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

ABSTRACT: Erythritol 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.The 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.In 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.

SUBMITTER: Janek T

PROVIDER: S-EPMC5504726 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Janek Tomasz T Dobrowolski Adam A Biegalska Anna A Mirończuk Aleksandra M AM

Microbial cell factories 20170711 1

<h4>Background</h4>Erythritol 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 studi ...[more]

PMID: 28693571

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Project description:BackgroundErythritol 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.ResultsIn 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.ConclusionsOur 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.

Project description:BackgroundThe oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica.ResultsThis study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter's strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use.ConclusionsIn this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

Project description:BackgroundSugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica.ResultsY. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production.ConclusionsWe demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

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

Publications

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

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