Abstract
BackgroundErythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.ResultsThe gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.ConclusionsA novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.
Highlights
Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor
C. magnoliae JH110 ER gene identification By utilizing the recently constructed expressed sequence tag (EST) library of C. magnoliae JH110 in our lab, we discovered a clone containing the ER-encoding gene from the randomly sequenced 912 EST clones
A 1041-bp C. magnoliae JH110 ER (CmER) with 5'- and 3'-untranslated regions was obtained from the C. magnoliae JH110 genomic DNA and it harbored an open reading frame (ORF) of 849 bp with an ATG initiation codon and a TGA termination codon
Summary
Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. Because of its sweetness and mouthfeel-enhancing property, erythritol has been used as a low-calorie alternative sweetener in the food industry. Erythritol can be synthesized by bioconversion that uses osmophilic yeasts and bacteria [7,8] and it has been produced commercially using an Trichosporonoides megachiliensis SNG-42 that produces erythritol in high yield [9]. This whole-cell biocatalytic production process is superior to chemical substrate conversion because it is a simpler process requiring only a few steps and is less expensive because a raw material is cheap and readily available. Various biological processes using C. magnoliae JH110 and its chemical mutant have been developed for maximizing erythritol production [11,12,13]
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