In vivo cloning of proline genes and its expression in Escherichia coli

The wild type proA+, B+ genes of E. coli were cloned in vivo using a plasmid containing a mini-Mu replicon, pEG5005. The cloning frequency was about 1.46 x 10(-3)/Kanr transductant. Genetic and biochemical analysis of these clones indicated that the proA+, B+ genes are on the plasmid pEG5005. The secretion of proline were assayed for 500 Pro+ clones. However, no proline accumulation was detected. A Pro+ clone pPR3 was mutagenized in vivo by NTG and the mutants resistant to D-proline were obtained. One of the Dpr mutants pPR7 was found to produce 0.35 mg/ml proline in a proA B deletion strain. When pPR7 was transferred into a proline producing strain, the yield of proline increased up to 2.5 mg/ml, which is 7 and 2.5 times higher than that of the donor and recipient respectively. The physical maps of pEG5005 and pPR3 were roughly established.