The Overexpression of YALI0B07117g Results in EnhancedErythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h ) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

Overexpression of YALI0B07117g Encoding Erythrose Reductase Homolog Results in Enhanced Erythritol Synthesis From Glycerol by the Yeast Yarrowia Lipolytica.

BackgroundPolyols are a group of sweet alcohols, frequently used as food additives. The constantly rising demand for polyols requires the application of new strategies to increase the production. Erythritol is synthesized by the yeast Yarrowia lipolytica under high osmotic pressure as an osmoprotectant. The metabolic pathway resulting in erythritol production remains partially unknown. However, the last reaction resulting in erythritol synthesis is conducted by an erythrose reductase (ER).ResultsThe Y. lipolytica strain was genetically modified to increase the erythritol yield and productivity, using glycerol as a sole carbon source. The modification focused on the ER homologue YALI0B07117g after the in silico analysis of the protein sequences of all reported ER homologues. Initial results in shake-flask experiments proved the influence of the gene YALI0B07117g in erythritol synthesis. Deletion of the gene resulted in 3-fold and 2-fold increased production of mannitol and arabitol, respectively. Overexpression of the native ER homologue gene showed a positive influence on erythritol production. Bath cultures were conducted and the obtained strain reached the yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the engineered strain, while the productivity of erythritol increased from 0.28 g/L/h for the A101 strain to 0.41 g/L/h for the modified strain.ConclusionsOverexpression of the gene YALI0B07117g resulted in increased production of erythritol in the yeast Y. lipolytica. Disruption of the metabolic pathway by deletion of the gene results in higher production titers of mannitol and arabitol. Application of the research may prove positive for shortening the cultivation time due to the increased consumption rate of the substrate combined with increased parameters of erythritol synthesis.

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

ABSTRACT 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 Cu2+ 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.

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.