Characterization of erythrose reductase from Yarrowia lipolytica and its influence on erythritol synthesis

Abstract

BackgroundErythritol is a natural sweetener that is used in the food industry. It is produced as an osmoprotectant by bacteria and yeast. Due to its chemical properties, it does not change the insulin level in the blood, and therefore it can be safely used by diabetics. Previously, it has been shown that erythrose reductase (ER), which catalyzes the final step, plays a crucial role in erythritol synthesis. ER reduces erythrose to erythritol with NAD(P)H as a cofactor. Despite many studies on erythritol synthesis by Yarrowia lipolytica, the enzymes involved in this metabolic pathway have ever been described.ResultsThe gene YALI0F18590g encoding the predicted erythrose reductase from Y. lipolytica was overexpressed, and its influence on erythritol synthesis was studied. The amino acid sequence of the Y. lipolytica ER showed a high degree of similarity to the previously described erythrose reductases from known erythritol producers, such as Candida magnoliae and Moniliella megachiliensis. Here, we found that the gene overexpression results in an enhanced titer of erythritol of 44.44 g/L (20% over the control), a yield of 0.44 g/g and productivity of 0.77 g/L/h. Moreover, on purification and characterization of the enzyme we found that it displays the highest activity at 37 °C and pH 3.0. The effects of various metal ions (Zn2+, Cu2+, Mn2+, Fe2+) on erythrose reductase were investigated. The addition of Zn2+ ions at 0.25 mM had a positive effect on the activity of erythrose reductase from Y. lipolytica, as well as on the erythritol production.ConclusionsIn this study we identified, overexpressed and characterized a native erythrose reductase in Y. lipolytica. Further optimizations of this strain via metabolic pathway engineering and media optimization strategies enabled 54 g/L to be produced in a shake-flask experiment. To date, this is the first reported study employing metabolic engineering of the native gene involved in the erythritol pathway to result in a high titer of the polyol. Moreover, it indicates the importance of environmental conditions for genetic targets in metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0733-6) contains supplementary material, which is available to authorized users.

Erythritol production by yeasts: a snapshot of current knowledge

“…A correlation was also found between transketolase activity and erythritol production, and transketolase overexpression was found to increase both erythritol yield and productivity (Carly et al, ; Sawada, Taki, Yamakawa, & Seki, ). A similar correlation between ER activity and erythritol production was found in Y. lipolytica (Janek et al, ).…”

“…Moreover, this strain showed higher glycerol uptake (Mirończuk, Rzechonek, Biegalska, Rakicka, & Dobrowolski, ). Strains overexpressing both transketolase and ER also showed important benefits as compared with wild‐type strains, highlighting the central gene of these genes in erythritol synthesis (Janek et al, ; Mirończuk et al, ). Gene deletion has also been used for strain improvement.…”

“…Various explanations were formulated to explain this positive effect of metal ions. In Y. lipolytica , the addition of metal ions was found to increase ER activity, with Mn 2+ or Zn 2+ having the most significant effect depending on the study considered (Janek et al, ; Tomaszewska, Rymowicz, & Rywińska, ). In Torula sp.…”

“…This enzyme, shown to have a preference for NADH as cofactor, is overexpressed in the presence of high concentrations of salts or sugar, demonstrating its role in osmotic stress (Lee, Kim, Ryu, Seo, & Kim, 2003a;Park, 2011). In Y. lipolytica, the gene encoding ER has been recently identified as gene YALI0F18590g (Janek, Dobrowolski, Biegalska, & Mirończuk, 2017).…”

Sugar Alcohols and Organic Acids Synthesis in Yarrowia lipolytica: Where Are We?

“…The latter is dephosphorylated by a still non-characterized erythrose-4P phosphatase (E4PP) before being reduced by an erythrose reductase (ER) into erythritol. In Y. lipolytica, three genes encoding erythrose reductase have been characterized so far (namely, YALI0F18590g, YALI0D07634g and YALI0C13508g) [99,100]. We also recently elucidated the full EOL catabolic pathway [101].…”