CONSTRUCTION OF RECOMBINANT PLASMIDS FOR EFFICIENT EXPRESSION OF THE PYRUVATE DECARBOXYLASE GENE (PDK) FROM ZYMOMONAS-MOBILIS IN BACILLUS-SUBTILIS

The pdk gene from Zymomonas mobilis localized in a 4.7-kbp SphI fragme nt of plasmid pB201 was subcloned into the SmaI site of the M13mp19 ve ctor using the DraI restriction endonuclease. The M13mp19 derivatives obtained, carrying a 1.8-kbp DraI fragment in opposite orientations, w ere used to sequence the pdk gene beginning and end (about 250 bp each ) and for site-directed mutagenesis. Using polymerase chain reaction w ith synthetic oligonucleotide primers, a BamHI site was created in fro nt of the pdk gene initiating codon. The BamHI fragment harboring the pdk gene was cloned into shuttle vector pCB20 under the control of ''e xpression unit'' EU19035 containing bacillar vegetative promoter and r ibosome-binding site (RBS). The pdk gene expression was studied in the recombinant plasmid pCB20pdkI, a derivative of pCB20, which was shown to yield a high level of pyruvate decarboxylase [EC 4.1.1.1] synthesi s in Bacillus subtilis. However, this plasmid strongly inhibited the E scherichia coli cell growth and was eliminated from the cells at a hig h frequency.