| Linoleic acid(LA)is one kind of polyunsaturated fatty acids,which belongs to essential fatty acid family and participates in many important physiological processes in the body.However,the high concentration of linoleic acid may cause cell toxicity since it is susceptible to oxidation.The oleaginous yeast can be used to synthesize intracellular lipids by using a variety of carbon sources.In order to study the toxic effects of endogenous linoleic acid on Yarrowia lipolytica yeast cells,this research was performed as follows:The ?12-fatty acid dehydrogenase gene was successfully transferred into Y.lipolytica yeast and the ratio of intracellular linoleic acid was increased.Firstly,the ?12-fatty acid dehydrogenase gene was ligated to expression vectors p INA1312 and p INA1292 by genetic engineering to obtain recombinant expressing plasmids p INA1312-?12 and p INA1292-?12,respectively.Then,the recombinant expression vector was transferred into the competent cells of Y.lipolytica by lithium acetate.The correct strains were verified by PCR to amplify the target gene fragment,which was integrated into the genome of the strain by gene replace method.And screening was performed to obtain a higher linoleic acid content strain.The selected recombinant strain and control strain were used to investigate the effects of the over-expression of ?12-fatty acid dehydrogenase gene on the growth,lipid accumulation and cell morphology of Y.lipolytica.The results showed that the over-expression of ?12-fatty acid dehydrogenase gene in Y.lipolytica had influence on biomass from 8.50 g/L to 12.75 g/L,the content of linoleic acid increased from 8.72% to 30.85%,and fatty acid profile,cell life,and the morphology of yeast cells changed.The cell morphology of recombinant strain 1292-12 was not filamentous,but in a larger single cell form.In order to investigate the toxic effect of linoleic acid on yeast cells,fish oil or rapeseed oil was added to culture medium at the start of fermentation.The added oil and tween 80 were emulsified with a homogenizer as a raw carbon source and the final oil concentration was 10 g/L.Fermentation experiments of each strains were carried out under the same conditions,and the biomass and oil content and the growth viability were measured and the cell morphology was observed.The results showed that when fish oil or rapeseed oil was used as the carbon source for fermentation,the ratio of C18:2 in the cells of the recombinant strain and the control strain was relatively decreased,and the number of cell death of the recombinant strain was decreased,but the biomass was higher than that of the common medium.Cell morphology was not greatly affected.The addition of fish oil or rapeseed oil to the medium has a positive effect on cell viability and lipid accumulation,but it is still insufficient to significantly reverse the toxic effects of linoleic acid on yeast cells.In order to further explore the toxic mechanism of linoleic acid in the cell of Y.lipolytica,vitamin C(also known as ascorbic acid)was added to the culture medium at a concentration of 250 ?g/m L,and the activity level of MDA,ROS,CAT and SOD were determined in both the gene overexpressing strains and the control strain.Hydrogenase CAT and superoxide dismutase SOD are indicators related to lipid oxidation levels.When treated with vitamin C,MDA contents were deceased from 1.05 nmol/mg protein to 0.61 nmol/mg protein,ROS content was lower,and CAT and SOD activities were increased by 40% and 87% compared to normal fermentation experiments.Therefore,it can be concluded that high content of endogenous linoleic acid has toxic effects on Y.lipolytic yeast,and it is closely related to lipid peroxidation process,which provides a theoretical basis for studying the toxic mechanism of polyunsaturated fatty acids on cells. |
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