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关系。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。
To characterize Sut2 gene in Pichia pastoris which can be induced by methanol,the function of Sut2 and its relationship with methanol metabolism was studied.By homologous recombination,Sut2 was deficientor overexpressed in Pichia pastoris.Then,GS115-ΔSut2,GS115-Sut2 and GS115 wild-type strains were cultured,and the transcription and translation levels of Aox1 were detected and compared.To identify the inducer of Sut2,methanol or formaldehyde was used as the sole carbon source for culturing Pichia pastoris,and the transcription levels of Sut2 were compared.High performance liquid chromatography(HPLC)was used to detect intracellular and extracellular ergosterol content of the three strains,GS115-ΔSut2,GS115-Sut2 and GS115 wild-type.The results showed that Sut2 was mainly induced by methanol,and Sut2 had a certain degree of positive regulation on the expression of Aox1 with indirect interaction,which might be involved in complex regulatory networks and feedback loops.It was inferred that Sut2 was involved in methanol metabolism by participating in sterol biosynthesis based on the HPLC results.This study would explore the regulatory mechanism of Sut2 on methanol metabolism and further enrich the methanol metabolism pathway in Pichia pastoris.
作者
郑雅婷
李翔
王鹏程
杨艳坤
白仲虎
ZHENG Yating;LI Xiang;WANG Pengcheng;YANG Yankun;BAI Zhonghu(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China)
出处
《生物学杂志》
CAS
CSCD
北大核心
2021年第4期29-33,共5页
Journal of Biology
基金
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)。
