Yarrowia Research Articles

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

Immobilization of N,N‐diallyl‐N,N‐dimethylammonium chloride on polyethylene. Biocidal properties of the material

AbstractThe immobilization of N,N‐diallyl‐N,N‐dimethylammonium chloride (DADMAC) on low‐density polyethylene (LDPE) films was carried out by the method of postradiation grafting using x‐ray radiation. The introduction of polycation exchanger links provided the appearance of hydrophilicity of the surface of the modified film. By measuring the wetting contact angle, IR and SEM, the island‐type character of the coating from grafted polyDADMAC was detected. The death of Gram‐negative bacteria Pseudomonas aeruginosa, Gram‐positive bacteria Staphylococcus aureus, and yeast (eukaryotes) Yarrowia lipolytica on the surface of the film with grafted polyDADMAC was demonstrated.

Yarrowia lipolytica CMGB32 Biosurfactants Produced Using n-Hexadecane: Developing Strategies for Environmental Remediation

The yeast Yarrowia lipolytica degrades petroleum compounds, including alkanes, via the monoterminal oxidation pathway, the hydrophobic carbon substrate assimilation is mediated by biosurfactants, and extracellular amphiphilic molecules are produced by the yeast cell. This study focuses on the ability of the strain Y. lipolytica CMGB32 to degrade n-hexadecane by producing biosurfactants with high potential for bioremediation. The hydrocarbon-degrading potential of the yeast strain was observed via a 2,6-dichlorophenolindophenol (DCPIP) test in Bushnell–Hass medium with 1% n-hexadecane, and cell hydrophobicity was expressed as microbial adhesion to hydrocarbons (MATH). Biosurfactant production on yeast peptone (YP) with 1% n-hexadecane was estimated after 72 h using the emulsification index (E24%) against toluene. Crude biosurfactant (cell-free broth) stability tests were performed at different temperatures (4 °C, 70 °C) and NaCl concentrations (2–10%). The effects of a biosurfactant on synthetic wastewater remediation comprised the growth curves (OD measurements) of natural heavy metal degrader Rhodotorula mucilaginosa, determination of nutrients (spectrophotometrically), physico-chemical parameters, and removal capacity of lead and cadmium ions (via inductively coupled plasma mass spectrometry—ICP-MS). The antimicrobial and anti-adherence activities of 20 mg/mL and 40 mg/mL of the biosurfactant against pathogenic Candida krusei strains involved growth observations and the crystal violet microtiter method. The DCPIP decolorization occurred after six days, corresponding to the maximum growth phase of the Y. lipolytica culture. After 72 h, the cells presented high hydrophobicity (82.61% MATH) and stable biosurfactant production (E24% 47%). The crude biosurfactant (5%) increased the growth of R. mucilaginosa strains cultivated on synthetic wastewater cultures contaminated with Pb2+ and Cd2+, increased the conductivity and COD (86%) of the samples, and determined Pb2+ (66%) and Cd2+ (42%) ions reduction. The concentrated biosurfactant inhibited C. krusei growth (70%) and biofilm adherence. In conclusion, Y. lipolytica CMGB32 shows important potential for development of biosurfactant-based technologies for the remediation of heavy-metal- and emerging pathogen-contaminated wastewaters.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids

BackgroundShort-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth.ResultsIn this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain.ConclusionsA more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

Biosurfactants production by marine yeasts isolated from zoanthids and characterization of an emulsifier produced by Yarrowia lipolytica LMS 24B

The present study investigates the potential for biosurfactant production of 19 marine yeast species obtained from zoanthids. Using the emulsification index test to screen the samples produced by the marine yeasts, we verified that five isolates exhibited an emulsification index ≥50%. Additional tests were performed on such isolates, including oil displacement, drop collapse, Parafilm M assay, and surface tension measurement. The tolerance of produced biosurfactants for environmental conditions was also analyzed, especially considering the media's temperature, pH, and salinity. Moreover, the surfactant's ability to emulsify different hydrocarbon sources and to metabolize kerosene as the sole carbon source was evaluated in vitro. Our results demonstrate that yeast biosurfactants can emulsify hydrocarbon sources under different physicochemical conditions and metabolize kerosene as a carbon source. Considering the Yarrowia lipolytica LMS 24B as the yeast model for biosurfactant production from the cell's wall biomass, emulsification indexes of 61.2% were obtained, even at a high temperature of 120 °C. Furthermore, the Fourier-transform middle infrared spectroscopy (FTIR) analysis of the biosurfactant's chemical composition revealed the presence of distinct functional groups assigned to a glycoprotein complex. Considering the status of developing new bioproducts and bioprocesses nowadays, our findings bring a new perspective to biosurfactant production by marine yeasts, especially Y. lipolytica LMS 24B. In particular, the presented results validate the relevance of marine environments as valuable sources of genetic resources, i.e., yeast strains capable of metabolizing and emulsifying petroleum derivatives.

Experimental validation off-line a nonlinear controller for removal of chromium using non-living cells of Yarrowia lipolytica

The removal of hexavalent chromium [Cr (VI)] using non-living cells of Yarrowia lipolytica was investigated. Batch and continuous studies on removal of Cr (VI) achieved 97% and 99% removal from aqueous phase, respectively. The specific uptake values at pH of 2 in batch process were 40.73 ± 1.3 mg/g and 30.09 ± 0.23 mg/g on non-living cells, when 100 and 200 mg/L of metal Cr (VI) concentrations were used. In order to investigate the regulation of Cr (VI) under continuous operation based on reaction volume numerically a new class of feedback controller from structure polynomial was designed. The proposed methodology was used to an experimentally kinetic model for a removal Cr (VI) from Yarrowia lipolytica biomass was showed satisfactory closed-loop performance the proposed controller. Starting from an off-line optimization performed in simulation, we present the controller implementation, focussing on the methodology required to could be suitable for implementation in real time. In our experimental results, we highlight some discrepancies between simulation and reality despite these differences, the controller managed to perform convergence to removal Cr (VI). Finally, the results validated with off-line samples suggest that the proposed control could be suitable for in application in potential scenarios for wastewater treatment.

First-class - biosynthesis of 6-MSA and bostrycoidin type I polyketides in Yarrowia lipolytica.

Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast Yarrowia lipolytica, an easily cultivable heterologous host. As an oleaginous yeast, Y. lipolytica displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, Y. lipolytica has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of Y. lipolytica by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (fsr1, fsr2, and fsr3) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid β-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in Saccharomyces cerevisiae (2.2 mg/L), this work demonstrates the potential of Y. lipolytica as a platform for heterologous production of complex fungal polyketides.

Adsorption of azo dye by biomass and immobilized Yarrowia lipolytica; equilibrium, kinetic and thermodynamic studies

One of the major environmental problems we have today is dye pollution, primarily caused by the textile industry. This pollution has detrimental effects on aquatic life, soil fertility, and human health. Many microbial biosorbents have been documented in the literature for the removal of a wide range of azo dyes commonly employed in the textile industry. However, Yarrowia lipolytica NBRC1658 is firstly used as both free and immobilized sorbents for the removal of Reactive yellow 18 (RY18), acid red 18 (AR18) and basic blue 41 (BB41) in this study. The effect of experimental conditions such as pH, biosorbent quantity, dye concentration, contact time, and temperature on dye removal capacity are examined. The research findings demonstrate that the adsorption capacity is higher in biomass compared to immobilized cells. The highest adsorption capacities are observed at pH 2 for RY18 and AR18, while pH 9 is optimal for BB41. Increasing the adsorbent dosage and initial concentration significantly improves the adsorption capacity. The Langmuir model best describes the adsorption process, indicating that the dye attaches to the biosorbent in a single layer, with a uniform biosorbent surface. The removal of the dye occurs through a chemical process on the biosorbent surface, as evidenced by the pseudo-second-order kinetic model. According to thermodynamic analysis, higher temperatures promote greater adsorption of dyes. Our study shows the effectiveness of Yarrowia lipolyica NBRC1658 as a biosorbent in the removal of a wide range of industrial dyes.Graphical

Construction and Regulation of the Abscisic Acid Biosynthesis Pathway in Yarrowia lipolytica.

Abscisic acid (ABA) is an important plant hormone with a variety of physiological functions such as regulating plant growth and helping plants to resist an adverse growth environment. However, at present, the ABA yield of heterologous biosynthesis by metabolic engineering is still low for industrial production. Therefore, five Botrytis cinerea genes (bcaba1, bcaba2, bcaba3, bcaba4, and bccpr1) related to ABA biosynthesis were expressed in Yarrowia lipolytica PO1h; its ABA production was 24.33 mg/L. By increasing the copy number of IDI and ERG12S, ERG20YMT, and bcaba3, bcaba1 genes, the yield of ABA was increased to 54.51 mg/L. By locating HMG-CoA reductase and HMG-CoA synthase in mitochondria, acetyl-CoA in mitochondria was converted into mevalonate; this increased the ABA yield to 102.12 mg/L. Finally, in the fed-batch fermentation process with the addition of dodecane, the ABA yield was up to 1212.57 mg/L, which is the highest yield of heterologous production of ABA by metabolic engineering.

Adaptive responses of erythritol-producing Yarrowia lipolytica to thermal stress after evolution

Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains.Key points• ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica• Genes encoding global regulators are mutated during thermal adaptive evolution• Ceramide and BCAA are critical molecules for cells to tolerate thermal stress

Carotenoids production and extraction from Yarrowia lipolytica cells: A biocompatible approach using biosolvents

The use of carotenoids, pigments with significant importance in the food and pharmaceutical industries, has been widely recognized because of their functions as colorants and antioxidants. The production of carotenoids from oleaginous yeasts is an efficient and eco-friendly alternative to that of synthetic carotenoids. Yarrowia lipolytica, due to its ability to synthesize high-value-added compounds, including carotenoids and lipids, has emerged as a promising biotechnological platform for this purpose. This study aimed to evaluate the efficiency of biosolvents for carotenoid extraction with the goal of replacing toxic organic solvents that pose risks to both human health and the environment. The Solid-Liquid Extraction (SLE) technique was employed to disrupt the rigid cell wall and extract intracellular pigments. Subsequent extractions were conducted using a 2:3:1 (w/w/w) ratio of ethanol:ethyl acetate:water, resulting in a total β-carotene concentration of approximately 5 mg β-carotene equivalent/g dry biomass. The COSMO-SAC model was utilized to elucidate the solute–solvent affinity, and the results obtained were consistent with the experimental findings. To improve the process, various ratios of biosolvents were investigated, and a subsequent Solid-Liquid-Liquid Extraction (SLLE) was introduced. Process integration and adjustments to the mixed biosolvent composition achieved yields ranging from 50 to 100 % in just four consecutive extractions. The proposed integrative platform not only enhances the efficiency of the extraction process but also enables the fractionation and purification of bioproducts from Y. lipolytica cells, thereby maximizing their biotechnological potential.

Yarrowia lipolytica as a promising protein source for Pacific white shrimp (Litopenaeus vannamei) diet: impact on growth performance, metabolism, antioxidant capacity, and apparent digestibility

Exploring alternatives to fishmeal (FM) in aquaculture nutrition remains a hot topic in this area. Yarrowia lipolytica (YL), a promising single-cell protein source, has yet to be evaluated for its use on the Pacific white shrimp (Litopenaeus vannamei) diet. This investigation assessed the effects of YL on growth performance, metabolism, antioxidant capacity, and nutrient digestibility in shrimp. Seven diets with equal protein and lipid contents were prepared, incorporating gradient YL (dry matter basis): 0% (control), 1.50%, 3.00%, 4.50%, 6.00%, 9.00%, and 12.00%. During a 56-day feeding trial involving shrimp, with an initial average weight of 0.32 ± 0.02 g, each kind of diet was randomly assigned to six tanks. It was observed that the growth performance improved initially but then exhibited a declining trend as the dietary inclusion of YL increased. Regression analysis indicated 2.68% as the optimal YL percentage for a specific growth rate (SGR), accounting for 10.54% of the dietary FM content. A higher YL (over 6.00%), which is equivalent to replacing over 23.6% of the FM, led to increased feed intake and conversion ratios, possibly correlating with decreased YL digestibility. However, YL inclusion across the studied range had no detrimental impact on intestinal or hepatopancreatic morphology. Notably, higher YL levels significantly enhanced hepatopancreatic trypsin activity and reduced serum triglyceride and cholesterol levels. The inclusion of YL notably augmented antioxidant defense mechanisms in shrimp, as evidenced by increased total antioxidant capacity and catalase activities, as well as reduced malondialdehyde levels in the hepatopancreas, possibly due to the presence of β-glucan and mannan oligosaccharides in YL. These findings support YL being viable as a protein source in shrimp feeds.

Transcriptome analysis reveals multiple targets of erythritol-related transcription factor EUF1 in unconventional yeast Yarrowia Lipolytica

BackgroundErythritol is a four-carbon polyol with an unclear role in metabolism of some unconventional yeasts. Its production has been linked to the osmotic stress response, but the mechanism of stress protection remains unclear. Additionally, erythritol can be used as a carbon source. In the yeast Yarrowia lipolytica, its assimilation is activated by the transcription factor Euf1. The study investigates whether this factor can link erythritol to other processes in the cell.ResultsThe research was performed on two closely related strains of Y. lipolytica: MK1 and K1, where strain K1 has no functional Euf1. Cultures were carried out in erythritol-containing and erythritol-free media. Transcriptome analysis revealed the effect of Euf1 on the regulation of more than 150 genes. Some of these could be easily connected with different aspects of erythritol assimilation, such as: utilization pathway, a new potential isoform of transketolase, or polyol transporters. However, many of the upregulated genes have never been linked to metabolism of erythritol. The most prominent examples are the degradation pathway of branched-chain amino acids and the glyoxylate cycle. The high transcription of genes affected by Euf1 is still dependent on the erythritol concentration in the medium. Moreover, almost all up-regulated genes have an ATGCA motif in the promoter sequence.ConclusionsThese findings may be particularly relevant given the increasing use of erythritol-induced promoters in genetic engineering of Y. lipolytica. Moreover, use of this yeast in biotechnological processes often takes place under osmotic stress conditions. Erythritol might be produce as a by-product, thus better understanding of its influence on cell metabolism could facilitate processes optimization.

Efficient production of isomaltulose using engineered Yarrowia lipolytica strain facilitated by non-yeast signal peptide-mediated cell surface display.

Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. In the present study, a simple and efficient surface display platform mediated by a non-yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi-site integrating genes into chromosome. The final strain gained 451.7 g L-1 isomaltulose with a conversion rate of 90.3% and a space-time yield of 50.2 g L-1  h-1 . The present study provides an efficient way for manufacturing isomaltulose with a high space-time yield. This heterogenous signal peptide-mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole-cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry.

Engineering Yarrowia lipolytica as a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose.

De novo biosynthesis of high-value added food additive p-coumaric acid (p-CA) direct from cellulose/hemicellulose is a more sustainable route compared to the chemical route, considering the abundant cellulose/hemicellulose resources. In this study, a novel factory was constructed for the production of p-CA in Yarrowia lipolytica using cellulose/hemicellulose as the sole carbon source. Based on multicopy integration of the TAL gene and reprogramming the shikimic acid pathway, the engineered strain produced 1035.5 ± 67.8 mg/L p-CA using glucose as a carbon source. The strains with overexpression of cellulases and hemicellulases produced 84.3 ± 2.4 and 65.3 ± 4.6 mg/L p-CA, using cellulose (carboxymethyl-cellulose) or hemicellulose (xylan from bagasse) as the carbon source, respectively. This research demonstrated the feasibility of conversion of cost-effective cellulose/hemicellulose into a value-added product and provided a sustainable cellulolytic cell factory for the utilization of cellulose/hemicellulose.

Abstract 2048 Loss of ATP-dependent Citrate Lyase Abrogates Phosphatidate Phosphatase Activity in the oleaginous yeast Yarrowia lipolytica

Abstract 2048 Loss of ATP-dependent Citrate Lyase Abrogates Phosphatidate Phosphatase Activity in the oleaginous yeast Yarrowia lipolytica

De Novo Biosynthesis of Lutein in Yarrowia lipolytica.

Lutein is a high-value tetraterpenoid carotenoid that is widely used in feed, cosmetics, food, and drugs. Microbial synthesis of lutein is an important method for green and sustainable production, serving as an alternative to plant extraction methods. However, an inadequate precursor supply and low catalytic efficiency of key pathway enzymes are the main reasons for the low efficacy of microbial synthesis of lutein. In this study, some strategies, such as enhancing the MVA pathway and localizing α-carotene synthase OluLCY within the subcellular organelles in Yarrowia lipolytica, were adopted to enhance the synthesis of precursor α-carotene, which resulted in a 10.50-fold increase in α-carotene titer, reaching 38.50 mg/L. Subsequently, by improving hydroxylase activity with truncated N-terminal transport peptide and locating hydroxylases to subcellular organelles, the final strain L9 producing 75.25 mg/L lutein was obtained. Eventually, a lutein titer of 675.40 mg/L (6.13 mg/g DCW) was achieved in a 5 L bioreactor by adding the antioxidant 2,6-ditert-butyl-4-methylphenol. This study realizes de novo synthesis of lutein in Y. lipolytica for the first time and achieves the highest lutein titer reported so far.

Identification of lipid synthesis genes in Schizochytrium sp. and their application in improving eicosapentaenoic acid synthesis in Yarrowia lipolytica

BackgroundEicosapentaenoic acid (EPA) is widely used in the functional food and nutraceutical industries due to its important benefits to human health. Oleaginous microorganisms are considered a promising alternative resource for the production of EPA lipids. However, the storage of EPA in triglyceride (TG) becomes a key factor limiting its level.ResultsThis study aimed to incorporate more EPA into TG storage through metabolic engineering. Firstly, key enzymes for TG synthesis, the diacylglycerol acyltransferase (DGAT) and glycerol-3-phosphate acyltransferase (GPAT) genes from Schizochytrium sp. HX-308 were expressed in Yarrowia lipolytica to enhance lipid and EPA accumulation. In addition, engineering the enzyme activity of DGATs through protein engineering was found to be effective in enhancing lipid synthesis by replacing the conserved motifs “HFS” in ScDGAT2A and “FFG” in ScDGAT2B with the motif “YFP”. Notably, combined with lipidomic analysis, the expression of ScDGAT2C and GPAT2 enhanced the storage of EPA in TG. Finally, the accumulation of lipid and EPA was further promoted by identifying and continuing to introduce the ScACC, ScACS, ScPDC, and ScG6PD genes from Schizochytrium sp., and the lipid and EPA titer of the final engineered strain reached 2.25 ± 0.03 g/L and 266.44 ± 5.74 mg/L, respectively, which increased by 174.39% (0.82 ± 0.02 g/L) and 282.27% (69.70 ± 0.80 mg/L) compared to the initial strain, respectively.ConclusionThis study shows that the expression of lipid synthesis genes from Schizochytrium sp. in Y. lipolytica effectively improves the synthesis of lipids and EPA, which provided a promising target for EPA-enriched microbial oil production.

Biomass and microbial lipids production by Yarrowia lipolytica W29 from eucalyptus bark hydrolysate

Using lignocellulosic biomass hydrolysate as a renewable and abundant feedstock for microbial lipids production is a sustainable and economic high-potential approach. This study investigated the potential of the oleaginous yeast Yarrowia lipolytica to produce lipids-rich biomass from eucalyptus bark hydrolysate (EBH) obtained by enzymatic hydrolysis of the biomass pretreated by steam explosion. The effect of EBH concentration (undiluted and 1:3 v/v diluted) and medium supplementation (CSL and KH2PO4) was evaluated in Erlenmeyer flasks and lab-scale stirred tank bioreactor, respectively. Additionally, the effect of volumetric oxygen transfer coefficient (kLa) and mode of operation (batch and two-stage repeated batch) was also assessed in the bioreactor. Under the best experimental conditions (undiluted EBH, 2 g⋅L−1 CSL, 1.8 g⋅L−1 (NH4)2SO4, and kLa of 66 h−1), Y. lipolytica W29 grown in batch cultures accumulated 26 % (w/w) of intracellular lipids, corresponding to 5.6 g⋅L−1 of concentration. Lipids of Y. lipolytica were highly unsaturated and mainly composed of oleic acid (48 %), followed by palmitoleic (20 %), linoleic (17 %) and palmitic acids (14 %). This composition of Y. lipolytica lipids suggests their potential use as feedstock for biodiesel (a renewable biofuel). This work demonstrated the robust features of Y. lipolytica W29 as a potential lipids production platform to implement lignocellulose-based biorefineries.