Yeast Yarrowia Lipolytica Research Articles

Agri-Food and Food Waste Lignocellulosic Materials for Lipase Immobilization as a Sustainable Source of Enzyme Support—A Comparative Study

Enzyme immobilization is a crucial method in biotechnology and organic chemistry that significantly improves the stability, reusability, and overall effectiveness of enzymes across various applications. Lipases are one of the most frequently applied enzymes in food. The current study investigated the potential of utilizing selected agri-food and waste materials—buckwheat husks, pea hulls, loofah sponges, and yerba mate waste—as carriers for the immobilization of Sustine® 121 lipase and Yarrowia lipolytica yeast biomass as whole-cell biocatalyst and lipase sources. Various lignocellulosic materials were pretreated through extraction processes, including Soxhlet extraction with hexane and ethanol, as well as alkaline and acid treatments for loofah sponges. The immobilization process involved adsorbing lipases or yeast cells onto the carriers and then evaluating their hydrolytic and synthetic activities. Preparations’ activities evaluation revealed that alkaline-pretreated loofah sponge yielded the highest hydrolytic activity (0.022 U/mg), while yerba mate leaves under brewing conditions demonstrated superior synthetic activity (0.51 U/mg). The findings underscore the potential of lignocellulosic materials from the agri-food industry as effective supports for enzyme immobilization, emphasizing the importance of material selection and pretreatment methods in optimizing enzymatic performance through giving an example of circular economy application in food processing and waste management.

Efficient Production of the Colorless Carotenoid Phytoene in Yarrowia lipolytica through Metabolic Engineering.

Phytoene, a colorless carotenoid with unique ultraviolet (UV)-B absorption properties, offers potential for applications in functional food, cosmetics, and therapeutics. However, their low natural yield poses a challenge for large-scale production. This study aims to enhance phytoene production in the oleaginous yeast Yarrowia lipolytica by introducing a heterologous phytoene synthase gene combined with metabolic engineering approaches. We enhanced phytoene synthesis by overexpressing key genes in the mevalonate pathway and compartmentalizing the biosynthetic pathway within peroxisomes. Moreover, we inhibited the glyoxylate cycle to increase the accumulation of peroxisomal acetyl-CoA available for phytoene production. Our engineered strains demonstrated a significant increase in phytoene production, reaching up to 1.34 g/L titer and 58.74 mg/gDCW yield in the flask-scale fed-batch culture, which are the highest levels reported to date. These results underscore the potential of Y. lipolytica as a robust platform for producing phytoenes and other terpenoids on an industrial scale, offering valuable insights for future efforts in metabolic engineering.

Potential role of alginate in marine bacteria-yeast interactions.

The ability of microorganisms to decompose brown algae has attracted attention. This study aims to clarify the characteristics of marine microbial communities in which prokaryotic and eukaryotic microorganisms interact via the metabolism of brown algae carbohydrates. Amplicon-based microbiome analysis revealed the predominance of the genera Marinomonas and Vibrio in seawater and seaweed samples mixed with alginate and mannitol, which are the primary carbohydrates in brown algae. Three Vibrio species and Candida intermedia were isolated via alginate enrichment culture. Although C. intermedia did not utilize alginate as a nutrient source, the yeast grew in the spent alginate medium in which Vibrio algivorus had been cultured. Coculture with C. intermedia and the Vibrio isolates, especially V. algivorus, also enhanced the growth of the yeast on alginate. These results suggested that C. intermedia grew because of the supply of nutrients via alginate metabolism by Vibrio species. In the coculture medium, the amount of phosphatidylserine increased in the early phase but decreased with the growth of C. intermedia, indicating that phosphatidylserine secreted by Vibrio is involved in the putative mutualistic interaction. We examined whether such interaction is applicable to the production of useful substances and succeeded in lipid production by oleaginous marine yeast Yarrowia lipolytica through coculture with V. algivorus. Our study suggested the potential of mutualistic interaction via degradation of alginate by marine Vibrio for production of industrially useful substances in yeast cells.IMPORTANCEIn this study, we analyzed the microbiome of seawater and seaweed in the presence of brown algae carbohydrates and reconstructed the putative mutualistic relationship of marine Vibrio and Candida intermedia mediated by metabolism of brown algae in the ocean.

Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Balanced Training Sets Improve Deep Learning-Based Prediction of CRISPR sgRNA Activity.

CRISPR-Cas systems have transformed the field of synthetic biology by providing a versatile method for genome editing. The efficiency of CRISPR systems is largely dependent on the sequence of the constituent sgRNA, necessitating the development of computational methods for designing active sgRNAs. While deep learning-based models have shown promise in predicting sgRNA activity, the accuracy of prediction is primarily governed by the data set used in model training. Here, we trained a convolutional neural network (CNN) model and a large language model (LLM) on balanced and imbalanced data sets generated from CRISPR-Cas12a screening data for the yeast Yarrowia lipolytica and evaluated their ability to predict high- and low-activity sgRNAs. We further tested whether prediction performance can be improved by training on imbalanced data sets augmented with synthetic sgRNAs. Lastly, we demonstrated that adding synthetic sgRNAs to inherently imbalanced CRISPR-Cas9 data sets from Y. lipolytica and Komagataella phaffii leads to improved performance in predicting sgRNA activity, thus underscoring the importance of employing balanced training sets for accurate sgRNA activity prediction.

The Key Enzymes of Carbon Metabolism and the Glutathione Antioxidant System Protect Yarrowia lipolytica Yeast Against pH-Induced Stress.

In this study, we first thoroughly assayed the response of the key enzymes of energy metabolism and the antioxidant system in Yarrowia lipolytica yeast at extreme pH. The activity of the tricarboxylic acid cycle enzymes, namely NAD-dependent isocitrate dehydrogenase, aconitate hydratase, NAD-dependent malate dehydrogenase, and fumarate hydratase, NADPH-producing enzymes of glucose-6-P dehydrogenase and NADP-dependent isocitrate dehydrogenase, and the enzymes of the glutathione system was assessed. All the enzymes that were tested showed a significant induction contrary to some decrease in the aconitate hydratase activity with acidic and alkaline stress. It is probable that a change in the enzyme activity in the mitochondria matrix is involved in the regulation of the cellular metabolism of Y. lipolytica, which allows the species to prosper at an extreme ambient pH. It distinguishes it from any other type of ascomycete. A close relationship between the induction of the Krebs cycle enzymes and the key enzymes of the glutathione system accompanied by an increased level of reduced glutathione was shown. The assumption that the increased activity of the Krebs cycle dehydrogenases and promotion of the pentose phosphate pathway at pH stress launches a set of events determining the adaptive response of Y. lipolytica yeast.

Boosting succinic acid production of Yarrowia lipolytica at low pH through enhancing product tolerance and glucose metabolism

BackgroundSuccinic acid (SA) is an important bio-based C4 platform chemical with versatile applications, including the production of 1,4-butanediol, tetrahydrofuran, and γ-butyrolactone. The non-conventional yeast Yarrowia lipolytica has garnered substantial interest as a robust cell factory for SA production at low pH. However, the high concentrations of SA, especially under acidic conditions, can impose significant stress on microbial cells, leading to reduced glucose metabolism viability and compromised production performance. Therefore, it is important to develop Y. lipolytica strains with enhanced SA tolerance for industrial-scale SA production.ResultsAn SA-tolerant Y. lipolytica strain E501 with improved SA production was obtained through adaptive laboratory evolution (ALE). In a 5-L bioreactor, the evolved strain E501 produced 89.62 g/L SA, representing a 7.2% increase over the starting strain Hi-SA2. Genome resequencing and transcriptome analysis identified a mutation in the 26S proteasome regulatory subunit Rpn1, as well as genes involved in transmembrane transport, which may be associated with enhanced SA tolerance. By further fine-tuning the glycolytic pathway flux, the highest SA titer of 112.54 g/L to date at low pH was achieved, with a yield of 0.67 g/g glucose and a productivity of 2.08 g/L/h.ConclusionThis study provided a robust engineered Y. lipolytica strain capable of efficiently producing SA at low pH, thereby reducing the cost of industrial SA fermentation.Graphical abstract

Synthesis of β-ionone from xylose and lignocellulosic hydrolysate in genetically engineered oleaginous yeast Yarrowia lipolytica.

β-ionone, an apocarotenoid derived from a C40 terpenoid has an intense, woody smell and a low odor threshold that has been widely used in as an ingredient in food and cosmetics. Yarrowia lipolytica is a promising host for β-ionone production because of its oleaginous nature, its ability to produce high levels of acetyl-CoA (an important precursor for terpenoids), and the availability of synthetic biology tools to engineer the organism. In this study, β-carotene-producing Y. lipolytica strain XK17 was employed for β-ionone biosynthesis. First, we explored the effect of different sources of carotenoid cleavage dioxygenase (CCD) genes on β-ionone production. A high-yielding strain rUinO-D14 with 122mg/L of β-ionone was obtained by screening promoters combined with rDNA mediated multi-round iterative transformations to optimize the expression of the CCD gene of Osmanthus fragrans. Second, to further develop a high-level production strain for β-ionone, we optimized key genes in the mevalonate pathway by multi-round iterative transformations mediated by non-homologous end joining, combined with a protein tagging strategy. Finally, the introduction of a heterologous oxidoreductase pathway enabled the engineered Y. lipolytica strain to use xylose as a sole carbon source and produce β-ionone. In addition, the potential for use of lignocellulosic hydrolysate as the carbon source for β-ionone production showed that the NHA-A31 strain had a high β-ionone productivity level. This study demonstrates that engineered Y. lipolytica can be used for the efficient, green and sustainable production of β-ionone.

Developing polycistronic expression tool in Yarrowia lipolytica

Unconventional oleaginous yeast Yarrowia lipolytica has gained widespread applications as a microbial cell factory for synthesizing various chemicals and natural products. The construction of efficient cell factories requires intricate metabolic engineering. However, multi-gene expression in Y. lipolytica is labor-intensive. To facilitate multi-gene expression, we developed the polycistronic expression tool using 2A peptides. We first compared different 2A peptides in Y. lipolytica and identified two 2A peptides with high cleavage efficiency: P2A and ERBV-1. The effect of 2A peptides on the expression level of upstream and downstream genes was then determined. Ultimately, we applied the identified 2A peptides to express four genes in canthaxanthin biosynthetic pathway within one expression cassette for canthaxanthin production. This study enriches the multi-gene expression tools of Y. lipolytica, which will facilitate the cell factory construction of Y. lipolytica.

An Inducible Whole-Cell Biosensor for Detection of Formate Ions

Ten strains of the yeast Yarrowia lipolytica were constructed, the genomes of which contain the hrGFP gene under regulation of the formate dehydrogenase promoters. The resulting strains can act as whole-cell biosensors for the detection of formate ions in various media. By visual assessment of the biomass fluorescence, we selected the three most promising yeast strains. The main biosensor characteristics (threshold sensitivity, amplitude, and response time) of the selected strains were measured. As a result, in terms of characteristics, the B26 strain was recognized as the most suitable for the detection of formate ions. A carbon source for the nutrient medium that does not reduce the activation of the biosensor was selected. Furthermore, we showed that unlike formate and formaldehyde, methanol practically does not induce the biosensor fluorescence response.

Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of ∼50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.

Combinatorial iterative method for metabolic engineering of Yarrowia lipolytica: Application for betanin biosynthesis

Combinatorial library-based metabolic engineering approaches allow lower cost and faster strain development. We developed a genetic toolbox EXPRESSYALI for combinatorial engineering of the oleaginous yeast Yarrowia lipolytica. The toolbox enables consecutive rounds of engineering, where up to three combinatorially assembled gene expression cassettes can be integrated into each yeast clone per round. The cassettes are integrated into distinct intergenic sites or an open reading frame of a target gene if a simultaneous gene knockout is desired. We demonstrate the application of the toolbox by optimizing the Y. lipolytica to produce the red beet color betanin via six consecutive rounds of genome editing and screening. The library size varied between 24 and 360. Library screening was facilitated by automated color-based colony picking. In the first round, betanin pathway genes were integrated, resulting in betanin titer of around 20 mg/L. Through the following five consecutive rounds, additional biosynthetic genes were integrated, and the precursor supply was optimized, resulting in a titer of 70 mg/L. Three beta-glucosidases were deleted to prevent betanin deglycosylation, which led to a betanin titer of 130 mg/L in a small scale and a titer of 1.4 g/L in fed-batch bioreactors. The EXPRESSYALI toolbox can facilitate metabolic engineering efforts in Y. lipolytica (available via AddGene Cat. Nr. 212682–212704, Addgene kit ID # 1000000245).

Dynamically Reconfigurable Micro-Patterned Hydrogels Based on Magnetic Pickering Emulsion Droplets.

Reconfigurability within hydrogels has emerged as an attractive functionality that can be used in information encryption, cargo/delivery, environmental remediation, soft robotics, and medicine. Here micro-patterned polymer hydrogels capable of temperature-dependent reconfigurability are fabricated. For this, the hydrogels are provided with micron-sized Pickering emulsion droplets stabilized by magnetic particles, which are capable of harnessing energy from external force fields. The droplets can both migrate under magnetic field gradients and heat the environment when laser irradiated. These functions not only affect a single compartment but have higher-order effects on the mesoscale, thanks to the temperature-responsiveness of the polymeric network. This double responsiveness is exploited to control the spatial organization of hundreds of droplets within the hydrogel matrix and form predesigned and sophisticated patterns. Furthermore, pattern self-reconfiguration driven by the droplets themselves upon laser irradiation is induced. Finally, we show that due to their internal liquid phase, the droplets can be used as reservoirs of hydrophobic nutrients for living cells (i.e., Yarrowia lipolytica yeast) in the solid-like environment of the polymeric network, and demonstrate communication between the droplets and the cells to facilitate nutrient uptake. Altogether, the results provide opportunities for the development of stimuli-sensitive polymer hydrogels with post-synthesis reprogrammable response using micro-compartments as building blocks.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction

The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROGfbr and EcPheAfbr increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT, ylARO1, and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/gglu, which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semipilot scale.

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD+-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.

Efficient synthesis of limonene production in Yarrowia lipolytica by combinatorial engineering strategies

BackgroundLimonene has a variety of applications in the foods, cosmetics, pharmaceuticals, biomaterials, and biofuels industries. In order to meet the growing demand for sustainable production of limonene at industry scale, it is essential to find an alternative production system to traditional plant extraction. A promising and eco-friendly alternative is the use of microbes as cell factories for the synthesis of limonene.ResultsIn this study, the oleaginous yeast Yarrowia lipolytica has been engineered to produce d- and l-limonene. Four target genes, l- or d-LS (limonene synthase), HMG (HMG-CoA reductase), ERG20 (geranyl diphosphate synthase), and NDPS1 (neryl diphosphate) were expressed individually or fused together to find the optimal combination for higher limonene production. The strain expressing HMGR and the fusion protein ERG20-LS was the best limonene producer and, therefore, selected for further improvement. By increasing the expression of target genes and optimizing initial OD, 29.4 mg/L of l-limonene and 24.8 mg/L of d-limonene were obtained. We also studied whether peroxisomal compartmentalization of the synthesis pathway was beneficial for limonene production. The introduction of d-LS and ERG20 within the peroxisome improved limonene titers over cytosolic expression. Then, the entire MVA pathway was targeted to the peroxisome to improve precursor supply, which increased d-limonene production to 47.8 mg/L. Finally, through the optimization of fermentation conditions, d-limonene production titer reached 69.3 mg/L.ConclusionsIn this work, Y. lipolytica was successfully engineered to produce limonene. Our results showed that higher production of limonene was achieved when the synthesis pathway was targeted to the peroxisome, which indicates that this organelle can favor the bioproduction of terpenes in yeasts. This study opens new avenues for the efficient synthesis of valuable monoterpenes in Y. lipolytica.

Bioreduction of 4'-Hydroxychalcone in Deep Eutectic Solvents: Optimization and Efficacy with Various Yeast Strains.

4'-dihydrochalcones are secondary metabolites isolated from many medicinal plants and from the resin known as 'dragon's blood'. Due to their biological potential, our research objective was to determine the possibilities of using biocatalysis processes carried out in deep eutectic solvents (DESs) to obtain 4'-dihydrochalcones as a model compound. The processes were carried out in a culture of the yeast Yarrowia lipolytica KCh 71 and also in cultures of strains of the genera Rhodotorula and Debaryomyces. Based on the experiments carried out, an optimum process temperature of 35 °C was chosen, and the most suitable DES contained glycerol as a hydrogen bond donor (HBD). For a medium with 30% water content (DES 11), the conversion observed after 24 h exceeded 70%, while increasing the amount of water to 50% resulted in a similar level of conversion after just 1 h. A fivefold increase in the amount of added substrate resulted in a reduction in conversion, which reached 30.3%. Of the other yeast strains tested, Rhodotorula marina KCh 77 and Rhodotorula rubra KCh 4 also proved to be good biocatalysts for the bioreduction process. For these strains, the conversion reached 95.4% and 95.1%, respectively. These findings highlight the potential of yeast as a biocatalyst for the selective reduction of α,β-unsaturated ketones and the possibility of using a DESs as a reaction medium in this process.

Broadening the application of Yarrowia lipolytica synthetic biology tools to explore the potential of Yarrowia clade diversity.

Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the Golden Gate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least five members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the uncharacterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.

Streamlining biological recycling of poly(ethylene terephthalate) via pre-treatment methods

In this study, we validated various methods of pre-treatment of poly(ethylene terephthalate) (PET) by the engineered yeast Yarrowia lipolytica. This research compares both the effect of the type of plastic used, the processing method and enzymes with different mechanisms of action (PETase and cutinase). The investigation demonstrated that the degradation efficiency varies depending on the type of plastic used, the processing methods and the applied enzyme. Moreover, it indicated that during prolonged yeast culture under the applied conditions, enzyme activity is not impaired. Among all the methods tested, the artificial aging process had the greatest impact on the degradation level by PETase, where the amount of TPA released from commercial PET film was the highest, and yielded over 2 gL-1. The maximum yield of TPA (0.59 gL-1), for the Y. lipolytica strain overexpressing cutinase, was observed during the process with recycled PET bottles shredded into 1 mm fragments. The maximum recorded weight loss of plastic film is over 70% for commercial PET film subjected to artificial ageing process.