Cloning Of Fatty Acid Synthesis-related Enzyme Genes And Their Expression In Cyanobacterial

Facing the rapid consumption of global fossil fuels and global ecological protection,it is extremely urgent to develop a renewable clean energy.Biodiesel is regarded as an important product to reduce human dependence on traditional fossil energy,but its low output value and high production cost are the main bottlenecks restricting its industrialization development.Microalgae is called the raw material of"the third generation"biomass energy because it does not occupy agricultural farmland and has a short growth cycle.While using microalgae to develop new biomass energy,it has also developed many high value-added products with broad development prospects.In this study,molecular biological methods were used to improve the content of medium and long-chain fatty acids in Synechocystis sp.PCC6803 and construct EPA-producing strains,which laid a foundation for further exploring the synthesis mechanism of fatty acids in Synechocystis sp.PCC 6803 and provided a new possibility for peopel to obtain EPA.In this paper,the fatty acid metabolism pathway of Synechocystis sp.PCC6803 was explored,and the overexpression of related genes in Synechocystis sp.PCC6803 was studied,and the production of medium and long chain fatty acids in algae was further promoted by regulating the culture conditions.The main research contents and achievements are as follows:1.Two homologous recombination transformation vectors slr0646UD and slr1704UD were constructed by homologous recombination.2.The sequence of Phosphopantetheinyl transferas gene of Synechocystis sp.PCC6803,?5 fatty acid desaturase gene and?6 fatty acid elongase gene of Phaeodactylum tricornutum and the related information of the protein to be expressed were analyzed by bioinformatics tools.The results showed that the proteins to be expressed by the three gene were hydrophilic,hydrophilic and hydrophobic,respectively,the number of transmembrane helices is 0,4 and 7,respectively,which are unstable protein,stable protein and stable protein respectively.3.The strain slr0495⁽⁺⁾,which had been screened out for complete transformation,was cultured under stress,and the changes of growth state,photosynthetic pigment,Fv/Fm and fatty acid components of algae cells under different treatment conditions were measured.The results showed that the low temperature and nitrogen deficiency culture conditions promoted the TFA content in algae strains.Under normal culture conditions,the contents of C12:0,C16:0 and C18:0 in mutant strain slr0495⁽⁺⁾were 1.31,8.07 and 1.35 mg·g^-1respectively,which were0.24,0.25 and 0.13 times higher than those in wild type strain.Under the conditions of light intensity of 50?·mol·photons·m^-2s^-1,low temperature(20?)and nitrogen deficiency(50%Na NO₃),the contents of C16:0 and C18:0 in mutant slr0495⁽⁺⁾were 1.44 and 1.42 times of those in Synechocystis sp.PCC6803.4.The mutant strain DM-slr0495 obtained after screening and identification was subjected to stress culture,The results showed that the mutant was exposed to nitrogen deficiency at 20?,50%N.The maximum TFA content was 32.16 mg·g^-1,and the medium and long chain fatty acids C16:0 and C18:0 increased significantly,which were 15.91 and 4.26 mg·g^-1,respectively,which were 1.09 and 1.38 times higher than the C16:0 and C18:0 contents of Synechocystis PCC6803 treated under the same treatment conditions,respectively.5.The genes?5 fatty acid desaturase and?6 fatty acid elongase from Phaeodactylum tricornutum were inserted into slr0646UD platform and slr1704UD platform respectively after codon optimization,and the overexpression vectors p D5D and p D6E of D5D and D6E genes were constructed,and then p D5D and p D6E were integrated into the genome of synechococcus sp.PCC6803 by natural transformation,the results showed that mutant strains Pcpc560+D5D+slr0646UD+Cm and Pcpc560+D6E+slr1704UD+Gm were successfully obtained.