Lipolytica Wratislavia K1 Research Articles

Enhancing erythritol production from crude glycerol in a wild-type Yarrowia lipolytica by metabolic engineering.

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.

Waste Conversion into a Sweetener—Development of an Innovative Strategy for Erythritol Production by Yarrowia lipolytica

The study proposed the innovative low-cost strategy for erythritol production by Yarrowia lipolytica through developing a simple medium based on industrial waste by-products and a natural method for culture broth purification. Results obtained proved that corn steep liquor might successfully replace traditional sources of nitrogen and other nutrients without compromising activities of the enzymes responsible for erythritol production and its production level. As a consequence, a production process was performed where Y. lipolytica A-6 was able to produce 108.0 g/L of erythritol, with a production rate of 1.04 g/Lh and a yield of 0.45 g/g of the medium containing exclusively 220 g/L of crude glycerol derived from biodiesel production and 40 g/L of corn steep liquor. Moreover, a comparable concentration of erythritol (108.1 g/L) was obtained when a part of crude glycerol was exchanged for the crude fraction of fatty acids in the two-steps process. Next, the collected post-fermentation broths were used in the culture with Y. lipolytica Wratislavia K1 for natural purification. The process resulted in a high increase of erythritol selectivity from 72% to 97% and in the production of 22.0 g/L of biomass with 40.4% protein content, which enables its use as an attractive animal feedstuff.

Polyol production from waste materials by genetically modified Yarrowia lipolytica

Sugar alcohols (polyols) are sweeteners with many industrial applications. In this study, a fermentation process of polyol production based on waste substrates – raw industrial molasses and crude glycerol – was tested. The yeast strain Yarrowia lipolytica Wratislavia K1 was genetically modified by overexpression of the Saccharomyces cerevisiae SUC2 gene and overexpression of the native GUT1 gene. This process allowed for sucrose utilization and rapid glycerol assimilation by the engineered strain. In this study, the obtained strain AIB pAD-UTGut1 produced 100.65±3.75g/l of polyols, with productivity of 1.09±0.9g/lh and yield of 0.67±0.2g/g. This is the first study describing efficient polyol production by the modified Y. lipolytica strain from industrial raw molasses and crude glycerol. By process optimization, we established conditions for abundant polyol synthesis from low-value substrates.

Technology of efficient continuous erythritol production from glycerol

Erythritol production in chemostat culture from pure and crude glycerol with different nitrogen sources by Yarrowia lipolytica Wratislavia K1 was investigated. Moreover, a process of erythritol purification based on ion exchange is proposed. The highest erythritol production (103.4 g L−1 with a volumetric erythritol production rate of 1.12 g L−1 h−1 and a yield of 0.52 g g−1) was obtained by applying an inorganic nitrogen source (4.6 g L−1 of ammonium sulfate). Very promising results were also obtained when pure glycerol was replaced with crude glycerol as a carbon source. During this culture yeast produced 81.9 g L−1 of erythritol, which corresponded to a 0.9 g L−1 h−1 volumetric erythritol production rate and a yield of erythritol production of 0.4 g g−1. The fermentation broth was desalinated and decolorized by ion exclusion, ion exchange and sorption on activated carbon. The final solution contained only erythritol. The presented technology follows the vision of a circular economy by turning waste into products and using renewable materials as a feedstock. This study supports the potential of the industrially relevant wild type Yarrowia lipolytica strain for intensified and efficient erythritol production. The proposed technology is ecofriendly, requires a small number of purification steps and generates low amount of wastes. The final solution is rich in erythritol and can be applied directly as a sweet low-calorie food additive.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants.

Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L−1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L−1 after 5 days, which corresponded to 0.47 g g−1 yield and productivity of 1.1 g L−1 h−1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Newly isolated mutant of Yarrowia lipolytica MK1 as a proper host for efficient erythritol biosynthesis from glycerol

Mutants of Yarrowia lipolytica Wratislavia K1 were generated by ultraviolet mutagenesis to enhance erythritol production and to minimize by-products formation. One of the mutants named as MK1 was selected for further investigation. In batch culture strain Y. lipolytica MK1 was able to produce up to 82.2gl−1 erythritol with 0.55gg−1 yield and productivity of 0.84gl−1h−1. The concentration of by-products was below 5% of all synthesized metabolites. In order to enhance the production of erythritol and to inhibit the production of by-products the repeated batch culture (RBC) was performed. In RBC strain Y. lipolytica MK1 gave maximum erythritol production 224gl−1 with 0.77gg−1 yield and productivity of 0.54gl−1h−1. The concentration of by-products was below 2.3% of all synthesized metabolites. Moreover, the lipid and fatty acid content in the biomass was determined. This is the first study on newly isolated Y. lipolytica mutant. It demonstrates that Y. lipolytica MK1 is able to produce significantly high erythritol with strong inhibition of by-products formation.

Optimization of Medium Composition for Erythritol Production from Glycerol by Yarrowia lipolytica Using Response Surface Methodology

Several factors affecting erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 strain were examined in batch fermentations. Ammonium sulfate, monopotassium phosphate, and sodium chloride were identified as critical medium components that determine the ratio of polyols produced. The central composite rotatable experimental design was used to optimize medium composition for erythritol production. The concentrations of ammonium sulfate, monopotassium phosphate, and sodium chloride in the optimized medium were 2.25, 0.22, and 26.4 g L−1, respectively. The C:N ratio was found as 81:1. In the optimized medium with 100 g L−1 of glycerol the Wratislavia K1 strain produced 46.9 g L−1 of erythritol, which corresponded to a 0.47 g g−1 yield and a productivity of 0.85 g L−1 hr−1. In the fed-batch mode and medium with the total concentration of glycerol at 300 g L−1 and C:N ratio at 81:1, 132 g L−1 of erythritol was produced with 0.44 g g−1 yield and a productivity of 1.01 g L−1 hr−1.

High selectivity of erythritol production from glycerol by Yarrowia lipolytica

The effect of different vitamins and nitrogen sources on the yield, productivity and selectivity of erythritol production from glycerol in batch and fed-batch cultures by Yarrowia lipolytica Wratislavia K1 strain was examined in the study.Thiamine was a vitamin necessary for efficient erythritol production and the yeast extract (YE) turned out to be its best source. The YE was additionally a very good source of nitrogen in this process. Erythritol production may be efficient in the media containing only yeast extract and crude glycerol. The application of a high substrate concentration (325 kg m−3) in the fed-batch system with pulsed addition of glycerol resulted in erythritol production of 201.2 kg m−3 after 168 h of cultivation. The mass fraction of by-products, such as other polyols and organic acids, did not exceed 10%. The study showed that by-products formation could be significantly reduced by the addition of a nitrogen source after glycerol depletion from the media. Wratislavia K1 strain does not utilize erythritol in the presence of other carbon sources and this feature was used in order to increase product selectivity up to 99.7% and protein content in yeast biomass up to 28.7%. The factors determining the use of biomass as animal feed, i.e. protein concentration and presence of metal ions, were discussed as well.

Optimization of erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1

Optimization of erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1

Effect of vitamins source on erythritol biosynthesis by Yarrowia lipolytica Wratislavia K1

Effect of vitamins source on erythritol biosynthesis by Yarrowia lipolytica Wratislavia K1

Comparison of citric acid production from glycerol and glucose by different strains of Yarrowia lipolytica

A wild type strain A-101 of Y. lipolytica and its three acetate-negative mutants (Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1) were compared for the production of citric acid from glucose and glycerol (pure and crude) in batch cultures. The substrates were used either as single carbon sources or as mixtures of glucose and pure or crude glycerol. The kinetic parameters, i.e., the volumetric citric acid production rate and yield obtained in the study show that the Wratislavia 1.31 and Wratislavia AWG7 strains produced the highest amount of citric acid from glycerol, with a yield from 0.40 to 0.53gg(-1). This substrate was found to be a better carbon source for the biosynthesis of citric acid than glucose. The results obtained with the same strains have shown low content of isocitric acid and polyols, such as erythritol and mannitol. Y. lipolytica A-101 strain produced the highest amount of isocitric acid, from 13.8 to 21% isocitric acid in the sum of citric acids. However, the highest concentrations of erythritol were found in cultures with Y. lipolytica Wratislavia K1, from 18.1 to 30gl(-1), for glucose and pure glycerol, respectively.

Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1

AbstractThis study shows a possible microbial process for utilization of crude glycerol generated by the biodiesel industry for citric acid and erythritol production. Simultaneous production of citric acid and erythritol under nitrogen-limited conditions with glycerol as the carbon source was achieved with an acetate negative mutant of Y. lipolytica Wratislavia K1 in fed-batch cultivations. The effect of the initial glycerol concentration (from 30–180 g dm−3) on the citrate and erythritol production was investigated. As a result of the experiments, maximum citric acid production (110 g dm−3) and a very high amount of erythritol (81 g dm−3) were determined after 168 h of fed-batch cultivation with the initial glycerol concentration of 150 g dm−3 and the total glycerol concentration of 250 g dm−3. In addition, the citric acid to isocitric acid ratio of the products from this strain was 35.5:1.