Construction of a proline-producing mutant of the extremely thermophilic eubacterium Thermus thermophilus HB27

Growth of Thermus thermophilus HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the proB gene) was inhibited by feedback inhibition in T. thermophilus. DHP-resistant mutants were reported previously for Escherichia coli (A.M. Dandekar and S.L. Uratsu, J. Bacteriol. 170:5943-5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693-699, 1992), and their mutated sites in the proB gene were identified. Comparison of the amino acid sequence of T. thermophilus γ-glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the proB gene as reported for the proline-producing mutant of S. marcescens. The mutant, TH401, excreted about 1 mg of L-proline per liter at 70°C after 12 h of incubation. It was also suggested that T. thermophilus has a proline degradation and transport pathway since it was able to grow in minimal medium containing L-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize L-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of L-proline per liter in minimal medium at 70°C after 12 h of incubation.