Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase

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Abstract
The gene for the major phosphofructokinase enzyme in E. coli, pfkA, was sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the 2nd, minor phosphofructokinase found in E. coli. The region which was sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes were identified from the nucleotide sequence: a gene for a periplasmic sulfate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene was subcloned into a high-copy-number plasmid under the control of a strong, chimeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialized recombinant was constructed which carries a 1.4 .times. 103-nucleotide insert containing just the pfkA gene flanked by 2 HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using reverse genetics.