Breeding of a mutant of Aureobasidium sp. with high erythritol production

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu 2؉ has been thoroughly reported, but the mechanism by which Cu 2؉ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu 2؉ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu 2؉ concentration. This suggests that supplemental Cu 2؉ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.

mentioning

confidence: 99%

Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

Lee¹,

Koo²,

Kim³

2002

“…This process has not been industrialized because of its low efficiency. Erythritol also can be produced by microbial methods that utilize osmophilic yeasts and some bacteria (42,48), and it has been produced commercially by using a mutant of Aureobasidium that produces erythritol in high yield (44% [wt/wt] glucose) (20). Recently, a high-erythritol-producing yeast strain was isolated from honeycombs and the new isolate was identified as Candida magnoliae KFCC 11023 (44).…”

mentioning

confidence: 99%

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Lee¹,

Kim²,

Ryu

et al. 2003

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K m ‫؍‬ 7.9 mM; k cat /K m ‫؍‬ 0.73 mM ؊1 s ؊1 ). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k cat /K m ‫؍‬ 450 mM ؊1 s ؊1 ) than with NADPH (k cat /K m ‫؍‬ 5.5 mM ؊1 s ؊1 ), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu 2؉ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.Erythritol, a four-carbon polyol, is widely distributed in nature (16). Like most other polyols, erythritol is a metabolite or storage compound and is found in seaweed, mushrooms, and fruits. It is a noncaloric, noncariogenic sweetener that is safe for diabetics (35). Erythritol has about 70% of the sweetness of sucrose in a 10% (wt/vol) solution and a very high negative heat capacity, providing a strong cooling effect when dissolved (16). Erythritol can be synthesized from dialdehyde starch by a high-temperature chemical reaction in the presence of a nickel catalyst (39). This process has not been industrialized because of its low efficiency. Erythritol also can be produced by microbial methods that utilize osmophilic yeasts and some bacteria (42, 48), and it has been produced commercially by using a mutant of Aureobasidium that produces erythritol in high yield (44% [wt/wt] glucose) (20). Recently, a high-erythritol-producing yeast strain was isolated from honeycombs and the new isolate was identified as Candida magnoliae KFCC 11023 (44). This strain can produce erythritol in high yield (43% [wt/wt] glucose) when cultured appropriately (42, 44).There are many repor...

“…However, this process has not been industrialized because of its low efficiency. Erythritol can also be produced by microbial methods with osmophilic yeasts and some bacteria (1,9,12,13,14,18,20,22,25,27,48) and has been produced commercially with a mutant of Aureobasidium that produces erythritol with a high yield (44%, wt/wt) (14). Erythritol is synthesized from erythrose-4-phosphate, which is an intermediate in the pentose phosphate cycle, by dephosphorylation and the subsequent reduction of erythrose.…”

mentioning

confidence: 99%

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

Lee¹,

Jung

Kim³

2003

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.