Excess production of Bacillus subtilis quercetin 2,3-dioxygenase affects cell viability in the presence of quercetin.

Bacillus subtilis quercetin 2,3-dioxygenase (QdoI) catalyzes the C-ring cleavage of quercetin to yield 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. The recombinant QdoI effectively decomposed several flavonols, including quercetin, whereas its activity toward fisetin was low, suggesting that the 5-hydroxyl group at the A-ring is critical for substrate recognition. A B. subtilis mutant with derepressed QdoI activity was much more sensitive to quercetin than the wild type, but did not exhibit similar sensitivity toward the other flavonoids tested. Further analysis, including co-cultivation with the wild type and the mutant, led to the assumption that intracellular accumulation of protocatechuic acid derived from the rapid decomposition of quercetin severely affects cell viability. Although protocatechuic acid is also produced by fisetin degradation, cell death was avoided, probably due to the lower activity of QdoI toward fisetin. The sensitivity of the B. subtilis mutant toward quercetin was quenched by repression of QdoI by the use of its authentic repressors. Moreover, this adverse effect of excess QdoI with quercetin was also exerted on Escherichia coli cells. This implies the availability of the QdoI regulatory system as a novel selection marker for genetic transformation without using antibiotic-resistant ones.