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Succinic acid is an essential bulk chemical with wide-ranging applications in materials, food, and pharmaceuticals. With the advancement of biotechnology, there has been a surge in focus on low-carbon sustainable microbial synthesis methods for producing biobased succinic acid. Due to its high intrinsic acid tolerance, Yarrowia lipolytica has gained recognition as a competitive chassis for the industrial manufacture of succinic acid. This review summarizes the research progress on succinic acid biomanufacturing using Y. lipolytica . First, it introduces the major metabolic routes for succinic acid biosynthesis and the pertinent engineering approaches for building efficient cell factories. Subsequently, we offer a review of methods employed for succinic acid synthesis by Y. lipolytica utilizing alternative substrates as well as the relevant optimization strategies for the fermentation process. Finally, future research directions for improving succinic acid biomanufacturing in Y. lipolytica are delineated in light of the recent progress, obstacles, and trends in this area.

7-Dehydrocholesterol (7-DHC) is an important precursor of vitamin D 3 . The microbial synthesis of 7-DHC has attracted substantial attention. In this study, multiple strategies were developed to create a sustainable green route for enhancing 7-DHC yield from kitchen waste by engineered Yarrowia lipolytica . Y. lipolytica strains were engineered and combined with various Δ 24 -dehydrocholesterol reductases. Overexpressing all the genes in the mevalonate pathway improved the precursor pool, increasing the 7-DHC titer from 21.8 to 145.6 mg/L. Additionally, optimizing medium components using the response surface method significantly raised the 7-DHC titer to 391.0 mg/L after shake flask cultivation. The engineered strain yielded a record 7-DHC titer of 3.5 g/L in a 5-L bioreactor when kitchen waste was used as a carbon source. Overall, these results demonstrate that engineered Y. lipolytica efficiently synthesizes 7-DHC from waste lipid feedstock, offering a promising route for its bioproduction.

Utilization of microbial hosts to produce natural plant products is regarded as a promising and sustainable approach. However, achieving highly efficient production of terpenoids using microorganisms remains a significant challenge. Here, mevalonate, a building block of terpenoids, was used as a demo product to explore the potential metabolic constraints for terpenoid biosynthesis in Yarrowia lipolytica . First, by regulation of the expression of ERG12 and HMGR , the mevalonate titer was improved by 7660%. Subsequently, the native mevalonate pathway (MVA pathway) was enhanced, and the production of mevalonate increased to 4.16 g/L. To ensure a sufficient supply of acetyl-CoA, the citrate route and TCA cycle were simultaneously engineered, and the mevalonate titer was further improved to 5.25 g/L in shake flasks. Ultimately, the citrate overflow metabolism of Y. lipolytica was eliminated by deleting CEX1 , resulting in the highest mevalonate titer of 101 g/L with a yield of 0.255 g/g of glucose in eukaryotes. These insights could be applied to the effective production of terpenoids and biochemicals derived from central carbon metabolic pathways.

Background: Crocetin is a multifunctional apocarotenoid natural product derived from saffron, holding significant promises for protection against various diseases and other nutritional applications. Historically, crocetin has been extracted from saffron stigmas, but this method is hindered by the limited availability of high-quality raw materials and complex extraction processes. To overcome these challenges, metabolic engineering and synthetic biology can be applied to the sustainable production of crocetin., Results: We constructed a Yarrowia lipolytica strain using hybrid promoters and copy number adjustment, which was able to produce 2.66 g/L of β-carotene, the precursor of crocetin. Next, the crocetin biosynthetic pathway was introduced, and we observed both the production and secretion of crocetin. Subsequently, the metabolite profiles under varied temperatures were studied and we found that low temperature was favorable for crocetin biosynthesis in Y. lipolytica. Therefore, a two-step temperature-shift fermentation strategy was adopted to optimize yeast growth and biosynthetic enzyme activity, bringing a 2.3-fold increase in crocetin titer. Lastly, fermentation media was fine-tuned for an optimal crocetin output of 30.17 mg/L, bringing a 51% higher titer compared with the previous highest report in shake flasks. Concomitantly, we also generated Y. lipolytica strains capable of achieving substantial zeaxanthin production, yielding 1575.09 mg/L, doubling the previous highest reported titer., Conclusions: Through metabolic engineering and fermentation optimization, we demonstrated the first de novo biosynthesis of crocetin in the industrial yeast Yarrowia lipolytica. In addition, we achieved a higher crocetin titer in flasks than all our known reports. This work not only represents a high production of crocetin, but also entails a significant simultaneous zeaxanthin production, setting the stage for sustainable and cost-effective production of these valuable compounds., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast Yarrowia lipolytica by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the SUR2 gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation. The Y. lipolytica SUR2 deletion (Ylsur2Δ) strain displayed retarded growth, increased pseudohyphal formation and stress sensitivity, along with the altered profiles of inositolphosphate-containing ceramides, GlcCers, and sterols. The subsequent disruption of the SLD1 gene, encoding a fungal/plant-specific Δ8 sphingolipid desaturase, restored filamentous growth in the Ylsur2Δ strain to a yeast-type form and further increased the production of human-type GlcCers. Additional introduction of mouse alkaline ceramidase 1 (maCER1) into the Ylsur2Δsld1Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the Ylsur2Δsld1Δ/maCER1 strain increased in proportion to the C/N ratio in the N-source optimized medium, leading to production of 1.4 g/L non-acetylated DHS at the 5 L fed-batch fermentation with glucose feeding. This study highlights the feasibility of using the engineered Y. lipolytica strains as a cell factory for valuable sphingolipid derivatives for pharmaceuticals, cosmeceuticals, and nutraceuticals., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sakuranetin, a flavonoid phytoalexin, has demonstrated neuroprotective properties and exhibits tyrosinase inhibitory activities, making it highly valuable in the cosmetics and pharmaceutical industries. In this study, we engineered a Yarrowia lipolytica strain for the high-titer de novo production of sakuranetin using glucose as a substrate. To effectively enhance sakuranetin production, we implemented a multimodule engineering strategy that included optimizing the sakuranetin synthesis pathway, designing a regeneration system for the methyl donor S-adenosyl methionine, increasing the malonyl-CoA precursor supplement, and constructing the feedback inhibition-relieved shikimate pathway. Moreover, a transcriptomic analysis was conducted to identify potential targets for further improving sakuranetin synthesis. As a result, the titer of de novo synthesized sakuranetin reached 344.0 mg/L from glucose in a 5 L bioreactor. These achievements hold significant promise for the sustainable and large-scale production of sakuranetin through industrial biomanufacturing.

This study explored the genomic alterations in Yarrowia lipolytica , a key yeast in industrial biotechnology, under both spontaneous and mutagen-induced conditions. Our findings reveal that spontaneous mutations occur at a rate of approximately 4 × 10 -10 events per base pair per cell division, primarily manifesting as single-nucleotide variations (SNVs) and small insertions and deletions (InDels). Notably, C-to-T/G-to-A transitions and C-to-A/G-to-T transversions dominate the spontaneous SNVs, while 1 bp deletions, likely resulting from template slippage, are the most frequent InDels. Furthermore, chromosomal aneuploidy and rearrangements occur, albeit at a lower frequency. Exposure to ultraviolet (UV) light, methylmethane sulfonate (MMS), and Zeocin significantly enhances the rates of SNVs and alters their mutational spectra in distinct patterns. Notably, Zeocin-induced SNVs are predominantly T-to-A and T-to-G substitutions, often occurring within the 5'-TGT * -3' motif ( * denotes the mutated base). Additionally, Zeocin exhibits a higher potency in stimulating InDels compared to UV and MMS. Translesion DNA synthesis is implicated as the primary mechanism behind most Zeocin-induced SNVs and some InDels, whereas non-homologous end joining serves as the main pathway for Zeocin-mediated InDels. Intriguingly, the study identifies the gene YALI1_E21053g , encoding a protein kinase, as negatively associated with Zeocin resistance. Overall, our results not only deepened our knowledge about the genome evolution in Y. lipolytica but also provided reference to develop innovative strategies to harness its genetic potential.IMPORTANCE Yarrowia lipolytica exhibits high environmental stress tolerance and lipid metabolism capabilities, making it a microorganism with significant industrial application potential. In this study, we investigated the genomic variation and evolutionary patterns of this yeast under both spontaneous and induced mutation conditions. Our results reveal distinctive mutation spectra induced by different mutagenic conditions and elucidate the underlying genetic mechanisms. We further highlight the roles of non-homologous end joining and translesion synthesis pathways in Zeocin-induced mutations, demonstrating that such treatments can rapidly confer drug resistance to the cells. Overall, our research enhances the understanding of how yeast genomes evolve under various conditions and provides guidance for developing more effective mutagenesis and breeding techniques.

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., Competing Interests: The authors declare no conflict of interest.

Taxifolin, also known as dihydroquercetin (DHQ), is a flavonoid recognized for its potent antioxidant properties and a wide range of biological activities, including anti-tumor, antiviral, and immunomodulatory effects. Conventional extraction and chemical synthesis methods for taxifolin are often limited by low yields and associated environmental concerns. In this study, we investigated the heterologous biosynthesis of taxifolin in Yarrowia lipolytica through a combination of metabolic engineering and genome-scale metabolic modeling (GSM), complemented by flux balance analysis (FBA). We engineered Yarrowia lipolytica by introducing key biosynthetic genes and successfully synthesized taxifolin using naringenin (NAR) as a substrate, chosen for its low cost. Fermentation experiments demonstrated an optimal taxifolin yield of 10% at a substrate concentration of 200 mg/L naringenin, with a maximum yield of 26.4 mg/L taxifolin at 1 g/L naringenin. To further enhance production, we applied a marker-free Cre-loxP-based gene integration method, allowing stable genomic integration of key genes, which increased taxifolin yield to 34.9 mg/L at 1 g/L naringenin. Additionally, intermediate metabolites eriodictyol (ERI) and dihydrokaempferol (DHK) accumulated to concentrations of 89.2 mg/L and 21.7 mg/L, respectively. Furthermore, we integrated metabolic data into a GSM and applied FBA to optimize the taxifolin biosynthetic pathway. Through Pareto frontier analysis, sensitivity analysis, flux variability analysis, and single gene deletion simulations, we identified key genetic modifications that significantly enhanced taxifolin yield. Overexpression of GND1 and IDP2 increased yields by 94% and 155%, respectively, while knockout of LIP2 led to a 46% increase. Using tri-baffled shake flasks to improve oxygen supply resulted in a 120% yield increase, whereas YPG medium decreased yield by 59%, validating our model's accuracy. To ensure stable and efficient gene expression, we integrated multi-copy constructs into the ribosomal DNA (rDNA) locus of Yarrowia lipolytica, doubling taxifolin production. These results demonstrate the effectiveness of GSM and FBA in addressing bottlenecks in microbial taxifolin biosynthesis and provide a basis for future optimization and large-scale production., Competing Interests: Declarations. Ethics Approval: The present research study does not involve any human participants, their data, or biological material. Consent to Participate: Not applicable. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 122 mg/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., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The oleaginous yeast Yarrowia lipolytica represents a potential microbial cell factory for the recombinant production of various valuable products. Currently, the commonly used selection markers for transformation in Y. lipolytica are limited, and successive genetic manipulations are often restricted by the number of available selection markers. In our study, we developed a dominant marker, dsdA, which encodes a D-serine deaminase for genetic manipulation in Y. lipolytica. In Y. lipolytica, this marker confers the ability to use D-serine as a nitrogen source. In addition, the selection conditions of several infrequently used dominant markers including bleoR (zeocin resistance), kanMX (G418 resistance), and guaB (mycophenolic acid resistance) were also analyzed. Our results demonstrated that these selection markers can be used for the genetic manipulation of Y. lipolytica and their selection conditions were different for various strains. Ultimately, the selection markers tested here will be useful to expand the genetic toolbox of Y. lipolytica. KEY POINTS: • The dsdA from Escherichia coli was developed as a dominant marker. • The applicability of several resistance markers in Y. lipolytica was determined. • We introduced the Cre/mutant lox system for marker recycling., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lipases are widely used in the modification of functional lipids, particularly in the enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In this study, a lipase named OUC-Sb-lip2 was expressed in Yarrowia lipolytica, achieving a promising enzyme activity of 472.6 U/mL by optimizing the culture medium, notably through olive oil supplementation. A significant proportion (58.8%) of the lipase activity was located in the cells, whereas 41.2% was secreted into the supernatant. Both whole-cell and immobilized OUC-Sb-lip2 were used to enrich DHA and EPA from fish oil. The whole-cell approach increased the DHA and EPA contents to 2.59 and 2.55 times that of the original oil, respectively. Similarly, the immobilized OUC-Sb-lip2 resulted in a 2.00-fold increase in DHA and an 1.99-fold increase in EPA after a 6-h hydrolysis period. Whole cell and the immobilized OUC-Sb-lip2 retained 48.7% and 52.7% of their activity after six cycles of reuse, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Polyphenols are powerful natural antioxidants with numerous biological activities. They change cell membrane permeability, interact with receptors, intracellular enzymes, and cell membrane transporters, and quench reactive oxygen species (ROS). Yarrowia lipolytica yeast, being similar to mammalian cells, can be used as a model to study their survival ability upon long-lasting cultivation, assaying the effect of dihydroquercetin polyphenol (DHQ). The complex assessment of the physiological features of the population assaying cell respiration, survival, ROS detection, and flow cytometry was used. Y. lipolytica showed signs of chronological aging by eight weeks of growth, namely a decrease in the cell number, and size, increased ROS generation, a decrease in colony-forming unit (CFU) and metabolic activity, and decreased respiratory rate and membrane potential. An amount of 150 µM DHQ decreased ROS generation at the 6-week growth stage upon adding an oxidant of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Moreover, it decreased CFU at 1-4 weeks of cultivation, inhibited cell metabolic activity of the 24-h-old culture and stimulated that on 14-56 days of growth, induced the cell respiration rate in the 24-h-old culture, and blocked alternative mitochondrial oxidase at growth late stages. DHQ serves as a mild pro-oxidant on the first day of age-stimulating anti-stress protection. In the deep stationary stage, it can act as a powerful antioxidant, stabilizing cell redox status and reducing free radical oxidation in mitochondria. It provides a stable state of population. The hormetic effects of DHQ using lower eukaryotes of Y. lipolytica have been previously discussed, which can be used as a model organism for screening geroprotective compounds of natural origin.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica , an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

This study evaluated the potential of Y. lipolytica (CBS 2075 and DSM 8218) to grow in waste motor oil (WMO) and produce valuable compounds, laying the foundation for a sustainable approach to WMO management. Firstly, yeast strains were screened for their growth on WMO (2-10 g·L -1 ) in microplate cultures. Despite limited growth, the CBS 2075 strain exhibited comparable growth to control conditions (without WMO), while DSM 8218 growth increased 2- and 3-fold at 5 g·L -1 and 10 g·L -1 WMO, respectively. The batch cultures in the bioreactor confirmed the best performance of DSM 8218. A two-stage fed-batch strategy-growth phase in aliphatic hydrocarbons, followed by the addition of WMO (one pulse of 5 g·L -1 or five pulses of 1 g·L -1 WMO), significantly increased biomass production and WMO assimilation by both strains. In experiments with five pulses, CBS 2075 and DSM 8218 strains reached high proteolytic activities (593-628 U·L -1 ) and accumulated high quantities of intracellular lipids (1.3-1.7 g·L -1 ). Yeast lipids, mainly composed of oleic and linoleic acids with an unsaturated/saturated fraction > 59%, meet the EU biodiesel standard EN 14214, making them suitable for biodiesel production.

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., (© 2024. The Author(s).)

Although less studied than bacterial biota, the presence of yeast during the artisanal cheese's production is of fundamental importance. Yeasts can prevent the growth of undesirable species in the cheese's core and surface and actively participate in the aromatic development of these products. On the other hand, reintroducing the most abundant yeast species can help mitigate potential health risks related to artisanal production. The main objective of this study was to carry out an extensive mapping of the cultivable yeast diversity present in the central Brazilian artisanal cheeses (n = 582 cheese samples). Torulasporadelbrueckii, Candidaparapsilosis, Candidazeylanoides, Yarrowialipolytica, Debaryomycesprosopidis, and Kluyveromyceslactis were amongst the 300 clusters detected, respectively. In Canastra, Cerrado, and Serro-type cheeses, the predominant species was T.delbrueckii, while in Coalho and Manteiga cheeses, Y.lipolytica was the main species detected. The findings of this study reveal the yeast cultivable diversity present in a large set of Brazilian artisanal cheeses. Furthermore, these data can serve as a basis for promoting new Brazilian origin designation policies based on microbiological data., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Omega fatty acids are important for human health. They are traditionally extracted from animals or plants but can be alternatively produced using oleaginous yeast. Current efforts are producing yeast strains with similar fatty acid distributions and powerful lipogenesis capacity. The next step is to further make the process more competitive., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters., (© 2024 American Institute of Chemical Engineers.)

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry., (© 2024 American Institute of Chemical Engineers.)

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 EcAROG fbr and EcPheA fbr 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/g glu , 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Background: Succinic 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., Results: An 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., Conclusion: This study provided a robust engineered Y. lipolytica strain capable of efficiently producing SA at low pH, thereby reducing the cost of industrial SA fermentation., (© 2024. The Author(s).)

Abscisic acid (ABA) is an important phytohormone with diverse applications. It currently relies on the fermentation of Botrytis cinerea , which suffers from limited availability of genetic engineering tools. Here, Yarrowia lipolytica was engineered to enable de novo biosynthesis of ABA. To overcome the rate-limiting P450 enzymes, systematic engineering strategies were implemented. Firstly, the dissolved oxygen was increased to boost the activity of P450 enzymes. Secondly, the expansion of endoplasmic reticulum was implemented to improve the functional expression of P450 enzymes. Lastly, rate-limiting enzymes were assembled to facilitate substrate trafficking. Moreover, ABA production was further improved by strengthening the mevalonate pathway. Finally, the engineered strain produced 1221.45 mg/L of ABA in a 5-L bioreactor. The study provides effective approaches for alleviating rate-limiting P450 enzymes to enhance ABA production and achieve competitive industrial-level ABA production in Y. lipolytica ., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Systematic metabolic engineering of Yarrowia lipolytica for efficient production of phytohormone abscisic acid”., (© 2024 The Authors.)

Astaxanthin, a lipophilic carotenoid renowned for its strong antioxidant activity, holds significant commercial value across industries such as feed, food, and cosmetics. Although astaxanthin can be synthesized through chemical methods, it may contain toxic by-products in the synthesized astaxanthin, limiting its application in medicine or functional food. Natural astaxanthin can be extracted from algae, however, the cultivation cycle of algae is relatively longer compared to microorganisms. With the advancement of synthetic biology and metabolic engineering, the method of microbial fermentation has emerged as a promising strategy for the large-scale production of astaxanthin. This article provides a comprehensive overview of the research progress in astaxanthin biosynthesis, highlighting the use of the natural host Xanthophyllomyces dendrorhous, and the heterologous hosts Yarrowia lipolytica and Saccharomyces cerevisiae. Additionally, future research prospects are also discussed., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Engineering the glycerol-3-phosphate pathway could enhance erythritol production by accelerating glycerol uptake. However, little work has been conducted on the alternative dihydroxyacetone (DHA) pathway in Yarrowia lipolytica. Herein, this route was identified and characterized in Y. lipolytica by metabolomic and transcriptomic analysis. Moreover, the reaction catalyzed by dihydroxyacetone kinase encoded by dak2 was identified as the rate-limiting step. By combining NHEJ-mediated insertion mutagenesis with a push-and-pull strategy, Y. lipolytica strains with high-yield erythritol synthesis from glycerol were obtained. Screening of a library of insertion mutants allows the identification of a mutant with fourfold increased erythritol production. Overexpression of DAK2 and glycerol dehydrogenase GCY3 together with gene encoding transketolase and transaldolase from the nonoxidative part of the pentose phosphate pathway led to a strain with further increased productivity with a titer of 53.1 g/L and a yield 0.56 g/g glycerol, which were 8.1- and 4.2-fold of starting strain., (© 2024 John Wiley & Sons Ltd.)

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ER K26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced K m values and increased k cat values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO 2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce β-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum β-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO 2 and light through co-culture systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 ., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana , but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L -1 of each alkane (dodecane and hexadecane), and 2 g·L -1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L -1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L -1 ) and lipids concentrations of 3.4 g·L -1 and 4.3 g·L -1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica., (© 2024. The Author(s).)

With the increasing demand for heavy metals due to the advancement of industrial activities, large proportions of heavy metals have been discharged into aquatic ecosystems, causing serious harm to human health and the environment. Existing physical and chemical methods for recovering heavy metals from wastewater encounter challenges, such as low efficiency, high processing costs, and potential secondary pollution. In this study, we developed a novel approach by engineering the endogenous sulphur metabolic pathway of Yarrowia lipolytica, providing it with the ability to produce approximately 550 ppm of sulphide. Subsequently, sulphide-producing Y. lipolytica was used for the first time in heavy metal remediation. The engineered strain exhibited a high capacity to remove various heavy metals, especially achieving over 90 % for cadmium (Cd), copper (Cu) and lead (Pb). This capacity was consistent when applied to both synthetic and actual wastewater samples. Microscopic analyses revealed that sulphide-mediated biological precipitation of metal sulphides on the cell surface is responsible for their removal. Our findings demonstrate that sulphide-producing yeasts are a robust and effective bioremediation strategy for heavy metals, showing great potential for future heavy metal pollution remediation practices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Numerous works have reported that magnetic fields serve as signals capable of influencing microbial metabolism. However, little is known about the effect of magnetic field on erythritol production by the model microorganism Yarrowia lipolytica (Y. lipolytica). Therefore, we investigated the effect of low-frequency alternating magnetic fields (LF-AMF) with different magnetic field intensities (0-1.5 mT) and different magnetic field treatment times (1-10 days) on the production of erythritol by Y. lipolytica -JZ204. The optimal treatment condition was 0.5 mT for 8 days. As a result, a maximal erythritol yield was achieved 63.74 g/L, the biomass was reached 37 g/L, and the specific erythritol yield per unit of biomass was 1.7227 g/g, which were 60.72%, 32.09%, and 24.85% higher than the control, respectively. We investigated the internal mechanism of magnetic fields impact by using transcriptomics and RT-qPCR technology. This study demonstrated the effectiveness of LF-AMF in enhancing erythritol production by Y. lipolytica JZ-204, providing insights for the application of magnetic field in assisting microbial fermentation and improving the synthesis of beneficial products., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13 C-metabolic flux analysis ( 13 C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13 C-MFA model construction and to validate the 13 C-MFA results. The 13 C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO 2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from β-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Yarrowia lipolytica is a well-characterized yeast with remarkable metabolic adaptability. It is capable of producing various products from different carbon sources and easily switching between planktonic and biofilm states. A biofilm represents a natural means of cell immobilization that could support continuous cultivation and production processes, such as perfusion cultivation. However, the metabolic activities of Y. lipolytica in biofilms have not yet been studied in detail. Therefore, this study aimed to compare the metabolic activities of Y. lipolytica in biofilm and planktonic states. Conventionally, a stirred tank bioreactor was used to cultivate Y. lipolytica in a planktonic state. On the other hand, a trickle bed bioreactor system was used for biofilm cultivation. The low pH at 3 was maintained to favor polyol production. The accumulation of citric acid was observed over time only in the biofilm state, which significantly differed from the planktonic state. Although the biofilm cultivation process has lower productivity, it has been observed that the production rate remains constant and the total product yield is comparable to the planktonic state when supplied with 42% oxygen-enriched air. This finding indicates that the biofilm state has the potential for continuous bioprocessing applications and is possibly a feasible option., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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 ( M orphology and n -alkane A dsorption R egulator 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.IMPORTANCE Yarrowia 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., Competing Interests: The authors declare no conflict of interest.

Background: 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., Results: 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 ., Conclusion: 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., (© 2024 Society of Chemical Industry.)

Research on the recovery of rare earth elements from wastewater has attracted increasing attention. Compared with other methods, biosorption is a simple, efficient, and environmentally friendly method for rare earth wastewater treatment, which has greater prospects for development. The objective of this study was to investigate the biosorption behavior and mechanism of Yarrowia lipolytica for five rare earth ions (La 3 ⁺, Nd 3 ⁺, Er 3 ⁺, Y 3 ⁺, and Sm 3 ⁺) with a particular focus on biosorption behavior, biosorption kinetics, and biosorption isotherm. It was demonstrated that the biosorption capacity of Y. lipolytica at optimal conditions was 76.80 mg/g. It was discovered that the biosorption process complied with the pseudo-second-order kinetic model and the Langmuir biosorption isotherm, indicating that Y. lipolytica employed a monolayer chemical biosorption process to biosorb rare earth ions. Characterization analysis demonstrated that the primary functional groups involved in rare earth ion biosorption were amino, carboxyl, and hydroxyl groups. The cooperative biosorption of rare earth ions by Y. lipolytica was facilitated by means of surface complexation, ion exchange, and electrostatic interactions. These findings suggest that Y. lipolytica has the potential to be an effective biosorbent for the removal of rare earth elements from wastewater., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cis-13, 16-docosadienoic acid (DDA) is an omega-6 polyunsaturated fatty acid with great potential for application in medicine and health. Using microbial cell factories for DDA production is considered a viable alternative to extracting DDA from plant seeds. In this study, using Yarrowia lipolytica Po1f (Δku70) as a chassis, firstly, the adaptation of three elongases in Po1f (Δku70) were explored. Secondly, the DDA biosynthetic pathway was redesigned, resulting in a DDA content of 0.046 % of total fatty acids (TFAs). Thirdly, through the "push-pull" strategy, the DDA content increased to 0.078 % of TFAs. By enhancing the supply of acetyl-CoA, the DDA production in the engineered strain YL-7 reached 0.391 % of the TFAs (3.19 mg/L). Through optimizing the fermentation conditions, the DDA titer of YL-7 reached 29.34 mg/L. This research achieves the sustainable biological production of DDA in Y. lipolytica., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Metabolically engineered microbial consortia can contribute as a promising production platform for the supply of polyamide monomers. To date, the biosynthesis of long-chain α,ω-diamines from n -alkanes is challenging because of the inert nature of n -alkanes and the complexity of the overall synthesis pathway. We combined an engineered Yarrowia lipolytica module with Escherichia coli modules to obtain a mixed strain microbial consortium that could catalyze an efficient biotransformation of n -alkanes into corresponding α,ω-diamines. The engineered Y. lipolytica strain was constructed (YALI10) wherein the two genes responsible for β-oxidation and the five genes responsible for the overoxidation of fatty aldehydes were deleted. This newly constructed YALI10 strain expressing transaminase (TA) could produce 0.2 mM 1,12-dodecanediamine (40.1 mg/L) from 10 mM n -dodecane. The microbial consortia comprising engineered Y. lipolytica strains for the oxidation of n -alkanes (O M ) and an E. coli amination module (A M ) expressing an aldehyde reductase (AHR) and transaminase (TA) improved the production of 1,12-diamine up to 1.95 mM (391 mg/L) from 10 mM n -dodecane. Finally, combining the E. coli reduction module (R M ) expressing a carboxylic acid reductase (CAR) and an sfp phosphopantetheinyl transferase with O M and A M further improved the production of 1,12-diamine by catalyzing the reduction of undesired 1,12-diacids into 1,12-diols, which further undergo amination to give 1,12-diamine as the target product. This newly constructed mixed strain consortium comprising three modules in one pot gave 4.1 mM (41%; 816 mg/L) 1,12-diaminododecane from 10 mM n -dodecane. The whole-cell consortia reported herein present an elegant "greener" alternative for the biosynthesis of various α,ω-diamines (C8, C10, C12, and C14) from corresponding n -alkanes.

Background: Limonene 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., Results: In 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., Conclusions: In 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., (© 2024. The Author(s).)

Abundant renewable resource lignocellulosic biomass possesses tremendous potential for green biomanufacturing, while its efficient utilization by Yarrowia lipolytica, an attractive biochemical production host, is restricted since the presence of inhibitors furfural and acetic acid in lignocellulosic hydrolysate. Given deficient understanding of inherent interactions between inhibitors and cellular metabolism, sufficiently mining relevant genes is necessary. Herein, 14 novel gene targets were discovered using clustered regularly interspaced short palindromic repeats interference library in Y. lipolytica, achieving tolerance to 0.35 % (v/v) acetic acid (the highest concentration reported in Y. lipolytica), 4.8 mM furfural, or a combination of 2.4 mM furfural and 0.15 % (v/v) acetic acid. The tolerance mechanism might involve improvement of cell division and decrease of reactive oxygen species level. Transcriptional repression of effective gene targets still enabled tolerance when xylose was a carbon source. This work forms a robust foundation for improving microbial tolerance to lignocellulose-derived inhibitors and revealing underlying mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

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.

With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in the parallelization of microbes cultivations are of great aid, however, for many species down-scaling leaves a metabolic footprint. Yarrowia lipolytica is one such demanding yeast species, for which scaling-down inevitably leads to perturbations in phenotype development. Strictly aerobic metabolism, propensity for filamentation and adhesion to hydrophobic surfaces, spontaneous flocculation, and high acidification of media are just several characteristics that make the transfer of the micro-scale protocols developed for the other microbial species very challenging in this case. It is well recognized that without additional 'personalized' optimization, either MTP-based or single-cell-based protocols are useless for accurate studies of Y. lipolytica phenotypes. This review summarizes the progress in the scaling-down and parallelization of Y. lipolytica cultures, highlighting the challenges that occur most frequently and strategies for their overcoming. The problem of Y. lipolytica cultures down-scaling is illustrated by calculating the costs of micro-cultivations, and determining the unintentionally introduced, thus uncontrolled, variables. The key research into culturing Y. lipolytica in various MTP formats and micro- and pico-bioreactors is discussed. Own recently developed and carefully pre-optimized high-throughput cultivation protocol is presented, alongside the details from the optimization stage. We hope that this work will serve as a practical guide for those working with Y. lipolytica high-throughput screens., (© 2024. The Author(s).)

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis , 4-coumarate CoA ligase from Nicotiana tabacum , and stilbene synthase from Vitis vinifera . This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana , malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens . These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.