| As the largest edible organic acid in China,citric acid is widely used.The safety and applicability of Aspergillus niger as the main citric acid-producing strain in the industry have been confirmed.As the global demand for citric acid increases,it is important to develop a high-yield citric acid strain that is genetically stable for industrial fermentation.In this study,A.niger strain S916 was used as the chassis cell to construct a genetically stable A.niger strain with high expression of citric acid by enhancing the citric acid secretion pathway,enhancing glycolysis activity and affinity to oxygen.The main results are as follows:(1)Enhanced citric acid secretion pathway: Strain S1633 was obtained by strengthening gene cex A of strain S916 using PEG-mediated protoplasts.The expression of cex A gene increased the exocrine capacity of citric acid,and the 3-day citric acid production was 11.49% higher than that of control strain S563.(2)Enhanced glycolysis activity: By enhancing the low-affinity glucose transporter gene mst C,the ability of the strain cells to take glucose under high glucose concentration was enhanced.Strain S2118 was obtained by co-enhancing genes hxk A and pfk A,which increased the flux of the glycolysis pathway and made more precursor substances flow to citric acid synthesis.Compared with strain S1633,the yield of citric acid in 3 days of strain S2118 increased by 12.6%.(3)Enhanced affinity to oxygen: The co-enhancement of genes mst A and vgb improved the cell uptake of glucose at low concentrations and the ability to obtain oxygen.And finally,a high-yield citric acid strain S2547 with stable genetic properties was obtained.Compared with strain S2118,the yield of citric acid in 3 days of strain S2547 increased by 6.3%.(4)By shaking flask fermentation analysis.The citric acid yield of strain S2547 reached 62.7g/L in 3 days,which was 32.8% higher than that of control strain S563.The 5-day yield of strain S2547 reached 121.2g/L,which was 16.5% higher than that of S563.The production intensity reached 1.01g/L/h and the citric acid fermentation cycle was shortened by 10 h.This strain has the potential for industrial fermentation of citric acid. |
