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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. [ABSTRACT FROM AUTHOR]

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 (Ylsur2 Δ) 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 Ylsur2 Δ 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 Ylsur2 Δ sld1 Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the Ylsur2 Δ sld1 Δ/ 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. [Display omitted] • Yarrowia lipolytica was engineered for production of sphingolipid derivatives. • SUR2 deletion enhanced secretory production of sphingoid bases without acetylation. • Subsequent SLD1 deletion recovered yeast-type growth and boosted human-type GlcCers. • Mouse alkaline ceramidase 1 expression increased the production of DHS and sphingosine. • Fed-batch fermentation with glucose feeding produced 1.4 g/L non-acetylated DHS. [ABSTRACT FROM AUTHOR]

Erythritol is a four-carbon sugar alcohol naturally produced by microorganisms as an osmoprotectant. As a new sugar substitute, erythritol has recently been popular on the ingredient market because of its unique nutritional characteristics. Even though the history of erythritol biosynthesis dates from the turn of the twentieth century, scientific advancement has lagged behind other polyols due to the relative complexity of making it. In recent years, biosynthetic methods for erythritol have been rapidly developed due to an increase in market demand, a better understanding of metabolic pathways, and the rapid development of genetic engineering tools. This paper reviews the history of industrial strain development and focuses on the underlying mechanism of high erythritol production by strains gained through screening or mutagenesis. Meanwhile, we highlight the metabolic pathway knowledge of erythritol biosynthesis in microorganisms and summarize the metabolic engineering and research progress on critical genes involved in different stages of the synthetic pathway. Lastly, we talk about the still-contentious issues and promising future research directions that will help break the erythritol production bottleneck and make erythritol production greener and more sustainable. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cheese is a biologically active food product, characterised by its colour, texture, and taste. Due to its rich matrix of fats and proteins, as well as the fact that the cheese's surface acts as its own packaging, the cheese becomes more susceptible to contamination by microorganisms during the ripening process, particularly bacteria and fungi. The ripening of cheese involves several biochemical reactions, with the proteolytic activity of the cheese microbiota being particularly significant. Proteolysis results in the presence of free amino acids, which are precursors to various metabolic mechanisms that can cause discolouration (blue, pink, and brown) on the cheese rind. Surface defects in cheese have been documented in the literature for many years. Sporadic inconsistencies in cheese appearance can lead to product degradation and economic losses for producers. Over the past few decades, various defects have been reported in different types of cheese worldwide. This issue also presents opportunities for innovation and development in edible and bioactive coatings to prevent the appearance of colour defects. Therefore, this review provides a comprehensive analysis of cheese colour globally, identifying defects caused by microorganisms. It also explores strategies and innovation opportunities in the cheese industry to enhance the value of the final product. [ABSTRACT FROM AUTHOR]

The utilization of industrial biomanufacturing has emerged as a viable and sustainable alternative to fossil-based resources for producing functional chemicals. Moreover, advancements in synthetic biology have created new opportunities for the development of innovative cell factories. Notably, Yarrowia lipolytica, an oleaginous yeast that is generally regarded as safe, possesses several advantageous characteristics, including the ability to utilize inexpensive renewable carbon sources, well-established genetic backgrounds, and mature genetic manipulation methods. Consequently, there is increasing interest in manipulating the metabolism of this yeast to enhance its potential as a biomanufacturing platform. Here, we reviewed the latest developments in genetic expression strategies and manipulation tools related to Y. lipolytica, particularly focusing on gene expression, chromosomal operation, CRISPR-based tool, and dynamic biosensors. The purpose of this review is to serve as a valuable reference for those interested in the development of a Y. lipolytica microbial factory. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

(1) Background: Odd-chain fatty acids (OCFAs) have garnered attention for their potential health benefits and unique roles in various biochemical pathways. Yarrowia lipolytica, a versatile yeast species, is increasingly studied for its capability to produce OCFAs under controlled genetic and environmental conditions. However, optimizing the synthesis of specific OCFAs, such as cis-9-heptadecenoic acid (C17:1), remains a challenge. (2) Methods: The gene coding for the Δ9 fatty acid desaturase, YlOLE1, and the gene coding the diacylglycerol O-acyltransferase 2, YlDGA2, were overexpressed in Y. lipolytica. With the engineered strain, the main goal was to fine-tune the production of OCFA-enriched lipids by optimizing the concentrations of sodium propionate and sodium acetate used as precursors for synthesizing odd- and even-chain fatty acids, respectively. (3) Results: In the strain overexpressing only YlDGA2, no significant changes in fatty acid composition or lipid content were observed compared to the control strain. However, in the strain overexpressing both genes, while no significant changes in lipid content were noted, a significant increase was observed in OCFA content. The optimal conditions for maximizing the cell density and the C17:1 content in lipids were found to be 2.23 g/L of sodium propionate and 17.48 g/L of sodium acetate. These conditions resulted in a cell density (optical density at 600 nm) of 19.5 ± 0.46 and a C17:1 content of 45.56% ± 1.29 in the culture medium after 168 h of fermentation. (4) Conclusions: By overexpressing the YlOLE1 gene and optimizing the concentrations of fatty acid precursors, it was possible to increase the content of OCFAs, mainly C17:1, in lipids synthesized by Y. lipolytica. [ABSTRACT FROM AUTHOR]

Lipases are enzymes of great application importance in the food industry, in the cosmetic and detergent industries, in pharmacy and medicine, and in organic chemistry. Among lipases of various origins, those from microorganisms are currently the most commonly used. An excellent producer of lipases seems to be the nonconventional Yarrowia lipolytica yeast, but the biosynthesis of valuable metabolites depends on many factors. This study aimed to investigate the biodiversity of extracellular enzymes produced by four strains of Y. lipolytica, and to determine the optimal conditions of catalysis for the enzymes, according to temperature and pH, in a model hydrolysis reaction. Based on the obtained results, the biodiversity and strain dependence in lipase biosynthesis were observed. Using a Central Composite Design, it was found that temperature is the main factor in determining lipase activity. The enzymes produced by four different strains exhibited other substrate specificity, which was investigated using Latin square design methodology. Only two examined yeast strains, KKP 379 and W29, produced extracellular lipases at a high activity level towards medium- and long-chain fatty acid esters. Moreover, extracellular lipase from wild-type strain KKP 379 was further characterized, followed by exploring the activity of whole-cell biocatalyst and lyophilized enzyme solutions, and it was acknowledged that it was a "true" lipase with the highest affinity to p-nitrophenyl oleate. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

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.

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.

Cheese is a biologically active food product, characterised by its colour, texture, and taste. Due to its rich matrix of fats and proteins, as well as the fact that the cheese’s surface acts as its own packaging, the cheese becomes more susceptible to contamination by microorganisms during the ripening process, particularly bacteria and fungi. The ripening of cheese involves several biochemical reactions, with the proteolytic activity of the cheese microbiota being particularly significant. Proteolysis results in the presence of free amino acids, which are precursors to various metabolic mechanisms that can cause discolouration (blue, pink, and brown) on the cheese rind. Surface defects in cheese have been documented in the literature for many years. Sporadic inconsistencies in cheese appearance can lead to product degradation and economic losses for producers. Over the past few decades, various defects have been reported in different types of cheese worldwide. This issue also presents opportunities for innovation and development in edible and bioactive coatings to prevent the appearance of colour defects. Therefore, this review provides a comprehensive analysis of cheese colour globally, identifying defects caused by microorganisms. It also explores strategies and innovation opportunities in the cheese industry to enhance the value of the final product.

Abstract 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.

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

Abstract 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. Graphical abstract

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. [ABSTRACT FROM AUTHOR]

The bioconversion of agricultural and industrial wastes is considered a green and sustainable alternative method for producing high-value biochemicals. As a major catalytic product of greenhouse gases and a by-product in the fermentation and lignocellulose processing industries, acetate is a promising bioconversion raw material. In this work, endogenous and heterologous enzymes were manipulated in Yarrowia lipolytica to achieve the conversion of acetate to high-value citric acid and itaconic acid, respectively. After the combinational expression of the key enzymes in the acetate metabolic pathway, the citric acid synthesis pathway, and the mitochondrial transport system, acetate could be efficiently converted to citric acid. Coupled with the down-regulation of fatty acid synthase expression in the competitive pathway, more acetyl-CoA flowed into the synthesis of citric acid, and the titer reached 15.11 g/L with a productivity of 0.51 g/g acetate by the engineered Y. lipolytica, which is comparable to the results using glucose as the substrate. On this basis, the heterologous cis-aconitate decarboxylase from Aspergillus terreus was introduced into the engineered Y. lipolytica to achieve the catalytic synthesis of itaconic acid from acetate. Combined with investigating the effects of multiple enzymes in the synthesis pathway, the titer of itaconic acid reached 1.87 g/L with a yield of 0.43 g/g DCW by the final engineered strain, which is the highest reported titer of itaconic acid derived from acetate by engineered microbes in shake flasks. It is demonstrated that acetate has the potential to replace traditional starch-based raw materials for the synthesis of high-value organic acids and our work lays a foundation for the rational utilization of industrial wastes and the catalytic products of greenhouse gases. [ABSTRACT FROM AUTHOR]

The yeast microflora of various dairy products sold in Russia was studied using RFLP analysis of the 5.8S-ITS rDNA fragment and sequencing of the D1/D2 domain of 26S rDNA. Most of the fermented milk products studied were dominated by lactose-utilizing yeasts Kluyveromyces and Debaryomyces and by lactose-negative yeasts Saccharomyces, Monosporozyma, Pichia, Geotrichum, and Yarrowia. The yeast Kluyveromyces marxianus was present in most of the fermented milk products studied, while the related species K. lactis was found only in some samples of ayran, curds, and cheese. The dominance of K. marxianus is apparently associated with their physiological characteristics (thermo- and osmotolerance), which provide these yeasts with better adaptation to industrial fermentation conditions. The dominant species in mixed-fermentation dairy products, Saccharomyces cerevisiae and Monosporozyma unispora, were completely absent in cheeses and lactic acid fermentation products. In general, the species composition of yeasts largely depended on the fermented milk product, the type of milk, and the specific manufacturer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ERK26N/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. [ABSTRACT FROM AUTHOR]

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. Take Away: The genes involved in the alternative dihydroxyacetone route were detected and then analyzed by GC/MS and RNA‐seq studies.Dihydroxyacetone kinase DAK2 catalyzes the rate‐limited step in the alternative glycerol uptake route.Diverse Yarrowia lipolytica strains had different adaptability to the alternative glycerol uptake route.53.1 g/L of erythritol production and a yield of 0.56 g/g glycerol were achieved by performing a push‐pull strategy in Yarrowia lipolytica M09. [ABSTRACT FROM AUTHOR]

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 13C-metabolic flux analysis (13C-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 13C-MFA model construction and to validate the 13C-MFA results. The 13C-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. • 13C-MFA, RNA-seq, and GSM investigate Y. lipolytica W29 triacylglycerol catabolism. • The glyoxylate shunt shows 7-fold higher flux in oleic acid vs. glucose or glycerol. • The oxidative pentose phosphate pathway is the primary route for NADPH generation. • The mannitol cycle and alternative oxidase may help balance excess NADH. • The carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool. [ABSTRACT FROM AUTHOR]

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 (La3⁺, Nd3⁺, Er3⁺, Y3⁺, and Sm3⁺) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

In this study, waste sunflower oils were evaluated as substrates for citric acid (CA) production by Yarrowia lipolytica IFP29 (ATCC 20460). This strain was selected based on its capacity to produce organic acids in a selective medium. Attempts were made to optimize the process using the Taguchi statistical method in terms of the oil polarity, oil concentration, fermentation time, and Triton X-100 concentration. The results indicated that Y. lipolytica IFP29 utilized waste sunflower oil as a substrate and produced a maximum CA of 32.17 ± 1.44 g/L. Additionally, Triton X-100 inhibited the production of CA. For this reason, this process could not be optimized. These results were obtained by periodically adjusting the pH with NaOH during the fermentation period. On the other hand, a new experimental design was created without Triton X-100. As a buffering agent, 2-morpholinoethanesulfonic acid monohydrate (MES) was used to prevent a drop in pH; the maximum concentration of CA was found to be 20.31 ± 2.76. The optimum conditions were as follows: 90 g/L of waste sunflower oil with a polarity of 16 and 12 days of fermentation. According to the analysis of variance results, the effects of factors other than polarity on CA production were found to be significant (p < 0.05). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]