Engineering oleaginous yeast Yarrowia lipolytica for enhanced limonene production from xylose and lignocellulosic hydrolysate

ABSTRACT Limonene, a valuable cyclic monoterpene, has been broadly studied in recent decades due to its wide application in the food, cosmetics and pharmaceutical industries. Engineering of the yeast Yarrowia lipolytica for fermentation of renewable biomass lignocellulosic hydrolysate may reduce the cost and improve the economics of bioconversion for the production of limonene. The aim of this study was to engineer Y. lipolytica to produce limonene from xylose and low-cost lignocellulosic feedstock. The heterologous genes XR and XDH and native gene XK encoding xylose assimilation enzymes, along with the heterologous genes tNDPS1 and tLS encoding orthogonal limonene biosynthetic enzymes, were introduced into the Po1f strain to facilitate xylose fermentation to limonene. The initially developed strain produced 0.44 mg/L of limonene in 72 h with 20 g/L of xylose. Overexpression of genes from the mevalonate pathway, including HMG1 and ERG12, significantly increased limonene production from xylose to ∼9.00 mg/L in 72 h. Furthermore, limonene production peaked at 20.57 mg/L with 50% hydrolysate after 72 h when detoxified lignocellulosic hydrolysate was used. This study is the first to report limonene production by yeast from lignocellulosic feedstock, and these results indicate the initial steps toward economical and sustainable production of isoprenoids from renewable biomass by engineered Y. lipolytica.

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Increased accumulation of squalene in engineered Yarrowia lipolytica through deletion of PEX10 and URE2

Applied and Environmental Microbiology ◽

10.1128/aem.00481-21 ◽

2021 ◽

Author(s):

Liu-Jing Wei ◽

Xuan Cao ◽

Jing-Jing Liu ◽

Suryang Kwak ◽

Yong-Su Jin ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Catabolite Repression ◽

Oleaginous Yeast ◽

Mevalonate Pathway ◽

Culture Media ◽

Yeast Cells ◽

Nitrogen Catabolite Repression ◽

Peroxisomal Membrane ◽

Cell Factories ◽

Pharmaceutical Industries

Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene as the yeast provides ample storage space for hydrophobic products. Here we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica . First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG, HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 encoding for diacylglycerol acyltransferase and deleting PEX10 for peroxisomal membrane E3 ubiquitin ligase. Third, we deleted URE2 coding for a transcriptional regulator in charge of nitrogen catabolite repression (NCR) to induce lipid accumulation regardless of carbon to nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g DCW) than a parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in S. cerevisiae , and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica . URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells on boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

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Stable isotope and chemical inhibition analyses suggested the existence of a non-mevalonate-like pathway in the yeast Yarrowia lipolytica

Scientific Reports ◽

10.1038/s41598-021-85170-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sivamoke Dissook ◽

Tomohisa Kuzuyama ◽

Yuri Nishimoto ◽

Shigeru Kitani ◽

Sastia Putri ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Oleaginous Yeast ◽

Isoprenoid Biosynthesis ◽

Mep Pathway ◽

Authentic Standard ◽

Chemical Inhibition ◽

Chromatographic Peaks ◽

Methyl Erythritol Phosphate ◽

Limiting Condition ◽

Yeast Yarrowia Lipolytica

AbstractMethyl erythritol phosphate (MEP) is the metabolite found in the MEP pathway for isoprenoid biosynthesis, which is known to be utilized by plants, algae, and bacteria. In this study, an unprecedented observation was found in the oleaginous yeast Yarrowia lipolytica, in which one of the chromatographic peaks was annotated as MEP when cultivated in the nitrogen limiting condition. This finding raised an interesting hypothesis of whether Y. lipolytica utilizes the MEP pathway for isoprenoid biosynthesis or not, because there is no report of yeast harboring the MEP pathway. Three independent approaches were used to investigate the existence of the MEP pathway in Y. lipolytica; the spiking of the authentic standard, the MEP pathway inhibitor, and the 13C labeling incorporation analysis. The study suggested that the mevalonate and MEP pathways co-exist in Y. lipolytica and the nitrogen limiting condition triggers the utilization of the MEP pathway in Y. lipolytica.

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