Analysis Of Extracellular Enzyme Secretion Mechanism Of Aspergillus Oryzae And Its Regulatory Application

Aspergillus oryzae is the initial strain for soy sauce brewing and plays an important role in the utilization of raw materials and the formation of product quality.However,there are significant differences in the extracellular enzymes of Aspergillus oryzae under different species and cultivation conditions.For a long time,Aspergillus oryzae 3.042 has been mainly used for soy sauce fermentation in China,but its research has not been in-depth,and is even less well-known internationally.At present,most studies of extracellular enzyme secretion in Aspergillus oryzae have focued on a single enzyme family or gene,with few reports on the overall extracellular enzyme secretion mechanism and transcriptional regulation.In this study,Aspergillus oryzae 3.042 was used to analyze the extracellular enzyme secretion mechanism by RNA-Seq transcriptomics from time,culture factors(carbon source,nitrogen source and initial p H values).Daqu was prepared with different raw materials and applied to soy sauce fermentation to further explore the effect of the extracellular enzymes of Aspergillus oryzae on soy sauce quality.To improve the advantages of Aspergillus oryzae in soy sauce fermentation,a new brewing process was designed with an optimized culture environment according to the secretion characteristics of extracellular enzymes.Finally,CRISPRi(inhibition)andCRISPRa(activation)systems were obtained to achieve the perturbation of transcription factors to extracellular enzymes,providing a theoretical basis for the regulation of extracellular enzyme transcription level of Aspergillus oryzae.Firstly,the effect of culture time on extracellular enzymes of Aspergillus oryzae was studied.By analyzing the extracellular enzyme secretion mechanism of Aspergillus oryzae in different stages under both solid and liquid cultivation modes,it was found that Aspergillus oryzae completing transcription and translation of extracellular enzyme genes on ribosomes.on the ribosome at the early stage of growth,and in a logarithmic growth period from 24 h to 48 h,genes related to the protein secretion mechanism began to express in large numbers,and reached a stable growth period at 48 h.After modified and sorted in Golgi apparatus,the protein was secreted and transported to the extracellular sapce,and was in a long-term protein secretion cycle in stable growth.In liquid culture,Aspergillus oryzae completed cell development earlier and began to secrete extracellular enzymes in the logarithmic phase from12 h to 36 h and it reached a stable growth phase at 36 h,but autophagy appeared at the late stage of 72 h,resulting in a shorter protein secretion cycle.The expression changes of carbon source transcription factors Amy R,Xln R,and nitrogen source transcription factor Are A demonstrated that solid state was more conducive to the utilization of raw materials.Secondly,the effect of culture factors on extracellular enzymes of Aspergillus oryzae was studied.By analyzing the extracellular enzyme secretion mechanism of maltose,glucose and and sucrose,it was found that the presence of glucose was more conducive to glycosylation of endoplasmic reticulum and vesicular transport,disaccharides mainly activated UPR mechanism to improve protein secretion efficiency,and sucrose had the best extracellular enzyme secretion ability because it was beneficial to the synthesis of GPI ankyrin.By analyzing the extracellular enzyme secretion mechanism of organic nitrogen,mixed nitrogen and inorganic,it was found that the main reason for the decline of extracellular enzymes of mixed nitrogen and inorganic nitrogen was the protein degradation caused by excessive accumulation of misfolded proteins.By analyzing the extracellular enzyme secretion mechanism of initial p H values of 6,7,and 8,it was found that neutral and weakly alkaline initial conditions were more conducive to the secretion of extracellular enzymes by Aspergillus oryzae 3.042,mainly for the upregulation of the dolichol pathway.Moreover,the acidification degree of solid culture was lower,which was more conducive to the activation of transcription factor Pac C and synthesis of alkaline protease.Pearson correlation analysis of carbon source,nitrogen source,and p H showed a strong positive correlation between carbon source and extracellular enzymes,a strong negative correlation between nitrogen source and extracellular enzymes,and a positive correlation between initial p H and extracellular enzymes.After that,the effect of extracellular enzyme families of Aspergillus oryzae on the quality of soy sauce was studied,it was found that Daqu with soybean meal and bran as raw materials was more favorable for the improvement of protease activities(acid protease,neutral protease and alkaline protease)and promoted the transformation of free amino acids into aldehyde flavor compounds during soy sauce brewing.Daqu with soybean and fried wheat as raw materials was more conducive to improving the activities of carbohydrate enzymes(α-amylase,glucoamylase,cellulase),promoting the synthesis of more umami amino acids in soy sauce brewing,and at the same time obtaining higher contents of alcohol and acid flavor compounds.The new brewing process of soy sauce utilized a relatively high p H and solid-liquid combination fermentation environment to compensate the insufficient activities Daqu protease,and enhanced the utilization rate of raw materials in the brewing process.The total nitrogen content(2.15 g/100 m L)and amino acid nitrogen content(1.22g/100 m L)all met the standard of special-grade soy sauce,and the free amino acid content was increased by 26%,and the proportion of flavor compounds such as esters,pyrazines and sulfur compounds was also obviously increased.Finally,the application of CRISPR/dCas9 system to regulate Aspergillus oryzae was realized.The CRISPR/dCas9 system was constructed by introducing D10 A and H840 A point mutations into the Ruv C and HNH nuclease domains of Cas9 gene,respectively,and the marker gene of Aspergillus oryzae 3.042 pyr G was introduced,which successfully verified the feasibility of the application of CRISPR/dCas9 system in Aspergillus oryzae 3.042.CRISPRi system was constructed on the basis of dCas9 gene.The successful fusion of dCas9 and VPR activation domain constructed the CRISPRa system.The sg RNA of transcription factor Amy R was fused with CRISPRi/CRISPRa system,respectively,and then transformed into Aspergillus oryzae 3.042 uracil auxotrophic strain,and two new strains were obtained,namely,i5 with 59.57% decrease in α-amylase activity and a6 with 16.04% increase in α-amylase activity,which successfully controlled the secretion ability of extracellular enzymes in Aspergillus oryzae by regulating transcription level.These above results provide a theoretical basis and reference value for optimizing the application of extracellular enzyme secretion by Aspergillus oryzae and disturbing the expression level of extracellular enzyme through transcripts.