGK1 promoter (PCR product 4) from yeast was amplified with primers

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PGK-F and PGK(PE)-R, while the ETR1 gene (PCR product 5) from S. cerevisiae

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was amplified with primers ETR1(PE)-F and ETR1(K)-R. Subsequently, the PR

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(PGK1+ETR1) fragment (PCR product PR) was obtained by fusing PCR products 4

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and 5 via fusion PCR using primers PGK-F and ETR1(K)-R containing restriction

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sites Apa I and Kpn I. This PCR product was digested with Apa I/Kpn I and then

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ligated into the Apa I/Kpn I sites of plasmid pYPGE15 to construct the pYPGE-PR

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plasmid. The plasmid pEEB1 was digested with BamH I/Sma I to obtain the ECK

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(EEB1+CYC1+KanMX) fragment, which was then ligated into the BamH I/Sma I

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sites of plasmid pYPGE-PR to construct pEEB1_ETR1. All plasmid constructions

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were performed with E. coli strain DH5α and all the constructed plasmids were

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confirmed by sequencing analysis (Genewiz, Inc., Suzhou, China). 7



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2.3 Yeast transformants and construction of recombinant yeast strains

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Yeast strains were transformed using the lithium acetate/single-stranded carrier

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DNA/polyethylene glycol (PEG) method19. The transformants were screened on

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YEPD plates containing 80 mg/L of G418 antibiotic. The recombinant strains were

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verified via PCR with specific primers.

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In order to investigate the effects of overexpression of either EEB1 or ETR1 in S.

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pastorianus on beer fermentation, ethanol O-acyltransferase-encoding EEB1

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overexpression strain (31194::EEB1) and 2-enoyl thioester reductase-encoding ETR1

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overexpression strain (31194::ETR1) were constructed by transforming the plasmids

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pEEB1 and pETR1, respectively, into the industrial strain S. pastorianus a. Similarly,

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EEB1/ETR1 co-overexpression strain (31194::EEB1::ETR1) was constructed by

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transforming pEEB1_ETR1 into S. pastorianus a, and was used to determine the

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effects of overexpression of both EEB1 and ETR1 in S. pastorianus on beer

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fermentation. The transformants were screened on G418 selective plates and verified

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through PCR with specific primers.

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2.4 Beer fermentation

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2.4.1 Wort preparation

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Malt wort was prepared by mixing 1 kg of crushed pale malt and 4 L of water.

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Wort preparation was carried out at the following temperatures: 37℃ for 30 min, 45℃

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for 30 min for protease activity, 63℃ for 60 min to allow for β-amylase activity, 70℃

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for 60 min to allow for α-amylase activity, and 78℃ for 15 min to inactivate all the

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enzymes. The resulting mash was then filtered to separate the wort, and the residual 8


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mash was washed with approximately 2 L of warm water. Subsequently, the wort, to

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which 0.5 g/L of hops was added, was allowed to boil for 1 h. The hops were added in

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two installments; two-thirds of it was added after the wort had been boiling for 5 min,

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and the rest was added after boiling the wort for 40 min. After cooling the wort to

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room temperature, it was filtered through a filter cloth and the sugar content of the

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filtrate was adjusted to 12°Plato.

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2.4.2 Fermentation conditions

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Yeast cells were precultured in 4 mL of wort medium (12°Plato) for 24 h at 28℃

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with shaking at 180 rpm. These cells were then transferred to 50 mL of wort medium

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(12°Plato) in a 150 mL conical flask and incubated for 12 h at 20℃ with shaking at

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180 rpm. This precultured yeast cell suspension (15 mL) was transferred to a 500 mL

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conical flask containing 150 mL of wort medium (12°Plato) and incubated for 12 h at

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15℃ with shaking at 180 rpm. After this, samples were all withdrawn and centrifuged

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at 1700 ｘ g for 10 min at 4℃. The yeast cells were then resuspended in wort to

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achieve a cell concentration of 4×107 cfu/mL. Subsequently, 40 mL of this inoculum

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was transferred into a 1000 mL conical flask containing 400 mL of wort medium

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(12°Plato) and incubated at 10℃ for 8–10 d. All fermentations were carried out in

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triplicates.

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2.5 Determination of fermentation performance

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Fermentation performance indicators, wort density and ethanol content, were

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measured using a pycnometer (Chinese National Standard for Beer Analysis GB/T

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4928-2008) and residual sugar was analyzed via the Fehling titration method20. The 9



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real degree of fermentation (RDF) was calculated using formula (1).

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(1) where RDF is expressed in %, A denotes ethanol content (%), and Z represents wort density (%).

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Volatile flavor compounds (higher alcohols and esters) were analyzed using a

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gas chromatograph (Agilent 7890N, GC System, Palo Alto, CA, USA) with a Gerstel

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multi- purpose sampler (Gerstel, Mülheim an der Ruhr, Germany) and flame

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ionization detector (FID). Ethyl formate was used as the internal standard. The

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column used for separation was a DB-WAXETR column (length, 60m; internal

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diameter, 0.32mm; film thickness, 0.25; Palo Alto, CA, USA). The chemicals were

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purchased from Alfa Aesar (Karlsruhe, Germany). The quantification of targeted

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compounds was carried out using the internal standard calibration curve. A series of

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50, 100, 200, 300, 400 µg/L mixture standards with 1 mg/L internal standard,

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respectively, was analyzed. Based on these, the calibration curve was established by

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linear regression.

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2.6 RNA extraction and real-time quantification-PCR

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Yeast cells were harvested by centrifugation (4℃, 2650 ｘ g, 2 min) after beer

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fermentation for 48 h, and immediately stored in liquid nitrogen until RNA extraction.

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The cells were ground into a powder, and RNA was extracted and purified using a

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yeast RNA kit (Omega, USA). RNA concentrations and quality were determined

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using a BioSpectrometer® basic (Eppendorf, Germany) and through agarose gel

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electrophoresis. Total RNA was used to synthesize oligo (dT) 18-primer cDNA using 10


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a Revert aid first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA,

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USA), according to manufacturer’s instructions. Real-time quantification-PCR

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(RT-qPCR) was performed on a StepOne™ real-time PCR system (Applied

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Biosystems, Foster City, CA) and using a DyNAmo color flash SYBR green qPCR kit

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(Thermo Fisher Scientific, Waltham, MA, USA). All reactions were performed in

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triplicates. No-template controls were included in each PCR, and the data were

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normalized to gene ACT1 rRNA21, 22. The thermal profile used for all PCRs is 95°C

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for 2 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. Amplicon dissociation

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curves were obtained after cycle 40 by heating from 60°C to 95°C at 0.3°C/s. The

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specificity of PCR amplification was confirmed by the presence of dissociation curves

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with single peaks and unique amplicons of the expected size upon electrophoresis on

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agarose gels. The RT-qPCR data were analyzed using StepOne Software v2.3

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(Thermo Fisher Scientific, Waltham, MA, USA). Gene expression of the parental

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strain was used as control. All quantifications were normalized to that of ACT1 rRNA,

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used as an internal standard21-23. The △△Ct method was used for relative

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quantification. The sequences of the primers used are listed in Table 2. The coding

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sequences of these genes were retrieved from the National Center for Biotechnology

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Information (NCBI) nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore).

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2.7 Statistical Analyses

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Raw data were further analyzed by Student’s test (*indicates p

Enhancing Medium-Chain Fatty Acid Ethyl Ester Production During

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