Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete

Abstract

Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.