Research On The Function Of Carnitine Acetyltransferase In Aspergillus Niger

Aspergillus niger is an important strain used for industrial fermentation of citric acid. Citric acid produced by Aspergillus niger is widely used in food, chemical, pharmaceutical, textile and other industries. Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules.Camitine acetyltransferase (CAT) is located in mitochondria, peroxisomal and cytosol, which catalyzes the reversible transfer of short chain acyl groups between CoA and carnitine, so that they can cross intracellular membranes. In A. niger,Carnitine acetyltransferase is encoded by two genes, whose physiological functions are still unclear.In this paper, the plasmid pFGL59Ble was constructed which has hygr and bler markers. We choosed pFGL59Ble as the vector for knocking out genes. Upstream and downtream fragments of target genes were amplified by PCR,Then they were ligased into the T-DNA area of the pFGL59Ble vector sequentially. The recombinant plasmid was obtained by being transformed into E.coli and they were named as pCATl and pCAT2 which were then electroporated into Agrobacterium tumefaciens, respecticely. By Agrobacterium tumefaciens-mediated transformation of Aspergillus niger, single-stranded linear T-DNA fragments carrying the homologous fragmens were transferred into the host cell. The transformants were selected by PCR.Through homologous recombination, target genes were knockout successfully, which were named as △CAT1 and △CAT2.This research revealed that these gene deletion strains had obvious defects:1) the mutants grew on PDA slowly; 2) the capacity of spore production was weakened significantly,especially △CAT2 mutant, with almost no spore generation; 3) pigment can not be synthesized normally. By fermentation experiments and HPLC analysis, the results showed that the citric acid production of the mutants were less than that of the wild strains. The citric acid production of WT、△CAT1 and △CAT2 were 31 g/L,20 g/L,21 g/L respectively. Compared to the wild type, citrate production of ACAT1 was reduced by 35% and ACAT2 was reduced by about 32%。...