Production of pyruvic acid from glycerol by Yarrowia lipolytica.

The aim of the study was to screen Yarrowia lipolytica strains for keto acid production and determine optimal conditions for pyruvic acid biosynthesis from glycerol by the best producer. The analyzed parameters were thiamine concentration, medium pH, stirring speed, and substrate concentration. The screening was performed in flask cultures, whereas pyruvic acid production was carried out in 5-L stirred-tank reactor with 2 L of working volume. In total, 24 Y. lipolytica strains were compared for their abilities to produce pyruvic and α-ketoglutaric acids. The total concentration of both acids ranged from 0.1 to 15.03 g/L. Ten strains were selected for keto acid biosynthesis in bioreactor. The Y. lipolytica SKO 6 strain was identified as the best producer of pyruvic acid. In the selected conditions (thiamine concentration 1.5 µg/L, pH 4.0, stirring speed 800 rpm, 150 g/L of glycerol), the strain Y. lipolytica SKO 6 produced 99.3 g/L of pyruvic acid, with process yield of 0.63 g/g and volumetric production rate of 1.18 g/L/h. Higher titer of pyruvic acid was obtained during fed-batch culture with 200 g/L of glycerol, reaching 125.8 g/L from pure glycerol (yield 0.68 g/g) and 124.4 g/L from crude glycerol (yield 0.62 g/g). Results obtained for the strain Y. lipolytica SKO 6 proved the suitability of microbial production of pyruvic acid at industrial scale.

Production of high titer of citric acid from inulin.

BACKGROUND: Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide - inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy-repeated-batch culture was applied. RESULTS: The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm- 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm- 3 h- 1 and a yield of 0.85 g g- 1. CONCLUSIONS: The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.

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.

An Effective Method of Continuous Production of Erythritol from Glycerol by Yarrowia lipolytica MK1.

This study demonstrates the potential applicability of the UV mutant Yarrowia lipolytica MK1 for the valorisation of glycerol and erythritol production in a chemostat culture. The aim of this research is to investigate the optimal C:N ratio in the feeding medium in order to enhance erythritol production. The highest erythritol concentration, at 113.1 g/L with a volumetric erythritol production rate of 1.1 g/(L·h) and a yield of 0.57 g/g, was obtained in the feeding medium with a C:N ratio of 80:1. Moreover, no residual glycerol was observed in the culture broth during cultivation. The chemical composition of the biomass was analysed. The contents of lysine and threonine in the biomass protein amino acid profile were higher than those required by the FAO/WHO for fodder yeast.

Transforming sugars into fat - lipid biosynthesis using different sugars in Yarrowia lipolytica.

In an era of ever-increasing energy demands, a promising technology is being developed: the use of oleaginous microorganisms such as Yarrowia lipolytica to convert waste materials into biofuels. Here, we constructed two Y. lipolytica strains that displayed both increased lipid accumulation and more efficient use of biomass-derived sugars, including glucose, fructose, galactose and inulin. The first strain, Y. lipolytica YLZ150, was derived from the French wild-type strain W29. It had inhibited triacylglycerol mobilization (∆tgl4) and ß-oxidation (∆pox1-6), and it overexpressed GPD1, DGA2, HXK1, the native Leloir pathway, SUC2 from Saccharomyces cerevisiae and INU1 from Kluyveromyces marxianus. The second strain, Y. lipolytica Y4779, was derived from the Polish A-101 strain. It had inhibited ß-oxidation (∆mfe2) and overexpressed GPD1, DGA1, HXK1, YHT3, SUC2 and INU1. In the first experiment, strain YLZ150 was batch-cultured in media containing different hexoses; the highest values for lipid concentration and yield of lipids from the substrate were obtained using fructose (20.3 g dm-3 and 0.14 g g-1 , respectively). In the second experiment, we grew the two strains in fed-batch cultures to examine lipid biosynthesis from inulin (a fructose polymer). For Y4779, the lipid concentration was 10.3 g dm-3 and the yield of lipids from substrate was 0.07 g g-1 ; in contrast, for YLZ150, these values were 24 g dm-3 and 0.16 g g-1 , respectively. The YLZ150 strain is thus able to efficiently exploit glucose, fructose, galactose, sucrose and inulin for lipid biosynthesis. Copyright © 2017 John Wiley & Sons, Ltd.

A novel strain of Yarrowia lipolytica as a platform for value-added product synthesis from glycerol.

BACKGROUND: Increasing interest of non-conventional yeasts has been observed for many years due to their biochemical characteristics and potential applications. Well-studied, oleaginous yeast Y. lipolytica is an attractive host for converting a low-cost glycerol, into value-added products such as erythritol (sweetener) or citric acid. Glycerol is an important renewable feedstock and is the main co-product of biodiesel production, which is nowadays applied on a large commercial scale. To this end, we engineered the yeast Y. lipolytica to increase the productivity of this strain. RESULTS: In this light, we enhanced glycerol assimilation by over-expression of the YALI0F00484g gene encoding glycerol kinase (GK) and gene YALI0B02948g encoding glycerol-3-P dehydrogenase (GDH). The modified strains have been tested for glycerol consumption rate and erythritol and citric acid synthesis under various conditions. Here, we show that the overexpression of GK and GDH, increased glycerol consumption resulting in rapid erythritol and citric acid synthesis. Next, we combined the two genes in the tandem gene construct for the simultaneous co-expression of GK and GDH, which further increased the desired product synthesis. The glycerol consumption was explored in a 5-L bioreactor and the engineered strains were able to utilize 150 g/L glycerol within 44-48 hours. The erythritol productivity for GK overexpression and co-expression of GK and DGH was 24 and 35 %, respectively, over the control strain. Moreover, we established conditions for the production of citric acid at pH 3.0, the engineered strains increased citric acid production 14-fold over the control. CONCLUSION: This work demonstrates the excellent capacity of the engineered strains as a starting platform for further modification for broad-range value-added product biosynthesis from glycerol. This study presents the highest reported titer citric acid at low pH to date. The process parameters such as productivity and yield of erythritol and citric acid were significantly elevated, what is valuable for industrial applications.

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose.

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content. Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.

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

Abstract 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.

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 (300gL(-1)), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142gL(-1) after 5 days, which corresponded to 0.47gg(-1) yield and productivity of 1.1gL(-1)h(-1). Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

A two-stage fermentation process of erythritol production by yeast Y. lipolytica from molasses and glycerol.

In this study, a two-stage fermentation process of erythritol production based on molasses and glycerol was investigated. During the first stage, the biomass of Yarrowia lipolytica was grown on medium containing sucrose as the sole carbon source. In the second stage, production of erythritol was initiated by glycerol addition. To use molasses as a substrate for erythritol synthesis, sucrose utilization was established by expressing the Saccharomyces cerevisiae SUC2 gene. In this study, cultivation of yeast Y. lipolytica could produce 52-114 g/L of erythritol. The productivity was 0.58-1.04 g/L/h, and yield was 0.26-0.57 g/g; the final biomasses yield ranged 17-41 g/L. This is the first report describing erythritol production via industrial raw molasses and glycerol by Y. lipolytica. This work uses genetically modified strains of Y. lipolytica as tool for the direct conversion of affordable raw industrial molasses and glycerol into the value-added erythritol product.

Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions.

BACKGROUND: Microbial lipid production using renewable feedstock shows great promise for the biodiesel industry. RESULTS: In this study, the ability of a lipid-engineered Yarrowia lipolytica strain JMY4086 to produce lipids using molasses and crude glycerol under different oxygenation conditions and at different inoculum densities was evaluated in fed-batch cultures. The greatest lipid content, 31% of CDW, was obtained using a low-density inoculum, a constant agitation rate of 800 rpm, and an oxygenation rate of 1.5 L/min. When the strain was cultured for 450 h in a chemostat containing a nitrogen-limited medium (dilution rate of 0.01 h(-1); 250 g/L crude glycerol), volumetric lipid productivity was 0.43 g/L/h and biomass yield was 60 g CDW/L. The coefficient of lipid yield to glycerol consumption (Y L/gly) and the coefficient of lipid yield to biomass yield (Y L/X ) were equal to 0.1 and 0.4, respectively. CONCLUSIONS: These results indicate that lipids may be produced using renewable feedstock, thus providing a means of decreasing the cost of biodiesel production. Furthermore, using molasses for biomass production and recycling glycerol from the biodiesel industry should allow biolipids to be sustainably produced.

Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis.

The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.

A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells.

Citric acid and erythritol biosynthesis from pure and crude glycerol by three acetate-negative mutants of Yarrowia lipolytica yeast was investigated in batch cultures in a wide pH range (3.0-6.5). Citric acid biosynthesis was the most effective at pH 5.0-5.5 in the case of Wratislavia 1.31 and Wratislavia AWG7. With a decreasing pH value, the direction of biosynthesis changed into erythritol synthesis accompanied by low production of citric acid. Pathways of glycerol conversion into erythritol and citric acid were investigated in Wratislavia K1 cells. Enzymatic activity was compared in cultures run at pH 3.0 and 4.5, that is, under conditions promoting the production of erythritol and citric acid, respectively. The effect of pH value (3.0 and 4.5) and NaCl presence on the extracellular production and intracellular accumulation of citric acid and erythritol was compared as well. Low pH and NaCl resulted in diminished activity of glycerol kinase, whereas such conditions stimulated the activity of glycerol-3-phosphate dehydrogenase. The presence of NaCl strongly influenced enzymes activity - the effective erythritol production was correlated with a high activity of transketolase and erythrose reductase. Therefore, presented results confirmed that transketolase and erythrose reductase are involved in the overproduction of erythritol in the cells of Y. lipolytica yeast.

Enhanced production of erythritol by Yarrowia lipolytica on glycerol in repeated batch cultures.

Erythritol is an important natural sweetener, industrially produced only by fermentation on glucose media. Glycerol is an important renewable feedstock as it is the major by-product of the biodiesel production process; here we present an alternative way to convert this low-cost substrate into value-added products, such as erythritol. Repeated batch cultures (RBC) were performed to improve the productivity of erythritol from pure and crude glycerol. An acetate negative mutant of Yarrowia lipolytica Wratislavia K1 was found to be applicable for the production of high amounts of erythritol in RBC. When 20 % of fresh replaced medium was added, the strain Wratislavia K1 was able to produce 220 g l (-1) erythritol, which corresponded to a 0.43 g g(-1) yield and a productivity of 0.54 g l(-1) h(-1). Additionally, the activity of the culture remained stable for more than 1,000 h, i.e., 11 cycles of the repeated batch bioreactors.