AUTHOR=Cui Dan-Yao , Wei Ya-Nan , Lin Liang-Cai , Chen Shi-Jia , Feng Peng-Peng , Xiao Dong-Guang , Lin Xue , Zhang Cui-Ying 

TITLE=Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through Saccharomyces cerevisiae Metabolic Engineering

JOURNAL=Frontiers in Microbiology

VOLUME=11

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.596306

DOI=10.3389/fmicb.2020.596306

ISSN=1664-302X

ABSTRACT=<p>Baijiu is a traditional distilled beverage in China with a rich variety of aroma substances. 2,3,5,6-tetramethylpyrazine (TTMP) is an important component in Baijiu and has the function of promoting cardiovascular and cerebrovascular health. During the brewing of Baijiu, the microorganisms in <italic>jiuqu</italic> produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene <italic>BDH1</italic> and another <italic>BDH2</italic> gene were deleted or overexpressed to evaluate the effect on the content of acetoin and TTMP in <italic>Saccharomyces cerevisiae</italic>. The results showed that the acetoin synthesis of strain α5-D1B2 was significantly enhanced by disrupting <italic>BDH1</italic> and overexpressing <italic>BDH2</italic>, leading to a 2.6-fold increase of TTMP production up to 10.55 mg/L. To further improve the production level of TTMP, the α-acetolactate synthase (ALS) of the pyruvate decomposition pathway was overexpressed to enhance the synthesis of diacetyl. However, replacing the promoter of the <italic>ILV2</italic> gene with a strong promoter (<italic>PGK1p</italic>) to increase the expression level of the <italic>ILV2</italic> gene did not result in further increased diacetyl, acetoin and TTMP production. Based on these evidences, we constructed the diploid strains AY-SB1 (Δ<italic>BDH1:loxP/</italic>Δ<italic>BDH1:loxP</italic>) and AY-SD1B2 (Δ<italic>BDH1:loxP-PGK1p-BDH2-PGK1t/</italic>Δ<italic>BDH1:loxP-PGK1p-BDH2-PGK1t</italic>) to ensure the fermentation performance of the strain is more stable in Baijiu brewing. The concentration of TTMP in AY-SB1 and AY-SD1B2 was 7.58 and 9.47 mg/L, respectively, which represented a 2.3- and 2.87-fold increase compared to the parental strain. This work provides an example for increasing TTMP production in <italic>S. cerevisiae</italic> by genetic engineering, and highlight a novel method to improve the quality and beneficial health attributes of Baijiu.</p>