Yarrowia Lipolytica Strains Research Articles

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.

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.

Unlocking the potential of one-carbon gases (CO2, CO) for concomitant bioproduction of β-carotene and lipids

This study investigates the use of a Yarrowia lipolytica strain for the bioconversion of syngas-derived acetic acid into β-carotene and lipids. A two-stage process was employed, starting with the acetogenic fermentation of syngas by Clostridium aceticum, metabolising CO, CO2, H2, to produce acetic acid, which is then utilized by Y. lipolytica for simultaneous lipid and β-carotene synthesis. The research demonstrates that acetic acid concentration plays a pivotal role in modulating lipid profiles and enhancing β-carotene production, with increased acetic acid consumption leading to higher yields of these compounds. This approach showcases the potential of using one-carbon gases as substrates in bioprocesses for generating valuable bioproducts, providing a sustainable and cost-effective alternative to more conventional feedstocks and substrates, such as sugars.

Studies of polyol production by the yeast Yarrowia lipolytica growing on crude glycerol under stressful conditions

Crude glycerol, the principal by-product of biodiesel production process, was employed as substrate by three wild-type Yarrowia lipolytica strains (ACA-YC 5030, LMBF 20 and NRRL Y-323). Stressful conditions (low pH value = 2.0 ± 0.3, low incubation temperature T = 20 ± 1 °C, non-aseptic conditions) were employed. Interesting production of yeast biomass and polyols (viz. erythritol, mannitol and arabitol) was noted at pH = 2.0 ± 0.3 and T = 20 ± 1 °C. Strains failed to produce significant quantities of cellular lipid, while variable quantities of intra-cellular polysaccharides were produced. Fermentations under previously pasteurized media supported significant biomass and polyols production for most of the tested strains, while only one strain (NRRL Y-323), managed to produce polyols at media that were not previously thermally treated at all. The production of mannitol was favored at low initial glycerol (Glol0) concentrations, whereas higher Glol0 quantities favored the biosynthesis of erythritol. For the strain NRRL Y-323, highly aerated / agitated bioreactor trials showed different physiological profiles as compared to the respective flask experiments. Finally, in flask experiments with the strain NRRL Y-323 at high Glol0 amounts (≈140 g/L) at low medium pH (=2.0 ± 0.3), a significant production of polyols (=84.2 g/L) with the corresponding remarkable conversion yield on glycerol consumed = 62 % w/w was achieved. Practical applicationRenewable and biodegradable fuels, such as biodiesel, are safer and environmentally friendlier than the conventional petroleum diesel. Glycerol is a cost-effective substrate obtained as the main side-product from biodiesel production process and is currently being employed in the realm of Industrial Microbiology and Biotechnology to produce metabolic products with added value. Current research focuses on using glycerol as a starting substrate for biotechnological conversions aiming at producing, amongst other compounds, polyols, microbial biomass, citric acid, etc. from selected strains of the Generally Recognized Αs Safe (GRAS) yeast Yarrowia lipolytica. In the current investigation therefore, we examined the capacity of new wild-type non-extensively studied strains of this yeast to grow and assimilate this inexpensive substrate. Specifically, we have performed the acclimatization of the mentioned strains to stressful environments (i.e., low pH, low incubation temperature, non-aseptic conditions, etc.) and remarkable quantities of the added-value compounds (polyols, yeast mass, citric acid) were produced.

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.

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.

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.

Two-stage syngas fermentation into microbial oils and β-carotene with Clostridium carboxidivorans and engineered Yarrowia lipolytica

An engineered Yarrowia lipolytica strain was able to simultaneously accumulate microbial oils and β-carotene from both acetic and butyric acids produced from syngas fermentation by Clostridium carboxidivorans, through the Wood-Ljungdahl pathway, with CO and CO2 as carbon sources. It was first determined that Y. lipolytica showed no inhibition at concentrations < 16 g/L of a mixture of VFAs (Volatile Fatty Acids) containing acetic, butyric, and even hexanoic acids. The yeast was then grown in a bioreactor with a culture medium derived from acetogenic syngas (CO, CO2, H2) fermentation containing all three acids. Y. lipolytica consumed a total of 91 g/L VFAs, progressively supplied through eight successive additions of these acids to the reactor. A maximum lipid content of 36.18% g/g cell was achieved at 61 g/L VFAs consumed and with an airflow rate of 2.0 vvm. Besides, a total of 88.4 mg/g cell and 759 mg/L β-carotene content and concentration were achieved at about 80 g/L VFAs consumed and with a 2.0 vvm airflow rate. It was concluded that; a non-limiting air flow favors the metabolic pathway for β-carotene formation against lipid accumulation.

Integrated fermentative process for lipid and β-carotene production from acetogenic syngas fermentation using an engineered oleaginous Yarrowia lipolytica yeast

An engineered Yarrowia lipolytica strain was successfully employed to produce β-carotene and lipids from acetic acid, a product of syngas fermentation by Clostridium aceticum. The strain showed acetic acid tolerance up to concentrations of 20 g/L. Flask experiments yielded a peak lipid content of 33.7 % and β-carotene concentration of 13.6 mg/g under specific nutrient conditions. The study also investigated pH effects on production in bioreactors, revealing optimal lipid and β-carotene contents at pH 6.0, reaching 22.9 % and 44 mg/g, respectively. Lipid profiles were consistent across experiments, with C18:1 being the dominant compound at approximately 50 %. This research underscores a green revolution in bioprocessing, showing how biocatalysts can convert syngas, a potentially polluting byproduct, into valuable β-carotene and lipids with a Y. lipolytica strain.

Biotransformation of protein-rich waste by Yarrowia lipolytica IPS21 to high-value products-amino acid supernatants.

The yeast strain Yarrowia lipolytica IPS 21 was tested for its ability to degrade potentially toxic chrome-tanned leather shavings (CTLS) in a liquid environment. Biological and chemical parameters were monitored during a 48-h period of biotransformation of the protein-rich waste. CTLS was added at a concentration of 0.1-4% (wt/wt) to a modified YPG medium (15 g L-1 yeast extract and 5 g L-1 NaCl). Biodegradation and bioconversion were performed in a one-step process. It was found that the higher degradation rate depended on the activity of the proteases and the pH of the medium, but not on the initial inoculum ratio and the activity of the dehydrogenase. The highest efficiency of the process was obtained for 4% (wt/wt) CTLS on day 2 (degradation rate 58-67%, biomass production 2.11-2.20 g L-1, protease activity 312 U mg-1 protein, and pH 9.20). Our results showed that total chromium was probably not transported across the cytoplasmic membrane of Y. lipolytica IPS21 and that chromium (III) was not oxidized to chromium (VI). The phytotoxicity of selected amino acid supernatants [2.5% (vol/vol)] was tested after the bioconversion process. It was found that the supernatants had a stimulating effect on the plants tested. The root elongation was 29-28% higher than that of the reference samples. This result makes Y. lipolytica IPS21 a potential candidate for safely converting potentially toxic protein-rich wastes into valuable products without enzyme isolation, e.g., amino acid fertilizers. IMPORTANCE Enzyme technologies have the greatest practical relevance to environmental trends. Overcoming the barrier of the high cost of carbon substrates used for biotransformation is the main challenge of these methods. The huge potential of the use of extracellular proteases of Yarrowia species or amino acids in various industries indicates the need for the extension of basic research on waste as a carbon source for this yeast. The experiments demonstrated that it is possible to use Y. lipolytica IPS21 for bioconversion of chrome-tanned leather shavings (CTLS) in a single-step process and to produce high-value amino acid supernatant without having an isolated enzyme. In our study, we show the effect of 2.5% (vol/vol) CTLS supernatant obtained from Y. lipolytica IPS21 on the elongation of the root system of selected plants and provide information on the effect of environmental factors on the efficiency of the bioconversion and the migration of chromium.

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 2: Citric Acid Production and Circular-Oriented Valorization of Glucose-Enriched Olive Mill Wastewaters Using Novel Yarrowia lipolytica Strains.

The global market for citric acid (CA) is one of the biggest and fastest expanding markets in the food industry. The CA production employing microbial bioprocessing with efficient GRAS strains and renewable waste streams is in line with the European Union binding targets for resource efficiency, sustainable consumption-production, and low-carbon technologies. In this work, the potential of three novel wild-type Yarrowia lipolytica strains (namely LMBF Y-46, LMBF Y-47 and ACA-YC 5033) regarding the production of CA and other valuable metabolites was tested on glucose-based media, and the most promising amongst the screened strains (viz. the strain ACA-YC 5033) was cultured on glucose-based media, in which part of the fermentation water had been replaced by olive-mill wastewaters (OMWs) in a novel approach of simultaneous OMW valorization and bioremediation. In the first part of this study, the mentioned strains were cultured under nitrogen-limited conditions with commercial (low-cost) glucose employed as a sole carbon source in shake-flask cultures at an initial concentration (S0) ≈ of 50 g/L. Variable quantities of secreted citric acid (CA) and intra-cellular compounds (viz. polysaccharides and lipids) were produced. All strains did not accumulate significantly high lipid quantities (i.e., maximum lipid in dry cell weight [DCW] values ≈30% w/w were noted) but produced variable CA quantities. The most promising strain, namely ACA-YC 5033, produced CA up to c. 24 g/L, with a yield of CA produced on glucose consumed (YCA/S) ≈ 0.45 g/g. This strain in stirred tank bioreactor experiments, at remarkably higher S0 concentrations (≈110 g/L) and the same initial nitrogen quantity added into the medium, produced notably higher CA quantities, up to 57 g/L (YCA/S ≈ 0.52 g/g). The potential of the same strain (ACA-YC 5033) to bioremediate OMWs and to produce value-added compounds, i.e., yeast cells, CA, and intra-cellular metabolites, was also assessed; under nitrogen-limited conditions in which OMWs had partially replaced tap water and significant glucose concentrations had been added (S0 ≈ 100 g/L, simultaneous molar ratio C/N ≈ 285 g/g, initial phenolic compounds [Phen0] adjusted to ≈1.0 g/L; these media were similar to the OMWs generated from the traditional press extraction systems) the notable CA quantity of 60.2 g/L with simultaneous YCA/S = 0.66 g/g, was obtained in shake flasks, together with satisfactory phenolic compounds removal (up to 19.5% w/w) and waste decolorization (up to 47.0%). Carbon-limited conditions with Phen0 ≈ 1.0 g/L favored the production of yeast DCW (up to 25.3 g/L), with equally simultaneous interesting phenolic compounds and color removal. The fatty acid profile showed that cellular lipids were highly unsaturated with oleic, linoleic and palmitoleic acids, accounting for more than 80% w/w. This study proposed an interesting approach that could efficiently address the biotreatment of toxic effluents and further convert them into circular-oriented bioproducts.

Techno-economic and environmental sustainability assessment of succinic acid production from municipal biowaste using an electrochemical membrane bioreactor

The techno-economic and environmental performance of succinic acid (SA) production by an engineering Yarrowia lipolytica strain has been evaluated in a process using the organic fraction of municipal solid waste (OFMSW) as feedstock and a novel electrochemical membrane bioreactor (EMB) for simultaneous SA production and extraction. The minimum selling price (MSP) of SA ($2.70–3.71/kg) considering 50,000 tSA/year production capacity using the EMB-based process was slightly lower than the conventional bioprocess depending on OFMSW management fees ($0–35/tOFMSW). Life Cycle Assessment was carried out for the EMB-based bioprocess and the conventional bioprocess considering either conventional electricity production mix (grid) or renewable electricity production from photovoltaics. The use of renewable electricity in the EMB-based bioprocess led to lower Global Warming Potential (0.81 kg CO2-eq/kgSA), Abiotic Depletion Potential (15.71 MJ/kgSA), Acidification Potential (1.87 kg SO2-eq/kgSA) and Eutrophication Potential (0.25 kg PO4-eq/kgSA) than the conventional bioprocess when renewable electricity was used, while higher Human Toxicity Potential (0.29 kg DCB-eq/kgSA) was observed due to the raw materials (e.g. heavy metals) and utilities (e.g. electricity) used in photovoltaics production. It has been demonstrated that the EMB-based system could improve the sustainability of SA production.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol

BackgroundYarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance.ResultsY. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9–20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation.ConclusionALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable “chassis” for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

Upgrading Major Waste Streams Derived from the Biodiesel Industry and Olive Mills via Microbial Bioprocessing with Non-Conventional Yarrowia lipolytica Strains

This study reports the development of a bioprocess involving the valorization of biodiesel-derived glycerol as the main carbon source for cell proliferation of Yarrowia lipolytica strains and production of metabolic compounds, i.e., citric acid (Cit), polyols, and other bio-metabolites, the substitution of process tap water with olive mill wastewater (OMW) in batch fermentations, and partial detoxification of OMW (up to 31.1% decolorization). Increasing initial phenolics (Phen) of OMW-glycerol blends led to substantial Cit secretion. Maximum Cit values, varying between 64.1–65.1 g/L, combined with high yield (YCit/S = 0.682–0.690 g Cit/g carbon sources) and productivity (0.335–0.344 g/L/h) were achieved in the presence of Phen = 3 g/L. The notable accumulation of endopolysaccharides (EPs) on the produced biomass was determined when Y. lipolytica LMBF Y-46 (51.9%) and ACA-YC 5033 (61.5%) were cultivated on glycerol-based media. Blending with various amounts of OMW negatively affected EPs and polyols biosynthesis. The ratio of mannitol:arabitol:erythritol was significantly affected (p &lt; 0.05) by the fermentation media. Erythritol was the major polyol in the absence of OMW (53.5–62.32%), while blends of OMW-glycerol (with Phen = 1–3 g/L) promoted mannitol production (54.5–76.6%). Nitrogen-limited conditions did not favor the production of cellular lipids (up to 16.6%). This study addressed sustainable management and resource efficiency enabling the bioconversion of high-organic-load and toxic waste streams into valuable products within a circular bioeconomy approach.

Modular Coculture to Reduce Substrate Competition and Off-Target Intermediates in Androstenedione Biosynthesis.

Substrate competition within a metabolic network constitutes a common challenge in microbial biosynthesis system engineering, especially if indispensable enzymes can produce multiple intermediates from a single substrate. Androstenedione (4AD) is a central intermediate in the production of a series of steroidal pharmaceuticals; however, its yield via the coexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17α-hydroxylase/17,20-lyase (CYP17A1) in a microbial chassis affords a nonlinear pathway in which these enzymes compete for substrates and produce structurally similar unwanted intermediates, thereby reducing 4AD yields. To avoid substrate competition, we split the competing 3β-HSD and CYP17A1 pathway components into two separate Yarrowia lipolytica strains to linearize the pathway. This spatial segregation increased substrate availability for 3β-HSD in the upstream strain, consequently decreasing the accumulation of the unwanted intermediate 17-hydroxypregnenolone (17OHP5) from 94.8 ± 4.4% in single-chassis monocultures to 24.8 ± 12.6% in cocultures of strains expressing 3β-HSD and CYP17A1 separately. Orthologue screening to increase CYP17A1 catalytic efficiency and the preferential production of desired intermediates increased the biotransformation capacity in the downstream pathway, further decreasing 17OHP5 accumulation to 3.9%. Furthermore, nitrogen limitation induced early 4AD accumulation (final titer, 7.71 mg/L). This study provides a framework for reducing intrapathway competition between essential enzymes during natural product biosynthesis as well as a proof-of-concept platform for linear steroid production.

Combination of a Push-Pull-Block Strategy with a Heterologous Xylose Assimilation Pathway toward Lipid Overproduction from Lignocellulose in Yarrowia lipolytica.

The production of biodiesel using microbial lipids derived from renewable lignocellulosic biomass is considered a promising strategy to reduce environmental pressure and promote the green energy transition. The hydrolysates of lignocellulosic biomass are rich in glucose and xylose, which makes it crucial to efficiently utilize both sugars. Here, we combined metabolic engineering and adaptive laboratory evolution (ALE) to construct an engineered Yarrowia lipolytica strain that can efficiently produce lipids from glucose and xylose. First, the "Push-Pull-Block" strategy was adopted to increase lipid content to 73.42% of the dry cell weight (DCW). Then, a heterologous xylose-utilization pathway was integrated into the engineered strain, which was subjected to ALE. The final evolved strain could accumulate 53.64% DCW of lipids from xylose, and the lipid titer reached 16.25 g/L. This work sheds light on the potential of microbial lipid overproduction from lignocellulose using engineered Y.lipolytica.

Evolving tolerance of Yarrowia lipolytica to hydrothermal liquefaction aqueous phase waste.

Hydrothermal liquefaction (HTL) is an emerging method for thermochemical conversion of wet organic waste and biomass into renewable biocrude. HTL also produces an aqueous phase (HTL-AP) side stream containing 2-4% light organic compounds that require treatment. Although anaerobic digestion (AD) of HTL-AP has shown promise, lengthy time periods were required for AD microbial communities to adapt to metabolic inhibitors in HTL-AP. An alternative for HTL-AP valorization was recently demonstrated using two engineered strains of Yarrowia lipolytica, E26 and Diploid TAL, for the overproduction of lipids and the polyketide triacetic acid lactone (TAL) respectively. These strains tolerated up to 10% HTL-AP (v/v) in defined media and up to 25% (v/v) HTL-AP in rich media. In this work, adaptive laboratory evolution (ALE) of these strains increased the bulk population tolerance for HTL-AP to up to 30% (v/v) in defined media and up to 35% (v/v) for individual isolates in rich media. The predominate organic acids within HTL-AP (acetic, butyric, and propionic) were rapidly consumed by the evolved Y. lipolytica strains. A TAL-producing isolate (strain 144-3) achieved a nearly 3-fold increase in TAL titer over the parent strain while simultaneously reducing the chemical oxygen demand (COD) of HTL-AP containing media. Fermentation with HTL-AP as the sole nutrient source demonstrated direct conversion of waste into TAL at 10% theoretical yield. Potential genetic mutations of evolved TAL production strains that could be imparting tolerance were explored. This work advances the potential of Y. lipolytica to biologically treat and simultaneously extract value from HTL wastewater. KEY POINTS: • Adaptive evolution of two Y. lipolytica strains enhanced their tolerance to waste. • Y. lipolytica reduces chemical oxygen demand in media containing waste. • Y. lipolytica can produce triacetic acid lactone directly from wastewater.

Comparative chemical and non-contact bioleaching of ion-adsorption type rare earth ore using ammonium sulfate and metabolites of Aspergillus niger and Yarrowia lipolytica to rationalise the role of organic acids for sustainable processing

Ion-adsorption type rare earth ore mainly contains medium and heavy REEs, which are regarded as strategic mineral resources. At present, leaching REEs with chemical reagents has been restricted, while bioleaching is better for environmental protection. It is also found that if strains directly act on ore for in situ leaching, the harsh conditions will adversely affect the growth of strains. Non-contact bioleaching can effectively solve this problem, however, it is rarely reported and compared with chemical leaching. In this study, taking Aspergillus niger and Yarrowia lipolytica as research objects, the feasibility of using the metabolites produced by two strains to leach REEs is determined, and the mechanism is investigated and compared with that of ammonium sulfate. In addition, this study is the first to leach REEs by the Yarrowia lipolytica strain under neutral conditions, while the Aspergillus niger strain is more adaptable to acidic environment. The experimental results showed that Aspergillus niger (glucose as the carbon source) reached the highest leaching efficiency of 91%, Aspergillus niger (sucrose as the carbon source) reached 66%, and Yarrowia lipolytica reached only 34%. Furthermore, both strains could produce different concentrations of organic acids that are effective for REE leaching. The obtained results are relevant for the future development of ion-adsorption type rare earth ore.

How do engineered Yarrowia lipolytica strains secrete free fatty acids: hints from comparative transcriptomics.

Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.