The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.
Background Yarrowia lipolytica is an unconventional yeast with a huge industrial potential. Despite many advantages for biotechnological applications, it possesses enormous demand for oxygen, which is a bottleneck in large scale production. In this study a codon optimized bacterial hemoglobin from Vitreoscilla stercoraria (VHb) was overexpressed in Y. lipolytica for efficient growth and erythritol synthesis from glycerol in low-oxygen conditions. Erythritol is a natural sweetener produced by Y. lipolytica under high osmotic pressure and at low pH, and this process requires high oxygen demand. Results Under these conditions the VHb overexpressing strain showed mostly yeast-type cells resulting in 83% higher erythritol titer in shake-flask experiments. During a bioreactor study the engineered strain showed higher erythritol productivity (QERY = 0.38 g/l h) and yield (YERY = 0.37 g/g) in comparison to the control strain (QERY = 0.30 g/l h, YERY = 0.29 g/g). Moreover, low stirring during the fermentation process resulted in modest foam formation. Conclusions This study showed that overexpression of VHb in Y. lipolytica allows for dynamic growth and efficient production of a value-added product from a low-value substrate.
Erythritol is a polyol produced by Yarrowia lipolytica under hyperosmotic stress. In this study, the osmo-sensitive strain Y. lipolytica yl-hog1Δ was subjected to stress, triggered by a high concentration of carbon sources. The strain thrived on 0.75 M erythritol medium, while the same concentrations of glucose and glycerol proved to be lethal. The addition of 0.1 M erythritol to the medium containing 0.75 M glucose or glycerol allowed the growth of yl-hog1Δ. Supplementation with other potential osmolytes such as mannitol or L-proline did not have a similar effect. To examine whether the osmoprotective effect might be related to erythritol accumulation, we deleted two genes involved in erythritol utilization, the transcription factor Euf1 and the enzyme erythritol dehydrogenase Eyd1. The strain eyd1Δ yl hog1Δ, which lacked the erythritol utilization enzyme, reacted to the erythritol supplementation significantly better than yl-hog1Δ. On the other hand, the strain euf1Δ yl-hog1Δ became insensitive to supplementation, and the addition of erythritol could no longer improve the growth of this strain in hyperosmotic conditions. This indicates that Euf1 regulates additional, still unknown genes involved in erythritol metabolism.
The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.
Background Yarrowia lipolytica is a dimorphic fungus, which switches from yeast to filament form in response to environmental conditions. For industrial purposes it is important to lock cells in the yeast or filamentous form depending on the fermentation process. yl-Hog1 kinase is a key component of the HOG signaling pathway, responsible for activating the osmotic stress response. Additionally, deletion of yl-Hog1 leads to increased filamentation in Yarrowia lipolytica, but causes significant sensitivity to osmotic stress induced by a high concentration of a carbon source. Results In this study, we tested the effect of point mutations on the function of yl-Hog1 protein kinase. The targets of modification were the phosphorylation sites (T171A-Y173A) and the active center (K49R). Introduction of the variant HOG1-49 into the hog1∆ strain partially improved growth under osmotic stress, but did not recover the yeast-like shape of the cells. The HOG1-171/173 variant was not functional, and its introduction further weakened the growth of hog1∆ strains in hyperosmotic conditions. To verify a genetic modification in filament form, we developed a new system based on green fluorescent protein (GFP) for easier screening of proper mutants. Conclusions These results provide new insights into the functions of yl-Hog1 protein in dimorphic transition and constitute a good starting point for further genetic modification of Y. lipolytica in filament form.
Background Yarrowia lipolytica is a dimorphic fungus, which switches from yeast to yeast-to-filament form in response to environmental conditions. For industrial purposes it is important to lock cells in the yeast or filamentous form depending on the fermentation process. yl-Hog1 kinase is a key component of the HOG signaling pathway, responsible for activating the osmotic stress response. Additionally, deletion of yl-Hog1 leads to increased filamentation in Yarrowia lipolytica, but causes significant sensitivity to osmotic stress induced by a high concentration of a carbon source. Results In this study, we tested the effect of point mutations on the function of yl-Hog1 protein kinase. The targets of modification were the phosphorylation sites (T171A-Y173A) and the active center (K49R). Introduction of the variant HOG1-49 into the hog1∆ strain partially improved growth under osmotic stress, but did not recover the yeast-like shape of the cells. The HOG1-171/173 variant was completely inactive, and its introduction further weakened the hog1∆ strains. To verify a genetic modification in filament form, we developed a new system based on green fluorescent protein (GFP) for easier screening of proper mutants. Conclusions These results provide new insights into the functions of yl-Hog1 protein in dimorphic transition and constitute a good starting point for further genetic modification of Y. lipolytica in filament form.
The unconventional yeast Yarrowia lipolytica produces erythritol as a response to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.
BackgroundPolyols are a group of sweet alcohols, frequently used as food additives. The constantly rising demand for polyols requires the application of new strategies to increase the production. Erythritol is synthesized by the yeast Yarrowia lipolytica under high osmotic pressure as an osmoprotectant. The metabolic pathway resulting in erythritol production remains partially unknown. However, the last reaction resulting in erythritol synthesis is conducted by an erythrose reductase (ER).ResultsThe Y. lipolytica strain was genetically modified to increase the erythritol yield and productivity, using glycerol as a sole carbon source. The modification focused on the ER homologue YALI0B07117g after the in silico analysis of the protein sequences of all reported ER homologues. Initial results in shake-flask experiments proved the influence of the gene YALI0B07117g in erythritol synthesis. Deletion of the gene resulted in 3-fold and 2-fold increased production of mannitol and arabitol, respectively. Overexpression of the native ER homologue gene showed a positive influence on erythritol production. Bath cultures were conducted and the obtained strain reached the yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the engineered strain, while the productivity of erythritol increased from 0.28 g/L/h for the A101 strain to 0.41 g/L/h for the modified strain.ConclusionsOverexpression of the gene YALI0B07117g resulted in increased production of erythritol in the yeast Y. lipolytica. Disruption of the metabolic pathway by deletion of the gene results in higher production titers of mannitol and arabitol. Application of the research may prove positive for shortening the cultivation time due to the increased consumption rate of the substrate combined with increased parameters of erythritol synthesis.
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