拟南芥WRI1转录因子对酿酒酵母脂肪酸合成的影响
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摘要
转录因子是植物体内一类应答环境胁迫所产生的蛋白,通过激活或抑制转录的方式发挥调控作用. WRI1转录因子是油脂合成的关键因子,主要在脂肪酸合成与糖酵解后期发挥调控作用.为了获得高产脂肪酸的酵母菌株,以拟南芥c DNA为模板,PCR扩增得到编码拟南芥WRI1转录因子的基因,经过Spe I/Bam H I双酶切后连接到表达载体上,得到重组质粒p416-Atwri1.将重组质粒用PEG/Li Ac化学转化法转入酿酒酵母BY4742中,通过营养缺陷型培养基进行筛选,挑取阳性菌落并进行摇瓶发酵,采用GC-MS对脂肪酸进行定性和定量分析.结果表明,Atwri1基因在酵母中的表达改变了脂肪酸的组成及含量,与空质粒酵母相比,重组酵母中长链脂肪酸的总含量增加了44. 8%,为以后酿酒酵母生产脂肪酸实现工业化打下基础.
Plant transcription factors are a kind of proteins produced in response to the environmental stress,which exhibit the regulatory function by activating or inhibiting the gene transcription. The WRI1 transcription factor is a key factor to regulate the fatty acid biosynthesis and the late glycolysis. To obtain the yeast strain with high productivity of fatty acids,the WRI1 gene from Arabidopsis thaliana was amplified by PCR,and digested by two restriction endonucleases Spe I and BamH I and ligated to the p416 plasmid for construction of recombinant expression plasmid p416-Atwri1. Then p416-Atwri1 was transformed into Saccharomyces cerevisiae BY4742 by PEG/Li Ac method. The positive colonies were screened through auxotrophic medium and then fermented under shake-flask conditions. Gas Chromatograph-Mass Spectrometer-computer was utilized to analyze the compositions and concentrations of fatty acids. The results revealed that the overexpression of Atwri1 gene changed both compositions and concentrations of fatty acids. Compared with the strain harboring the empty p416 plasmid,the longchain fatty acids in the strain harboring p416-Atwri1 were increased by 44. 8%. The engineered yeast provides a foundation for industrial production of fatty acids.
Plant transcription factors are a kind of proteins produced in response to the environmental stress,which exhibit the regulatory function by activating or inhibiting the gene transcription. The WRI1 transcription factor is a key factor to regulate the fatty acid biosynthesis and the late glycolysis. To obtain the yeast strain with high productivity of fatty acids,the WRI1 gene from Arabidopsis thaliana was amplified by PCR,and digested by two restriction endonucleases Spe I and BamH I and ligated to the p416 plasmid for construction of recombinant expression plasmid p416-Atwri1. Then p416-Atwri1 was transformed into Saccharomyces cerevisiae BY4742 by PEG/Li Ac method. The positive colonies were screened through auxotrophic medium and then fermented under shake-flask conditions. Gas Chromatograph-Mass Spectrometer-computer was utilized to analyze the compositions and concentrations of fatty acids. The results revealed that the overexpression of Atwri1 gene changed both compositions and concentrations of fatty acids. Compared with the strain harboring the empty p416 plasmid,the longchain fatty acids in the strain harboring p416-Atwri1 were increased by 44. 8%. The engineered yeast provides a foundation for industrial production of fatty acids.
引文
[1]刘伟.两个二倍体棉种CDPK和FAD基因家族的全基因组鉴定与基因结构功能分析[D].杭州:浙江大学,2015.
[2]刘强,张贵友.植物转录因子的结构与调控作用[J].科学通报,2000,45(14):1465-1474.DOI:10.3321/j.issn:0023-074X.2000.14.002.
[3]周宏,钱娇,李荣芳,等.拟南芥Trihelix转录因子基因家族研究[J].江苏科技大学学报(自然科学版),2017,31(2):231-236.DOI:10.3969/j.issn.1673-4807.2017.02.020.ZHOU Hong,QIAN Jiao,LI Rongfang,et al.Research of the Arabidopisis Trihelix transcrip tion factor gene family[J].Journal of Jiangsu Universlity of Science and Technology(Natural Science Edition),2017,31(2):231-236.DOI:10.3969/j.issn.1673-4807.2017.02.020.(in Chinese)
[4]BAUD S,WUILL ME S,TO A,et al.Role of WRIN-KLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis[J].Plant Journal,2010,60(6):933-47.DOI:10.1111/j.1365-313X.2009.04011.
[5]FUKUDA N,IKAWA Y,AOYAGI T,et al.Expression of the genes coding for plastidic acetyl-Co A carboxylase subunits is regulated by a location-sensitive transcription factor binding site[J].Plant Molecular Biology,2013,82(4/5):473-483.DOI:10.1007/s11103-013-0075-7.
[6]MARCHIVE C,NIKOVICS K,TO A,et al.Transcriptional regulation of fatty acid production in higher plants:Molecular bases and biotechnological outcomes[J].European Journal of Lipid Science&Technology,2015,116(10):1332-1343.DOI:10.1002/ejlt.201400027.
[7]ADHIKARI N D,BATES P D,BROWSE J.Wrinkledl rescues feedback inhibition of fatty acid synthesis in Hydroxylase-experessing seeds[J].Plant Physiology,2016,171(1):179.
[8]CURRAN B P G,BUGEJA V.Basic Investigations in Saccharomyces cerevisiae[J].Methods in Molecular Biology,2006,313:1-13.DOI:10.1385/1-59259-958-3:001.
[9]TANG X,CHEN W N.Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition[J].Bioresource Technology,2014,162:200-206.DOI:org/10.1016/j.biortech.2014.03.061.
[10]RUNGUPHAN W,KEASLING J D.Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals[J].Metabolic Engineering,2014,21(1):103-113.DOI:org/10.1016/j.ymben.2013.07.003.
[11]YU A Q,JUWONO N K P,FOO J L,et al.Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids[J].Metabolic Engineering,2016,34:36-43.
[12]TANG X,LEE J,CHEN W N.Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae for advanced biofuel production[J].Metabolic Engineering Communications,2015,2(C):58-66.DOI:10.1016/j.meteno.2015.06.005.
[13]BAUD S,LEPINIEC L.Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis[J].Plant Physiology&Biochemistry,2009,47(6):448-455.DOI:10.1016/j.plaphy.2008.12.006.
[14]BAUD S,WUILL ME S,TO A,et al.Role of WRIN-KLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis[J].Plant Journal for Cell&Molecular Biology,2010,60(6):933-947.DOI:10.1111/j.1365-313X.2009.04011.x.
[15]POUVREAU B,BAUD S,VERNOUD V,et al.Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis[J].Plant Physiology,2011,156(2):674-686.DOI:10.1104/pp.111.173641.
[16]BATES P D,JOHNSON S R,CAO X,et al.Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly[J].Proceedings of the National Academy of Sciences,2014,111(3):1204.DOI:10.1073/pnas.1318511111.
[17]GUO D,ZHU J,DENG Z,et al.Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways[J].Metabolic Engineering,2014,22(22):69-75.
[2]刘强,张贵友.植物转录因子的结构与调控作用[J].科学通报,2000,45(14):1465-1474.DOI:10.3321/j.issn:0023-074X.2000.14.002.
[3]周宏,钱娇,李荣芳,等.拟南芥Trihelix转录因子基因家族研究[J].江苏科技大学学报(自然科学版),2017,31(2):231-236.DOI:10.3969/j.issn.1673-4807.2017.02.020.ZHOU Hong,QIAN Jiao,LI Rongfang,et al.Research of the Arabidopisis Trihelix transcrip tion factor gene family[J].Journal of Jiangsu Universlity of Science and Technology(Natural Science Edition),2017,31(2):231-236.DOI:10.3969/j.issn.1673-4807.2017.02.020.(in Chinese)
[4]BAUD S,WUILL ME S,TO A,et al.Role of WRIN-KLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis[J].Plant Journal,2010,60(6):933-47.DOI:10.1111/j.1365-313X.2009.04011.
[5]FUKUDA N,IKAWA Y,AOYAGI T,et al.Expression of the genes coding for plastidic acetyl-Co A carboxylase subunits is regulated by a location-sensitive transcription factor binding site[J].Plant Molecular Biology,2013,82(4/5):473-483.DOI:10.1007/s11103-013-0075-7.
[6]MARCHIVE C,NIKOVICS K,TO A,et al.Transcriptional regulation of fatty acid production in higher plants:Molecular bases and biotechnological outcomes[J].European Journal of Lipid Science&Technology,2015,116(10):1332-1343.DOI:10.1002/ejlt.201400027.
[7]ADHIKARI N D,BATES P D,BROWSE J.Wrinkledl rescues feedback inhibition of fatty acid synthesis in Hydroxylase-experessing seeds[J].Plant Physiology,2016,171(1):179.
[8]CURRAN B P G,BUGEJA V.Basic Investigations in Saccharomyces cerevisiae[J].Methods in Molecular Biology,2006,313:1-13.DOI:10.1385/1-59259-958-3:001.
[9]TANG X,CHEN W N.Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition[J].Bioresource Technology,2014,162:200-206.DOI:org/10.1016/j.biortech.2014.03.061.
[10]RUNGUPHAN W,KEASLING J D.Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals[J].Metabolic Engineering,2014,21(1):103-113.DOI:org/10.1016/j.ymben.2013.07.003.
[11]YU A Q,JUWONO N K P,FOO J L,et al.Metabolic engineering of Saccharomyces cerevisiae for the overproduction of short branched-chain fatty acids[J].Metabolic Engineering,2016,34:36-43.
[12]TANG X,LEE J,CHEN W N.Engineering the fatty acid metabolic pathway in Saccharomyces cerevisiae for advanced biofuel production[J].Metabolic Engineering Communications,2015,2(C):58-66.DOI:10.1016/j.meteno.2015.06.005.
[13]BAUD S,LEPINIEC L.Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis[J].Plant Physiology&Biochemistry,2009,47(6):448-455.DOI:10.1016/j.plaphy.2008.12.006.
[14]BAUD S,WUILL ME S,TO A,et al.Role of WRIN-KLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis[J].Plant Journal for Cell&Molecular Biology,2010,60(6):933-947.DOI:10.1111/j.1365-313X.2009.04011.x.
[15]POUVREAU B,BAUD S,VERNOUD V,et al.Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis[J].Plant Physiology,2011,156(2):674-686.DOI:10.1104/pp.111.173641.
[16]BATES P D,JOHNSON S R,CAO X,et al.Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly[J].Proceedings of the National Academy of Sciences,2014,111(3):1204.DOI:10.1073/pnas.1318511111.
[17]GUO D,ZHU J,DENG Z,et al.Metabolic engineering of Escherichia coli for production of fatty acid short-chain esters through combination of the fatty acid and 2-keto acid pathways[J].Metabolic Engineering,2014,22(22):69-75.