[Hyperproduction of L-proline in Escherichia coli]

The proA and proB genes from Escherichia coli have been cloned using the plasmids pBR322 and pBR325 as vectors. The episome F128 (proAB, lac) was used as cloning DNA source. Both genes were firstly located within a 10 kilobases EcoRI DNA fragment of the episome. Subcloning experiments showed that both proteins were coded by a 3 kilobases PstI DNA fragment. Although, th recombinant plasmids containing the proA and proB genes were able to complement the Pro- phenotype of different E. coli strains, bacteria harboring these plasmids did not excrete L-proline to the culture medium. Nevertheless, an operon, proAB, able to confer to E. coli cells the property of excreting L-proline, was isolated from an UV-mutant of E. coli E5014 [F' (proAB, lac)] resistant to the L-proline analogue, thioproline. E. coli HB101 cells transformed with the plasmid pJABP (UV) carrying the mutated proAB operon excreted up to 5 g/l of L-proline, after 40 hours of fermentation at 37 degrees C in a modified M63 minimal medium. The production of L-proline was not increased when the proC gene was inserted in the plasmid pJABP (UV).