基于重组毕赤酵母的2-癸烯酸生物合成研究  

Expression of Thiesterase in Pichia Pastoris Cells

作　　者：张丽华 张子洋 王子睿 姜彦君 苏静[1] 李丕武[1] 范翰 王瑞明[1] 汪俊卿[1] ZHANG Li-hua;ZHANG Zi-yang;WANG Zi-rui;JIANG Yan-jun;SU Jing;LI Pi-wu;FAN Han;WANG Rui-ming;WANG Jun-qing(School of Biological Engineering,State Key Laboratory of Biomaterial and Green Papermaking,Qilu University of Technology(Shandong Academy of Sciences),Jinan 250353,China)

机构地区：[1]齐鲁工业大学(山东省科学院)生物工程学院,生物基材料与绿色造纸国家重点实验室,济南250353

出　　处：《齐鲁工业大学学报》2021年第3期1-6,共6页Journal of Qilu University of Technology

基　　金：国家自然科学基金(31801527);山东省重点研发计划(重大创新工程)(2020CXGC010603、2019JZZY011003);泰山产业领军人才(tscy20180103)。

摘　　要：2-癸烯酸是一种具有2位不饱和键的中链脂肪酸,是合成功能化合物10-羟基-2-癸烯酸的重要前体物质。本研究首先将硫脂酶基因YdiI和脂肪酸CoA脱氢酶基因FadA分别连接至具有pGAP启动子的pGAPZαA载体上,并电转化至毕赤酵母GS115感受态中,经过Zeocin抗性筛选及基因组PCR鉴定后,成功获得重组载体同源重组至毕赤酵母GS115基因组上的阳性重组子。采用气相色谱-质谱联用方法对发酵产物进行检测,结果发现培养12 h后发酵产物中有2-癸烯酸出现,在发酵24 h产量可达33.7 mg/L,这表明YdiI和FadA基因已成功转入毕赤酵母GS115中。本研究成功构建了能够合成2-癸烯酸的重组毕赤酵母GS115工程菌,为下一步利用毕赤酵母进一步合成10-羟基-2-癸烯酸打下基础。2-decenoic acid is a medium-chain fatty acid with 2-position unsaturated bond and an important precursor for the synthesis of functional compound 10-hydroxy 2-decenoic acid.In this study,the thiolipase gene YdiI and the fatty acid CoA dehydrogenase gene FadA were respectively linked to the pGAPZαA vector with GAP promoter,and electrotransformed into the Pichia pastoris GS115 competence.After Zeocin resistance screening and genome PCR identification,the positive recombinant vector homologously recombined into the Pichia pastoris,GS115 genome was successfully obtained.The fermentation products were detected by gas chromatography-mass spectrometry.The results showed that 2-decenoic acid appeared in the fermentation products after 12 hours of culture,and the yield could reach 33.7 mg/L after 24 hours of fermentation,which indicated that YdiI and FadA genes had been successfully transferred to Pichia pastoris GS115.In this study,a recombinant Pichia pastoris GS115 engineering strain capable of synthesizing 2-decenoic acid was successfully constructed,which laid the foundation for the further synthesis of 10-hydroxy 2-decenoic acid using Pichia pastoris.

关 键 词：2-癸烯酸 毕赤酵母GS115 YdiI FadA 基因重组表达 

分 类 号：Q786[生物学—分子生物学]