Magdalena Rakicka

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.

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.

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.

Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using Yarrowia lipolytica expressing inulinase

Inulin and glycerol were used as substrates for efficient erythritol and citric acid production by newly engineered Yarrowia lipolytica strains. Hydrolysis of inulin by the Y. lipolytica Wratislavia K1 strain was established by expressing the Kluyveromyces marxianus INU1 gene. Erythritol was produced in two stages: inulin was used for biomass formation, followed by erythritol biosynthesis initiated by glycerol addition. The highest titer of erythritol obtained, 120.9 g L−1 with the yield of 0.6 g g−1, was produced by the K1 INU 6 strain. Moreover, the K1 INU 6 strain in fed-batch culture produced a high amount of citric acid: 105.2 g L−1 after 235 h from 200 g L−1 of inulin. Maximum activity of inulinase during this culture was 14000 U g−1 of cell dry mass. The presented study proves the potential of new Y. lipolytica transformants for efficient erythritol and citric acid production from inexpensive raw materials such as inulin and glycerol.

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.

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 β‐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 β‐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

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.