Optimization Of Synthetic Medium For Glucoamylase Production By Aspergillus Niger And Preliminary Exploration Of Metabolic Profiling Analysis Approach

Glucoamylase is one of the largest enzyme production in the world, the average yield of which can reach20g/L, but it is still in short supply. Glucoamylase synthesis is closely related to the metabolism of Aspergillus niger. In order to further understand the bottleneck of glucoamylase synthesis, the metabolic flux analysis and metabolic profiling analysis of Aspergillus niger for glucoamylase production during fermentation process were carried out.Metabolic flux analysis and metabolic profiling analysis should be carried out in synthetic medium. Based on this, the synthetic medium was constructed and optimized in this paper. The effects of inorganic nitrogen sources, ratios between carbon and nitrogen source, phosphorus source concentrations on glucoamylase production were studied.In addition, metal ions were optimized to get the optimum conditions for prediction.In terms of intracellular metabolite detection, a method of metabolic profiling analysis for Aspergillus niger was established based on GC-MS in this paper. The influence of different solute and derivative agent on the response to standard substance was conducted Then, with Aspergillus niger as the research object, the effect of different quenching agents and extract solution on intracellular metabolites were investigated and also the quenching effect of quenching agent was surveyed. In addition, the method established was verified scientifically, the results showed that the method had a good selectivity, accuracy, stability and repeatability. The method established in this research laid a good foundation for subsequent metabolite profiling analysis of Aspergillus niger in this paper.The methods of metabolic flux analysis and metabolic profiling analysis were employed to systematically study the differences of the two strains (wild type and mutant) both macro and micro metabolic differences. The substrate and oxygen consumption rate of mutant strain were close to that of wild strains, but glucoamylase production in mutant strain were7.88times as high as that in wild strain and concentration of byproducts in mutant strain was10%of that in wild strains. By means of principal component analysis, four biomarkers were determined. According to intracellular metabolic flux analysis, we found that the fluxes of mutant strains in PP (Pentose Phosphate) pathway, oxidative phosphorylation, amino acid synthesis pathway were strengthened to some extent, and substrate utilization in mutant strain increased by nearly30%to that of wild strain. The above mentioned results explained metabolic response rules of Aspergillus niger to glucoamylase synthesis. It provided new insights for design of Aspergillus niger strains and fermentation process optimization.