3种不同植物来源的tHMGR对于酿酒酵母MVA代谢流通量的比较研究  

Effects of tHMGR from three different plant sources on mevalonate(MVA)pathway flux in Saccharomyces cerevisiae

作　　者：张晓丽 晁二昆[2,3] 孙梦楚 赵宏飞 王彩霞[2] 张柏林[1] ZHANG Xiao-li;CHAO Er-kun;SUN Meng-chu;ZHAO Hong-fei;WANG Cai-xia;ZHANG Bo-lin(College of Biological Sciences and Technology,Beijing Forestry University,Beijing 100083,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;College of Life Sciences,Qufu Normal University,Qufu 273165,China)

机构地区：[1]北京林业大学生物科学与技术学院,北京100083 [2]中国中医科学院中药研究所,北京100700 [3]曲阜师范大学生命科学学院,山东曲阜273165

出　　处：《中国中药杂志》2022年第10期2614-2622,共9页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2019YFC19066);中国中医科学院科技创新工程项目(C12021A04111,C12021A04116)。

摘　　要：该研究对来自甘草Glycyrrhiza uralensis、黄花蒿Artemisia annua和拟南芥Arabidopsis thaliana的HMGR基因编码蛋白分别进行生物信息学分析,发现GuHMGR和AaHMGR蛋白各包含2个跨膜区,AtHMGR蛋白具有3个跨膜区。且GuHMGR、AaHMGR和AtHMGR3个蛋白均具有HMGR催化作用的活性中心结构域。将甘草、黄花蒿和拟南芥的3个截短HMGR基因(tHMGRs)连接到pYES3载体上,成功构建pYES3-tGuHMGR、pYES3-tAaHMGR、pYES3-tAtHMGR质粒。将对照pYES3质粒和重组质粒pYES3-tGuHMGR、pYES3-tAaHMGR、pYES3-tAtHMGR分别转化酿酒酵母Cen.pk2-1D,成功构建酿酒酵母菌株Y0、Y1、Y2、Y3。挑取阳性单克隆,菌株发酵后通过GC-MS检测其鲨烯、羊毛甾醇、麦角固醇的含量。采用实时荧光定量PCR检测tGuHMGR、tAaHMGR、tAtHMGR分别在菌株Y1、Y2、Y3中的相对表达情况。结果显示,过表达tAaHMGR的菌株,其鲨烯的产量以及萜类前体物质鲨烯、麦角固醇和羊毛甾醇的总产量都是最高的。实时荧光定量PCR结果发现,tAaHMGR在菌株中的相对表达量比tGuHMGR高,与菌株发酵结果一致。该研究通过比较3种不同植物来源的tHMGR对于酿酒酵母MVA途径代谢通量的影响,挑选出一个较优的tHMGR,可为今后构建酿酒酵母底盘细胞,在提高其鲨烯及萜类前体物质积累的优势基因选择上提供一些参考。In this study,we used bioinformatic tools to analyze the 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR)genes from Glycyrrhiza uralensis,Artemisia annua,and Arabidopsis thaliana.The results indicated that GuHMGR and AaHMGR contained two transmembrane regions while AtHMGR had three transmembrane regions.GuHMGR,AaHMGR,and AtHMGR all had the active center for catalysis.Three truncated HMGR genes(tHMGRs)of G.uralensi,A.annua,and A.thaliana were respectively ligated to pYES3 vector to construct the recombinant plasmids pYES3-tGuHMGR,pYES3-tAaHMGR,and pYES3-tAtHMGR.Afterwards,the control plasmid pYES3 and the three plasmids and were respectively introduced into Saccharomyces cerevisiae Cen.pk2-1 D,which yielded strains Y0,Y1,Y2,and Y3,respectively.The content of squalene,lanosterol,and ergosterol in these strains was measured by GC-MS.The relative expression of tGuHMGR,tAaHMGR,and tAtHMGR in strains Y1,Y2,and Y3 was determined by quantitative real-time PCR.The results showed that the strain overexpressing tAaHMGR had the highest yield of squalene and the highest total yield of squalene,ergosterol,and lanosterol.The quantitative real-time PCR showed higher relative expression of tAaHMGR than tGuHMGR,consistent with the strain fermentation result.We selected a superior tHMGR by comparing the effects of different tHMGRs on the mevalonate(MVA)pathway flux in S.cerevisiae.The findings can provide a reference for the construction of S.cerevisiae strains with high yields of squalene and terpenoid precursors.

关 键 词：截短的3-羟基-3-甲基戊二酰辅酶A还原酶 MVA 鲨烯 酿酒酵母 

分 类 号：S567[农业科学—中草药栽培]