Identification, characterization of two NADPH-dependent erythrose reductases in the yeast Yarrowia lipolytica and improvement of erythritol productivity using metabolic engineering

Abstract

Background: Erythritol is a four-carbon sugar alcohol with sweetening properties that is used by the agro-food industry as a food additive. In the yeast Yarrowia lipolytica, the last step of erythritol synthesis involves the reduction of erythrose by specific erythrose reductase(s). In the earlier report, an erythrose reductase gene (YALI0F18590g) from erythritol-producing yeast Y. lipolytica MK1 was identified (Janek et al. in Microb Cell Fact 16:118, 2017). However, deletion of the gene in Y. lipolytica MK1 only resulted in some lower erythritol production but the erythritol synthesis process was still maintained, indicating that other erythrose reductase gene(s) might exist in the genome of Y. lipolytica.

Results: In this study, we have isolated genes g141.t1 (YALI0D07634g) and g3023.t1 (YALI0C13508g) encoding two novel erythrose reductases (ER). The biochemical characterization of the purified enzymes showed that they have a strong affinity for erythrose. Deletion of the two ER genes plus g801.t1 (YALI0F18590g) did not prevent erythritol synthesis, suggesting that other ER or ER-like enzymes remain to be discovered in this yeast. Overexpression of the newly isolated two genes (ER10 or ER25) led to an average 14.7% higher erythritol yield and 31.2% higher productivity compared to the wild-type strain. Finally, engineering NADPH cofactor metabolism by overexpression of genes ZWF1 and GND1 encoding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, allowed a 23.5% higher erythritol yield and 50% higher productivity compared to the wild-type strain. The best of our constructed strains produced an erythritol titer of 190 g/L in baffled flasks using glucose as main carbon source.

Conclusions: Our results highlight that in the Y. lipolytica genome several genes encode enzymes able to reduce erythrose into erythritol. The catalytic properties of these enzymes and their cofactor dependency are different from that of already known erythrose reductase of Y. lipolytica. Constitutive expression of the newly isolated genes and engineering of NADPH cofactor metabolism led to an increase in erythritol titer. Development of fermentation strategies will allow further improvement of this productivity in the future.

Keywords: Erythritol; Erythrose reductase; Metabolic engineering; NADPH; Yarrowia lipolytica.

Fig. 1

HPLC analysis of reaction product catalyzed by the crude extracts of E. coli overexpressing putative ER enzymes. a standard of d-erythrose; b–d reaction product obtained with a cell extract of E. coli strain HCE102, HCE110, and HCE111, respectively; e standard of erythritol

Fig. 2

Phylogenetic analysis of the three erythrose reductases (ER10, ER25, and ER27, underlined in blue) from Y. lipolytica and other selected reductases (ADR aldose reductase, LAR l-arabinose reductase, XR xylose reductase). The phylogenetic tree was constructed based on the alignment of full amino acid sequences. All the analyzed sequences of aldose reductase enzymes were retrieved from GenBank and SWISS-PROT databases

Fig. 3

HPLC analysis of the reaction product of ER10 reductase in the presence of erythritol and NADP⁺. a erythritol standard; b reaction mixture containing erythritol, NADP⁺, and ER10; c erythrulose standard; d erythrose standard

Fig. 4

Effect of pH on the specific activity of ER10 and ER25 reductases. Purified enzymes were mixed with Mcilvaine’s buffer and assayed at various pH values ranging from 3.0 to 8.0. The values provided are the means of three independent replicates; the standard deviations represented less than 10% of the means