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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 Mr of A. niger citrate synthase is 48 501, 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.

In our paper, we analysed the effects of overproduction of citrate synthase on citric acid production by Aspergillus niger in addition to some descriptive work on the enzyme itself and the citA gene. Dr Ratledge argues that our attempt to increase productivity by amplifying citrate synthase was 'a complete waste of time and effort’. He states that we could have known beforehand that this was not going to work by looking at biochemical data. I oppose his reasoning and will explain my view. Dr Ratledge performs a rough calculation to show that, in an unmodified strain, citrate synthase is well in excess of the amount required to sustain the metabolic flux observed during fermentation. A comparison between the maximal …