Cloning of ATP-Citrate Lyase (acl1) from Aspergillus niger and its Expression in Escherichia coli
Aspergillus niger is an important strain used for industrial fermentation of citrate. The production of citrate is closely related to the growth and metabolism of A. niger. ATP-citrate lyase (ACL) is responsible for catalyzing the conversion of citrate in
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Cloning of ATP-Citrate Lyase (acl1) from Aspergillus niger and its Expression in Escherichia coli Fang Sun, Hong Chen, Xihong He and Hao Liu
Abstract Aspergillus niger is an important strain used for industrial fermentation of citrate. The production of citrate is closely related to the growth and metabolism of A. niger. ATP-citrate lyase (ACL) is responsible for catalyzing the conversion of citrate into oxaloacetate and acetyl-CoA, which is a bridge between glucose metabolism and fatty acid synthesis. In A. niger, tandem divergently transcribed genes (acl1 and acl2) encode the subunits of ACL, whose physiological function is unclear. In this study, acl1 was obtained from A. niger by RT-PCR. The sequencing result was consistent with the sequence of genome database. We constructed the expression vector pET28a+-acl1-his6 which was suitable for the efficient expression in Escherichia coli BL21. After purification with Ni2+ chelating chromatography column, SDS-PAGE analysis showed that the molecular mass of the ACL1 was 66 KDa. The result laid the foundation for further research about protease characteristics and physiological functions of ACL1 in A. niger. Keywords Aspergillus niger expression RT-PCR
ATP-citrate lyase
Acetyl-CoA
Protein
15.1 Introduction Aspergillus niger is an important industrial workhorse with extensive application in the sectors of industrial enzymes, heterogeneous proteins, organic acids, and so on [1]. A. niger is economically important as a fermentation organism used for the F. Sun H. Chen X. He H. Liu (&) College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China e-mail: [email protected] H. Liu Tianjin University of Science and Technology, No. 29 13th Avenue, TEDA, Tianjin 300457, People’s Republic of China
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 249, DOI: 10.1007/978-3-642-37916-1_15, Ó Springer-Verlag Berlin Heidelberg 2014
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production of citric acid. Industrial citric acid production by A. niger represents one of the most efficient, highest yield bioprocesses in use currently by industry. At present, the annual output of citric acid is more than 1.5 million tons in the world, of which 99 % citric acid is fermented from A. niger. The production of citric acid is closely related to the growth and metabolism of A. niger [2–4]. For a long time, researchers have focused on the genes related to the metabolism of A. niger. The researches have clarified the significant roles of the glycolytic pathway, the tricarboxylic acid cycle, and the glyoxylate cycle in the citric acid metabolism and revealed that pyruvate dehydrogenase, pyruvate carboxylase, and citrate synthase directly involved in the function of key enzyme encoding the genes in the citric acid metabolism [5, 6]. The disclosure of the genomic sequence to the public brought the study of A. niger into the post-genomic era [7
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