AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was use...AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.展开更多
Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The...Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.展开更多
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
基金Supported by the National Natural Science Foundation of China, No. 30200167 Tianjin Natural Science Foundation, No. 013802511
文摘AIM： To clone △^12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.METHODS： Reverse transcriptional polymerase chain reaction （RT-PCR） was used to clone the open reading frame of △^12-fatty acid desaturase gene （D12D） of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli（E.coli） strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain IN- VSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain IN- VSc1.RESULTS： Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E. coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of △^12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active △^12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.CONCLUSION： Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.
基金Supported by the National Natural Science Foundation of China(31171657)Heilongjiang Youth Science Foundation(QC2010093)+1 种基金Heilongjiang Natural Science Foundation(20100481037)Key Projects on Ministry of Education in Heilongjiang(12511Z022)
文摘Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.
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.
