Construction of a Proline-producing Mutant from an Extremely Thermophilic Eubacterium Thermus thermophilus HB27
Takehide KOSUGE* and Takayuki HOSHINO
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305, Japan
The proB, proA and proC genes coding forγ-glutamyl kinase (GK), γ-glutamyl phosphate reductase (GPR) and Δ'-pyrroline-5-carboxylate reductase (P5CR), respectively, were cloned and sequenced from an extremely thermophilic eubacterium Thermus thermophilus HB27 (1,2). The proB and proA genes existed in tandem and overlapped by 2 bp. The proC gene existed apart from the proBA genes. Comparison of the amino acid sequences of the T. thermophilus GK, GPR and P5CR proteins with those of the GK, GPR and P5CR proteins of Escherichia coli revealed 58.5%, 57.2% and 47.0% similarities, respectively. The proA and proC gene expression plasmids, designated as pKK-proA and pPROK-proC, respectively, were constructed. A thermostable GPR or P5CR activity was detected in the crude extract of E. coli transformed with the plasmid pKK-proA or pPROK-proC. The polypeptide bands of 48.7 kDa and 27.8 kDa which were consistent with the deduced molecular weight of the ProA and ProC proteins were detected by SDS-PAGE of heat-treated crude extracts from the E. coli transformants.
In the proline biosynthetic pathway of T. thermophilus HB27, GK was subjected to the feed-back inhibition by proline since the growth of T. thermophilus was inhibited by a proline analogue, 3,4-dehydroproline. It was also indicated that T. thermophilus had the proline dedradation pathway since T. thermophilus was able to grow by uptaking L-proline as a sole nitrogen source. A 3,4-dehydroproline-resistant mutant whose GK protein had an amino acid substitution from Ala to Val at position 125 was constructed fromT. thermophilus HB27. The mutant was named as TH401. A further mutant TH4017 which was not able to utilize the L-proline for the growth was constructed from TH401. This strain produced about 2 mg/1 of L-proline in minimal medium after 12 hr incubation at 70ーC. This is the first report about the construction of an amino acid-producing mutant in extreme thermophiles.
1. Hoshino, T., Kosuge, T., Hidaka, Y., Tabata, K., and Nakahara, T. (1994) Biochem. Biophys. Res. Commun., 199, 410-417.
2. Kosuge, T., Tabata, K., and Hoshino, T. (1994) FEMS Microbiol. Lett., 123, 55-62.
