[Design of recombinant plasmids for effective Zymomonas mobilis pyruvate decarboxylase (pdk) gene expression in...

The pdk gene from Z. mobilis localized on the 4.7-kb SpHI DNA fragment in plasmid pB201 was subcloned using DraI restriction endonuclease into the SmaI site of the phage cloning vector M13mp19. The derivatives of M13mp19 obtained, containing 1.8-kb inserts of the pdk gene in two opposite orientations, were used for DNA sequencing and site-directed mutagenesis. The latter was performed using polymerase chain reaction (PCR) and synthetic deoxyribonucleotides of appropriate structure as primers. In this way a BamHI site near the initial (formylmethionine) codon of the pdk gene was created. After amplification the pdk gene was treated by restriction endonuclease BamHI and cloned into pUC19, and then recloned into shuttle vector pCB20 capable of replicating in both Gram negative and Gram positive bacteria. A recombinant plasmid pCB20pdkI--a derivative of pCB20 carrying the pdk gene under control of the "expression unit" EU19035 containing a bacillar vegetative promoter and an RBS site was obtained. The properties of the pCB20pdkI in E. coli and Bac. subtilis cells were studied. It was shown that pCB20pdkI determines a high level of PDK synthesis in Bac. subtilis. At the same time, it strongly inhibits E. coli cell growth and segregates rapidly from this host.