Yarrowia Research Articles

Improving Precursor Supply and Optimizing the Fermentation Process for High-Level Production of Docosapentaenoic Acid in Yarrowia Lipolytica.

Docosapentaenoic acid (DPA) is widely applied in medicine and health products because of its important physiological functions. Using microbial cell factories for DPA production is considered a viable alternative to extracting DPA from seal oil. In this study, an engineering strategy for the efficient production of DPA was developed. First, the DPA biosynthesis pathway was successfully established in Yarrowia lipolytica. Then, the increase of acetyl-CoA by engineering citrate metabolism and malonyl-CoA by introducing a new orthogonal malonyl-CoA synthesis pathway was to further enhance DPA production. Furthermore, overexpression of glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME) enhanced NADPH availability. Finally, by optimizing the fermentation conditions, the DPA content of the engineered strain reached 40.0%, and the yield reached 13.0 g/L in the 5 L bioreactor, representing the highest levels reported so far in Y. lipolytica. This study provides a promising strategy to construct microbial cell factories for fatty acid biosynthesis.

Engineering novel Yarrowia lipolytica whole-cell biocatalysts by cell surface display of the native Lip2 lipase for biodiesel production

Yeast surface display (YSD) has proven to be a valuable tool in cellular engineering, particularly for conferring biocatalytic activities to cells, thereby enabling the creation of novel whole-cell biocatalysts. Compared to intracellular and secretion-based strategies, cell surface display of enzymes offers distinct advantages, including the elimination of enzyme recovery and purification steps, the resolution of substrate transport limitations, and enhanced activity, stability, and selectivity. In this work, YSD was employed to construct efficient display systems for Yarrowia lipolytica using the native lipase Lip2 fused with constitutive promoter-based anchoring vectors. The biocatalytic activity of the Lip2 biomass–bound recombinant strains was evaluated in transesterification reaction (butanolysis of p-nitrophenyl palmitate). The best-performing strain expressing the LIP2 gene under the UAS1B8-TMAL(250) hybrid promoter was cultivated in a lab-scale bioreactor to identify crucial parameters that have to be fine-tuned for a scalable bioprocess that yields biomass of both high density and Lip2 activity. Harvested biomass was used to catalyze the synthesis of biodiesel (olive oil methanolysis) yielding under optimal reaction conditions 71.4% conversion after 48 h.

Enhanced Immunity and Infection Resistance in Mice Through Co-Expression of Porcine IL-3, IL-7, and IL-15 Fusion Molecules in Yarrowia lipolytica

China’s livestock industry grapples with challenges posed by infectious diseases and the misuse of antibiotics, resulting in a heightened risk of drug-resistant pathogens. This study explored the immunomodulatory effects of co-expressing porcine interleukin 3, 7, and 15 in Yarrowia lipolytica, denoted as Po1h-IL-3/7/15. A 42-day experiment involving mouse immunization and pathogen challenge was conducted, during which in vivo assessments of antibodies, immune-related cells, and gene expression were detected following oral administration of Po1h-IL-3/7/15. Immunological alterations in mice were analyzed using flow cytometry, qRT-PCR, ELISA, and HE staining. Notably, the serum IgG and intestinal sIgA levels in the Po1h-IL-3/7/15 group were substantially elevated compared to the control groups (p < 0.01), so were the contents of IL-7, IL-15, IFN-γ, IL-22, IL-23, and TNF-α. Furthermore, there was a marked increase in naïve T cells and central memory T cells, accompanied by a significant decrease in regulatory T cells in peripheral blood. Post-challenge with Staphylococcus aureus or Salmonella typhimurium, the expression levels of BD2, IL-1β, IL-8, Jak1, RegⅢ, S100A8, STAT1, and TNF-α genes in the intestines of the Po1h-IL-3/7/15 group were markedly higher than those in the control groups (p < 0.01). Following the challenges, the survival rate of the Po1h-IL-3/7/15 group was 100%, a significant increase compared to the 20% and 40% survival rates observed in the control groups (p < 0.05). These results confirm that IL-3/7/15 significantly boosts innate immunity, humoral and cell-mediated immune responses, and intestinal mucosal immunity in mice, enhancing resistance to bacterial infections and exhibiting potent protective effects.

Transcriptomic Studies on the Product Stress Response Revealed that YCF1 is a Beneficial Factor for Progesterone Production in Yarrowia lipolytica

Transcriptomic Studies on the Product Stress Response Revealed that YCF1 is a Beneficial Factor for Progesterone Production in Yarrowia lipolytica

DM9CP-8 upon binding microbes activates MASPL-1-C3 axis to regulate the mRNA expressions of IL17s in oysters.

DM9CP-8 upon binding microbes activates MASPL-1-C3 axis to regulate the mRNA expressions of IL17s in oysters.

De novo biosynthesis of cannabinoid and its analogs in Yarrowia lipolytica

De novo biosynthesis of cannabinoid and its analogs in Yarrowia lipolytica

Waste cooking oils as a sustainable feedstock for bio-based application: A systematic review.

Waste cooking oils as a sustainable feedstock for bio-based application: A systematic review.

Enhanced Lipid Yield from Olive-Mill Wastewater by Yarrowia lipolytica NRRL YB-423

Lipid production from olive-mill wastewater (OMW) by Yarrowia lipolytica NRRL YB-423 was optimized (biomass concentration and lipid yield based on dry cell weight) using multi-response criteria based on the Taguchi orthogonal array. Sixteen experimental runs were performed using the L16 orthogonal array. Dilution rates of OMW (15, 30, 45, and 60%), Tween 80 (0, 0.2, 0.4, and 0.6%), sodium chloride (NaCl; 0, 1, 2, and 3%), and sterility were selected as factors. The significance of the parameters was determined using analysis of variance (ANOVA). The effects of all factors on the lipid yield were statistically significant (p<0.05). The results showed that sterility had a maximum contribution of 48.12% to lipid yield. The highest lipid yield (40.88 %) was achieved in sterile medium supplemented with 15% diluted OMW, 0.6% Tween 80, and 3% NaCl.

Engineered yeast Yarrowia lipolytica as a chassis for biosynthesis of fatty acids from mannitol and macroalgal biomass extracts

BackgroundYarrowia lipolytica possesses the capability to utilize many unconventional carbon sources, such as crude glycerol, alkanes and fatty acids. Despite producing polyols, such as erythritol, arabitol and mannitol, the re-utilization of mannitol is not as efficient as erythritol utilization. Genes involved in mannitol uptake and metabolism in Y. lipolytica remain undescribed. However, deletion of the EYD1 gene (YALI0F01650g), believed to encode erythritol dehydrogenase, has been found to result in a high rate of growth on media containing mannitol as the sole carbon source. Therefore this unique feature was used for further fermentation studies on media containing macroalgal mannitol extracts, obtained from the brown alga Fucus vesiculosus, to produce value-added products.ResultsThe obtained strain AJD Δeyd1Dga1 was able to uptake pure and algal mannitol efficiently and produce high amounts of lipids, thanks to overexpression of the DGA1 gene (YALI0E32769g), encoding diacylglycerol (DAG) acyltransferase. The lipid content reached almost 32% of the overall dry biomass as compared to the wild type strain, where this value was more than 4 times lower. Additionally, the biomass at the end of the experiment was the highest among all of the tested strains, reaching 12.67 g/L, more than 50% higher than the control strain.ConclusionsThe results of this study shed new light on the potential for the yeast Y. lipolytica to utilize macroalgae biomass as a carbon source for production of value-added products, including biomass and lipids. Moreover, the increased mannitol utilization capabilities can provide new insight into mannitol metabolism, including its uptake, which is especially crucial, as the metabolic pathways for all polyols produced by this organism seem to be closely intertwined.

Synergistic effect of transporter and pathway engineering on the key performance indicators of erythritol synthesis by the yeast Yarrowia lipolytica.

The expansion of the erythritol market attracted excessive capital injection, resulting in overcapacity, operational difficulties, and even bankruptcy of erythritol manufacturers. Technology upgrades in the industry are imminent. However, the production technology of existing enterprises is seriously homogenized, and there is a lack of competitive core-producing strains. In this study, the industrial erythritol-producing strain Y. lipolytica CGMCC7326 was genetically modified by integrating substrate transport and pathway modification, which improved the conversion of glucose and significantly improved KPIs, thereby reducing the erythritol production cost.

Engineering Yarrowia lipolytica for Enhanced Gastrodin Production via High-Throughput Screening and Transcriptomics-Guided Optimization.

Gastrodin, the principal bioactive component of the renowned herb Gastrodia elata, is extensively utilized in medicinal drugs and nutraceuticals. This study seeks to enhance microbial production of gastrodin through high-throughput screening (HTS) and transcriptomics-guided optimization. Initially, atmospheric pressure and room temperature plasma (ARTP)-mediated mutagenesis were employed to develop a library of mutant strains. Furthermore, a transcription factor-based biosensor and a high-throughput solid-phase extraction mass spectrometry (HP-SPE-MS) were evaluated to establish an HTS method for gastrodin. Consequently, mutant strain MT8 was isolated, producing 9.8 g/L of gastrodin in YPD medium, which represents a 55.6% increase compared to the control strain. Next, key genes identified via transcriptomics were overexpressed in strain MT8, with the overexpression of gene YALI2E01737g, a gene involved in the synthesis of aromatic amino acids, significantly enhancing gastrodin production to reach 10.1 g/L. In addition, fermentation process optimization further improved gastrodin titer up to 13.1 g/L in shaking flasks. This study demonstrated the utility of HTS techniques to enhance gastrodin production and paved the way for its future industrial application.

Metabolic Engineering of Yarrowia lipolytica for Enhanced De Novo Biosynthesis of Icaritin.

Icaritin (ICT) is a naturally occurring flavonoid compound with notable anticancer properties, recently recognized for its efficacy in treating advanced hepatic carcinoma. Traditional methods of ICT production, including plant extraction and chemical synthesis, face challenges such as low yield and environmental concerns. This study leverages synthetic biology to construct a microbial cell factory using Yarrowia lipolytica for de novo ICT synthesis. We engineered the yeast by integrating the ICT synthesis pathway involving EsPT from Epimedium sagittatum and OsOMTm from Oryza sativa. By optimizing the metabolic pathways, including enhancing the supply of DMAPP via mevalonate pathway modifications, and fine-tuning the expression and catalytic efficiency of EsPT through truncation strategies, we significantly improved ICT yield to 247.02 mg/L─the highest microbial ICT titer reported to date. These findings lay a solid foundation for the large-scale industrial production of ICT and offer valuable insights into the biosynthesis of other flavonoid plant natural products.

The Effect of Agitation and the Use of Perfluorodecalin on Lipase Production by Yarrowia lipolytica in a Bioreactor

Lipase production by the strictly aerobic yeast Yarrowia lipolytica is closely related to the content of dissolved oxygen in the culture medium. Some strategies to improve oxygen transfer to microorganisms have already been used, such as the use of perfluorocarbons (PFCs). The present work investigates the influence of agitation speed and the use of perfluorodecalin (PFC) on the profile of the produced lipases. Lipase production increased 2.5-fold with a higher agitation speed (550 to 650 rpm) without PFCs in the medium. The presence of an oxygen carrier led to a significant 91% increase in lipase production at lower shaking speeds compared to the assay without PFC; however, an increase in lipase production was not detected with PFC at 650 rpm. The protein profiles exhibited typical bands for two lipases produced (near 40 and 60 kDa), and these bands became more intense when PFC was added during production, as a result of the large enhancement in lipolytic activity. Additionally, the protein profiles obtained from extracts at 650 rpm were clearer and more selective regardless of the presence of PFC, suggesting an enhancement in specific activity associated with increased shaking. These findings highlight the significant impact of oxygen availability on lipase production, offering valuable insights for industrial applications.

Waste-Free Glucose to Erythritol Conversion—Innovations with Yarrowia lipolytica Wratislavia K1 UV15

This study investigates the waste-free bioconversion of glucose to erythritol using the UV-mutagenized strain Yarrowia lipolytica Wratislavia KI UV15. This research focuses on optimizing fermentation parameters to enhance erythritol yield, with a key emphasis on utilizing post-crystallization erythritol filtrate as a primary carbon source, promoting a cost-effective and sustainable approach to erythritol production. The experimental design included systematic variations in ammonium sulfate concentration, yeast extract supplementation, and initial glucose concentration. The results demonstrate that the UV15 strain achieves high erythritol production efficiency. An optimal nitrogen source concentration (3.1 g/L) and reduced yeast extract levels (0.25 g/L) provided the best results, achieving a maximum erythritol concentration of 174.8 g/L with a yield of 58.2%. Furthermore, lowering the initial glucose concentration further improved process efficiency, confirming the feasibility of using post-crystallization filtrate as an effective and sustainable carbon source. These findings highlight the biotechnological potential of Y. lipolytica UV15 in erythritol production, demonstrating its adaptability to waste-derived substrates and advancing the development of economically viable, environmentally sustainable production methods.

Recent advances in genetic engineering and chemical production in yeast species.

Yeasts have emerged as well-suited microbial cell factory for the sustainable production of biofuels, organic acids, terpenoids, and specialty chemicals. This ability is bolstered by advances in genetic engineering tools, including CRISPR-Cas systems and modular cloning in both conventional (Saccharomyces cerevisiae) and non-conventional (Yarrowia lipolytica, Rhodotorula toruloides, Candida krusei) yeasts. Additionally, genome-scale metabolic models (GEMs) and machine learning approaches have accelerated efforts to create a broad range of compounds that help reduce dependency on fossil fuels, mitigate climate change, and offer sustainable alternatives to petrochemical-derived counterparts. In this review, we highlight the cutting-edge genetic tools driving yeast metabolic engineering and then explore the diverse applications of yeast-based platforms for producing value-added products. Collectively, this review underscores the pivotal role of yeast biotechnology in efforts to build a sustainable bioeconomy.

Biotechnological Production of Carotenoids Using Yarrowia lipolytica.

Carotenoids are a group of tetraterpenoid natural products with a variety of physiological activities, which led to their application in food, cosmetics, agriculture, and other industries with broad market prospects. The fermentation of carotenoids using engineered microbial hosts has emerged as an efficient, sustainable, and environmentally friendly production method with significant potential for further development. Yarrowia lipolytica (Y. lipolytica), an unconventional oleaginous yeast, has intrinsic advantages as a host strain for the production of carotenoids. This review outlines the functions of some well-studied carotenoids, including lycopene, β-carotene, and astaxanthin. Furthermore, the biotechnological strategies for carotenoid production in Y. lipolytica are categorized and summarized. Finally, potentially feasible future strategies for further improvement of carotenoid production in Y. lipolytica are also prospected.

YlRax1 and YlRax2 Play a Partial and Functionally Interdependent Role in Regulating Bipolar Budding in the Yeast Yarrowia lipolytica.

Rax1 and Rax2 proteins provide the spatial landmark signal during the bipolar budding of Saccharomyces cerevisiae for the proper assembly of the new bud. The nonconventional yeast Yarrowia lipolytica also undergoes bipolar budding, and its genome encodes YlRax1 (YALI0E10329) and YlRax2 (YALI0A04609), the orthologs of Rax1 and Rax2, respectively. In this study, we explored the roles of YlRax1 and YlRax2 in the bipolar budding of Y. lipolytica. Deletion of YlRax1 and YlRax2 caused a proportion of Y. lipolytica cells to exhibit unipolar budding. Furthermore, our results revealed that YlRax1 and YlRax2 were localized at the mother-bud neck as well as the previous division sites, and their localization to the division sites was persistent through multiple cell cycles. Moreover, our study demonstrated that the 100 amino acids at the N-terminal of YlRax1 are not essential for its function. In contrast, the transmembrane domains at the C-terminal of YlRax1 and YlRax2 are essential for their normal function, as the truncated protein fragments with deleted TM domains could not restore the normal functioning of the corresponding strains with knocked-out YlRax1 or YlRax2. These results indicate that YlRax1 and YlRax2 are involved in partially regulating bipolar budding and that these two proteins are interdependent in localization and function. Furthermore, our results indicate that there will be novel landmark proteins regulating its bipolar budding in Y. lipolytica.

De Novo Biosynthesis of Chlorogenic Acid in Yarrowia lipolytica through Cis-Acting Element Optimization and NADPH Regeneration Engineering.

Chlorogenic acid (CGA) is a natural hydroxycinnamic acid ester with significant applications in food preservation and nutritional health. However, extraction of CGA from plants is challenging, resulting in low purity that fails to meet increasing market demands. Furthermore, the broad substrate specificity of hydroxycinnamoyl-CoA:quinic acid transferase catalysis generating a plethora of byproducts, lack of NADPH regeneration, and the presence of degrading proteins impede microbial synthesis of CGA. This study achieved de novo synthesis of CGA in Yarrowia lipolytica by introducing hydroxylation and condensation modules based on screening synthetic pathway genes and optimizing parallel promoters. Additionally, an NADPH regeneration system was incorporated to enhance the efficiency of hydroxylation, thereby increasing the titer of CGA to 333.16 mg/L. From transcriptome data, 528 significantly upregulated genes were identified, and deletion of YALI0_B21824g significantly slowed the rate of CGA degradation, which increased the titer of CGA to 351.33 mg/L in shake flasks. Applying fed-batch fermentation in a 5 L bioreactor further increased CGA production to 4837.32 mg/L (64 mg/g DCW). This study established de novo synthesis of CGA in Y. lipolytica, providing a foundation for microbial production of coumaric acid and its derivatives.

CarveAdornCurate: a versatile cloud-based platform for constructing multiscale metabolic models.

CarveAdornCurate: a versatile cloud-based platform for constructing multiscale metabolic models.

Engineering Xylose Isomerase and Reductase Pathways in Yarrowia lipolytica for Efficient Lipid Production.

Xylose is a common monosaccharide in lignocellulosic residues that Yarrowia lipolytica cannot naturally metabolise for lipid production and therefore, heterologous xylose metabolic pathways must be engineered in this yeast to facilitate its consumption. We have compared the metabolic efficiency of two xylose metabolic pathways by developing three recombinant Y. lipolytica strains: one harbouring a xylose reductase pathway, one with a xylose isomerase pathway, and one combining both pathways, and the strains were tested for xylose consumption and lipid production at different scales. The recombinant strain with the reductase pathway that was directly isolated in selective xylose medium showed the highest lipid yield, producing up to 12.8 g/L of lipids, or 43% of the biomass dry weight, without requiring any other xylose consumption adaptive evolution process. This strain achieved a lipid yield of 0.13 g lipids/g xylose, one of the highest yields in yeast reported so far using xylose as the sole carbon and energy source. Although the strain harbouring the isomerase pathway performed better under oxygen-limiting conditions and led to higher lipid intracellular accumulation, it showed a lower xylose uptake and biomass production, rendering a lower yield under non-limiting oxygen conditions. Unexpectedly, the combination of both pathways in the same strain was less effective than the use of the reductase pathway alone.