Study On The Response Mechanism Of Aspergillus Oryzae Under Salt Stress

Aspergillus oryzae is a fermenting microorganism widely used in the production of soybean meal and brewing soy sauce.Soy sauce brewing can be divided into salt-free solid-state fermentation,low-salt solid-state fermentation and high-salt dilute fermentation according to different fermentation processes.Now the latter two fermentation methods are more commonly used.The presence of salt can not only inhibit the growth of bacteria,but also closely related to the formation of special flavor of soy sauce.However,the salt treatment during fermentation also inhibits the growth of A.oryzae,so it is necessary to conduct further studies on it.So far,the response mechanism of A.oryzae under salt stress has rarely been reported,and it is necessary to carry out more in-depth research to provide a practical basis for the transformation and selection of more suitable A.oryzae strains.Based on the above,this subject has carried out the following researches:1.Analysis of growth and metabolites of A.oryzae under salt stress: fresh spores of A.oryzae were inoculated in PDA medium containing 0%,5%,10%,15% sodium chloride,and cultured at 30°C for 72 h.The growth of A.oryzae was observed with a salt concentration of 0% as a control,and the dry biomass and spore amount of A.oryzae were determined.The results showed that the dry biomass of the control group had been at the highest value at the same time point.Salt stress caused the decrease of A.oryzae biomass,especially after 48 h,the change of dry biomass was more obvious and the higher the salt concentration,the more pronounced the inhibitory effect.Under the condition of 15% salt concentration,no growth was observed before 36 h,but the growth trend of A.oryzae in the experimental group of each salt concentration was basically the same.After 60 h of culture,the biomass did not change and reached a stable period.Under the salt stress,the production of conidia of A.oryzae was inhibited,and the high salinity group was more obvious.The A.oryzae mycelium after 72 hours of culture was collected,and the intracellular fatty acid species and content were determined,and transcriptome sequencing was performed.The results showed that the content of 18-carbon fatty acid and 16-carbon fatty acid accounted for more than 95% of the total fatty acid content of A.oryzae.The main fatty acids in A.oryzae are palmitic acid(C16:0),stearic acid(C18:0),oleic acid(C18:1)and linoleic acid(C18:2).Compared with the 0% control group,the C18 content in the salt stress group was slightly higher,the C16 content was slightly lower,and the unsaturated fatty acid content was significantly higher than the saturated fatty acid content.Transcriptome data showed that a total of 4578 genes in the three salinity treatment groups showed differential expression levels compared to the 0% control group,of which 848 genes were shared by the three salt concentration groups,and the number of differential genes in 15% salt concentration was significantly greater than the 5% and 10% salt concentration groups,indicating a more complex biological metabolic response in the 15% salinity group.Combined with metabolic pathways,differentially expressed genes were involved in arginine biosynthesis and linoleic acid biosynthesis in A.oryzae,and the stearic acid desaturase gene expression level was significantly up-regulated at 5% salt concentration.2.Functional identification of salt stress related genes in yeast: qRT-PCR was used to analyze the expression patterns of the rate-limiting enzyme genes AoD9D1 and AoD9D2 in the oleic acid synthesis pathway by desaturation of stearic acid in A.oryzae.The results showed that the expression of AoD9D1 gradually increased from the adaptation phase to the log phase and the stationary phase,while AoD9D2 had the highest expression level in the adaptation phase and less expression in the log phase and stationary phase.The expression levels of AoD9D1 and AoD9D2 increased with increasing salt concentration.Gene structure analysis revealed that both AoD9D1 and AoD9D2 contain FAD and Cyt-b5 domains.Evolutionary analysis found that AoD9D1 and AoD9D2 were clearly in the two groups,and both groups were closely related to D9 D in A.oryzae RIB40.Analysis of protein properties revealed that the AoD9D1 gene encodes 456 amino acids with a molecular weight of approximately 51.9 kDa,theoretical isoelectric point is 9.10,hydrophilic average coefficient is negative,and contains three transmembrane regions.The AoD9D2 gene encodes 469 amino acids with a molecular weight of approximately 53.6 kDa,theoretical isoelectric point is 9.01,and hydrophilic average coefficient is negative,which also has three transmembrane regions.Vectors were constructed for six genes including AoD9D1 and AoD9D2 and their domains.The wild type and D9 D knockout Saccharomyces cerevisiae were transformed by PEG/LiAc method,and the yeast fermentation plants were subjected to liquid fermentation and fatty acid content determination.The results showed that heterologous expression of AoD9D1 and AoD9D2 gene increased the salt tolerance of wild-type S.cerevisiae,and the salt tolerance of the strain heterologously expressing AoD9D1 gene was slightly stronger than that of the strain heterologously expressing AoD9D2 gene.Compared with the wild type,the D9 D knockout yeast strain showed lower salt tolerance,and when AoD9D1 and AoD9D2 were transformed into the D9 D knockout strain,the salt tolerance was significantly enhanced at 7% and higher concentrations,and the AoD9D1 transformed strain has higher salt tolerance than the AoD9D2 transformed strain.At 5% low salt concentration,the growth of the domain gene-transformed yeast was inhibited to varying degrees,but with the increase of salt concentration,the two domain-transformed strains of AoD9D1 gene showed stronger salt stress tolerance.However,the two domain-transformed strains of the AoD9D2 gene did not have this effect.Analysis of fatty acid data of the above yeast strains showed that the content of unsaturated fatty acids,including palmitoleic acid and oleic acid,was significantly increased in heterologously expressed AoD9 D S.cerevisiae,while the content of saturated fatty acids,mainly palmitic acid and stearic acid was decreased.The change in fatty acid content in the heterologously expressed AoD9D1 strain was more pronounced than in the heterologous expression of the AoD9D2 strain.The AoD9D1 domain transformed strain exhibited a higher level of unsaturated fatty acid content than the AoD9D2 domain transformed strain.3.Overexpression and phenotypic analysis of AoD9D1 gene in A.oryzae: In view of the positive effect of AoD9D1 gene in improving salt tolerance of yeast,the overexpression vector in A.oryzae was constructed,and the vector contained red fluorescence,which facilitated the localization of gene expression.The recombinant plasmid was transformed into A.oryzae by Agrobacterium tumefaciens Ti plasmid-mediated method and the overexpression strain was subjected to gene expression localization observation and stress tolerance test.The results showed that the overexpression of AoD9D1 gene in A.oryzae was localized on the cell membrane,and the gene overexpressing strain showed stronger reproductive ability and salt stress tolerance than wild type.4.Study on the transcriptional regulation mechanism of AoD9D1 gene action: ten proteins interacting with the desaturase encoding by AoD9D1 gene were predicted using the STRING database.Then,the related vectors were constructed,and the transcriptional regulation mechanism of AoD9D1 gene was studied by yeast two-hybrid system.The results showed that the inorganic phosphate transporter and the peptidyl-prolyl cis-trans isomerase interacted with the protein encoded by the AoD9D1 gene.