酿酒酵母JP2代谢苹果酸途径及其关键基因解析

苏昊; 梁璋成; 李维新; 任香芸; 何志刚; 林晓姿; 鞠延仑

doi:10.19303/j.issn.1008-0384.2023.05.015

酿酒酵母JP2代谢苹果酸途径及其关键基因解析

doi: 10.19303/j.issn.1008-0384.2023.05.015

苏昊^{1, 2, 3,},
梁璋成^{1, 2, 3},
李维新^{1, 2, 3, ,},
任香芸^{1, 2, 3},
何志刚^{1, 2, 3},
林晓姿^{1, 2, 3},
鞠延仑⁴

1.
福建省农业科学院农业工程技术研究所，福建福州　350003
2.
农业农村部亚热带特色果蔬菌加工重点实验室（部省共建），福建福州　350003
3.
福建省农产品（食品）加工重点实验室，福建福州　350003
4.
西北农林科技大学葡萄酒学院，陕西杨陵　712100

基金项目: 福建省自然科学基金项目（2020J011373）；福建省科技计划公益类专项（2020R1032008）；福建省农业高质量发展超越“5511”协同创新工程项目（XTCXGC2021019-GCS01）；泉州市科技计划项目（2021N043）；福建省农业科学院农业科技创新联盟专项（CXLM202204）；福建省星火项目（2020S0035）

详细信息

作者简介:
苏昊（1984−），男，博士，助理研究员，主要从事食品发酵与酿造方面的研究（E-mail：suhaoxp@163.com）

通讯作者:
李维新（1970−），男，博士，研究员，主要从事食品发酵与酿造方面的研究（E-mail：lwx406x@163.com）

中图分类号: TS262.7
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出版历程
- 收稿日期: 2023-03-10
- 修回日期: 2023-05-04
- 网络出版日期: 2023-07-06
- 刊出日期: 2023-05-28

Metabolism pathway of malic acid in Saccharomyces cerevisiae JP2 and analysis of its key genes

SU Hao^{1, 2, 3
,},
LIANG Zhangcheng^{1, 2, 3},
LI Weixin^{1, 2, 3
, ,},
REN Xiangyun^{1, 2, 3},
HE Zhigang^{1, 2, 3},
LIN Xiaozi^{1, 2, 3},
JU Yanlun⁴

1.
Institute of Agricultural Engineering Technology, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China
2.
Key Laboratory of Subtropical Characteristic Fruits, Vegetables and Edible Fungi Processing (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs, Fuzhou, Fujian　350003, China
3.
Fujian Key Laboratory of Agricultural Products (Food) Processing, Fuzhou, Fujian　350003, China
4.
College of Enology, Northwest A & F University, Yangling, Shanxi　712100, China

摘要

摘要: 目的酿酒酵母JP2（Saccharomyces cerevisiae JP2）是从自然发酵枇杷酒醪中分离得到一株具有较好代谢苹果酸的酵母菌。为深入研究其内在降酸机理，对其进行全基因组测序和基因组序列信息解析，并分析其可能的代谢苹果酸途径，找出影响降酸的基因。方法基于Illumina Miseq和PacBio测序平台，联用第二代和第三代测序技术对酿酒酵母JP2和模式菌株酿酒酵母S288c进行全基因组测序，采用生物信息学方法对其进行序列组装、基因预测与功能注释，并挖掘JP2可能的苹果酸代谢路径。结果（1）通过对酿酒酵母JP2基因组组装共得到52个重叠群，整个基因组大小为11.98 Mbp，GC含量 38.07%；JP2和S288c二者的全基因组大小和GC含量差异不大。（2）JP2代谢苹果酸的途径有3个：苹果酸→草酰乙酸→丙酮酸；苹果酸→丙酮酸；苹果酸→富马酸。相关的主要基因分别为maeA、MDH及fumC，其中苹果酸→草酰乙酸→丙酮酸是主要代谢路径，关键基因是maeA。（3）模拟发酵过程苹果酸代谢及其关键基因表达量变化显示，JP2的苹果酸含量下降了35.8%，下降程度显著高于S288c；与S288c相比，JP2的maeA基因呈现表达极显著上调（P＜0.01），在发酵后期，其相对表达量达到起始值的130%。结论 JP2在果酒发酵过程中呈现maeA基因高效表达，从而显著提高了苹果酸降解效率。研究结果可为酿酒酵母JP2降酸功能基因的研究以及降酸工程菌的研发提供技术支撑。
- 酿酒酵母 /
- 生物降酸 /
- 苹果酸代谢路径 /
- 基因表达
Abstract: Objective Saccharomyces cerevisiae JP2, isolated from naturally fermented loquat, is a yeast strain with good metabolism of malic acidwine mash. To further investigate its intrinsic acid lowering mechanism, whole genome sequencing and genome sequence information analysis were conducted, and possible metabolic pathways of malic acid were analyzed to identify genes that affect acid lowering. Method Based on the Illumina Miseq and PacBio sequencing platforms, second and third generation sequencing techniques were used to sequence the entire genome of S. cerevisiae JP2 and the type strain S. cerevisiae S288c. Bioinformatics methods were used to assemble their sequences, predict their genes, and annotate their functions, while exploring the possible malic acid metabolism pathways of JP2. Result (1) A total of 52 contigs were obtained by assembling the genome of S. cerevisiae JP2, with a total genome size of 11.98 Mbp and a GC content of 38.07%; There is no significant difference in overall genome size and GC content between JP2 and S288c. (2) There are three pathways for JP2 to metabolize malic acid: malic acid → oxaloacetate → pyruvate; malic acid → pyruvate; and malic acid → fumaric acid. The main genes involved are maeA, MDH, and fumC, among which malic acid → oxaloacetate → pyruvate is the main metabolic pathway with the key gene maeA. (3) The simulated fermentation process showed a decrease of 35.8% in malic acid metabolism and key gene expression levels in JP2, with a significant decrease compared to S288c; Compared with S288c, the maeA gene of JP2 was significantly upregulated (P＜0.01), and its relative expression reached 130% of the initial value in the late fermentation stage. Conclusion JP2 exhibits high expression of maeA gene during fruit wine fermentation, significantly improving the efficiency of malic acid degradation. The research results can provide technical support for the study of the acid reducing functional gene of S. cerevisiae JP2 and the development of acid reducing engineering bacteria.
- Saccharomyces cerevisiae /
- biological acid degradation /
- malic acid metabolic pathway /
- gene expression

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图 1 KEGG功能注释基因

Figure 1. KEGG functional annotation gene

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图 2 酵母菌JP2代谢苹果酸路径

Figure 2. Metabolism pathway of malic acid by yeast JP2

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图 3 JP2与S288c全基因组对比分析

Figure 3. Comparative analysis of the whole genome of JP2 and S288c

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图 4 JP2与S288c Venn图

Figure 4. JP2 and S288c Venn diagram

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图 5 苹果酸代谢相关基因PCR检测

Figure 5. PCR detection of malate metabolism-related genes

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图 6 酿酒酵母JP2与S288c在降酸过程中maeA相对表达量的变化

Figure 6. Changes in the relative expression of maeA gene in S. cerevisiae JP2 and S288C during acid reduction

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表 1 基因组基本信息

Table 1. Basic information of genome

特征 Property	酿酒酵母JP2 S. cerevisiae JP2	酿酒酵母S288c S. cerevisiae S288c
序列总长 Total sequence length/Mbp	11.98	12.16
GC含量 GC content/%	38.07	38.15
预测基因占比 Predicted gene proportion/%	66.50	75.37
预测基因数量 Predicted number of genes	5205	5906
预测基因长度 Predicted Gene Length/bp	1545	1369
tRNA数量 tRNA quantity	289	299
rRNA数量 rRNA quantity	4	10

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表 2 酿酒酵母JP2和S288c发酵过程有机酸代谢

Table 2. Organic acid metabolism during the fermentation process of S. cerevisiae JP2 and S. cerevisiae S288c　（单位：μg·mL⁻¹）

有机酸 Organic acid	发酵时间 Fermentation time	JP2试验组 JP2 group	S288c试验组 S288c group
草酸 Oxalate	发酵起点	0.00±0.00	0.00±0.00
草酸 Oxalate	发酵结束	2.58±0.02	2.76±0.04
酒石酸 Tartaric acid	发酵起点	0.00±0.00	0.00±0.00
酒石酸 Tartaric acid	发酵结束	6.39±0.05	6.81±0.08
苹果酸 Malic acid	发酵起点	4500.00±0.00	4500.00±0.00
苹果酸 Malic acid	发酵结束	2886.87±150.21	4365.36±143.45^*
乳酸 Lactic acid	发酵起点	0.00±0.00	0.00±0.00
乳酸 Lactic acid	发酵结束	44.19±3.14	47.12±4.86
乙酸 Acetic acid	发酵起点	0.00±0.00	0.00±0.00
乙酸 Acetic acid	发酵结束	556.26±25.01	213.14±18.24
富马酸 Fumaric acid	发酵起点	0.00±0.00	0.00±0.00
富马酸 Fumaric acid	发酵结束	1.71±0.04	1.98±0.06
：同一发酵时间不同试验组之间差异显著（P＜0.05）。 : Significant difference between different groups at the same fermentation time (P＜0.05).

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表 3 苹果酸代谢关键基因表达量比较

Table 3. Comparison of expression of key genes of malic acid metabolism

菌株 Strain	maeA基因 maeA gene	MDH基因 MDH gene	fumC基因 fumC gene
JP2	1.25^**	1.02	0.97
S288C	1.00	1.00	1.00
：不同菌株之间差异极显著（P＜0.01）。 : Significant difference between different strains (P＜0.01).

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参考文献(18)

[1]	GRANDVALET C. Oenococcus oeni: Queen of the cellar, nightmare of geneticists [J]. Microbiology, 2017, 163(3): 297−299. doi: 10.1099/mic.0.000456
[2]	JIANG J, SUMBY K M, SUNDSTROM J F, et al. Directed evolution of Oenococcus oeni strains for more efficient malolactic fermentation in a multi-stressor wine environment [J]. Food Microbiology, 2018, 73: 150−159. doi: 10.1016/j.fm.2018.01.005
[3]	FERNÁNDEZ-PÉREZ R, TENORIO RODRÍGUEZ C, RUIZ-LARREA F. Fluorescence microscopy to monitor wine malolactic fermentation [J]. Food Chemistry, 2019, 274: 228−233. doi: 10.1016/j.foodchem.2018.08.088
[4]	VILELA A. Use of nonconventional yeasts for modulating wine acidity [J]. Fermentation, 2019, 5(1): 27. doi: 10.3390/fermentation5010027
[5]	李华, 刘延琳, 蒋思欣, 等. Oenococcus oeni苹果酸-乳酸发酵关键酶基因在酿酒酵母中的转化与表达 [J]. 农业生物技术学报, 2006, 14(4):606−611. LI H, LIU Y L, JIANG S X, et al. Cloning of two key enzyme genes from Oenococcus oeni for malolactic fermentation and their expressions in Saccharomyces cerevisiae [J]. Journal of Agricultural Biotechnology, 2006, 14(4): 606−611.(in Chinese)
[6]	BENITO Á, JEFFARES D, PALOMERO F, et al. Selected Schizosaccharomyces pombe strains have characteristics that are beneficial for winemaking [J]. PLoS One, 2016, 11(3): e0151102. doi: 10.1371/journal.pone.0151102
[7]	PETRUZZI L, CAPOZZI V, BERBEGAL C, et al. Microbial resources and enological significance: Opportunities and benefits [J]. Frontiers in Microbiology, 2017, 8: 995. doi: 10.3389/fmicb.2017.00995
[8]	BENITO S, PALOMERO F, CALDERÓN F, et al. Selection of appropriate Schizosaccharomyces strains for winemaking [J]. Food Microbiology, 2014, 42: 218−224. doi: 10.1016/j.fm.2014.03.014
[9]	MYLONA A E, DEL FRESNO J M, PALOMERO F, et al. Use of Schizosaccharomyces strains for wine fermentation—Effect on the wine composition and food safety [J]. International Journal of Food Microbiology, 2016, 232: 63−72. doi: 10.1016/j.ijfoodmicro.2016.05.023
[10]	MINNAAR P P, JOLLY N P, PAULSEN V, et al. Schizosaccharomyces pombe and Saccharomyces cerevisiae yeasts in sequential fermentations: Effect on phenolic acids of fermented Kei-apple (Dovyalis caffra L. ) juice [J]. International Journal of Food Microbiology, 2017, 257: 232−237. doi: 10.1016/j.ijfoodmicro.2017.07.004
[11]	VAUDANO E, COSTANTINI A, GARCIA-MORUNO E. An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR [J]. International Journal of Food Microbiology, 2016, 234: 15−23. doi: 10.1016/j.ijfoodmicro.2016.06.017
[12]	BENITO Á, CALDERÓN F, BENITO S. Schizosaccharomyces pombe biotechnological applications in winemaking [J]. Methods in Molecular Biology, 2018, 1721: 217−226.
[13]	何志刚, 李维新, 梁璋成, 等. 优良降酸酿酒酵母的分离和鉴定 [J]. 中国食品学报, 2013, 13(5):191−197. doi: 10.16429/j.1009-7848.2013.05.028 HE Z G, LI W X, LIANG Z C, et al. Separation and identification of selected yeast with deacidification ability for brewing in fruit wine [J]. Journal of Chinese Institute of Food Science and Technology, 2013, 13(5): 191−197.(in Chinese) doi: 10.16429/j.1009-7848.2013.05.028
[14]	李维新, 魏巍, 何志刚, 等. 酿酒酵母JP2发酵枇杷汁的有机酸代谢及其产香特征分析 [J]. 中国食品学报, 2016, 16(2):251−257. doi: 10.16429/j.1009-7848.2016.02.034 LI W X, WEI W, HE Z G, et al. Analysis of organic acids metabolism and fragrant characteristic for fermentated loquat juice using Saccharomyces cerevisiae JP2 [J]. Journal of Chinese Institute of Food Science and Technology, 2016, 16(2): 251−257.(in Chinese) doi: 10.16429/j.1009-7848.2016.02.034
[15]	LIANG Z C, LIN X Z, HE Z G, et al. Amino acid and microbial community dynamics during the fermentation of Hong Qu glutinous rice wine [J]. Food Microbiology, 2020, 90: 103467. doi: 10.1016/j.fm.2020.103467
[16]	COMITINI F, AGARBATI A, CANONICO L, et al. Yeast interactions and molecular mechanisms in wine fermentation: A comprehensive review [J]. International Journal of Molecular Sciences, 2021, 22(14): 7754. doi: 10.3390/ijms22147754
[17]	DARBANI B, STOVICEK V, VAN DER HOEK S A, et al. Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae [J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(39): 19415−19420. doi: 10.1073/pnas.1900287116
[18]	BOVO B, NADAI C, VENDRAMINI C, et al. Aptitude of Saccharomyces yeasts to ferment unripe grapes harvested during cluster thinning for reducing alcohol content of wine [J]. International Journal of Food Microbiology, 2016, 236: 56−64. doi: 10.1016/j.ijfoodmicro.2016.07.022