Construction Of Citric Acid Production Engineering Strain By Aspergillus Niger

Citric acid is popular in the food industry due to its unique sour taste and safe and nontoxic characteristics.Moreover,citric acid is widely used in medicine,cosmetics,chemical industry and other fields.,and it is the most consumed organic acid in the world.China has rich sources of citric acid raw materials and good production prospects,and has become the world's largest producer and exporter of citric acid.The main method of citric acid production is submerged fermentation by Aspergillus niger.Although the citric acid fermentation titer and sugar acid conversion rate have reached a high level at present,there is still room for further improvement.Therefore,it is particularly important to construct Aspergillus niger fermentation engineering strain with high citric acid production.In this paper,Aspergillus niger ATCC1015 was used as the initial strain,and the effects of oxaloacetate hydrolase gene(oahA),malate dehydrogenase gene(cmdh),pyruvate carboxylase gene(cpyc),fumarase gene(fumr),fumarate reductase gene(frds)and phosphofructokinase gene(pfk)in EMP pathway on citric acid synthesis were analyzed by means of molecular biology,so as to construct citric acid high-yield strain.The main results are as follows:(1)pfk,fumr,frds expression vectors were constructed using the expression plasmid pLH454 constructed in laboratory as the starting vector.(2)Agrobacterium-mediated transformation of Aspergillus niger,oahA knockout bacteria were obtained as starting strains,and then over-expressed strains such as pfk,fumr,frds,mdh,pyc were constructed respectively.(3)Citric acid production was analyzed by shake flask fermentation.It was found that the over-expressed strains that overexpressed pfk,fumr,frds,mdh and pyc genes alone increased their production by 4.16%,4.89%,4.98%,1.17%and 1.03%respectively compared with the original strains.The strain with double overexpression of fumr and frds genes is 6.13%higher than the original strain.(4)RNA was extracted to analyze the transcription of over-expressed genes.Heterologous fumr gene and frds gene were expressed in Aspergillus niger.The expression levels of pfk gene,mdh gene and pyc gene are all increased to different degrees compared with the original strain,wherein the expression level of pfk gene is increased up to 3.03 times as much as that of the original strain S549,and the expression levels of mdh gene and pyc gene are 2.42 times and 1.28 times as much as that of the original strain respectively.(5)Enzymatic analysis showed that compared with the original strain S549,the enzymatic activities of strains S642,S703 and S706 which overexpressed the pfk gene,mdh gene and pyc gene increased by 1.23,1.59 and 1.34 times,respectively.