Oleaginous Yeast Research Articles

Production of genetically engineered designer biodiesel from yeast lipids

Biodiesels constitute a growing class of fuel in a world that is increasingly inclined towards more ecological and sustainable energy. Despite their many advantages, biodiesels have limited cold flow properties and larger NOX emissions. These limitations are mostly attributed to the chemical compositions of biodiesels which are dictated by the chemical compositions of their feedstock oils. Accordingly, this study presents a novel approach to produce Genetically Engineered Biodiesel (GEB) whose chemical composition can be controlled by the genetic manipulation of oleaginous yeast oils for the production of designer biodiesels with improved properties and performances. Using full-factorial central composite design, the best chemical composition of an optimal biodiesel was predicted. Then, simple and combined MFE1, PEX10 and POX2 mutants of the oleaginous yeast Yarrowia lipolytica were constructed. These mutants showed interesting lipid profiles where their biodiesels are predicted to have better cold flow properties. These mutants showed also higher lipid titers by 2–3 folds compared to the parent strain. This study provides an approach for tailor designing of biodiesel properties and performances via genetic engineering. Moreover, it provides solutions potentially enabling biodiesel to be used as a standalone fuel in cold climates without any mixing with petrodiesel.

Constructing a green oleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol

This study provides a green oleaginous yeast cell factory for sustainable production of sclareol, a significant precursor of fragrance molecules.

Tracking trash to treasure: in situ monitoring of single microbial cell oil biosynthesis from waste cooking oil using Raman spectroscopy and imaging.

Waste cooking oil is a major pollutant that contaminates terrestrial and aquatic bodies which is generated from household kitchens and eateries. The bioremediation of waste cooking oil (WCO) into microbial oil, also known as single microbial cell oil (SMCO), can be accomplished by oleaginous microbes. Conventional methods excel in SMCO analysis but lack efficacy for in situ or lysis-free monitoring of nascent SMCO synthesis and turnover. To bridge this knowledge gap, this study shows the applicability of Raman reverse stable isotope probing (RrSIP) in monitoring time-dependent nascent SMCO synthesis and assimilation in Yarrowia lipolytica, an oleaginous yeast grown in hydrophilic (glucose) as well as hydrophobic carbon sources (cooking oil and waste cooking oil). This study also combines the RrSIP approach with Raman imaging for temporal visualization of the distribution and turnover dynamics of the SMCO pool in a single cell. Our finding provides a unique perspective utilizing optical spectroscopy methods for lysis-free SMCO analysis and holds potential for prospective utility as an adjunct tool in bioprocess and biofuel industries.

Biodiesel production of oleaginous yeast isolated from the Mount Makiling Forest Reserve

Biodiesel production of oleaginous yeast isolated from the Mount Makiling Forest Reserve

Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.

Mushrooms for enhanced agriculture sustainability – the MUSA concept

The project MUSA – MUshrooms for Sustainable Agriculture is an effort to use mushroom-based processes to enhance agriculture sustainability in Nordic and Baltic countries. The project covers both the production of fruitbodies of edible fungi and the upgrading of the exhausted substrate from mushroom cultivation. The suitability of residues generated locally for producing edible mushrooms is investigated. Residues from Nordic agriculture and sub-utilized streams from forestry management, as well as wood processing by-products, are evaluated as the substrate base for producing shiitake (Lentinula edodes) and oyster (Pleurotus spp.) mushrooms. The project explores the potential of spent mushroom substrate (SMS) to support food production. SMS prospective as source of bioactive compounds and sugars is evaluated. MUSA investigates the suitability of SMS hydrolysates as carbon sources for cultivating oleaginous yeast to produce microbial oil suitable for human consumption. Using SMS for substituting mineral fertilizers and providing wastewater bioremediation solutions is also assessed.

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.

Simultaneous enhancement of lignin-derived inhibitor tolerance and lipid accumulation of oleaginous yeast Trichosporon cutaneum by adaptive evolution

Inhibitory compounds should be removed from the pretreated lignocellulose feedstock before fermentative production of microbial lipid by oleaginous yeast. Biodetoxification effectively degrades furan aldehydes and weak organic acids, but is less efficient against complex and various lignin-derived phenolic aldehydes. Residual phenolics in lignocellulose hydrolysates require a tolerant fermentation strain. This study adopted the long-term adaptive evolution combined with centrifugal enrichment in corn stover hydrolysate to simultaneously improve the phenolic inhibitors tolerance and lipid synthesis of Trichosporon cutaneum. The obtained mutant T. cutaneum MS28 showed significant improved performances of phenolic aldehydes tolerance and lipid accumulation. The final cellulosic lipid production of T. cutaneum MS28 utilizing corn stover reached 33.8 ± 0.1 g/L, approximately 6-fold higher than that of the starting strain. Transcriptional analysis suggested that T. cutaneum MS28 had more active lignocellulose-derived sugars metabolism and lipid synthesis shunts. The intracellular contents of lipid synthesis precursors including NADPH and acetyl-CoA increased 1.7–3.0 folds. This adaptive evolution in lignocellulose hydrolysate combined with centrifugal enrichment is an efficient tool to targeted enhancement of inhibitors tolerance and cellulosic lipid production of oleaginous yeast.

Oleaginous Yeast Biorefinery: Feedstocks, Processes, Techniques, Bioproducts

The world climate crisis has triggered the search for renewable energy sources. Oleaginous yeasts are a potential renewable source of biofuels. However, the yeast-derived biofuels cost is still non-competitive with the fossil fuel prices. To improve the sustainability of yeast-derived biofuels, it is necessary to valorize all yeast biomass fractions, an approach based on the biorefinery concept. This review describes the present situation of the oleaginous yeast biorefinery research, emphasizing the feedstock, processes and techniques involved in this concept, as well as on potential bioproducts that can be obtained from oleaginous yeast biomass.

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.

Simultaneous Lipid and Carotenoid Production via Rhodotorula paludigena CM33 Using Crude Glycerol as the Main Substrate: Pilot-Scale Experiments.

Rhodotorula paludigena CM33 is an oleaginous yeast that has been demonstrated to accumulate substantial quantities of intracellular lipids and carotenoids. In this study, crude glycerol, a by-product of biodiesel production, was used as a carbon source to enhance the accumulation of lipids and carotenoids in the cells. The culture conditions were first optimized using response surface methodology, which revealed that the carotenoid concentration and lipid content improved when the concentration of crude glycerol was 40 g/L. Different fermentation conditions were also investigated: batch, repeated-batch, and fed-batch conditions in a 500 L fermenter. For fed-batch fermentation, the maximum concentrations of biomass, lipids, and carotenoids obtained were 46.32 g/L, 37.65%, and 713.80 mg/L, respectively. A chemical-free carotenoid extraction method was also optimized using high-pressure homogenization and a microfluidizer device. The carotenoids were found to be mostly beta-carotene, which was confirmed by HPLC (high pressure liquid chromatography), LC-MS (liquid chromatography-mass spectrometry), and NMR (nuclear magnetic resonance). The results of this study indicate that crude glycerol can be used as a substrate to produce carotenoids, resulting in enhanced value of this biodiesel by-product.

Palmitoleic acid-rich oleaginous yeast Scheffersomyces segobiensis DSM 27193 exerts anti-obesity effects by ameliorating hepatic steatosis and adipose tissue hypertrophy.

Yeast biomass, encompassing fatty acids, terpenoids, vitamins, antioxidants, enzymes, and other bioactive compounds have been extensively utilized in food-related fields. The safety and potential bioactivities of Scheffersomyces segobiensis DSM 27193, an oleaginous yeast strain, are unclear. Scheffersomyces segobiensis DSM 27193 accumulated large palmitoleic acid (POA) levels (43.4 g kg-1 biomass) according to the results of whole-cell components. We annotated the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and predicted the categories and host of the pathogen-host interactions (PHI) genes in S. segobiensis DSM 27193. However, S. segobiensis DSM 27193 did not exert toxic effects in mice. Administration of S. segobiensis DSM 27193 led to substantial weight reduction by diminishing food intake in an obesity mouse model. Additionally, it reversed hepatic steatosis and adipose tissue hypertrophy, and improved abnormalities in serum biochemical profiles such as triglyceride, total cholesterol, low-density lipoprotein cholesterol, lipopolysaccharide, tumor necrosis factor-α, interleukin-1β, and interleukin-6. This study is the first to illustrate the safety and effects of S. segobiensis DSM 27193 against obesity and offers a scientific rationale for its application in functional food supplements. © 2023 Society of Chemical Industry.

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.

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.

Boosting concurrent yeast oil production and secretion by Rhodosporidium toruloides through non-ionic surfactant addition and gene expression monitoring

Yeast oil (YO) secretion derived from glycerol was facilitated by non-ionic surfactant-enhanced conditions, leading to extracellular yeast oil formation and expression of the ACC and DGA genes. The kinetic parameters of oleaginous yeast cultivation were estimated by employing through mathematical modeling. Seven yeast strains were cultivated on a glycerol-based oil production medium. Rhodosporidium toruloides TISTR 5186 demonstrated the highest efficacy in YO production and was chosen to investigate the impact of non-ionic surfactant supplementation on releasing intracellular YO into the extracellular space. Adding Triton X-100, Tween 20, and Brij 58 induced the release of YO by R. toruloides. Highest total YO formation and oil content were observed at 4.00 ± 0.07 g/L and 78.38 ± 0.37% (w/w) for 0.7% (w/v) Tween 20, respectively. Under this condition, the expression of the ACC and DGA genes was significantly increased by 3.4 and 3.6-fold, respectively compared to the control treatment. The YO fatty acids were consistently dominated by C16:0 and C18:1 under different yeast strains and cultural conditions. Modulation of YO secretion by non-ionic surfactants is a simple technique for enhancing oil recovery without the need for cell digestion. This strategy is a promising and straightforward process that can be employed for simultaneous oil production and recovery.

Comprehensive Exploration of the Growth and Lipid Synthesis Phases of T. oleaginosus Cultures Implementing Design of Experiments and Response Surface Methodology.

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Environmental assessment of Rhodosporidium toruloides-1588 based oil production using wood hydrolysate and crude glycerol

Rhodosporidium toruloides, an oleaginous yeast, is a potential feedstock for biodiesel production due to its ability to utilize lignocellulosic biomass-derived hydrolysate with a considerably high lipid titer of 50–70 % w/w. Hence, for the first-time environmental assessment of large-scale R. toruloides-based biodiesel production from wood hydrolysate and crude glycerol was conducted. The global warming potential was observed to be 0.67 kg CO2 eq./MJ along with terrestrial ecotoxicity of 1.37 kg 1,4-DCB eq./MJ and fossil depletion of 0.13 kg oil eq./MJ. The highest impacts for global warming (∼45 %) and fossil depletion (∼37 %) are attributed to the use of chloroform for lipid extraction while fuel consumption for transportation contributed more than 50 % to terrestrial ecotoxicity. Further, sensitivity analysis revealed that maximizing biodiesel yield by increasing lipid yield and solid loading could contribute to reduced environmental impacts. In nutshell, this investigation reveals that environmental impact varies with the type of chemical utilized.

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.

Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.