Yarrowia Research Articles

Reconfiguration of the reductive TCA cycle enables high-level succinic acid production by Yarrowia lipolytica

Succinic acid (SA) is an important C4-dicarboxylic acid. Microbial production of SA at low pH results in low purification costs and hence good overall process economics. However, redox imbalances limited SA biosynthesis from glucose via the reductive tricarboxylic acid (TCA) cycle in yeast. Here, we engineer the strictly aerobic yeast Yarrowia lipolytica for efficient SA production without pH control. Introduction of the reductive TCA cycle into the cytosol of a succinate dehydrogenase-disrupted yeast strain causes arrested cell growth. Although adaptive laboratory evolution restores cell growth, limited NADH supply restricts SA production. Reconfiguration of the reductive SA biosynthesis pathway in the mitochondria through coupling the oxidative and reductive TCA cycle for NADH regeneration results in improved SA production. In pilot-scale fermentation, the engineered strain produces 111.9 g/L SA with a yield of 0.79 g/g glucose within 62 h. This study paves the way for industrial production of biobased SA.

Citric Acid: Properties, Microbial Production, and Applications in Industries.

Citric acid finds broad applications in various industrial sectors, such as the pharmaceutical, food, chemical, and cosmetic industries. The bioproduction of citric acid uses various microorganisms, but the most commonly employed ones are filamentous fungi such as Aspergillus niger and yeast Yarrowia lipolytica. This article presents a literature review on the properties of citric acid, the microorganisms and substrates used, different fermentation techniques, its industrial utilization, and the global citric acid market. This review emphasizes that there is still much to explore, both in terms of production process techniques and emerging new applications of citric acid.

Investigation of the Yeast Yarrowia lipolytica Cultivation on Tuna Head Protein Hydrolysate

Tuna (Thunnus spp.) is a significant commodity within the fishing industry. However, up to half of the total tuna weight ends up as waste, including tuna heads, which contain valuable nutrients such as protein, polyunsaturated fatty acids, enzymes, minerals, and trace elements. The purpose of this study was to investigate an environmentally sustainable approach for tuna head processing by yeast cultivation.  The oleaginous yeast Yarrowia lipolytica was successfully cultured on tuna head protein hydrolysate and the effects of culture conditions including inoculum size, hydrolysate pH and incubation volume on nutrition utilization and on yeast growth were investigated. Y. lipolytica showed good biomass accumulation when cultured on tuna head protein hydrolysates. The yeast biomass increased with decreasing incubation volume and reached 8.6 g/L. The yeast also showed the ability to reduce up to 83.7% of hydrolysate lipid content while remaining 70.7% of nitrogen content. The culture conditions had more effect on lipid reduction than on nitrogen reduction and a good correlation between the specific growth rate and lipid reduction was observed. The yeast biomass was 64.0% in protein content and 9.6% in lipid content, without optimization. The investigation results demonstrated the potential improvement of tuna head utilization as well as the potential application of yeast biomass cultivation.

A Review on Citric Acid Production by Yarrowia lipolytica Yeast: Past and Present Challenges and Developments

The biosynthesis of citric acid (CA) and its derivatives is of great interest due to its wide range of applications in various manufacturing sectors. The fungus Aspergillus niger is mainly used for the commercial production of CA, using sucrose and molasses as the primary carbon sources. Since the 1960s, intensive research has been underway to introduce Yarrowia lipolytica yeast as an alternative to traditional fungal technology. This review discusses the practical uses of CA and its derivatives. Also, the challenges and developments that have led to efficient and green CA synthesis technologies using Y. lipolytica are outlined. The nutrient medium requirements and the use of various carbon sources, encompassing pure substrates and industry, agriculture, and food waste are considered. Additionally, the choice and improvement of strain producers, including efficient mutagenesis, genetic modification, and screening methods, are discussed.

The biopreservative properties of Metschnikowia pulcherrima LMA 2038 and Trichosporon asahii LMA 810 in a model fresh cheese, are presented

The diversity of microorganisms present in cheeses contributes to their typicity and organoleptic properties. Inhibitory strains found in these ecosystems could be used as biopreservative agents in order to replace chemicals, which are increasingly decried by consumers. Here, we isolated from cheese two yeasts named, Metschnikowia pulcherrima LMA 2038 and Trichosporon asahii LMA 810, which are active against fungi, such as Yarrowia lipolytica LMA 800, Rhodotorula mucilaginosa LMA 808, Cladosporium cladosporioides LMA 31, and Penicillium commune LMA 72, as well as against bacteria, such as Listeria innocua ATCC 33090 and Clostridium tyrobutyricum LMG 1285. When cultivated on model fresh cheese, these antagonistic yeasts significantly reduced the number of Y. lipolytica LMA 800 and R. mucilaginosa LMA 808. Preliminary data enabled us to establish a snapshot of the molecules responsible for these antifungal and antibacterial inhibitions. The molecules presumed to be present in the culture supernatant were subjected to various treatments, including heat, pH variations and enzymatic treatments. Thus, we hypothesize that different antimicrobial compounds were produced by the aforementioned antagonistic yeasts, and these molecules act in a target strain-specific manner. In summary, our findings demonstrate that cheeses possess an innumerable supply of antagonistic microbial strains, which can be exploited in the field of biopreservation to substitute chemicals and extend the shell-life of food products, including cheese.

Revealing the Mechanisms of Enhanced β-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

β-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing β-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of β-farnesene, the changes in intracellular metabolites in the β-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the β-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the β-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Unconventional Extraction Methods of Oleaginous Yeast Cell Pretreatment and Disruption

Extraction is one of the most commonly used methods for obtaining and purifying chemical compounds for commercial usage. The aim of this study was to evaluate the effect of unconventional permeabilization and cell disruption methods on the yield of lipid extraction from cells of the oleaginous yeast Yarrowia lipolytica. Batch cultures in a medium with molasses and waste post-frying oil were carried out. The biomass was subjected to pulsed electric field (PEF), high-pressure processing (HPP), ultrasounds (US), and several conventional processing techniques with chemical and mechanical agents (glass beads, acetone, Triton and Tween surfactants). The effectiveness of the applied methods, either on cell permeabilization or cell disruption, was investigated by analyzing the oil and total protein extraction yield and oil leaching efficiency, as well as by using microscope images. The PEF and US treatments proved to be effective permeabilization methods as a step of sample pretreatment for extraction. These unconventional physical methods could efficiently increase intracellular lipid extraction yield in solvent applications.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.

Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.

Inoculation of Yarrowia lipolytica promotes the growth of lactic acid bacteria, Debaryomyces udenii and the formation of ethyl esters in sour meat

Yarrowia lipolytica N12 and A13 with high lipase activity obtained by mutagenesis were inoculated into sour meat, and their effects on physicochemical properties, microbial community succession, free amino acids, and volatile compounds of sour meat were investigated. Inoculation fermentation increased the contents of free amino acids observably, rapidly reduced pH, promoted the accumulation of total acids, decreased 2-thiobarbituric acid reactive substances (TBARS) values. In addition, the addition of Y. lipolytica might contribute to the growth of lactic acid bacteria, Candida spp., and Debaryomyces udenii, which play an important role in production of volatile compounds. It was shown that inoculation promoted the production of esters, aldehydes, and alcohols, especially ethyl esters, giving sour meat a better meat flavor. Besides, it was found that Y. lipolytica A13 had better fermenting property. Sample of A13 group had higher contents of ethyl esters, free amino acids and dominant microorganisms. The results may help to provide new strains for sour meat fermentation.

Efficient Production of Triacetic Acid Lactone from Lignocellulose Hydrolysate by Metabolically Engineered Yarrowia lipolytica.

Lignocellulose is a promising renewable feedstock for the bioproduction of high-value biochemicals. The poorly expressed xylose catabolic pathway was the bottleneck in the efficient utilization of the lignocellulose feedstock in yeast. Herein, multiple genetic and process engineering strategies were explored to debottleneck the conversion of xylose to the platform chemical triacetic acid lactone (TAL) in Yarrowia lipolytica. We identified that xylose assimilation generating more cofactor NADPH was favorable for the TAL synthesis. pH control improved the expression of acetyl-CoA carboxylase and generated more precursor malonyl-CoA. Combined with the suppression of the lipid synthesis pathway, 5.03 and 4.18 g/L TAL were produced from pure xylose and xylose-rich wheat straw hydrolysate, respectively. Our work removed the bottleneck of the xylose assimilation pathway and effectively upgraded wheat straw hydrolysate to TAL, which enabled us to build a sustainable oleaginous yeast cell factory to cost-efficiently produce green chemicals from low-cost lignocellulose by Y. lipolytica.

Extracellular activity of proteases from Yarrowia lipolytica IPS21 as a function of the carbon and nitrogen source

Abstract The yeast strain Yarrowia lipolytica IPS 21 was tested for its ability to produce the protease enzyme on analytically pure carbon sources as well as on waste carbon sources. It was confirmed that the yeast Y. lipolytica IPS21 can have a higher proteolytic activity in the presence of waste carbon sources in chrome-tanned leather shavings (CTLS) than on yeast extract alone. This is confirmed by the high concentration of amino acids in samples with CTLS, suggesting increased degradation of CTLS by Y. lipolytica or secretion of proteases into the medium. It was also confirmed that metals accumulate mainly in the biomass and not in the supernatant. The biomass was also found to contain high levels of Ca, K and P, which are essential for plant growth. These results show that Y. lipolytica strain IPS21 can be used for the production of extracellular alkaline proteases and for the degradation of protein waste.

New pAraDH2 Promoter for Metabolic Engineering of the Yarrowia lipolytica Yeast

New pAraDH2 Promoter for Metabolic Engineering of the Yarrowia lipolytica Yeast

Optimization of Cultivation Conditions of Yarrowia lipolytica Po1f pUV3-Op Transformant to Get the Encapsulated Phytase as The Feed Supplement

Optimization of Cultivation Conditions of Yarrowia lipolytica Po1f pUV3-Op Transformant to Get the Encapsulated Phytase as The Feed Supplement

Safety evaluation of the food additive steviol glycosides, predominantly Rebaudioside M, produced by fermentation using Yarrowia lipolyticaVRM.

The EFSA Panel on Food Additive and Flavourings (FAF Panel) provides a scientific opinion on the safety of a new process to produce steviol glycosides by fermentation of simple sugars using a genetically modified strain of Yarrowia lipolytica (named Y. lipolytica VRM). The manufacturing process may result in impurities different from those that may be present in the other steviol glycosides E 960a-d, therefore the Panel concluded that separate specifications are required for the food additive produced as described in the current application. Viable cells and DNA from the production strain are not present in the final product. The Panel considered that the demonstration of the absence of kaurenoic acid in the proposed food additive, using a method with a limit of detection (LOD) of 0.3 mg/kg, is adequate to dispel the concerns for potential genotoxicity. Given that all steviol glycosides follow the same metabolic pathways, the Panel considered that the current steviol glycosides would fall within the same group of substances. Therefore, the Panel considered that the already existing data on rebaudioside M and structurally related steviol glycosides are sufficient, and a similar metabolic fate and toxicity is expected for the food additive. The results from the bacterial reverse mutation assay and the invitro micronucleus assay were negative and indicated absence of genotoxicity from the food additive. The existing acceptable daily intake (ADI) of 4 mg/kg body weight (bw) per day, expressed as steviol equivalents, was considered to be applicable to the proposed food additive. The Panel concluded that there is no safety concern for steviol glycosides, predominantly Rebaudioside M, produced by fermentation using Y. lipolytica VRM, to be used as a food additive at the proposed uses and use levels.

BIOCONVERSION OF MEAT PROCESSING FATTY WASTE USING YEAST Y.LIPOLYTICA

A technology for the utilization of fat waste from meat processing based on a combination of physical and chemical effects and biological oxidation is proposed. The main chemical and microbiological parameters of the fat-containing phase of effluents of meat processing enterprises were determined. It was established that in the process of holding waste in a sludge collector (1…13 days) as a result of the vital activity of autochthonous microorganisms, the fat content decreases from 87 % to 58 %. The dominant organism of the autochthonous microflora of the waste is the fungus Geotrichum.sp. It was established that Yarrowia lipolytica yeast, selected for the substrate, is the most promising fat biodestructor. A method of pretreatment of the fat-containing solid phase of fatty effluents has been developed, which increases the efficiency of their consumption by the Yarrowia lipolytica culture. The basis of the method is the ultrasonic dispersion of fat mass, as a result of which the bioavailability of the substrate increases and, as a result, the specific growth rate and yield of microbial biomass increases by 11 % and 30 %, respectively. The methods of seed preparation are proposed, which allow to increase the efficiency of the main process according to the indicators: biomass yield (by 10.1 %); specific growth rate (from 0.20 to 0.26 h–1); protein content (by 16.7 %). The methods include the selection (5…7 passages) of high-performance clones, which is carried out in the directions of increasing affinity to the substrate and stress resistance to the action of hydrogen peroxide (2.5 g/l). The obtained results showed that when using yeast Yarrowia lipolytica for the biodestruction of fatty effluents, it is advisable to use both top-up and continuous methods of cultivation, which do not lead to significant changes in the quality of the obtained biomass. Yeast leaching was not observed in the top-up mode at 24 % withdrawal of culture liquid per hour, and in the case of continuous mode - at a flow rate of 0.24 h–1, while the protein and lipid content was 42.7 % and 7.2 %, 41.4 % and 7.9 %, respectively.

Beet red food colourant can be produced more sustainably with engineered Yarrowia lipolytica

Synthetic food colourants are widely used in the food industry, but consumer concerns about safety and sustainability are driving a need for natural food-colour alternatives. Betanin, which is extracted from red beetroots, is a commonly used natural red food colour. However, the betanin content of beetroot is very low (~0.2% wet weight), which means that the extraction of betanin is incredibly wasteful in terms of land use, processing costs and vegetable waste. Here we developed a sustainability-driven biotechnological process for producing red beet betalains, namely, betanin and its isomer isobetanin, by engineering the oleaginous yeast Yarrowia lipolytica. Metabolic engineering and fermentation optimization enabled production of 1,271 ± 141 mg l−1 betanin and 55 ± 7 mg l−1 isobetanin in 51 h using glucose as carbon source in controlled fed-batch fermentations. According to a life cycle assessment, at industrial scale (550 t yr−1), our fermentation process would require significantly less land, energy and resources compared with the traditional extraction of betanin from beetroot crops. Finally, we apply techno-economic assessment to show that betanin production by fermentation could be economically feasible in the existing market conditions.

Polyhydroxybutyrate (PHB) Biosynthesis by an Engineered Yarrowia lipolytica Strain Using Co-Substrate Strategy

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h−1. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica.

Installing xylose assimilation and cellodextrin phosphorolysis pathways in obese Yarrowia lipolytica facilitates cost-effective lipid production from lignocellulosic hydrolysates

BackgroundYarrowia lipolytica, one of the most charming chassis cells in synthetic biology, is unable to use xylose and cellodextrins.ResultsHerein, we present work to tackle for the first time the engineering of Y. lipolytica to produce lipids from cellodextrins and xylose by employing rational and combinatorial strategies. This includes constructing a cellodextrin-phosphorolytic Y. lipolytica by overexpressing Neurospora crassa cellodextrin transporter, Clostridium thermocellum cellobiose/cellodextrin phosphorylase and Saccharomyces cerevisiae phosphoglucomutase. The effect of glucose repression on xylose consumption was relieved by installing a xylose uptake facilitator combined with enhanced PPP pathway and increased cytoplasmic NADPH supply. Further enhancing lipid production and interrupting its consumption conferred the obese phenotype to the engineered yeast. The strain is able to co-ferment glucose, xylose and cellodextrins efficiently, achieving a similar μmax of 0.19 h−1, a qs of 0.34 g-s/g-DCW/h and a YX/S of 0.54 DCW-g/g-s on these substrates, and an accumulation of up to 40% of lipids on the sugar mixture and on wheat straw hydrolysate.ConclusionsTherefore, engineering Y. lipolytica capable of assimilating xylose and cellodextrins is a vital step towards a simultaneous saccharification and fermentation (SSF) process of LC biomass, allowing improved substrate conversion rate and reduced production cost due to low demand of external glucosidase.

Cutaneotrichosporon curvatum and Yarrowia lipolytica as key players for green chemistry: efficient oil producers from food waste via the carboxylate platform

ABSTRACT Cutaneotrichosporon curvatum and Yarrowia lipolytica can accumulate microbial oils using short-chain fatty acids (SCFA) as carbon sources. SCFAs-rich media often contain significant amounts of nitrogen that prevent high carbon:nitrogen (C:N) ratios necessary to boost lipid production. This work assessed the intrinsic ability of C. curvatum and Y. lipolytica to produce high amounts of microbial oils from these unusual carbon sources. Results demonstrated that minor differences in SCFA concentration (only 2 g/L) had a significant effect on yeast growth and lipid production. A C:N of 80 promoted yeast growth at all SCFA concentrations and favored SCFA consumption at 19 g/L SCFAs. The different SCFA uptake preferences in C. curvatum and Y. lipolytica highlighted the importance of considering the SCFA profile to select a suitable yeast strain for microbial oils production. At the most challenging SCFA concentration (19 g/L), 57.2% ±1.6% (w/w) and 78.4 ± 0.6% (w/w) lipid content were obtained in C. curvatum and Y. lipolytica, respectively. These values are among the highest reported for wild-type strains. To circumvent the challenges associated with media with high nitrogen content, this report also proved struvite precipitation as an effective method for increasing lipid production (from 17.9 ± 3.9% (w/w) to 41.9 ± 2.6% (w/w)) after nitrogen removal in food waste-derived media.