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Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree-negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg/L of ergothioneine in small-scale cultivation, with an additional copy of each gene improving the titer to 205 mg/L. The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate-limited fed-batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g/L ergothioneine in 220 hours, corresponding to an overall volumetric productivity of 7.41 mg L-1 h-1 , showing that Yarrowia lipolytica is a promising host for ergothioneine production.

Background Sugar alcohols are widely used as low-calorie sweeteners in the food and pharmaceutical industries. They can also be transformed into platform chemicals. Yarrowia lipolytica, an oleaginous yeast, is a promising host for producing many sugar alcohols. In this work, we tested whether heterologous expression of a recently identified sugar alcohol phosphatase (PYP) from Saccharomyces cerevisiae would increase sugar alcohol production in Y. lipolytica. Results Y. lipolytica was found natively to produce erythritol, mannitol, and arabitol during growth on glucose, fructose, mannose, and glycerol. Osmotic stress is known to increase sugar alcohol production, and was found to significantly increase erythritol production during growth on glycerol. To better understand erythritol production from glycerol, since it was the most promising sugar alcohol, we measured the expression of key genes and intracellular metabolites. Osmotic stress increased the expression of several key genes in the glycerol catabolic pathway and the pentose phosphate pathway. Analysis of intracellular metabolites revealed that amino acids, sugar alcohols, and polyamines are produced at higher levels in response to osmotic stress. Heterologous overexpression of the sugar alcohol phosphatase increased erythritol production and glycerol utilization in Y. lipolytica. We further increased erythritol production by increasing the expression of native glycerol kinase (GK), and transketolase (TKL). This strain was able to produce 27.5 ± 0.7 g/L erythritol from glycerol during batch growth and 58.8 ± 1.68 g/L erythritol during fed-batch growth in shake-flasks experiments. In addition, the glycerol utilization was increased by 2.5-fold. We were also able to demonstrate that this strain efficiently produces erythritol from crude glycerol, a major byproduct of the biodiesel production. Conclusions We demonstrated the application of a promising enzyme for increasing erythritol production in Y. lipolytica. We were further able to boost production by combining the expression of this enzyme with other approaches known to increase erythritol production in Y. lipolytica. This suggest that this new enzyme provides an orthogonal route for boosting production and can be stacked with existing designs known to increase sugar alcohol production in yeast such as Y. lipolytica. Collectively, this work establishes a new route for increasing sugar alcohol production and further develops Y. lipolytica as a promising host for erythritol production from cheap substrates such as glycerol.

Producing high value-added products from waste lipid feedstock by microbial cell factory has great advantages to minimize the pollution as well as improve the economic value of wasted oils and fats. Yarrowia lipolytica is a non-conventional oleaginous yeast and can grow on a variety of hydrophobic substrates. In this study, we explored its ability to synthesize α-farnesene, an important sesquiterpene, using lipid feedstock. Based on the α-farnesene production strain, we constructed previously, we identified that Erg12 was the key limiting factor to further increase the α-farnesene production. The α-farnesene production was improved by 35.8% through increasing the copy number of ERG12 and FSERG20 on oleic acid substrate. Expression of heterologous VHb further improved α-farnesene production by 12.7%. Combining metabolic engineering with the optimization of fermentation conditions, the α-farnesene titer and yield reached 10.2 g/L and 0.1 g/g oleic acid, respectively, in fed-batch cultivation. The α-farnesene synthesis ability on waste cooking oil and other edible oils were also explored. Compared with using glucose as carbon source, using lipid substrates obtained higher α-farnesene yield and titer, but lower by-products accumulation, demonstrating the advantage of Y. lipolytica to synthesize high value-added products using lipid feedstock.

Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable production of terpenoids. The non-conventional yeast, Yarrowia lipolytica, is a promising host for terpenoid biosynthesis due to its inherent mevalonate pathway, high fluxes of acetyl-CoA and NADPH, and the naturally hydrophobic microenvironment. In this review, we highlight progress in the engineering of Y. lipolytica as terpenoid biomanufacturing factories, describing the different terpenoid biosynthetic pathways and summarizing various metabolic engineering strategies, including progress in genetic manipulation, dynamic regulation, organelle engineering, and terpene synthase variants. ispartof: CRITICAL REVIEWS IN BIOTECHNOLOGY vol:42 issue:4 pages:618-633 ispartof: location:England status: published

Background Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers. L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers.

This study focuses on the development of a secondary model for Yarrowia lipolytica in a sewage treatment process. The raw data of Y. lipolytica growth were adjusted to the Buchanan model in order to obtain growth parameters such as initial count cells (Y0), maximum specific growth rate (μmax), latency phase (λ) and maximum cell population (Ymax). The µ values obtained at different pH levels (5.0 to 8.0) were used to build the secondary model based on a linear equation. The results showed a significant effect of pH on µmax values. The validation process of the developed models displays accuracy (Af) and bias factor (Bf) values close to one, while the values of root mean square error (RMSE) were low, confirming that such models can predict the growth of Y. lipolytica in dairy wastewater. This can be interesting to optimize sewage treatments that involve this kind of microorganism. Moreover, the dairy wastewater was a good substrate to support the Yarrowia lipolytica's growth and could be used to produce enzymes. Resumo Este estudo tem como foco o desenvolvimento de um modelo secundário para Yarrowia lipolytica em processos de tratamento de esgoto. Os dados brutos de crescimento de Y. lipolytica foram ajustados ao modelo de Buchanan a fim de obter parâmetros de crescimento como: contagem inicial de células (Y0), taxa de crescimento específico máximo (μmax), fase de latência (λ) e população máxima de células (Ymax). Os valores µ obtidos em diferentes níveis de pH (5,0 a 8,0) foram usados para construir o modelo secundário baseado em uma equação linear. Os resultados mostraram um efeito significativo do pH nos valores de µmax. O processo de validação dos modelos desenvolvidos apresenta valores de acurácia (Af) e fator de bias (Bf) próximos a um, enquanto os valores de root mean square error (RMSE) foram baixos, confirmando que tais modelos podem prever o crescimento de Y. lipolytica em águas residuais de laticínios. Isso pode ser interessante para otimizar tratamentos de esgoto que envolvem esse tipo de microrganismo. Além disso, as águas residuais do leite eram um bom substrato para apoiar o crescimento da Yarrowia lipolytica e poderiam ser utilizadas para produzir a produção de enzimas.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Four non-conventional yeast strains belonging to the genera Yarrowia and Rhodosporidium were cultivated under nitrogen-limited conditions in shake flasks with biodiesel-derived glycerol employed as sole substrate. The strains R. toruloides DSM 4444 and Y. lipolytica ACA-DC 5033 displayed better performances and were further studied. At initial glycerol (Gly0) ≈ 50 g/L, the strain ACA-DC 5033 produced dry cell weight (DCW) ≈7.0 g/L, and also secondary extra-cellular metabolites as citric acid (CA) at ≈16 g/L and polyols (Pol) (mannitol, erythritol and arabitol) at ≈21 g/L. In double-media (besides nitrogen also Mg was limiting) for Gly0 ≈ 50 g/L, a shift towards CA production occurred (CAmax ≈ 33 g/L). The strain DSM 4444 at Gly0 ≈ 50 g/L produced DCW = 18.1 g/L containing lipids = 30.3% in DCW. In single nitrogen-limited media with Gly0 ≈ 90 g/L, the strain ACA-DC 5033 produced Pol ≈ 48 g/L and CA ≈ 20 g/L, while the strain DSM 4444 produced DCW = 27.3 g/L containing lipid = 54.5% w/w. At the late growth phases, metabolites were re-consumed. Balanced growth phase (trophophase) and phase of secondary metabolite synthesis (idiophase) were successfully simulated with the aid of a modified Velhlust-Aggelis model. Lipid extraction process was studied for these two strains by using two different extraction methods. Yeast lipids contained mostly oleic acid, constituting suitable precursors for the synthesis of 2nd generation biodiesel.

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations.1,2 Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits.3 Here we demonstrate a biotechnological method for the production of pheromones of economically important moth pests using engineered yeast cell factories. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a synthetic pheromone mixture. We further demonstrated the production of the main pheromone component of the fall armyworm Spodoptera frugiperda. Our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control by yeast fermentation.Significance statementAgriculture largely relies on insecticides and genetically modified crops for pest control, however alternative solutions are required due to emerging resistance, toxicity and regulatory issues, and consumer preferences. Mating disruption with sex pheromones that act by preventing insect reproduction is considered the most promising and scalable alternative to insecticides. This method is highly efficient and safe for human health and environment. The likelihood of insect resistance development is very low and can be handled by adjusting the pheromone composition. The high cost of chemically synthesized pheromones is the major barrier for the wider adoption of pheromones. A novel method based on yeast fermentation enables the production of insect sex pheromones as a lower cost from renewable feedstocks.

Background Functional sugar alcohols have been widely used in the food, medicine, and pharmaceutical industries for their unique properties. Among these, erythritol is a zero calories sweetener produced by the yeast Yarrowia lipolytica. However, in wild-type strains, erythritol is produced with low productivity and yield and only under high osmotic pressure together with other undesired polyols, such as mannitol or d-arabitol. The yeast is also able to catabolize erythritol in non-stressing conditions. Results Herein, Y. lipolytica has been metabolically engineered to increase erythritol production titer, yield, and productivity from glucose. This consisted of the disruption of anabolic pathways for mannitol and d-arabitol together with the erythritol catabolic pathway. Genes ZWF1 and GND encoding, respectively, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were also constitutively expressed in regenerating the NADPH2 consumed during erythritol synthesis. Finally, the gene RSP5 gene from Saccharomyces cerevisiae encoding ubiquitin ligase was overexpressed to improve cell thermoresistance. The resulting strain HCY118 is impaired in mannitol or d-arabitol production and erythritol consumption. It can grow well up to 35 °C and retain an efficient erythritol production capacity at 33 °C. The yield, production, and productivity reached 0.63 g/g, 190 g/L, and 1.97 g/L·h in 2-L flasks, and increased to 0.65 g/g, 196 g/L, and 2.51 g/L·h in 30-m3 fermentor, respectively, which has economical practical importance. Conclusion The strategy developed herein yielded an engineered Y. lipolytica strain with enhanced thermoresistance and NADPH supply, resulting in a higher ability to produce erythritol, but not mannitol or d-arabitol from glucose. This is of interest for process development since it will reduce the cost of bioreactor cooling and erythritol purification.

Nowadays, there is an increase attention on the effect of selenium (Se) on metabolic processes of microorganisms. Strains belonging to the genus of Yarrowia are of great biotechnological interest for various industries. In this study, we evaluated the effect of 10 mg/L of Se on the growth and lipid production of two Yarrowia lipolytica strains: the ACA DC 50109 and one more with increased oleagenicity, derived after ALE methodology (referred here as Y. lipolytica ALE_70). The presence of Se in the growth medium negatively affected both cell mass production and total lipid accumulation, for both Y. lipolytica strains. Fractionation of total lipids showed an inhibition on neutral lipid (NL) synthesis and consequently, an increase of polar lipids (glycolipids plus sphingolipids, and phospholipids) on the lipids of the Se-enriched ACA DC 50109 strain; however, the NL/polar ratio of the Se-enriched ALE_70 indicated that Se, apart from the inhibition of NL synthesis, provoked also the accumulation of polar lipids in this strain. In addition, the fatty acid (FA) composition was differently affected by Se. Se-enriched total lipids of the ALE_70 strain were enriched in linoleic acid (C18:2 n-6), which resulted in increase of the unsaturated index. On the other hand, Se-enriched lipids of the ACA DC 50109 strain were more saturated, as the percentage of palmitic (C16:0) and stearic (C18:0) acids increased in the total FAs. Moreover, it seems that Se influenced the activity or the expression of desaturases and elongase in both strains. Finally, the supplementation of growth medium with Se affected cell morphology, as well as the size and distribution of lipid droplets inside the yeast cells. According to our opinion, Se caused stress conditions and the consequence of that was the occurrence of metabolic disorders that affected cell mass, lipid content, and/or morphological structures. The results of the present study suggest that further research should be carried out to understand the background of the lipogenesis process in yeast cells cultured under stress conditions.

Background Yarrowia lipolytica belongs to the normal human microbiota but is also found in substrates with high contents in lipids and used in biotechnological processes. It is sometimes reported as human pathogen and especially in catheter-related candidaemia. Objectives Two apparently grouped cases of infections and/or contamination were reported involving 3 and 9 patients, respectively, in two hospitals. The goal of this study was to design a molecular tool to study the genetic diversity of Y lipolytica and confirm or not the common source of contamination during these grouped cases. Methods Given that there is no genotyping method, we used genomic markers assessed on environmental isolates to determine intra-species relationship. We selected five highly polymorphic intergenic regions, totalling more than 3200 bp and sequenced them for clinical (n = 20) and environmental (n = 14) isolates. Antifungal susceptibility was determined by EUCAST broth microdilution method. Results Multiple alignment of the five sequences revealed divergence of 0%-5.8% between isolates as compared to approximately 0.2%-0.25% after alignment of whole genomes, suggesting their potential usefulness to establish genetic relatedness. The analysis showed the multiple origins of the isolates. It uncovered two grouped case of fungaemia involving 3 and 2 patients, respectively. It also revealed several unrelated sporadic cases despite their temporal relationship and one probable laboratory contamination by a common yet uncovered source, explaining several consecutive positive cultures without infection. All isolates had high minimal inhibitory concentration (MIC) for flucytosine, the majority (14/34) was susceptible to fluconazole, and all to the other antifungal agents tested. Conclusion This method could help elucidate cases related to the opportunistic pathogen Y lipolytica.

Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrates with different entry points into the metabolic network have different energetic and redox statuses. Generally, substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. Aiming to efficiently synthesize the high-value rose-smell 2-phenylethanol (2-PE) in Y. lipolytica, we analyzed the stoichiometric constraints of the Ehrlich pathway and identified that the selectivity of the Ehrlich pathway and the availability of 2-oxoglutarate are the rate-limiting factors. Stepwise refactoring of the Ehrlich pathway led us to identify the optimal catalytic modules consisting of l-phenylalanine permease, ketoacid aminotransferase, phenylpyruvate decarboxylase, phenylacetaldehyde reductase, and alcohol dehydrogenase. On the other hand, mitochondrial compartmentalization of 2-oxoglutarate inherently creates a bottleneck for efficient assimilation of l-phenylalanine, which limits 2-PE production. To improve 2-oxoglutarate (aKG) trafficking across the mitochondria membrane, we constructed a cytosolic aKG source pathway by coupling a bacterial aconitase with a native isocitrate dehydrogenase (ylIDP2). Additionally, we also engineered dicarboxylic acid transporters to further improve the 2-oxoglutarate availability. Furthermore, by blocking the precursor-competing pathways and mitigating fatty acid synthesis, the engineered strain produced 2669.54 mg/L of 2-PE in shake flasks, a 4.16-fold increase over the starting strain. The carbon conversion yield reaches 0.702 g/g from l-phenylalanine, 95.0% of the theoretical maximal. The reported work expands our ability to harness the Ehrlich pathway for production of high-value aromatics in oleaginous yeast species.

Background The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. Results In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. Conclusions An efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.

Transposable elements are DNA sequences capable of moving or copying and reinserting themselves in the genome of a host cell. The Hermes DNA transposon was originally found in the housefly Musca domestica. Since its discovery, Hermes has been adapted to multiple eukaryotic species for random mutagenesis or mapping of chromatin structure. The Ty3 retrotransposons of S. cerevisiae is a long terminal repeat (LTR) mobile element of the gypsy–like class. Ty3 is unique among retroelements for its highly specific targeting of RNAP3-transcribed genes for integration. In this dissertation, each of these mobile elements are utilized for different purposes. The Ty3 retrotransposon was used to investigate the relationship between integrase and host factors that direct insertion site selection. Ty3 integration events were quantitatively profiled genome-wide, demonstrating an unexplained bias within an already limited selection of RNAP3 targets. This bias was not defined by the host chromatin environment flanking target sites, and investigation of RNAP3 factors found poor agreement with Ty3 targeting bias. DNA sequence composition and structure of local target sequence were found to regulate integration frequency, in a motif-independent manner. The Hermes DNA transposon was used to establish a functional genomics system for the oleaginous yeast Yarrowia lipolytica, a quickly emerging and valuable microbe with many biotechnology applications. Identification of essential genes provides important information required for genome engineering. Hermes was used to mutagenize a population of Y. lipolytica. To classify essential and non-essential genes, transposon were profiled in surviving mutants by statistically testing under-representation of insertions relative to an expected number derived for each gene. The Y. lipolytica genome was categorized as 20.8 % essential, 71.1 % non-essential and 8.1 % poor essential. Many essential differed from S. cerevisiae, demonstrating the utility of a model obligate aerobe compared to current model yeasts. Pooled mutants were grown for 80 generations in glucose or glycerol, an industrial waste stream, to identify genes contributing to fitness. Glycerol media created a much more competitive environment than glucose. Gene classifications inform future metabolic studies by fine tuning flux analysis and genome scale models, while mutant libraries provide a pipeline for rescreening phenotypes relevant to industrial applications.

Yarrowia lipolytica belongs to the group of microorganisms widely used in scientific research for environmentally friendly biotransformation reactions. This yeast produces a number of compounds important from the point of view of the food and cosmetics industries, including flavor and fragrance compounds. The results of selected studies on the possibility of biosynthesis of fat-derived fragrances, namely gamma-decalactone (GDL) and hexanal, are presented in this article. The wild-type Yarrowia yeast strain KKP379 and the LOX/HPL mutant were used in the studies. With the aim of improving the synthesis yield of both aroma compounds, parameters such as concentration of lipid substrate, the type of culture medium, and the addition of surfactants, and, not yet verified in the available literature in this context, the concentration of inoculum, the addition of heptane, and the emulsification of culture medium were analyzed. The research showed that the concentration of the lipid substrate and the degree of emulsification of the medium had a significant influence on the amount of GDL production. The higher the content of castor oil in the medium, the higher the concentration of the synthesized aroma compound, with a significant extension of the reaction time. By varying the concentration of castor oil in the medium in the range of 10–100 g/L, an increase in the lactone concentration was obtained from 1.86 ± 0.15 g/L to 3.06 ± 0.2 g/L, with a simultaneous extension of the reaction from 3 to 7 days. It is noteworthy that the additional application of the emulsification of the medium allowed the efficiency of GDL biosynthesis to be significantly increased by over 2 g/L to a lactone concentration of approximately 5.25 ± 0.10 g/L. This is one of the highest reported concentrations for the production of this biofragrance by the wild strain. In the case of hexanal synthesis, the increase of the concentration of linoleic acid in the culture medium from 25 g/L to 100 g/L resulted in a 5-fold higher concentration of aldehyde in the cell extract. At a higher concentration of the lipid substrate in the medium, the modified whole-cell catalyst showed a higher activity of lipoxygenase.

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

International audience; Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D >mu max of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts., This study establishes Molybdenum cofactor biosynthesis in Yarrowia lipolytica to expand the nutrient range thereof.

Yarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504, which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing non-preferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, β-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation and degradation in biomass hydrolysates.IMPORTANCEYarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

Energy security and global climate change have necessitated the development of renewable energy with net-zero emissions. As alternatives to traditional fuels used in heavy-duty vehicles, advanced biofuels derived from fatty acids and terpenes have similar properties to current petroleum-based fuels, which makes them compatible with existing storage and transportation infrastructures. The fast development of metabolic engineering and synthetic biology has shown that microorganisms can be engineered to convert renewable feedstocks into these advanced biofuels. The oleaginous yeast Yarrowia lipolytica is rapidly emerging as a valuable chassis for the sustainable production of advanced biofuels derived from fatty acids and terpenes. Here, we provide a summary of the strategies developed in recent years for engineering Y. lipolytica to synthesize advanced biofuels. Finally, efficient biotechnological strategies for the production of these advanced biofuels and perspectives for future research are also discussed.

International audience; The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Alginate lyase possesses wide application prospects for the degradation of brown algae and preparation of alginate oligosaccharides, and its degradation products display a variety of biological activities. Although many enzymes of this type have been reported, alginate lyases with unique properties are still relatively rare. In the present work, an alginate lyase abbreviated as Alyw203 has been cloned from Vibrio sp. W2 and expressed in food-grade Yarrowia lipolytica. The Alyw203 gene consists of an open reading frame (ORF) of 1,566 bp containing 521 amino acids, of which the first 17 amino acids are considered signal peptides, corresponding to secretory features. The peak activity of the current enzyme appears at 45°C with a molecular weight of approximately 57.0 kDa. Interestingly, Alyw203 exhibits unique heat recovery performance, returning above 90% of its initial activity in the subsequent incubation for 20 min at 10°C, which is conducive to the recovery of current enzymes at low-temperature conditions. Meanwhile, the highest activity is obtained under alkaline conditions of pH 10.0, showing outstanding pH stability. Additionally, as an alginate lyase independent of NaCl and resistant to metal ions, Alyw203 is highly active in various ionic environments. Moreover, the hydrolyzates of present enzymes are mainly concentrated in the oligosaccharides of DP1–DP2, displaying perfect product specificity. The alkali suitability, heat recovery performance, and high oligosaccharide yield of Alyw203 make it a potential candidate for industrial production of the monosaccharide and disaccharide.

International audience; Yarrowia lipolytica strains PGC01003 and PGC202 engineered for succinic acid production were studied and compared to the wild type strain W29. For the first time, these two strains were characterized in a chemically defined medium. Strain growth and organic acid production were investigated in fed-batch mode with glycerol as carbon and energy source. This study evaluated the impact of nitrogen deficiency strategy to redirect carbon flux toward succinic acid synthesis. Strain PGC01003 produced 19 g L−1 succinic acid with an overall yield of 0.23 g g−1 and an overall productivity of 0.23 g L−1 h−1, while strain PGC202 produced 33 g L−1 succinic acid with an overall yield of 0.12 g g−1 and a productivity of 0.57 g L−1 h−1. Nitrogen limitation effectively stopped biomass growth and increased succinic acid yield of PGC01003 and PGC202 by 18 % and 62 %, respectively. However, the specific succinic acid production rate was reduced by 77 % and 66 %, respectively.

There is ever increasing evidence that isocitric acid can be used as a promising compound with powerful antioxidant activity to combat oxidative stress. This work demonstrates the possibility of using waste product from the alcohol industry (so-called ester-aldehyde fraction) for production of isocitric acid by yeasts. The potential producer of isocitric acid from this fraction, Yarrowia lipolytica VKM Y-2373, was selected by screening of various yeast cultures. The selected strain showed sufficient growth and good acid formation in media with growth-limiting concentrations of nitrogen, sulfur, phosphorus, and magnesium. A shortage of Fe2+ and Ca2+ ions suppressed both Y. lipolytica growth and formation of isocitric acid. The preferential synthesis of isocitric acid can be regulated by changing the nature and concentration of nitrogen source, pH of cultivation medium, and concentration of ester-aldehyde fraction. Experiments in this direction allowed us to obtain 65 g/L isocitric acid with a product yield (YICA) of 0.65 g/g in four days of cultivation.

Gamma-decalactone is an interesting flavuring compound with an intense oily-peachy aroma. It is commonly used in food and cosmetic industry. Biotechnological methods of its synthesis are based on the processes of fatty acid degradation, mainly ricinoleic acid, which is the main component of castor oil. The aim of this study was to compare the productivity of gamma-decalactone in batch cultures of two strains of Yarrowia lipolytica, wild strain W29 and its derived mutant MTLY40-2p, grown in flasks and a bioreactor. We analyzed the concentration of gamma-decalactone in the aqueous and oil phase, the optical density of the medium, the yield of yeast dry matter and the particle size distribution in the particular media. The modified strain had a higher aroma synthesis capacity. This strain grown in flasks produced thrice as much gamma-decalactone (5.5 ± 0.16 g/L) as compared to the wild-type strain (1.8 ± 0.03 g/L). During the 7-day biotransformation, the mutant strain did not have the ability to degrade gamma-decalactone, whereas in the biotransformation using the wild-type strain, reutilization was observed after the third day. Regardless of the type of culture, larger amounts of gamma-decalactone were accumulated in the lipid phase. Cultivation in a bioreactor resulted in a lower biomass yield and a lower concentration of lactone compared to flask culture, regardless of the strain used. The MTLY40-2p strain grew in mycelium form and tended to aggregate cells.

Objectives The construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited, including those of wild-type strains. Results Using the Golden Gate method, we assembled a set of six CRISPR/Cas9 vectors, each containing a different selection marker, to be used for editing the genome of the industrial yeast Y. lipolytica. This vector set is available via Addgene. Any guide RNA (gRNA) sequence can be easily and rapidly introduced in any of these vectors using Golden Gate assembly. We successfully edited six different genes in a variety of genetic backgrounds, including those of wild-type strains, with five of the six vectors. Use of these vectors strongly improved homologous recombination and cassette integration at a specific locus. Conclusions We have created a versatile and modular set of CRISPR/Cas9 vectors that will allow any Y. lipolytica strain to be rapidly edited; this tool will facilitate experimentation with any prototroph wild-type strains displaying interesting features.

Non-conventional yeasts are an excellent option for a number of different industrial bioprocesses. They possess beneficial natural phenotypes, which translates to several fermentation advantages when compared to traditional hosts, like Saccharomyces cerevisiae. The non-conventional yeasts Yarrowia lipolytica, Trichosporon oleaginosus, Kluyveromyces marxianus, Dekkera bruxellensis, Pichia kudriavzevii, Debaryomyces hansenii and Hansenula polymorpha, are considered desirable industrial hosts due to their natural characteristics, including tolerance to several by-products and inhibitors, thermotolerance, salt resistance or osmo- and xerotolerance. Therefore, they are a great alternative for the industrial production of bioethanol, fine chemicals, lipids and recombinant proteins, among others. In this review, we summarize the best natural characteristics of those seven non-conventional yeasts and their use in industrial biotechnology, as well as the molecular/synthetic biology tools available for their genetic modification. Moreover, possible limitations regarding their performance in industrial fermentations and a list of challenges to overcome in the future are also discussed.

BackgroundLimonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly.ResultsIn this proof-of-principle study, the oleaginous yeastYarrowia lipolyticawas successfully engineered to produce bothd-limonene andl-limonene by introducing the heterologousd-limonene synthase fromCitrus limonandl-limonene synthase fromMentha spicata, respectively. However, only 0.124 mg/Ld-limonene and 0.126 mg/Ll-limonene were produced. To improve the limonene production by the engineered yeastY. lipolyticastrain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis inY. lipolytica. Through the overexpression ofHMGRgene, the titers ofd-limonene andl-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineeredY. lipolyticastrains from glucose, and the final titers ofd-limonene andl-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved ford-limonene andl-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineeredY. lipolyticastrains was investigated. We showed thatd-limonene andl-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization.ConclusionsThis study represents the first report on the development of a new and efficient process to convert waste cooking oil intod-limonene andl-limonene by exploiting metabolically engineeredY. lipolyticastrains for fermentation. The results obtained in this study lay the foundation for more future applications ofY. lipolyticain converting waste cooking oil into various industrially valuable products.

Background Yarrowia lipolytica, an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica. The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture. Results In this study, we showed that environmental version of minimization of metabolic adjustment (eMOMA) can be used for predicting metabolic flux distribution of Y. lipolytica under the nitrogen-limited condition and identifying metabolic engineering strategies to improve lipid production in Y. lipolytica. Several well-characterized overexpression targets, such as diglyceride acyltransferase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, were successfully rediscovered by our eMOMA-based design method, showing the relevance of prediction results. Interestingly, the eMOMA-based design method also suggested non-intuitive knockout targets, and we experimentally validated the prediction with a mutant lacking YALI0F30745g, one of the predicted targets involved in one-carbon/methionine metabolism. The mutant accumulated 45% more lipids compared to the wild-type. Conclusion This study demonstrated that eMOMA is a powerful computational method for understanding and engineering the metabolism of Y. lipolytica and potentially other oleaginous microorganisms.

Background Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast. Methods The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387. Results The R 2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R 2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R 2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods. Conclusions Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.

Background Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. Results In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. Conclusions Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica. Electronic supplementary material The online version of this article (10.1186/s12934-019-1127-8) contains supplementary material, which is available to authorized users.

Background Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide – inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy–repeated-batch culture was applied. Results The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm− 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm− 3 h− 1 and a yield of 0.85 g g− 1. Conclusions The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol–oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 μg/g of kynurenic acid, which increases the health-promoting value of the product.

Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in Yarrowia lipolytica. We evaluated different propionate-activating enzymes and the overexpression of propionyl-CoA transferase gene from Ralstonia eutropha increased the accumulation of OCFAs by 3.8 times over control strain, indicating propionate activation is the limiting step of OCFAs synthesis. It was shown that acetate supplement was necessary to restore growth and to produce a higher OCFA contents in total lipids, suggesting the balance of the precursors between acetyl-CoA and propionyl-CoA is crucial for OCFA accumulation. To improve β-ketovaleryl-CoA pools for further increase of OCFA production, we co-expressed the bktB encoding β-ketothiolase in the producing strain, and the OCFA production was increased by 33% compared to control. Combining strain engineering and the optimization of the C/N ratio promoted the OCFA production up to 1.87 ​g/L representing 62% of total lipids, the highest recombinant OCFAs titer reported in yeast, up to date. This study provides a strong basis for the microbial production of OCFAs and its derivatives having high potentials in a wide range of applications., Graphical abstract Image 1, Highlights • The supply of propionyl-CoA by overexpressing propionate-activating enzymes improved OCFA production in Yarrowia lipolytica. • Balancing between acetyl-CoA and propionyl-CoA is crucial for higher production of OCFA. • Boosting the availability of precursor pools by introducing β-ketothiolase increased lipid accumulation.

Background Succinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low. Results In this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica. PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource. Conclusion Our results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica, which will promote the industrial production of bio-based SA.

The host microbiome plays an essential role in health and disease. Microbiome modification by pathogens or probiotics has been poorly explored especially in the case of probiotic yeasts. Next-generation sequencing currently provides the best tools for their characterization. Debaryomyces hansenii 97 (D. hansenii 97) and Yarrowia lipolytica 242 (Y. lipolytica 242) are yeasts that protect wildtype zebrafish (Danio rerio) larvae against a Vibrio anguillarum (V. anguillarum) infection, increasing their survival rate. We investigate the effect of these microorganisms on the microbiome and neutrophil response (inflammation) in zebrafish larvae line Tg(Bacmpx:GFP)i114. We postulated that preinoculation of larvae with yeasts would attenuate the intestinal neutrophil response and prevent modification of the larval microbiome induced by the pathogen. Microbiome study was performed by sequencing the V3-V4 region of the 16S rRNA gene and prediction of metabolic pathways by Piphillin in conventionally raised larvae. Survival and the neutrophil response were both evaluated in conventional and germ-free conditions. V. anguillarum infection resulted in higher neutrophil number in the intestinal area compared to non-infected larvae in both conditions. In germ-free conditions, infected larvae pre-inoculated with yeasts showed fewer neutrophil numbers than infected larvae. In both conditions, only D. hansenii 97 increased the survival of infected larvae. Beta diversity of the microbiota was modified by V. anguillarum and both yeasts, compared to non-inoculated larvae. At 3 days post-infection, V. anguillarum modified the relative abundance of 10 genera, and pre-inoculation with D. hansenii 97 and Y. lipolytica 242 prevented the modification of 5 and 6 of these genera, respectively. Both yeasts prevent the increase of Ensifer and Vogesella identified as negative predictors for larval survival (accounting for 40 and 27 of the variance, respectively). In addition, yeast pre-inoculation prevents changes in some metabolic pathways altered by V. anguillarum’s infection. These results suggest that both yeasts and V. anguillarum can shape the larval microbiota configuration in the early developmental stage of D. rerio. Moreover, modulation of key taxa or metabolic pathways of the larval microbiome by yeasts can be associated with the survival of infected larvae. This study contributes to the understanding of yeast–pathogen–microbiome interactions, although further studies are needed to elucidate the mechanisms involved.

Yarrowia lipolytica is one of the most studied non-conventional forms of yeast, exhibiting a high secretory capacity and producing many industrially important and valuable metabolites. The yeast conceals a great biotechnological potential to synthesize organic acids, sweeteners, microbial oil, or fragrances. The vast majority of bioprocesses are carried out in bioreactors, where suitable culture conditions are provided. In the current study, the effect of agitation speed (200–600 rpm) and air flow rate (0.0375–2.0 dm3/(dm3 × min)) on the biomass yield and lipase activity of Y. lipolytica KKP 379 is analyzed in a growth medium containing waste fish oil. The increase of aeration intensity limited the period of oxygen deficit in the medium. Simultaneously, an increase in lipolytic activity was observed from 2.09 U/cm3 to 14.21 U/cm3; however, an excessive agitation speed likely caused oxidative or shear stresses, and a reduction in lipolytic activity was observed. Moreover, it is confirmed that the synthesis of lipases is related to oxygen consumption, pH, and the yeast growth phase, and appropriate process selection may provide two advantages, namely, the maximum use of the waste carbon source and the production of lipolytic enzymes that are valuable in many industries.

The study aimed to evaluate the impact of selected factors of the freeze-drying process on the hydrolytic and synthetic activity of the extracellular lipases of Y. lipolytica KKP 379 and to attempt the use of the crude enzyme preparation as a biocatalyst in the synthesis of geranyl 4-hydroxyphenylpropanoate. Antioxidant and antibacterial properties of the geranyl ester derivative were also investigated in order to evaluate their usefulness as a novel food additive. The studies confirmed that freeze-drying was an effective method of dehydrating yeast supernatant and allowed for obtaining lyophilizates with low water activity from 0.055 to 0.160. The type and concentration of the additive (2–6% whey protein hydrolyzate, 0.5% and 1% ammonium sulphate) had a significant effect on the hydrolytic activity of enzyme preparations, while the selected variants of drying temperature during the freeze-drying process were not significant (10 °C and 50 °C). Low yield of geranyl 4-hydroxyphenylopropionate was shown when the lyophilized supernatant was used (5.3%), but the yield of ester synthesis increased when the freeze-dried Y. lipolytica yeast biomass was applied (47.9%). The study confirmed the antioxidant properties of the synthesized ester by the DPPH• and CUPRAC methods, as well as higher antibacterial activity against tested bacteria than its precursor with 0.125 mM MIC (minimal inhibitory concentration) against L. monocytogenes.

The current Research Topic provides an effective communication platform, collecting both original research articles and review papers examining explorations of the mechanism for lipid accumulation, biotechnological applications, and metabolic engineering efforts related to oleaginous fungi including the non-conventional yeasts. Microbes have been harnessed for the production of hydrocarbon with a high-energy density as “drop-in” fuels, renewable chemicals, and value-added compounds. In addition to the commonly used model organisms such as Escherichia coli and Saccharomyces cerevisiae, over the past few years, oleaginous yeasts that naturally accumulate high-content lipids have been directly used or genetically modified for producing diverse bioproducts, although early trials on the commercial production of microbial oil date back to World War I. This Research Topic concentrates on the advancement of bioengineering of oleaginous yeasts, including Yarrowia lipolytica and Rhodosporidium (Rhodotorula) toruloides, for producing biofuels and bioproducts, with particular emphasis on the establishment of synthetic biology tools and novel engineering strategies.

β-carotene is an increasingly sought-after organic pigment with antioxidant properties and a vitamin precursor. Yarrowia lipolytica, though unable to naturally synthesize carotenoids, can produce high amounts of the precursor acetyl-CoA making it a promising host for metabolic engineering towards novel biotechnological production of carotenoids. Here, we describe a synthetic biology methodology for Y. Lipolytica metabolic engineering based on Golden Gate DNA assembly for the generation of a multigene cassette, subsequent transformation enabling β-carotene biosynthesis, and quantification of the compound.

International audience; The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Volatile fatty acids (VFAs), which can be generated by acidogenesis of organic wastes, are important building blocks for chemicals production, and are intermediates in many bioprocesses such as microbial lipids production. Important factors affecting the bioconversion of VFAs (acetate, propionate and butyrate) by Yarrowia lipolytica W29 and NCYC 2904 for growth and lipids accumulation were studied. Yarrowia lipolytica grew efficiently in VFAs-based media, but lipids production was enhanced by the addition of co-substrates (glucose or glycerol) in batch cultures. A two-stage batch culture growth phase on glucose, followed by VFAs addition improved lipids accumulation. Lipids concentrations of 2.3?g·L-1 and 3.5?g·L-1 were obtained with this mode of operation, with addition of 18?g·L-1 VFAs, for Y. lipolytica W29 and NCYC 2904, respectively. For the first time, it was demonstrated that oxygen mass transfer is a crucial factor for lipids production by Y. lipolytica from VFAs. Intracellular lipids produced by Y. lipolytica strains were mainly composed by oleic and linoleic acids, similar to common vegetable oils, making these lipids suitable for biodiesel production. Moreover, margaric acid, which may improve biodiesel properties, was only detected in propionate medium. The strategies studied herein will contribute to the feasibility of using VFAs as low-cost feedstock for microbial lipids production by Y. lipolytica strains., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project TUBITAK 2014 (TUBITAK/0009/2014), the strategic funding of UIDB/04469/2020 unit and Doctoral grants (SFRH/BD/144188/2019; SFRH/BD/129592/2017)., info:eu-repo/semantics/publishedVersion

The sugar alcohols and functional sugars have wide applications in food, pharmaceutical, and chemical industries. However, the smaller quantities of natural occurring sugar alcohols and functional sugars restricted their applications. The enzymatic and whole-cell catalyst production is emerging as the predominant alternatives. The properties of Yarrowia lipolytica make it a promising sugar alcohol and functional sugar producer. However, there are still some issues to be resolved. As there exist reviews about the chemical structures, physicochemical properties, biological functions, applications, and biosynthesis of sugar alcohols and/or functional sugars in Y. lipolytica, this mini review will not only update the recent advances in enzymatic and microbial production of sugar alcohols (erythritol, D-threitol, and xylitol) and functional sugars (isomaltulose, trehalose, fructo-oligosaccharides, and galacto-oligosaccharides) by using recombinant Y. lipolytica but also focus on the studies of gene discovery, pathway engineering, expanding substrate scope, bioprocess engineering, and novel breeding methods to resolve the aforementioned issues.

Background Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. Results Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. Conclusion We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

β-carotene is a precursor of vitamin A and has multiple physiological functions. Producing β-carotene by microbial fermentation has attracted much attention to consumers’ preference for natural products. This study focused on improving β-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing β-carotene. Thereafter, the β-carotene biosynthetic pathway’s metabolic balance was accurately regulated to reduce the intermediates’ accumulation. Notably, the β-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of β-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing β-carotene. The β-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

A synthetic medium containing glucose, glycerol, yeast extract (YE), and ammonium sulfate (AS) was compared to several low-cost media in their ability to produce high emulsification index (EI). The goal was to reduce the production costs of an emulsifier with application in food oil-in-water emulsions. To this end, agro-industrial by-products were screened for bioemulsifier production from Yarrowia lipolytica. The statistical analysis showed that the EIs of media containing residual frying oil from palm oil (RFO_palm) or soybean oil (RFO_soy), residual liquid from butter production (butter whey, BWhey) or cheese production (cheese whey, CWhey), supplemented with YE and AS were similar to the EI of the synthetic medium. The replacement of YE by corn steep liquor (CSL) also resulted in similar EI, except for RFO_soy. BWhey was tested with CSL without AS and similar EI (66.8%) was detected in comparison to that of the same medium with AS (66.3%). The cell-free broth obtained after Y. lipolytica growth in BWhey+CSL was successfully used to obtain vegetable oil-in-water emulsions indicating its potential application in food products. RESUMO: O meio sintético contendo glicose, glicerol, extrato de levedura (YE) e sulfato de amônio (AS) foi comparado a meios de baixo custo para produzir alto índice de emulsificação (EI). Para este fim, os subprodutos agroindustriais foram rastreados quanto à produção de bioemulsificante por Yarrowia lipolytica. A análise estatística mostrou que EI de meios contendo óleo de fritura residual de óleo de palma (RFO_palm) ou óleo de soja (RFO_soy), líquido residual da produção de manteiga (soro de manteiga, BWhey) ou da produção de queijo (soro de queijo, CWhey), suplementado com YE e AS foram semelhantes ao EI do meio sintético. A substituição do YE por milhocina (CSL) também resultou em EI semelhante, exceto no RFO_soy. O BWhey foi testado com CSL sem AS e EI semelhante (66,8%) foi detectado em comparação com o mesmo meio com AS (66,3%). O meio isento de células obtido após o crescimento de Y. lipolytica no meio BWhey + CSL foi utilizado com sucesso para obter emulsões de óleo vegetal em água, indicando sua potencial aplicação em produtos alimentícios.