Overproduction of NAD⁺ and 5′-inosine monophosphate in the presence of 10 μM Mn²⁺ by a mutant of Corynebacterium ammoniagenes with thermosensitive nucleotide reduction (nrd ^ts) after temperature shift

Abstract

Corynebacterium ammoniagenes strain CH31 is thermosensitive due to a mutation in nucleotide reduction (nrd ^ts). The strain was examined for nucleotide overproduction upon shifting the culture temperature to a range of elevated temperatures. No overproduction of NAD⁺ was detected in the control maintained at 27°C whereas NAD⁺ was accumulated extracellularily by strain CH31 at 37°C and at 40°C. As a result of the temperature shift, division-inhibited cells displayed only limited elongation. This is a characteristic morphological feature of cell-cycle-arrested coryneform bacteria. Ribonucleotide reductase (RNR) activity was inactivated immediately after the temperature shift in the NAD⁺-proficient cultures, leading presumably to an exhaustion of deoxyribonucleotide pools and impairment of DNA replication. In contrast to the low extracellular accumulation of NAD⁺, at the non-permissive temperature of 35°C a distinct capacity for intracellular nucleotide overproduction was revealed by a new method using nucleotide-permeable cells. The approach of shifting the culture temperature was applied successfully to the overproduction of taste-enhancing nucleotides in the presence of 10 μM Mn²⁺. Concomitant with a dramatic loss of viability, the thermosensitive mutant CH31 accumulated 5.3 g 5′-inosine monophosphate per liter following the addition of hypoxanthine as precursor for the salvage pathway.