1,8-Dihydroxynaphthalene (DHN)-Melanin Biosynthesis Inhibitors Increase Erythritol Production in Torula corallina , and DHN-Melanin Inhibits Erythrose Reductase

Abstract

The yeast Torula corallina is a strong erythritol producer that is used in the industrial production of erythritol. However, melanin accumulation during culture represents a serious problem for the purification of erythritol from the fermentation broth. Melanin biosynthesis inhibitors such as 3,4-dihydroxyphenylalanine and 1,8-dihydroxynaphthalene (DHN)-melanin inhibitors were added to the T. corallina cultures. Only the DHN-melanin inhibitors showed an effect on melanin production, which suggests that the melanin formed during the culturing of T. corallina is derived from DHN. This finding was confirmed by the detection of a shunt product of the pentaketide pathway, flaviolin, and elemental analysis. Among the DHN-melanin inhibitors, tricyclazole was the most effective. Supplementation with tricyclazole enhanced the production of erythritol while significantly inhibiting the production of DHN-melanin and DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase. The erythrose reductase from T. corallina was purified to homogeneity by ion-exchange and affinity chromatography. Purified erythrose reductase was significantly inhibited in vitro in a noncompetitive manner by elevated levels of DHN-melanin. In contrast, the level of erythrose reductase activity was unaffected by increasing concentrations of tricyclazole. These results suggest that supplemental tricyclazole reduces the production of DHN-melanin, which may lead to a reduction in the inhibition of erythrose reductase and a higher yield of erythritol. This is the first report to demonstrate that melanin biosynthesis inhibitors increase the production of a sugar alcohol in T. corallina.

Biosynthesis of non-melanin pigment by a divergent polyketide synthase in Metarhizium robertsii

“…2A). Consistent with this, the supplementation of the DHN-melanin inhibitor pyroquilon (Lee et al, 2003) did not affect the WT or mutant phenotypes (Fig. 2B), confirming that the MrPks2 cluster is not involved in DHN-melanin production.…”

“…The wild-type (WT) and mutants of the M. robertsii strains ARSEF 2575 and ARSEF 23 were routinely cultured on potato dextrose agar (PDA, BD Difco, Radnor, PA) or PDA amended with 20 lg/ ml of the DHN-melanin inhibitor pyroquilon (Sigma, St. Louis, MO) (Lee et al, 2003) at 25°C. The cultures were alternatively incubated in Sabouraud dextrose broth (BD Difco) for DNA extraction or spore germination assays.…”

Characterization of melanin isolated from a dark septate endophyte (DSE), Exophiala pisciphila

“…The colonies growing on 10 lg mL -1 concentration of the inhibitor were reddish brown instead of the usual black, indicating that melanin biosynthesis was inhibited. Tricyclazole is a specific and effective inhibitor of DHN melanin and blocks its synthesis by inhibiting DHN-melanin biosynthetic enzymes, such as trihydroxynaphthalene reductase, involved in melanin synthesis (Bell Wheeler 1986;Lanisnik and Wheeler 2003;Lee et al 2003;Romero-Martinez et al 2000) and it has been used to detect DHN melanin in fungi (Ravishankar et al 1995;Lanisnik and Wheeler 2003). Colonies growing in the presence of kojic acid (DOPA melanin inhibitor) appeared normal and was indistinguishable from the controls (Fig.…”

Alternaria alternata transcription factor CmrA controls melanization and spore development

“…For RNA analysis, cultures were incubated in liquid mCDB medium for 3 or 7 days at 28 uC. To inhibit melanin biosynthesis, 3 mg tricyclazole was diluted in 100 ml DMSO and mixed with 100 ml mCDB agar to a final concentration of 30 mg ml 21 (Lee et al, 2003). Al.…”