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  • ISSN 1006-3080
  • CN 31-1691/TQ

NADH扰动对酿酒酵母葡萄糖效应的影响

徐亚英 李志敏

徐亚英, 李志敏. NADH扰动对酿酒酵母葡萄糖效应的影响[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220106006
引用本文: 徐亚英, 李志敏. NADH扰动对酿酒酵母葡萄糖效应的影响[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20220106006
XU Yaying, LI Zhimin. Influence of NADH disturbance on Crabtree effect of Saccharomyces cerevisiae[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220106006
Citation: XU Yaying, LI Zhimin. Influence of NADH disturbance on Crabtree effect of Saccharomyces cerevisiae[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20220106006

NADH扰动对酿酒酵母葡萄糖效应的影响

doi: 10.14135/j.cnki.1006-3080.20220106006
基金项目: 国家自然科学基金(32171478)
详细信息
    作者简介:

    徐亚英(1990-),女,江苏盐城人,博士生,酿酒酵母生产衣康酸。E-mail:y12170046@mail.ecust.edu.cn

    通讯作者:

    李志敏, E-mail:lizm@ecust.edu.cn

  • 中图分类号: Q591

Influence of NADH disturbance on Crabtree effect of Saccharomyces cerevisiae

  • 摘要: 利用不同拷贝数的载体在酿酒酵母表达NADH氧化酶NOX和可替代氧化酶AOX1,并通过线粒体定位信号减少AOX1对胞质NADH的影响,从而考察NOX和AOX1在分批发酵中对酿酒酵母葡萄糖效应的作用及其对葡萄糖生产衣康酸的影响。研究结果表明,高拷贝载体表达noxaox1的菌株均有明显的代谢变化,高拷贝表达nox菌株能够氧化胞质NADH,培养基中甘油分泌减少43.94%,衣康酸生产不受影响。而高拷贝表达aox1的菌株存在胞质残留AOX1,会氧化胞质NADH而减少甘油积累。利用线粒体定位信号AAC2和BCS1p将AOX1进一步定位至酿酒酵母线粒体,不再影响甘油合成,120 h时衣康酸产量增加至116.98 mg/L。但是AOX1、AAC2-AOX1和BCS1p-AOX1表达菌株都没有明显减少分批发酵中乙醇的积累。综上所述,在高糖培养基中进行分批发酵,NOX能够减少葡萄糖效应甘油的积累,而AOX1 、AAC2-AOX1和BCS1p-AOX1不能减少葡萄糖效应副产物乙醇的积累。

     

  • 图  1  利用NADH氧化酶NOX减少甘油生产

    Figure  1.  Reducing glycerol production with NADH oxidase NOX

    图  2  利用替代氧化酶AOX1减少乙醇生产

    Figure  2.  Reducing ethanol production by overexpressing an alternative oxidase AOX1

    Profiles of cell density, ethanol, glycerol, and itaconic acid concentrations in the cultivation of different Saccharomyces cerevisiae strains

    图  3  激光共聚焦显微镜观察NOX和AOX1在酿酒酵母中的定位

    Figure  3.  Location of NOX(A) and AOX1(B) in Saccharomyces cerevisiae

    图  4  利用线粒体定位信号将替代氧化酶AOX1定位至线粒体

    Figure  4.  Further location of AOX1 in mitochondria of Saccharomyces cerevisiae with mitochondrial localization signals

    Subcellular localization of AAC2-AOX1-mCherry(A5) and BCS1p-AOX1-mCherry(A7) fusion proteins in Saccharomyces cerevisiae. Strains expressing fusion protein were stained with MitoTracker® Green FM. Scale bar—5 μm.

    图  5  AOX1线粒体定位提高衣康酸滴度

    Figure  5.  Mitochondrial localization of AOX1 increases the titer of itaconic acid

    Expression of AAC2-AOX1 (XYY29) and BCS1p-AOX1(XYY31) fusion proteins with high-copy plasmids

    表  1  本研究所使用的菌株

    Table  1.   Strains used in this study.

    StrainsGenotypeSource or reference
    E.coli DH5a Our lab
    BY4741 MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 Professor
    Xin-Qing Zhao
    XYY2 BY4741, pRS415-PGPD-cadA-TCYC1 [17]
    XYY21 XYY2, pRS413 This study
    XYY22 XYY2, pRS423 [17]
    XYY23 XYY2, pRS413-PGPD2-nox-TCYC1 This study
    XYY24 XYY2, pRS423-PGPD2-nox-TCYC1 This study
    XYY25 XYY2, pRS413-PTEF1-aox1-TCYC1 This study
    XYY26 XYY2, pRS423-PTEF1-aox1-TCYC1 This study
    XYY29 XYY2, pRS423-PTEF1-aac2-aox1-TCYC1 This study
    XYY31 XYY2, pRS423-PTEF1-bcs1p-aox1-TCYC1 This study
    Z1 BY4741, HO△:: PGPD-cadA-TCYC1, pRS423 This study
    Z2 Z1, GPD2△:: nox This study
    L1 BY4741, pRS425 This study
    L2 BY4741, pRS425-PGPD2-mCherry-TCYC1 This study
    L3 BY4741, pRS425-PGPD2-nox-mCherry-TCYC1 This study
    A1 BY4741, pRS423 This study
    A2 BY4741, pRS423-PTEF1-mCherry-TCYC1 This study
    A3 BY4741, pRS423-PTEF1-aox1-mCherry-TCYC1 This study
    A4 BY4741, pRS423- PTEF1-aac2-mCherry-TCYC1 This study
    A5 BY4741, pRS423-PTEF1-aac2-aox1-mCherry-TCYC1 This study
    A6 BY4741, pRS423-PTEF1-bcs1p-mCherry-TCYC1 This study
    A7 BY4741, pRS423-PTEF1-bcs1p-aox1-mCherry-TCYC1 This study
    下载: 导出CSV

    表  2  本研究所使用的质粒

    Table  2.   Plasmids used in this study

    PlasmidsCharacteristic(s)Reference
    pRS415ARS/CEN, Amp, LEU[17]
    pRS413ARS/CEN, Amp, HISThis study
    pRS4252 µm ori, Amp, LEUThis study
    pRS4232 µm ori, Amp, HIS[17]
    pCAS9-NATCAS9 expression plasmidProfessor
    Qiang Hua
    pHYBgRNA cassetteProfessor
    Qiang Hua
    下载: 导出CSV

    表  3  本研究所使用的引物

    Table  3.   Primers used in this study.

    NameSequence
    zw-1Fcggccgctctagaactagtggatccgcaatgtttcgttggttataacattag
    zw-1Rctacaacgattttactcataagctttgataaggaaggggagcgaagg
    zw-2Ftcgctccccttccttatcaaagcttatgagtaaaatcgttgtagtcggtg
    zw-2Rcgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc
    zw-3Fggctgcccttacggctgaaaaataaaagcttatcgataccgtcgacctcg
    zw-3Rcgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc
    zw-4Fctaaagggaacaaaagctggagctccagcaatgtttcgttggttataacattag
    zw-4Rcgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc
    zw-5Fcggccgctctagaactagtggatccatagcttcaaaatgtttctactcctt
    zw-5Rcgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc
    zw-6Fctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcc
    zw-6Rgtaatggcagtgctgttttaactagttttgtaattaaaacttagattagattg
    zw-7Fgttttaattacaaaactagttaaaacagcactgccattactaatacacctc
    zw-7Rtaatgatgatgatgatgatggatccgttttgtttaagctgatgcaattttttg
    zw-8Ftctaagttttaattacaaaactagtatgtcttccaacgcccaagtcaaaa
    zw-8Rtcgcgcatcctgaggtcggatacattttgaacttcttaccaaacaagatc
    zw-9Fgatcttgtttggtaagaagttcaaaatgtatccgacctcaggatgcgcg
    zw-9Rtaatgatgatgatgatgatggatcctcatatcacctcatcccgttcccag
    zw-10Ftctaagttttaattacaaaactagtatgtcggataagccgattgacatac
    zw-10Rtcgcgcatcctgaggtcggatacattccattgtcatgttgtatgtagtttg
    zw-11Fcaaactacatacaacatgacaatggaatgtatccgacctcaggatgcgcgag
    zw-11Rtaatgatgatgatgatgatggatcctatcacctcatcccgttcccagccag
    zw-12Fctaaagggaacaaaagctggagctcgcaatgtttcgttggttataacatt
    zw-12Rccatggccatctttgcaaagcttggtgataaggaaggggagcgaaggaa
    zw-13Fatcaccaagctttgcaaagatggccatggtgagcaagggcgaggaggataac
    zw-13Rcgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgccg
    zw-14Fatcaccaagctttgcaaagatggccatgagtaaaatcgttgtagtcggtgc
    zw-14Rtatcctcctcgcccttgctcaccattttttcagccgtaagggcagccattg
    zw-15Fctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcct
    zw-15Rcgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgcc
    zw-16Fcaaaccaagctttgcaaagatggccatgtatccgacctcaggatgcgcga
    zw-16Rtatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag
    zw-17Fcaaaccaagctttgcaaagatggccatgtcttccaacgcccaagtcaaaac
    zw-17Rtatcctcctcgcccttgctcaccattttgaacttcttaccaaacaagatc
    zw-18Fzw-17F
    zw-18Rtatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag
    zw-19Fcaaaccaagctttgcaaagatggccatgtcggataagccgattgacatac
    zw-19Rtatcctcctcgcccttgctcaccattccattgtcatgttgtatgtagtttg
    zw-20Fzw-19F
    zw-20Rzw-18R
    zw-21Fcctgtgtgacatttatgacggttttagagctagaaatagcaag
    zw-21Rcgtcataaatgtcacacagggatcatttatctttcactgcgga
    zw-22Fcatccaaaatattaaattttacttttattacatacaactttttaaactaatatacacattagtttatcattatcaatactcgcca
    zw-22Rcaactattagctctaaatccatatcctcataagcagcaatcaattctatctatactttaaagcaaattaaagccttcgagcgtccc
    zw-23Fcgcactatctggtgcaaactgttttagagctagaaatagcaag
    zw-23Ragtttgcaccagatagtgcggatcatttatctttcactgcgga
    zw-24Fgtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcaatgcttgctgtcatgagtaaaatcgttgtagtcgg
    zw-24Rgtataatgataaattggttgggggaaaaagaggcaacaggaaagatcagagggggagggggggggagagtgtttatttttcagccgtaagggcagcc
    下载: 导出CSV
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  • 收稿日期:  2022-01-06
  • 网络出版日期:  2022-05-14

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