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

Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate car boxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzy me in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino acid sequences of four trypt ic peptides were determined. Synthetic oligonucleotides based on the s equences of two of the peptides hybridized to the same bands in variou s restriction-enzyme digests of S. coelicolor genomic DNA. This hybrid ization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could enc ode amino acid sequences similar to those of PEPC from other microorga nisms. A BamHI/PstI fragment was subcloned into the streptomycete high -copy-number plasmid vector pIJ486 and transferred into Streptomyces l ividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100000 M(r), the same as that of purified S. coelicolor PEPC.