The Study On Construction Of Aspergillus Niger Strain With The Multiple GlaA Gene And Citric Acid Production

Citric acid is an important organic acid,which is widely used in food,medicine and chemical industry.Currently,Aspergillus niger is the main strain for citric acid production via fermentation process,however,the strain possesses lower glucoamylase activity and higher energy charge for carbohydrate degradation and sugar metabolism,which limited the development of citric fermentation industry.From the perspective of practice,the gene coding glucoamylase was overexpressed by genetic engineering technology and intracellular energy charge was regulated to a suitable level for enhancing citric acid production in this study.The glaA-cDNA gene was obtained from A.niger 101?agdA RNA by the reverse transcription PCR(RT-PCR)method.Compared with A.niger CBS513.88 from NCBI database,the glaA-cDNA gene alignment result showed a 99.90%identity(GenBank:XM 001390493),the amino acid sequence alignment result showed a 99.84%identity(GenBank:XP 001390530).The plasmid p55 containing the overexpression elements were constructed by overlapping PCR method and the plasmid was transferred into Agrobacterium tumefaciens by electroporation method.By Agrobacterium tumefaciens-mediated transformantion method,the glucoamylase overexpression strains named TNA OG 1,TNA OG 17 and TNA OG 31 were obtained and the three overexpression strains had good genetic stability via cultivation for 15 generations.The measurement results of ?-glucosidase and glucoamylase activity of TNA 101?agdA and TNA OG 1 showed that the highest ?-glucosidase activity of the two strains was almost equal and TNA OG 1 possessed higher glucoamylase activity,increased by 34.48%.It was investigated that the glaA gene mRNA expression levels of strain TNA OG1 was 1.43-fold as much as that of TNA 101?agdA by the relative quantification real time RT-PCR method.The fermentation results revealed that the residual reducing sugar content in broth using strain TNA OG 1 decreased by 36.67%,the conversion ratio of sugar to acid increased by 2.72%,and the final citric acid production increased by 8.91%when compared with the fermentations using TNA 101?agdA.The fermentation results conducted on 30 L or 500 t scale fermenters with TNA OG 1 showed that the average acid production was 173.72 g/L and average conversion ratio of sugar to acid reached up to 101.51%.In summary,it was beneficial for improving the utilization of substrate and decreasing the residual sugar concentrations as well as the production cost by appropriately enhancing the glucoamylase activity.The change trends of intracellular ATP,NADH and NADH/NAD+ was measured for A.niger TNA OG 1 and the standard strain ATCC 1015.The results showed that the concentrations of intracellular ATP,NADH and NADH/NAD+ of TNA OG 1 were notably lower than those of ATCC 1015 at several fermentation stages.0.2 mg/L oxidative phosphorylation inhibitor(antimycin A)or 0.1 mg/L uncoupler of oxidative phosphorylation(DNP)was added to the fermentation medium at the 24-h time point for A.niger TNA OG 1,and the results revealed that intracellular ATP concentration declined and the oxidation rate of intracellular NADH were accelerated.Compared with the control group,the citric acid production of DNP-adding group and antimycin A-adding group was respectively increased by 8.93%,14.97%.In conclusion,the excessive ATP concentration has an important effect on citric acid accumulation in A.niger.Strengthening the ability of NADH oxidation and appropriately reducing the concentration of intracellular ATP can enhance citric acid yields.