生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (4): 29-.doi: 10. 3969 / j. issn. 2095-1736. 2021. 04. 029

• 研究报告 • 上一篇    下一篇

毕赤酵母Sut2对甲醇代谢的调控研究

  

  1. 江南大学粮食发酵工艺与技术国家工程实验室,无锡214122
  • 出版日期:2021-08-18 发布日期:2021-08-18
  • 通讯作者: 杨艳坤,副教授,硕士生导师,主要从事微生物学、分子生物学和发酵工程研究,E-mail:yangyankun@jiangnan.edu.cn
  • 作者简介:郑雅婷,硕士,主要从事发酵工程、分子生物学研究,E-mail:6170201056@stu.jiangnan.edu.cn
  • 基金资助:
    国家自然科学基金项目(31570034);江苏省第十五批六大人才高峰项目(SWYY-180)

Research of regulation of Sut2tome thanolmet abolismin Pichiapastoris

  1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China
  • Online:2021-08-18 Published:2021-08-18

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摘要： 根据毕赤酵母Sut2基因表达在甲醇培养基中显著升高的特性,进一步探究该基因的功能及其与甲醇代谢的摘关系要。在毕赤酵母基因组中,利用同源重组原理,分别通过双交换和单交换构建Sut2敲除菌株与Sut2过表达菌株。同时培养GS115-ΔSut2、GS115-Sut2和GS115野生型菌株,检测并比较Aox1基因的转录和翻译水平。以甲醇和甲醛分别作碳源培养菌株,检测Sut2的转录水平,从而鉴定该基因的诱导剂。用高效液相色谱法(HPLC)检测毕赤酵母菌株细胞内外麦角固醇含量。结果显示:Sut2主要由甲醇所诱导,对Aox1有一定程度的正调控作用,但两个基因可能参与复杂的调控网络和反馈回路,因而间接相互作用;HPLC结果表明,Sut2基因可能与麦角固醇合成有关。探究Sut2对甲醇代谢的调控机理,进一步丰富了巴斯德毕赤酵母甲醇代谢通路。

关键词: 毕赤酵母, Sut2基因, Aox1基因, 甲醇代谢, 麦角固醇

Abstract: To characterizeSut2gene inPichiapastoriswhich can be induced by methanol, the function ofSut2and its relationship with methanol metabolism was studied. By homologous recombination,Sut2was deficientor overexpressed inPichiapastoris. Then, GS115-ΔSut2, GS115-Sut2and GS115 wild-type strains were cultured, and the transcription and translation levels ofAox1were detec-ted and compared. To identify the inducer ofSut2, methanol or formaldehyde was used as the sole carbon source for culturingPichiapastoris, and the transcription levels ofSut2were compared. High performance liquid chromatography (HPLC) was used to detect in-tracellular and extracellular ergosterol content of the three strains, GS115-ΔSut2, GS115-Sut2and GS115 wild-type. The results showed thatSut2was mainly induced by methanol, andSut2had a certain degree of positive regulation on the expression ofAox1with indirect interaction, which might be involved in complex regulatory networks and feedback loops. It was inferred thatSut2was involved in methanol metabolism by participating in sterol biosynthesis based on the HPLC results. This study would explore the regulatory mechanism ofSut2on methanol metabolism and further enrich the methanol metabolism pathway inPichiapastoris.

Key words: Pichiapastoris;Sut2;Aox1, methanol metabolism, ergosterol

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