Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be...Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.展开更多
Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocys...Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocystis sp.PCC6803 under continuous white light at 30C for 5 days.The crude converted product was detected using thin-layer chromatography(TLC)and further analyzed using high-performance liquid chromatography(HPLC)as well as HPLC with electron spray ionization mass spectrometry(HPLC-ESI-MS).Results:TLC results showed that 6-deoxypseudoanisatin was converted into a less polar product.HPLC and MS data indicated that the retention time of the converted product increased in comparison with the standard of 6-deoxypseudoanisatin.Conclusion:Thus,the study appears to demonstrate that Synechocystis sp.PCC6803 can transform 6-deoxypseudoanisatin.The polarity of the converted product is less than that of 6-deoxypseudoanisatin.展开更多
【背景】蛋白酶能够降解细胞中错误折叠或是无功能的蛋白,Clp家族蛋白就是一类重要的蛋白酶复合物。Clp蛋白酶复合物的水解核心是ClpP,集胞藻PCC6803中存在4种不同的ClpP蛋白,分别为ClpP1-ClpP4。作为重要的蛋白水解复合物的功能组分,...【背景】蛋白酶能够降解细胞中错误折叠或是无功能的蛋白,Clp家族蛋白就是一类重要的蛋白酶复合物。Clp蛋白酶复合物的水解核心是ClpP,集胞藻PCC6803中存在4种不同的ClpP蛋白,分别为ClpP1-ClpP4。作为重要的蛋白水解复合物的功能组分,目前对集胞藻ClpP的研究十分有限,对其生理功能与调控底物的研究甚少。【目的】选择集胞藻为研究对象探究ClpP2蛋白的功能,鉴定其潜在底物,为集胞藻ClpP2作用机制提供实验支撑。【方法】构建集胞藻ClpP2突变株(ΔClpP2),进行其生长实验和光合生理功能研究。通过标记定量蛋白质组学技术(isobaric tag for relative absolute quantitation,iTRAQ)鉴定ClpP2调控的靶标蛋白,生物信息学分析底物蛋白参与的代谢通路,最后利用平行反应监测(parallel reaction monitoring,PRM)技术对部分定量数据进行验证。【结果】ΔClpP2可以在自然条件下光合自养生长至对数生长期,但高光或高温胁迫下则无法正常生长。相较于野生型,ΔClpP2有着显著降低的PSⅡ电子传递效率及PSⅠ环式电子传递活性。通过iTRAQ定量蛋白质组学手段,ΔClpP2相对于WT共鉴定到206个差异表达蛋白,其中131个上调、75个下调,为ClpP2蛋白酶提供了丰富的潜在底物库。基因本体论(gene ontology,GO)分析发现ClpP2主要参与各种物质的转运,其中ABC蛋白转运途径显著被富集。利用PRM技术对34个差异表达的蛋白进行了验证。【结论】ClpP2蛋白不是集胞藻生长所必需,但在高温或者高光胁迫下是必不可少的,其失活会降低集胞藻光合系统活性。ClpP2可能通过调控离子转运进而影响光合系统。ClpP2很可能与ClpX结合形成蛋白酶复合物。展开更多
基金Supported by the International S&T Cooperation Program of China(No.2012DFA30450)the National Natural Science Foundation of China(No.30871541)+1 种基金the Taishan Scholar Foundation of Shandong Province(No.tshw20091014)the Innovation Program of the University Institutes of Jinan,Shandong Province(No.201004044)
文摘Genetic modifi cation is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. S ynechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid(GLA) and stearidonic acid(SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6 D, Syd15 D and Syd6Dd15 D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in S ynechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.
基金Beijing University of Chinese Medicine(2013-JYBZZ-JS-139)Independent Project Topics Foundation.
文摘Objective:To explore the ability of Synechocystis sp.PCC6803 in transforming 6-deoxypseudoanisatin.Methods:The experiment was performed by incubating 6-deoxypseudoanisatin with the freshwater cyanobacterium Synechocystis sp.PCC6803 under continuous white light at 30C for 5 days.The crude converted product was detected using thin-layer chromatography(TLC)and further analyzed using high-performance liquid chromatography(HPLC)as well as HPLC with electron spray ionization mass spectrometry(HPLC-ESI-MS).Results:TLC results showed that 6-deoxypseudoanisatin was converted into a less polar product.HPLC and MS data indicated that the retention time of the converted product increased in comparison with the standard of 6-deoxypseudoanisatin.Conclusion:Thus,the study appears to demonstrate that Synechocystis sp.PCC6803 can transform 6-deoxypseudoanisatin.The polarity of the converted product is less than that of 6-deoxypseudoanisatin.
文摘【背景】蛋白酶能够降解细胞中错误折叠或是无功能的蛋白,Clp家族蛋白就是一类重要的蛋白酶复合物。Clp蛋白酶复合物的水解核心是ClpP,集胞藻PCC6803中存在4种不同的ClpP蛋白,分别为ClpP1-ClpP4。作为重要的蛋白水解复合物的功能组分,目前对集胞藻ClpP的研究十分有限,对其生理功能与调控底物的研究甚少。【目的】选择集胞藻为研究对象探究ClpP2蛋白的功能,鉴定其潜在底物,为集胞藻ClpP2作用机制提供实验支撑。【方法】构建集胞藻ClpP2突变株(ΔClpP2),进行其生长实验和光合生理功能研究。通过标记定量蛋白质组学技术(isobaric tag for relative absolute quantitation,iTRAQ)鉴定ClpP2调控的靶标蛋白,生物信息学分析底物蛋白参与的代谢通路,最后利用平行反应监测(parallel reaction monitoring,PRM)技术对部分定量数据进行验证。【结果】ΔClpP2可以在自然条件下光合自养生长至对数生长期,但高光或高温胁迫下则无法正常生长。相较于野生型,ΔClpP2有着显著降低的PSⅡ电子传递效率及PSⅠ环式电子传递活性。通过iTRAQ定量蛋白质组学手段,ΔClpP2相对于WT共鉴定到206个差异表达蛋白,其中131个上调、75个下调,为ClpP2蛋白酶提供了丰富的潜在底物库。基因本体论(gene ontology,GO)分析发现ClpP2主要参与各种物质的转运,其中ABC蛋白转运途径显著被富集。利用PRM技术对34个差异表达的蛋白进行了验证。【结论】ClpP2蛋白不是集胞藻生长所必需,但在高温或者高光胁迫下是必不可少的,其失活会降低集胞藻光合系统活性。ClpP2可能通过调控离子转运进而影响光合系统。ClpP2很可能与ClpX结合形成蛋白酶复合物。
