Fermentation Regulation Of Rhodotorula Sp.U13N3 For Unsaturated Fatty Acids Production

Unsaturated fatty acids are very important substances in life and have many important physiological activities.As the main sources of unsaturated fatty acids are plants and aquatic products,their production has been affected by uncontrollable factors such as climate conditions.The use of lipid-producing microorganisms to produce lipid rich in unsaturated fatty acids has a series of advantages,such as:short growth cycle,high production intensity,and not easily affected by external environmental factors,which is a relatively feasible alternative.In this study,a non-traditional lipid-producing yeast Rhodotorula sp.U13N3 is used as the original strain.Firstly,the triacylglycerol lipase ATG15 gene and peroxidase AOX2 gene,which affect lipid accumulation,are knocked out by CRISPR/Cas9 gene editing system.Then,mutant strains with high lipid yield are screened and fermentation conditions are optimized.On this basis,the effects ofΔ9 desaturase gene inducer（Fe）、osmotic pressure and acyl-Co A elongase substrates（stearic acid and oleic acid）related to the synthesis of very-long chain fatty acids on the lipid composition and yield of mutant strain are analyzed respectively,and finally the optimal conditions for producing unsaturated fatty acids are obtained.The main conclusions are as follows:1.The gene of Rhodotorula sp.U13N3 is edited by CRISPR/Cas9 system,and the plasmid containing g RNA of ATG15 and AOX2 is transformed by ATMT technology.A total of 27positive clones with two genes defect are screened,and finally a mutant strainΔATGΔAOX3with high lipid yield is successfully obtained.The relative content of unsaturated fatty acids in lipid produced by mutant strain decreased slightly,but the lipid yield increased by 52.89%compared with that of original strain U13N3.Further analysis show that maintaining the p H value of fermentation broth at a high level（p H 5～6）is helpful to increase the lipid yield.By adding Ca CO₃ for p H-controlled fermentation,the lipid yield of mutantΔATGΔAOX3 is further increased by 59.82%,reaching 13.84 g/L,in which the relative content of unsaturated fatty acids is 70.82%.2.The addition of exogenous Fe can affect the transcription level ofΔ9 desaturase gene ole1.After the addition of 50～200μM Fe,the ole1 transcription level is significantly increased in the middle of fermentation.With the increase of concentration,the induction of ole1transcription by Fe is gradually weakened.At the end of the fermentation,the addition of 50μM Fe make the relative content of monounsaturated fatty acid increased by 1.01%.In addition,Fe can induce up-regulation of the transcription levels of acyl-Co A elongase genes elo1 and elo2.After the addition of 50～200μM Fe,the transcription level of elo2 is increased more than two times as much as that of the control group during the middle stage of fermentation.However,the final relative contents of fatty acids with chain lengths of C20 and C22 in the lipid are not significantly changed.In the early stage of fermentation,enhancing osmotic pressure in fermentation broth can promote the transcription of ole1,elo1 and elo2 genes,and then increase the contents of unsaturated fatty acids and C18,but at the end of fermentation,the contents of unsaturated fatty acids and very-long chain fatty acids will not change significantly.3.Exogenous addition of sodium stearate can significantly increase the transcription levels of ole1,elo1 and elo2 in the middle and early stage of fermentation and promote the lipid yield.Among them,1 g/L is more favorable forΔATGΔAOX3 fermentation to produce lipid.At the end of fermentation,the lipid yield can reach 16.74 g/L,in which the unsaturated fatty acid content is 10.79 g/L and C20 fatty acid content is 102.11 mg/L,which is increased by 15.13%,18.02%and 23.20%respectively compared with the control group.In addition,exogenous oleic acid can also significantly increase the transcription levels of ole1,elo1 and elo2.Finally,after fermentation for 120 h with exogenous 5 g/L oleic acid,the intracellular contents of unsaturated fatty acids,polyunsaturated fatty acids and very-long chain fatty acids of mutantΔATGΔAOX3reach 12.41 g/L,5.42 g/L and 277.35 mg/L,respectively,which are 2.74 times,1.96 times and6.50 times higher than those of the original strain U13N3.