Gene Knockout Of Alpha-glycosidase In Aspergillus Niger Producing-citric Acid

Citric acid is a crucial and multifunctional organic acid,which is widely used in food,medicine,chemical industry and other fields.China is a major producer of citric acid,and covers 70%of the world total's.However,for the fermentation of citric acid,the residual sugar content of fermentation liquid(FL)was about 2%,which caused a great waste of raw materials.The analysis of the residual sugar content of FL showed that 69%was isomaltose,and 23%was alpha-D-glucopyranoside,which mainly catalyzed by alpha-glucosidase.Therefore,one of the key enzymes that majorly effect the fermentation of citric acid is the alpha-glucosidase.In this paper,with the aim that reduce the content of the residual sugar and increase the utilization rate of sugar and the yield of citric acid,the putative alpha-glucosidase genes were analyzed and knocked out via the analysis of the genome sequence of producing-citric acid Aspergillus niger.1.Two knockout vectors were constructed with respective alpha-glucosidase A gene 214233 and hypothetical alpha-glucosidase gene 40261,and then transformed into Aspergillus niger using agrobacterium-mediated methods,for the gene knockout with homologous recombination technique.And the mutant KO-214233 and KO-40261 were obtained.2.No obvious changes on the colony morphology,spore morphology and growth rate between knockout mutant strains and wild-type strain.But the morphology and size of mycelium pellets were changed,and the edge of mycelium pellets of the mutant KO-214233 was more smooth than the strain KO-40261 and wild-type.The results of circumference measurements showed that the knockout strain KO-214233 has the smallest mycelium pellet circumference,followed by KO-40261 and the wild-type.3.The contents of total sugar,glucose,isomaltose and citric acid were further detected.The results showed,as for the two mutant strains,the content of total sugar,reduecd sugar and isomaltose were all decreased by respective 10.11%,9.44%,3.03%and 3.93%,19.98%,6.37%,compared with the wild-type.However,the production of citric acid was increased by 9.54%,3.51%respectively.These results showed that the strains lack of gene 214233 and 40261 could reduce the content of residual sugar,increase the utilization rate of sugar and the yield of citric acid.This study lays the foundation for the utilization of sugar in the production of citric acid and other chemicals by Aspergillus niger.