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Dihydrofolate Reductase Thermosensitive (ts-dfrA) Mutant Induces Dihydrofolate Overproduction by Lactobacillus plantarum

ABSTRACT Dihydrofolate (dihydrofolic acid, vitamin B9) is one of the principle reduced forms of folates, which is converted to tetrahydrofolate by dihydrofolate reductase (DHFR). The aim of the present study was to overproduce total folate by inducing accumulation of dihydrofolate (DHF) due to inhibition of DHFR in a thermosensitive mutant (TS) of Lactobacillus plantarum. This mutant (TS34) was obtained by UV-mutagenesis of L. plantarum wild-type parent strain (HMA07) isolated from fermented sausage. The mutant TS34 was characterized by its ability to grow at 30°C but not at 40°C. Strain HMA07 and its mutant TS34 were identified by determining partial sequence of 16S rRNA gene. A single C→T exchange in the dfrA (dihydrofolate reductase gene) at nucleotide 184 was detected. This exchange led to the conversion of leucine (L) to phenylalanine (F) at the position 62 in amino acid sequence (L62F transition). TS43 showed significant (p < 0.05) reduction in DHFR activity from 1.51 to 0.06 Unit/mg protein, resulting in increasing DHF amount at the expense of THF. Accordingly, total folate production by the mutant TS34 (in two-stage fermentation process with temperature shift-up from 30°C to 40°C) increased by three-fold compared with the parental strain HMA07. In conclusion, thermosensitive mutation in dfrA caused inhibition of DHFR and resulted in accumulation of DHF. The technique described here could be considered as novel strategy for overproduction of DHF in two-stage fermentation process.