Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production.
Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated M(r) of A. niger citrate synthase is 48501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain.
Ruijter
G J
GJ
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands. george.ruijter@algemeen.mgim.wau.nl
Panneman
H
H
Xu
D
D
Visser
J
J
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D002950:Citrate (si)-Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D010071:Oxaloacetates / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014170:Transformation, Genetic
D018076:DNA, Complementary; D010071:Oxaloacetates; D019343:Citric Acid; D000105:Acetyl Coenzyme A; D002950:Citrate (si)-Synthase