食品与发酵工业 >

2025 , Vol. 51 >Issue 23: 20 - 29

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.042003

研究报告

鲁氏接合酵母2-苯乙醇发酵优化与转录组分析

  • 徐凌龙 ,
  • 上官玲玲 ,
  • 谢燿孺 ,
  • 陈胜 ,
  • 代俊
展开
  • 1(湖北工业大学 生命科学与健康工程学院,湖北省工业微生物重点实验室,湖北 武汉,430068)
    2(安琪酵母股份有限公司,湖北 宜昌,443008)
第一作者:硕士研究生(代俊副教授为通信作者,E-mail:jundai@hbut.edu.cn)

收稿日期: 2024-12-30

  修回日期: 2025-04-17

  网络出版日期: 2025-12-25

基金资助

国家自然科学基金项目(31871789,41876114);湖北省自然科学基金项目(2024AFB803)

收起

Optimization of 2-phenylethanol fermentation and transcriptomic analysis of Zygosaccharomyces rouxii

  • XU Linglong ,
  • SHANGGUAN Lingling ,
  • XIE Yaoru ,
  • CHEN Sheng ,
  • DAI Jun
Expand
  • 1(Hubei Key Laboratory of Industrial Microbiology, School of Life and Health Sciences, Hubei University of Technology, Wuhan 430068, China)
    2(Angel Yeast Co.Ltd., Yichang 443008, China)

Received date: 2024-12-30

  Revised date: 2025-04-17

  Online published: 2025-12-25

Fold

摘要

2-苯乙醇(2-phenylethanol,2-PE)作为一种广泛使用的有机化工原料,有着较高的应用价值。目前,生物合成法以其高效环保等优势,在2-PE的生产上逐渐取代天然提取法与化学合成法,展现出广阔的发展前景。然而目前合成2-PE的微生物资源有限,为了挖掘2-PE生物合成的新菌种资源。该文从前期筛选的高2-PE耐受鲁氏接合酵母出发,针对其合成2-PE的莽草酸途径与艾氏途径,通过单因素试验优化培养基,再结合转录组测序与荧光定量PCR技术验证菌株在不同培养基上转录水平的差异,最后在5 L发酵罐中进行补料发酵。结果表明,发酵2-PE的最佳培养基组成为60 g/L葡萄糖、6.6 g/L L-苯丙氨酸、0.71 g/L MgSO4·7H2O、1.7 g/L酵母基础氮源。在此条件下,转录组测序也证明鲁氏接合酵母在艾氏途径的相关基因显著表达以合成2-PE,并在发酵罐中通过工艺优化达到4.10 g/L产量,相较初始摇瓶产量提升了70.8%。为后续鲁氏接合酵母代谢工程的改造以及工业化生产奠定了基础。

关键词： 2-苯乙醇; 鲁氏接合酵母; 发酵工艺优化; 转录组测序

本文引用格式

徐凌龙 , 上官玲玲 , 谢燿孺 , 陈胜 , 代俊 . 鲁氏接合酵母2-苯乙醇发酵优化与转录组分析[J]. 食品与发酵工业, 2025 , 51(23) : 20 -29 . DOI: 10.13995/j.cnki.11-1802/ts.042003

Abstract

2-Phenylethanol(2-PE), a widely used organic chemical raw material, has significant application value in various industrial fields.Currently, biosynthesis has gradually surpassed traditional natural extraction and chemical synthesis methods for 2-PE production because of its advantages of high efficiency and environmental sustainability, thereby exhibiting promising development prospects.However, the current microbial resources for 2-PE synthesis remain limited.To explore new microbial resources for the biosynthesis of 2-PE, this study used the highly 2-PE-tolerant Zygosaccharomyces rouxii as the starting strain.To enhance the Shikimate and Ehrlich pathways responsible for 2-phenylethanol biosynthesis.The culture medium was optimized using single-factor experiments.Subsequently, transcriptome sequencing and quantitative real-time PCR analyses were conducted to validate transcription-level variations in the strain under different culture conditions.Finally, fed-batch fermentation was conducted in a 5 L fermenter.The results indicate that the optimal culture medium for fermenting 2-phenylethanol comprises 60 g/L glucose, 6.6 g/L L-phenylalanine, 0.71 g/L MgSO4·7H2O, and 1.7 g/L yeast nitrogen base.Under these conditions, transcriptome sequencing confirms that genes associated with the Ehrlich pathway in Z.rouxii are significantly expressed;consequently, the yield of 2-phenylethanol in the fermenter reaches 4.10 g/L by optimization the processes—an increase of 70.8% compared to initial flask yields.This research establishes a foundation for subsequent metabolic engineering modifications of Z.rouxii for industrial production.

Key words： 2-phenylethanol(2-PE); Zygosaccharomyces rouxii; optimization of fermentation processes; transcriptome sequencing

参考文献

[1] 卞一凡, 刘姝晗, 张贝萌, 等.微生物合成2-苯乙醇研究进展[J].中国生物工程杂志, 2022, 42(8):128-136.
BIAN Y F, LIU S H, ZHANG B M, et al.Advances in microbial synthesis of 2-phenylethanol[J].China Biotechnology, 2022, 42(8):128-136.
[2] WANG D K, HE Z X, LIU M M, et al.Exogenous fatty acid renders the improved salt tolerance in Zygosaccharomyces rouxii by altering lipid metabolism[J].LWT, 2023, 177:114579.
[3] FENG J P, MA W Y, MA Z B, et al.GhNHX3D, a vacuolar-localized Na+/H+ antiporter, positively regulates salt response in upland cotton[J].International Journal of Molecular Sciences, 2021, 22(8):4047.
[4] 钱莹, 宋娜, 高志明.鲁氏接合酵母盐胁迫适应机制及组学研究方法进展[J].生物技术, 2022, 32(6):779-786.
QIAN Y, SONG N, GAO Z M.Advances in salt stress adaptation mechanism and omics research methods of Zygosaccharomyces rouxii[J].Biotechnology, 2022, 32(6):779-786.
[5] 徐莹, 姜维, 何晓霞.耐盐性鲁氏酵母的研究进展[J].中国酿造, 2009, 28(10):1-4.
XU Y, JIANG W, HE X X.Research progress on salt-tolerant Zygosaccharomyces rouxii[J].China Brewing, 2009, 28(10):1-4.
[6] ZHU N Q, XIA W J, WANG G L, et al.Engineering Corynebacterium glutamicum for de novo production of 2-phenylethanol from lignocellulosic biomass hydrolysate[J].Biotechnology for Biofuels and Bioproducts, 2023, 16(1):75.
[7] ZHAN Y Y, SHI J, XIAO Y, et al.Multilevel metabolic engineering of Bacillus licheniformis for de novo biosynthesis of 2-phenylethanol[J].Metabolic Engineering, 2022, 70:43-54.
[8] QIAN T, WEI W P, DONG Y X, et al.Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction[J].Bioresource Technology, 2024, 411:131354.
[9] HASSING E J, DE GROOT P A, MARQUENIE V R, et al.Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae[J].Metabolic Engineering, 2019, 56:165-180.
[10] XU H, LI Z, LI L Y, et al.Sustainable production of 2-phenylethanol from agro-industrial wastes by metabolically engineered Bacillus licheniformis[J].LWT, 2023, 173:114414.
[11] WANG G L, WANG M Y, YANG J C, et al.De novo synthesis of 2-phenylethanol from glucose by metabolically engineered Escherichia coli[J].Journal of Industrial Microbiology and Biotechnology, 2023, 49(6):kuac026.
[12] HUA D L, XU P.Recent advances in biotechnological production of 2-phenylethanol[J].Biotechnology Advances, 2011, 29(6):654-660.
[13] GUO D Y, ZHANG L H, KONG S J, et al.Metabolic engineering of Escherichia coli for production of 2-phenylethanol and 2-phenylethyl acetate from glucose[J].Journal of Agricultural and Food Chemistry, 2018, 66(23):5886-5891.
[14] ZHU L H, WANG J H, XU S, et al.Improved aromatic alcohol production by strengthening the shikimate pathway in Saccharomyces cerevisiae[J].Process Biochemistry, 2021, 103:18-30.
[15] KONG S J, PAN H, LIU X Y, et al.De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris[J].Enzyme and Microbial Technology, 2020, 133:109459.
[16] GU Y, MA J B, ZHU Y L, et al.Engineering Yarrowia lipolytica as a chassis for de novo synthesis of five aromatic-derived natural products and chemicals[J].ACS Synthetic Biology, 2020, 9(8):2096-2106.
[17] 梅建凤, 闵航, 吕镇梅.利用酵母细胞生物催化合成2-苯乙醇[J].催化学报, 2007, 28(11):993-998.
MEI J F, MIN H, LYU Z M.Biocatalytic synthesis of 2-phenylethanol by yeast cells[J].Chinese Journal of Catalysis, 2007, 28(11):993-998.
[18] 贾薇, 白岩岩, 冯杰, 等.樟芝发酵产2-苯乙醇的条件优化[J].食用菌学报, 2016, 23(1):47-52.
JIA W, BAI Y Y, FENG J, et al.Optimization of selected growth parameters affecting 2-phenylethanol production in submerged cultures of Taiwanofungus camphoratus[J].Acta Edulis Fungi, 2016, 23(1):47-52.
[19] MORTAZAVI A, WILLIAMS B A, MCCUE K, et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nature Methods, 2008, 5(7):621-628.
[20] LI J G, WANG Y L, WANG L Q, et al.Integration of transcriptomic and proteomic analyses for finger millet[Eleusine coracana (L.) Gaertn.] in response to drought stress[J].PLoS One, 2021, 16(2):e0247181.
[21] 严伟, 高豪, 蒋羽佳, 等.2-苯乙醇生物合成的研究进展[J].合成生物学, 2021, 2(6):1030-1045.
YAN W, GAO H, JIANG Y J, et al.Research progress in 2-phenylethanol production through biological processes[J].Synthetic Biology Journal, 2021, 2(6):1030-1045.
[22] DAI J, XIA H L, YANG C L, et al.Sensing, uptake and catabolism of L-phenylalanine during 2-phenylethanol biosynthesis via the Ehrlich pathway in Saccharomyces cerevisiae[J].Frontiers in Microbiology, 2021, 12:601963.
[23] LEANDRO M J, CABRAL S, PRISTA C, et al.The high-capacity specific fructose facilitator ZrFfz1 is essential for the fructophilic behavior of Zygosaccharomyces rouxii CBS 732T[J].Eukaryotic Cell, 2014, 13(11):1371-1379.
[24] 陆军. 微生物转化L-苯丙氨酸生成2-苯乙醇的研究[D].无锡:江南大学, 2008.
LU J.Biotransformation of L-phenylalanine to 2-phenylethanol by microorganism[D].Wuxi:Jiangnan University, 2008.
[25] 唐育岐, 刘天明, 赵修报, 等.高产2-苯乙醇酵母菌株的筛选及培养基优化[J].中国食品添加剂, 2012, 23(3):137-142.
TANG Y Q, LIU T M, ZHAO X B, et al.Screening of high-yield yeast strains for 2-phenylethanol land its medium optimization[J].China Food Additives, 2012, 23(3):137-142.
[26] 王虎玄, 代春吉, 孙宏民.高糖胁迫下鲁氏接合酵母细胞膜生理应答机制研究[J].陕西科技大学学报, 2020, 38(6):40-47.
WANG H X, DAI C J, SUN H M.Physiological mechanism of cell membrane response to high sugar stress in Zygosaccharomyces rouxii[J].Journal of Shaanxi University of Science & Technology, 2020, 38(6):40-47.
[27] 韩燕祯, 王相际, 杨朔鹏, 等.咖啡因及其代谢物的检测及应用研究进展[J].化学研究与应用, 2020, 32(5):689-695.
HAN Y Z, WANG X J, YANG S P, et al.Recent advances on the detection and application of caffeine and its metabolites[J].Chemical Research and Application, 2020, 32(5):689-695.
[28] 吴俏槿, 杜冰, 蔡尤林, 等.α-亚麻酸的生理功能及开发研究进展[J].食品工业科技, 2016, 37(10):386-390.
WU Q J, DU B, CAI Y L, et al.Research development of alpha- linolenic acid[J].Science and Technology of Food Industry, 2016, 37(10):386-390.
[29] DAI J, LI K, SONG N, et al.Zygosaccharomyces rouxii, an aromatic yeast isolated from chili sauce, is able to biosynthesize 2-phenylethanol via the shikimate or Ehrlich pathways[J].Frontiers in Microbiology, 2020, 11:597454.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司