The Impact Of Malic Enzyme ETC. On Citric Acid Metabolism In A.Niger

Citric acid (C6H8O7), which is one of the most important organic acids, generally recognized as safe products (GRAS), is widely applied in food, chemical, pharmaceutical, textile and other industries. At present, most citric acid is produced by Aspergillus niger. In citric acid fermentation process by Aspergillus niger, citrate synthase can convert oxaloacetate and acetyl-CoA into citrate, in which oxaloacetate playes a key role in the synthesis of the citrate and NAD+-malic enzyme, pyruvate carboxylase, oxaloacetate transport protein in this paper are directly or indirectly involved in the synthesis of oxaloacetate. Therefore, this paper mainly studys the impact of oxaloacetate on the metabolism of citric acid by Aspergillus niger by deleting relative genes coding enzymes above.In this paper, we choose p44as the vector for knocking out genes. Upstream and downtream fragments of target genes were amplified by PCR. Then they were ligased to the T-DNA of the p44vecter sequentially. The recombinant plasmid was obtained by transformed into E.coli and they were named as p44-me, p44-mpyc and p44-oat which were then electroporated into Agrobacterium tumefaciens, respecticely. By Agrobacterium tumefaciens-mediated transformation of Aspergillus niger, single-stranded linear T-DNA fragment carrying the homologous fragmens was transferred into the host cell. The transfoemants were selected by PCR. And through homologous recombination, target genes was knockout successfully, which named as NAD+-ME△(malic enzyme deletant), mPYC△(mitochondrial pyruvate carboxylase deletant), OAT△(oxaloacetate transporter deletant).By fermentation experiments of the mutants and wild strain and HPLC for analysis, the results showed that when using sucrose at the concentration of140g/L as C-source, the weight of mycelia of all strains were about15g/L, and the residual sugar were about25g/L. The citric acid productions by the three mutants were less than the wild strain. The citric acid production of WT, NAD+-ME△, mPYC△and OAT△were75g/L,65g/L,61g/L and68g/L, respectively. Compared to the wild type, citrate production of mPYC△was reduced by18.67%, NAD+-ME△was reduced by about13.33%, OAT△was reduced by about9.33%...