Dataset Information

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

ABSTRACT: 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 jiuqu produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene BDH1 and another BDH2 gene were deleted or overexpressed to evaluate the effect on the content of acetoin and TTMP in Saccharomyces cerevisiae. The results showed that the acetoin synthesis of strain ?5-D1B2 was significantly enhanced by disrupting BDH1 and overexpressing BDH2, 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 ?-acetolactate synthase (ALS) of the pyruvate decomposition pathway was overexpressed to enhance the synthesis of diacetyl. However, replacing the promoter of the ILV2 gene with a strong promoter (PGK1p) to increase the expression level of the ILV2 gene did not result in further increased diacetyl, acetoin and TTMP production. Based on these evidences, we constructed the diploid strains AY-SB1 (?BDH1:loxP/?BDH1:loxP) and AY-SD1B2 (?BDH1:loxP-PGK1p-BDH2-PGK1t/?BDH1:loxP-PGK1p-BDH2-PGK1t) 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 S. cerevisiae by genetic engineering, and highlight a novel method to improve the quality and beneficial health attributes of Baijiu.

SUBMITTER: Cui DY

PROVIDER: S-EPMC7726194 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Cui Dan-Yao DY Wei Ya-Nan YN Lin Liang-Cai LC Chen Shi-Jia SJ Feng Peng-Peng PP Xiao Dong-Guang DG Lin Xue X Zhang Cui-Ying CY

Frontiers in microbiology 20201126

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 jiuqu produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene BDH1 and another BDH2 gene were deleted or ...[more]

PMID: 33324376

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Project description:Background7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin D3. Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae, the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ24-dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation.ResultsIn order to decouple 7-DHC production with cell growth, inducible GAL promoters was employed to control 7-DHC synthesis. Meanwhile, the precursor pool was increased via overexpressing all the mevalonate (MVA) pathway genes (ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20). Through screening DHCR24s from eleven tested sources, it was found that DHCR24 from Gallus gallus (Gg_DHCR24) achieved the highest 7-DHC production. Then 7-DHC accumulation was increased by 27.5% through stepwise fine-tuning the transcription level of Gg_DHCR24 in terms of altering its induction strategy, integration position, and the used promoter. By blocking the competitive path (ΔERG6) and supplementing another copy of Gg_DHCR24 in locus ERG6, 7-DHC accumulation was further enhanced by 1.07-fold. Afterward, 7-DHC production was improved by 48.3% (to 250.8 mg/L) by means of deleting NEM1 that was involved in lipids metabolism. Eventually, 7-DHC production reached to 1.07 g/L in 5-L bioreactor, which is the highest reported microbial titer as yet known.ConclusionsCombined engineering of the pathway and the host cell was adopted in this study to boost 7-DHC output in the yeast. 7-DHC titer was stepwise improved by 26.9-fold compared with the starting strain. This work not only opens large opportunities to realize downstream de novo synthesis of other steroids, but also highlights the importance of the combinatorial engineering of heterologous pathway and host to obtain microbial overproduction of many other natural products.

Dataset Information

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

Publications

Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through <i>Saccharomyces cerevisiae</i> Metabolic Engineering.

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