Cloning and Characterization of the Genes Related to High Production of L-malate in Aspergillus sp. N1-14’
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Abstract:
In order to understand the mechanism behind the high production of L-malate (LMA) by Aspergillus sp. N1-14’, its total DNA was extracted to be used as the template, homologous primers were designed to amplify the segment containing the full length of the pyruvate carboxylase gene (pyc) and the malate dehydrogenase gene (mdh), and sequencing was then performed. Based on the results of this sequencing, the coding regions of pyc and mdh were identified, their coding sequences were amplified from the total cDNA of N1-14’ using the designed primers, and sequencing was carried out through TA cloning. The results of the sequencing showed that the open reading frame (ORF) of pyc had 3582 bp and it encoded 1193 amino acids. The analysis of the results showed that the amino acid sequence of pyruvate carboxylase (PYC) was quite conserved in Aspergillus species as the sequence similarity with other Aspergillus species was higher than 90 %. Mutations occurred at two conserved sites of N1-14’, 833(A) and 1022(F), which were located in a loop and in the middle of α-helix, respectively, suggesting that those mutations may be relevant to the high production of LMA. The ORF of mdh had 1023 bp and it encoded 340 amino acids. The amino acid sequence of malate dehydrogenase (MDH) was also conserved across the Aspergillus species, and there were two amino acid mutation sites in the conserved domain. Both of them were situated in the α-helix. In this experiment, two genes related to the key process of producing LMA in N1-14’ were cloned, the specificity of these two genes was analyzed, and the function of the specific amino acid sites were predicted. This study provides a basis for further study on the mechanisms behind the high production of LMA and genetic manipulations required for increasing its production.
