Metabolic engineering of the oleaginous yeast Yarrowia lipolytica PO1f for production of erythritol from glycerol

Abstract

Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica. Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

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

“… Mirończuk et al (2017) functionally overexpressed TKL1 , TAL1 , ZWF1, and GND1 , among which TKL1 overexpression increased erythritol production by 2 times. However, Jagtap et al (2021) did not find that overexpression of the TKL1 gene in the Y. lipolytica PO1f strain increased erythritol, possibly because the Y. lipolytica PO1f strain is an uracil and leucine auxotrophic strain, which somehow limits erythritol production.…”

“… Mirończuk et al (2014) found that the mutant Y. lipolytica Wratislavia K1 obtained by acetate-negative mutation was suitable for producing large amounts of erythritol. In addition, a few records of Y. lipolytica producing erythritol from glycerol have been reported ( Rakicka et al, 2016b ; Carly et al, 2017 ; Mirończuk et al, 2019 ; Bilal et al, 2020 ; Mawire et al, 2020 ; Jagtap et al, 2021 ). Crude glycerol is the principal by-product of the manufacturing of biodiesel, an emerging alternative and sustainable fuel.…”

“…Recent studies have indicated that glycerol-induced high osmotic pressure can enhance erythritol production ( Yang et al, 2014 ). Jagtap et al (2021) revealed that using glycerol as a carbon source significantly increased erythritol yield. They also demonstrated that the use of glycerol was effective in reducing the by-product mannitol production.…”

Metabolomics and modelling approaches for systems metabolic engineering

“…These intracellular metabolites become diluted in the more voluminous extracellular medium, adding to significant variability in intracellular metabolite quantitation due to the presence of interfering extracellular metabolites and concentrated media components ( Faijes et al, 2007 ). Fast filtration has also been used to separate microbial cells from cell culture medium prior to quenching through liquid nitrogen immersion or cold/hot solvent ( Castaño-Cerezo et al, 2019 ; Wordofa et al, 2017 ; Kim et al, 2013 ; Jagtap et al, 2021 ). Even though this method overcomes the issue of intracellular metabolite leakage, the quenching of cell metabolism is not immediate due to the time required for the filtration process.…”

Near-Complete Genome Sequence of Zygosaccharomyces rouxii NRRL Y-64007, a Yeast Capable of Growing on Lignocellulosic Hydrolysates

“…The genomic DNA and total RNA of Z. rouxii NRRL Y-64007 were extracted using the Dr. GenTLE (from yeast) high recovery kit (TaKaRa Bio Inc., Shiga, Japan) and the RNeasy minikit (Qiagen, Hilden, Germany), respectively ( 11 , 12 ).…”