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.
Wang
A Q
AQ
Institute of Microbiology, Academia Sinica, Beijing.
Jiao
H
H
Chen
X Z
XZ
Huang
H R
HR
D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D011392:Proline / Q000096:biosynthesis / Q000235:genetics*; D014170:Transformation, Genetic
D011392:Proline