Stability and instability of a cloned mutant allele of the proline gene in Serratia marcescens. Use of a mini-F plasmid in the proline-overproducing mutant
Abstract For the purpose of improving proline production by a Serratia marcescens mutant, a mutant allele of the proline gene, dpr-1, that is responsible for proline overproduction, was cloned from the mutant genomic DNA into a mini-F plasmid. Furthermore, the dpr-1 allele was subcloned into plasmids pLG339 and pBR322 to investigate the effect of copy number of the hybrid plasmids on proline productivity and the plasmid stability. Strains carrying the dpr-1 hybrid plasmids grew more slowly than the control strain, indicating that the hybrid plasmids were deleterious to host cells. The dpr-1 alleles cloned on pBR322 and pLG339 were genetically unstable in the wild-type and the proline-overproducing strain, respectively. On the other hand, the allele cloned on the mini-F plasmid with the lowest copy number was considerably stable even in the proline-overproducing mutant. The mutant carrying the cloned dpr-1 on the mini-F plasmid produced 62 mg ml−1 of proline, which was approximately 10% more than the yield of the control strain.