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EUF1 - a newly identified gene involved in erythritol utilization in Yarrowia lipolytica.

ABSTRACT: The gene YALI0F01562g was identified as an important factor involved in erythritol catabolism of the unconventional yeast Yarrowia lipolytica. Its putative role was identified for the first time by comparative analysis of four Y. lipolytica strains: A-101.1.31, Wratislavia K1, MK1 and AMM. The presence of a mutation that seriously damaged the gene corresponded to inability of the strain Wratislavia K1 to utilize erythritol. RT-PCR analysis of the strain MK1 demonstrated a significant increase in YALI0F01562g expression during growth on erythritol. Further studies involving deletion and overexpression of the selected gene showed that it is indeed essential for efficient erythritol assimilation. The deletion strain Y. lipolytica AMM∆euf1 was almost unable to grow on erythritol as the sole carbon source. When the strain was applied in the process of erythritol production from glycerol, the amount of erythritol remained constant after reaching the maximal concentration. Analysis of the YALI0F01562g gene sequence revealed the presence of domains characteristic for transcription factors. Therefore we suggest naming the studied gene Erythritol Utilization Factor - EUF1.

SUBMITTER: Rzechonek DA

PROVIDER: S-EPMC5624910 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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EUF1 - a newly identified gene involved in erythritol utilization in Yarrowia lipolytica.

Rzechonek Dorota A DA Neuvéglise Cécile C Devillers Hugo H Rymowicz Waldemar W Mirończuk Aleksandra M AM

Scientific reports 20171002 1

The gene YALI0F01562g was identified as an important factor involved in erythritol catabolism of the unconventional yeast Yarrowia lipolytica. Its putative role was identified for the first time by comparative analysis of four Y. lipolytica strains: A-101.1.31, Wratislavia K1, MK1 and AMM. The presence of a mutation that seriously damaged the gene corresponded to inability of the strain Wratislavia K1 to utilize erythritol. RT-PCR analysis of the strain MK1 demonstrated a significant increase in ...[more]

PMID: 28970528

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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:Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains. KEY POINTS: • ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica • Genes encoding global regulators are mutated during thermal adaptive evolution • Ceramide and BCAA are critical molecules for cells to tolerate thermal stress.

Project description: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.

Project description:Background: Erythritol is a zero-calorie sweetener that is widely used in the food, pharmaceutical, and medical industries. Crude glycerol is the main by-product of biodiesel, and the effective utilization of crude glycerol will help to improve biodiesel viability. Previous studies on the production of erythritol from Y. lipolytica using crude glycerol as a carbon source have focused on optimizing the fermentation process of the mutant Y. lipolytica Wratislavia K1, while metabolic engineering has not been successfully applied. Results: To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain. Wild strains tolerant to high concentrations of crude glycerol were screened and identified. A series of rational metabolic approaches were employed to improve erythritol production. Among them, the engineered strain Y-04, obtained by tandem overexpression of GUT1 and GUT2, significantly increased glycerol assimilation by 33.3%, which was consistent with the results of RT-qPCR analysis. The effects of tandem overexpression of GUT1, GUT2, TKL1, and TAL1 on erythritol synthesis were also evaluated. The best results were obtained using a mutant that overexpressed GUT1, GUT2, and TKL1 and knocked out EYD1. The final Y-11 strain produced 150 g/l erythritol in a 5-L bioreactor with a yield and productivity of 0.62 g/g and 1.25 g/l/h, respectively. To the best of our knowledge, this is the highest erythritol yield and productivity from crude glycerol ever reported in Y. lipolytica. Conclusion: This work demonstrated that overexpression of GUT1, GUT2, and TKL1 and knockdown of EYD1 could be used to improve crude glycerol utilization and erythritol synthesis in Y. lipolytica. The process parameters such as erythritol yield and productivity were significantly elevated, which is valuable for industrial applications. Crude glycerol, as a carbon source, could efficiently restrict the synthesis of by-products while enhancing the generation of erythritol, compared to glucose. This indicates considerable potential for synthesizing value-added products from crude glycerol by Y. lipolytica.

Dataset Information

EUF1 - a newly identified gene involved in erythritol utilization in Yarrowia lipolytica.

Publications

EUF1 - a newly identified gene involved in erythritol utilization in Yarrowia lipolytica.

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