Yeast for increased lipid production

Abstract

The present invention relates to a Yarrowia lipolytica CBS 8108 strain for producing lipids, comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes. In a second aspect, the invention relates to a method for producing lipids using said Y. lipolytica strain. The invention further pertains to lipids obtainable by the method, a composition comprising said lipids, a composition comprising Y. lipolytica strain, and a food product obtainable therefrom.

Description

[0001] YEAST FOR INCREASED LIPID PRODUCTION

[0002] Field of the invention

[0003] The present invention relates to the field of molecular biology, more particularly the field of microbial engineering, even more particularly to field of improving lipid yields of microbial strains. The invention relates to yeast strains for increased lipid production comprising chimeric genes and constructs. Furthermore, the invention relates to methods for lipid production using the yeast strains of the invention.

[0004] Background

[0005] In the recent years, the use of microorganisms such as bacteria, yeasts, fungi and microalgae for production of microbial oils has been studied in many industrial branches, from the generation of renewable energy to the provision of value-added products in the pharmaceutical, cosmetics and food industries. The bottleneck is to achieve a less costly and more sustainable way for a biomass transformation, and to obtain highly versatile products, which can be applied in various industries.

[0006] Triacylglycerols (TAGs) are esters of glycerol with three fatty acids. TAGs are the main constituent of animal and vegetable fats and oils, and as such, have numerous commercial applications, including food, personal care, and oil- paints and coatings products. Current technologies for producing TAGs are typically via extraction from plant or animal sources, which is costly and leads to problems of sustainability of these plant or animal sources. It is hard to overstate the environmental burden of animal agriculture. It is the second largest contributor to human-associated greenhouse gas emissions (after fossil fuels) and is a leading cause of deforestation, biodiversity loss, and water and air pollution. For example, animal production takes up 77% of all agricultural land on earth despite supplying only 17% of humanity's food. On the other hand, the production of plant oils faces another challenges. For example, the production of coconut oil (as well as other alternatives, such as palm oil), is highly unsustainable, increasing deforestation and greatly reducing biodiversity, possibly even more so than palm oil production. Second, the current plant oil production is not sufficient for future demand.

[0007] Recent concerns about sustainability, food security, and the environmental and public health impacts of industrial agriculture accelerated the need for alternative sources for lipid production. Fungal cells, in particular oleaginous yeast cells could be a suitable source of lipids and triacylglycerols. For example, EP3388516, W02017070065, US20240043788 and US11692209 relate to a fungal cell capable of producing fatty acids, fatty acid derivatives, including triacylglycerols, for example by providing the fungal cell with at least one modification to the endogenous fatty acid metabolism.

[0008] There is a need to develop new microbial strains for the sustainable and cost-effective production of lipids, preferably by precision fermentation. There is a need for obtaining novel yeast strains, for production of lipids at high yield and within shorter period of time compared to known yeasts. The new strains should be suitable for lipid production on an industrial scale level. There is a need for an improved method for a large-scale production of yeast derived lipids. Finally, it is of interest to produce TAGs of specific structures and composition in order to get desired properties to fit various food, pharmaceutical and / or cosmetics applications. For example, new trends at producing sustainable plant-based alternatives to animal foods, such as plant-based meat and dairy, require fats that are similar to animal fats in order to mimic the taste and texture of animal food products.

[0009] Summary

[0010] The present invention aims to provide alternative industrially applicable oleaginous yeast strains, which could be used for lipid production in a more sustainable manner and produced at high yield, preferably with a desired chemical composition of lipids. Preferably said yeast could provide tailored, more animallike lipids, preferably triacylglycerols, which, for example, can mimic the taste of the animal fats and as such represent a versatile component for various applications such as food products. In an independent aspect, the present invention aims to provide a method for producing lipids using the yeast strains, which method would allow for a high yield of obtainable lipids, and relatively short lipid synthesis time. Furthermore said method is suitable for an industrial scale production.

[0011] In a first aspect, the application relates to a newly engineered Yarrowia lipolytica strain for producing lipids, in particular the Y. lipolytica CBS 8108 strain, comprising one or more chimeric genes, wherein each one of said chimeric genes comprises: i) a yeast-expressible promoter, ii) a DNA region encoding an ACC1, MCE2 and / or DGA1 protein; and iii) a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0012] The yeast strain according to a first aspect of the invention is engineered to allow an increased lipid synthesis. The combination of the overexpression of ACC1, MCE2 and / or DGA1 genes can lead to an increased lipid synthesis compared to the known wild type strain, and a preferred oleaginous yeast alternative, when a fast and / or efficient production of high and / or industrial-scale quantities of triacylglycerols (TAGs) is preferred.

[0013] In a particularly preferred embodiment, the invention relates to the Y. lipolytica CBS 8108 strain for producing lipids, the strain comprising three chimeric genes, a first one comprising a nucleic acid sequence to encode the ACC1 protein, a second one comprising a nucleic acid sequence to encode the MCE2 protein, a third one comprising a nucleic acid sequence to encode the DGA1 protein. This preferred strain allows for a particularly increased TAGs production. The lipid yield expressed as grams of lipids produced by one liter of the yeast culture and / or during fermentation was increased compared to other known oleaginous Y. lipolytica strains, and the fatty acid profile is preferred as there is an increased content of polyunsaturated fatty acids, compared to a known, reference Y. lipolytica strain. Moreover, said new strain is capable of growing on alternative carbon sources, such as, for example, glycerol, xylose and / or arabinose.

[0014] The present invention relates to a Y. lipolytica CBS 8108 strain for producing lipids, comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes.

[0015] In a second aspect, the invention relates to a method for producing lipids using said Y. lipolytica strain. According to a second aspect, the invention relates to a method for producing lipids using the strain according to a first aspect of the invention, the method comprising the steps of:

[0016] -culturing the strain according a first aspect of the invention in a culture medium and in culturing conditions suitable for production of lipids, and

[0017] -optionally, extracting said lipids from said culture medium and / or said strain.

[0018] The method according to the second aspect is particularly suited for a large-scale TAGs production, and it allows reaching the plateau phase of lipid production faster than the known methods in the art. Said method is particularly suited for large scale, industrial yeast production, wherein methods are to be robust, reproducible and fast.

[0019] The invention further pertains to lipids obtainable by the method, a composition comprising said lipids, a composition comprising Y. lipolytica strain, and a food product obtainable therefrom.

[0020] Brief description of the Figures

[0021] The figures described are only schematic and are non-limiting. In the figures, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0022] Figure 1. A) Phenotypic characterization of two Y. lipolytica strains: the wild type reference strain ATCC20460 (herein referred to as W29) and the newly selected strain CBS 8108 (herein referred to as YI51). The strains were characterized on their ability to grow to alternative carbon sources and their resistance towards industrially relevant inhibitors. The assays were performed in agar plates and mean colony size was determined. Normalized growth (arbitrary units) is presented in the spider plots. B) Performance of the wild type strains W29 and YI51 as well as of their "obese" variants, which were engineered to recombinantly overexpress ACC1, DGA1 and MCE2 proteins for improved lipid production, in shake flasks fermentations. The strains were grown in a low nitrogen (N) medium supplemented with 6% glucose, at 30 ° C (220 rpm) for 5 days. At the end of the fermentations the lipids were extracted and lipid titer was determined. C) Comparison of the performance of the "obese" W29 with "obese" YI51 variants engineered for overexpression of heterologous DGA1 proteins, as further clarified in Example 2. The strains were grown in a low N medium supplemented with 6 % glucose, at 30° C (220 rpm) for 5 days. At the end of the fermentations the lipids were extracted and lipid titer was determined. In B and C, data are shown from at least 3 repeats of the fermentations, and the bars indicate the standard deviation (SD) between the replicates.

[0023] Figure 2. A) Comparison of the "obese" W29 and YI51 performance in fed-batch fermentations. The strains were grown in 8 L bioreactors for 4 days. Lipid titers and dried cell weight (DCW) were monitored every day. B) Comparison of the pseudohyphae formation, productivity of lipids, and fatty acid (FA) composition between the two strains in fed-batch fermentations.

[0024] Figure 3. Comparison of lipogenic activity between "obese" strains ("obese" YI51 and "obese" W29 obtained in Example 1) in shake flask fermentations. The two strains were first cultivated in rich bioreactor medium (3.8 g / L yeast nitrogen base without amino acids and ammonium sulfate, 3 g / L yeast extract, 5 g / L ammonium sulfate, 50 g / L glucose) for 48 hours to generate biomass. After 48h the generated biomass from each of the strains was harvested, washed twice with 5% glucose and then transferred, at a starting dry cell weight (DCW) of 4 g / l, to shake flasks containing 5% glucose, as the sole nutrient, to induce lipid production. At the indicated time points (24, 68, 96 hours post-inoculation, i.e., 24, 68, 96 h after the fermentations have been started), A) the DCW, B) the lipid titer and C) lipid yield of the "obese" strains were measured (based on the consumed glucose). The cultures were incubated at 30 °C with shaking at 200 rpm. The experiment was conducted in triplicate with error bars representing standard deviation (SD) between the replicates. Statistical significance of differences between the "obese" YI51 and W29 strains at each timepoint was evaluated using t-test (p-values are shown in the graphs A-C of Fig 3.).

[0025] Detailed description

[0026] Definitions

[0027] The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. It is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0028] The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0029] Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments, of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0030] The term "recombinant expression" as used herein, means expression of a protein of interest by introducing a vector or expressing cassette which comprises the nucleotide sequence required for expression of a chimeric gene, as defined below, into a host organism, and culture said organism as to obtain host cells expressing said protein of interest. Some non, limiting examples of the model organism are: yeast, bacteria, algae, procaryotic organisms, mammals, insects. The recombinant expression may allow, for example, that once that the recombinant DNA is introduced into a recipient host cell, said DNA gets multiplied and is upon expression translated into recombinant protein.

[0031] A "chimeric gene" or "chimeric construct" or "chimeric gene construct", used herein as synonyms, is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.

[0032] The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other known yeast genes, or from T-DNA. The terminator to be added may be derived from, for example, any yeast derived gene, or less preferably from any other eukaryotic gene.

[0033] "Nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analog. By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.

[0034] "Coding sequence" is a nucleotide sequence, which is transcribed into mRNA and / or translated into a polypeptide and / or protein when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.

[0035] "Promoter region of a gene" as used here refers to a functional DNA sequence unit that, when operably linked to a coding sequence and possibly placed in the appropriate inducing conditions, is sufficient to promote transcription of said coding sequence.

[0036] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A promoter sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence. The term "operably linked" as used herein also refers to a functional linkage between the promoter sequence and the gene of interest, such that said promoter sequence is able to initiate transcription of the gene of interest that has been linked to.

[0037] "Gene" as used here includes both the promoter region of the gene as well as the coding sequence. It refers both to the genomic sequence (including possible introns) as well as to the cDNA derived from the spliced messenger, operably linked to a promoter sequence.

[0038] The term "terminator" or "transcription termination signal" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other microorganism genes, preferably yeast genes, or from T-DNA.

[0039] An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. Such cassettes can be constructed into a "vector".

[0040] The term "vector", "vector construct," "expression vector," or "gene transfer vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, and includes any vector known to the skilled person, including any suitable type. The term vector, as used herein, may include, but is not limited to, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Expression vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Suitable vectors have regulatory sequences, such as promoters, enhancers, terminator sequences, and the like as desired and according to a particular host organism (e.g. bacterial cell, yeast cell). Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art.

[0041] The term "expression cassette" refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including, in addition to plant cells, prokaryotic, yeast, fungal, insect or mammalian cells. The term includes linear and circular expression systems. The term includes all vectors. The cassettes can remain episomal or integrate into the host cell genome. The expression cassettes can have the ability to selfreplicate or not (i.e., drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimum elements needed for transcription of the recombinant nucleic acid.

[0042] The term "overexpression", as used herein, refers to a recombinant expression method wherein the protein of interest is produced in higher levels as compared to the native protein level of said protein of interest, and thus means the genetic modification which includes a nucleic acid construct that increases the expression of the gene products and / or the nucleic acid construct. The nucleic acid construct can, for example, include (a) an expression cassette comprising a nucleic acid sequence encoding the gene products under the control of a suitable homologous or heterologous promoter, and / or (b) a nucleic acid sequence that modulates the level of expression of the gene products when inserted into the genome of the cell. In some embodiments, the nucleic acid construct inhibits or disrupts the natural regulation of a native gene encoding the gene product resulting in overexpression of the native gene. In some embodiments, inhibition or disruption of the natural regulation of the native gene is mediated by deletion, disruption, mutation and / or substitution of a regulatory region, or a part of a regulatory region regulating expression of the gene.

[0043] The term "yield" as used herein generally refers to a measurable product from a microorganism, particularly a yeast. Yield and yield increase (in comparison to a non-transformed , non-engineered strain or wild-type yeast) can be measured in a number of ways, and it is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular microbial oil production concerned and the specific purpose or application concerned. The terms "improved yield" or "increased yield" can be used interchangeable. As used herein, the term "improved yield" or the term "increased yield" means any improvement in the yield of any measured yeast and / or yeast isolate. In accordance with the invention, the expression of the chimeric genes such as ACC1, DGA1 and / or MCE2 allows obtaining of an increased yield of lipids, in particular triacylglycerols in, for example, yeast fermentation cultures. For example, and without limitation, parameters such as an increased lipid titer are the possible way to express and / or quantify said increased lipid yield. For example, enhanced or increased "yield" refers to one or more yield parameters selected from the group consisting of biomass yield, dry biomass yield, and the like. Any increase in yield is an improved yield in accordance with the invention. For example, the improvement in yield can comprise a 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or greater increase in any measured parameter, comprising lipid production. For example, an increased yield can mean 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or greater increase in a lipid titer or a dry cell weight (DCW) values. In embodiments, an increased yield may also concern an increase in quantity of obtained and / or harvested triacylglycerols.

[0044] "Higher" or "increased" as used herein refers to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold or at least 10 fold higher quantity or an effect. In some embodiments, "higher" or "increased" refers to a statistically significant difference. "Predominantly" as used herein, means at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of quantity or an effect.

[0045] "Lower" or "decreased" as used herein is defined herein as a statistically significantly decreased, more particularly an at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.5 fold, at least 2 fold, at least 3 fold, at least 5 fold or at least 10 fold lower quantity or an effect. In some embodiments, "lower" or "decreased" refers to a statistically significant difference.

[0046] The "lipid titer" as used herein, is a measure of the production of the lipids by a yeast, for example achieved by increased expression of the chimeric gene(s) which confers a phenotype of increased lipid titer, increased lipid productivity and / or increased lipid yield. The lipid titer as used herein in the context of yeast lipid synthesis, e.g., in the context of a fatty acid synthesis by an oil-producing Y. lipolytica strains described herein, refers to an amount of lipid synthesized per volume of a microbial culture comprising the oil-producing microbe. The lipid titer can be, for example, at least 25 g / L. In some embodiments, the lipid titer can be, for example, 25-150 g / L.

[0047] The production of the lipids can be defined as "the lipid productivity", and can be, for example, at least 0.25 g / L / h. In some embodiments, the lipid productivity is 0.25 g / L / h to 1.5 g / L / h. In some embodiments, the lipid yield is at least 0.220 g-fatty acid methyl esters / g-glucose. In some embodiments, the lipid yield is 0.220 g-fatty acid methyl esters / g-glucose to 0.280 g-fatty acid methyl esters / g-glucose.

[0048] The term "lipid" refers to fatty acids and their derivatives. Accordingly, examples of lipids include fatty acids (FA, both saturated and unsaturated), glycerides or glycerolipids, also referred to as acylglycerols (such as monoglycerides (monoacylglycerols), diglycerides (diacylglycerols), triglycerides (triacylglycerols, TAGs, or neutral fats), phosphoglycerides (glycerophospholipids), nonglycerides (sphingolipids, sterol lipids, including cholesterol and steroid hormones, prenol lipids including terpenoids, fatty alcohols, waxes, and polyketides), and complex lipid derivatives (sugar-linked lipids or glycolipids, and protein-linked lipids). Lipids are an essential part of the plasma membrane of living cells and microbes. Some cells and microbes also produce lipids to store energy, for example in the form of triacylglycerols in lipid bodies, lipid droplets, or vacuoles.

[0049] Some aspects relate to engineered microbes for biofuel or biofuel precursor production. In some embodiments, the microbes provided herein are engineered to optimize their lipid metabolism for lipid production. The term "lipid metabolism" refers to the molecular processes that involve the creation or degradation of lipids. Fatty acid synthesis, fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAG storage and TAG degradation are examples of processes that are part of the lipid metabolism of a cell. Accordingly, the term "fatty acid metabolism" refers to all cellular or organismic processes that involve the synthesis, creation, transformation or degradation of fatty acids. Fatty acid synthesis, fatty acid oxidation, TAG synthesis, and TAG degradation are examples of processes are part of the fatty acid metabolism of a cell.

[0050] The term "triacylglycerol" (TAG, sometimes also referred to as triglyceride) refers to a molecule comprising a single molecule of glycerol covalently bound to three fatty acid molecules, aliphatic monocarboxylic acids, via ester bonds, one on each of the glycerol molecule's three hydroxyl (OH) groups.

[0051] As used herein, the terms "bioreactor", refers to an enclosure, or partial enclosure, in which a biological and / or chemical reaction takes place, at least part of which involves a living organism or part of a living organism.

[0052] A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product, for example a biofuel or biofuel precursor, for example a lipid, fatty acid and / or TAG, on a commercial or quasi-commercial scale. Large scale bioreactors can have volumes for example in the range of liters, hundreds of liters, thousands of liters, or more.

[0053] The term ACC1 or ACC1 protein as used herein, refers to Acetyl-CoA carboxylase (referred to as class E.C. 6.4.1.2), biotin containing enzyme; catalyses carboxylation of cytosolic acetyl-CoA to form malonyl-CoA and regulates histone acetylation by regulating the availability of acetyl-CoA; rate-limiting step for de novo biosynthesis of fatty acids. Malonyl-CoA is precursor of lipid biosynthesis, and this phase of lipid biosynthesis is also referred to as a "PUSH" phase.

[0054] The term DGA1 or DGA1 protein, as used herein, refers to a diacylglycerol acyltransferase (referred to as class E.C.2.3.1.20); which enzyme catalyses the terminal, i.e., terminal step of triacylglycerol (TAG) formation, acylates diacylglycerol using acyl-CoA as an acyl donor. Said acylating diacylglycerol using acyl-CoA as an acyl donor helps to increase the ratio of fatty acids incorporation to TAGs, thus leading to an increased lipid production ("PULL" phase of the lipid biosynthesis).

[0055] The term MCE2 or MCE2 protein, as used herein, refers to a cytosolic NADP+-dependent malic enzyme (referred to as class 1.1.40), which can catalyse the oxidative decarboxylation of malate to pyruvate thereby effectively restoring the NADPH homeostasis during lipogenesis. Said enzyme activates the pyruvate / oxaloacetate / malate (POM) cycle, which can convert 1 mol NADH to 1 mol NADPH at a cost of 1 mol ATP, thereby overexpression the malic enzyme was demonstrated to be effective in lipid yield improvement. The endogenous malic enzyme in Y. lipolytica is herein referred to as "yIMAE" and is a mitochondria-associated NAD+-dependent enzyme, whose overexpression and knockout has little impact on lipid production.

[0056] In an initial step of a fatty acid synthesis, acetyl-CoA is carbonylated by the addition of CO2 to malonyl- CoA, by the enzyme acetyl-CoA carboxylase. Biotin is an essential cofactor in this reaction, and is covalently attached to the ACC apoprotein, by the enzyme biotin: apoprotein ligase. ACC is a trifunctional enzyme, harbouring a biotin carboxyl carrier protein domain, a biotin-carboxylase domain, and a carboxyl-transferase domain. Eukaryotic ACC, including mitochondrial ACC variants harbour these functions on a single polypeptide. Malonyl-CoA produced by ACC serves as a two carbon donor in a cyclic series of reactions catalysed by fatty acid synthase, and elongases. De novo synthesis of fatty acids utilizes substantial amounts of metabolites, acetyl-CoA, ATP and NADPH, and thus competes with other cellular processes that are dependent on these compounds. NADPH is required for two reduction steps in the fatty acid elongation cycle, linking fatty acid synthesis to the metabolic state of the cell and results in fatty acid synthesis being restricted to conditions of high energy load of the cells, indicated by increased ATP / AMP ratio, elevated reduction equivalents and elevated acetyl-CoA pool. Almost all subcellular organelles are involved in fatty acid metabolism, indicating that maintenance of fatty acid homeostasis requires regulation at multiple levels.

[0057] Detailed description of embodiments

[0058] In the present invention we have identified a Yarrowia lipolytica strain CBS 8108 with superior properties, especially suitable in facilitating the production of different lipid compositions at high yields. Moreover, it was shown that genetically engineering this Y. lipolytica strain in view of its use in lipid productions resulted in an increased and / or synergistic lipid yield as compared to the Y. lipolytica strains known in the art. Hence, the invention relates to said engineered Y. lipolytica strain recombinantly expressing chimeric gene constructs coding for ACC1, MCE2 and / or DGA1 enzymes, preferably Y. lipolytica CBS 8108, as to increase the lipid production compared to lipid production observed in yeasts currently used for lipid production known in the prior art. The present invention provides new engineered Y. lipolytica strains which are suitable for increased lipid production and / or production of tailored lipids, for example lipids that can mimic animal fat. Particularly, the invention relates to a Y. lipolytica CBS 8108 strain for producing lipids, comprising one or more of chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes.

[0059] In a first aspect, the invention provides for Y. lipolytica CBS 8108 strain for producing lipids, comprising one or more chimeric gene constructs, wherein each one of said chimeric gene constructs comprises: i) a yeast-expressible promoter, ii) a DNA region encoding an ACC1, MCE2 and / or DGA1 protein; and iii) a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0060] In other words, the invention aims to provide Y. lipolytica CBS 8108 strain for producing lipids, wherein in said strains one or more of the following genes is recombinantly expressed: ACC1, MCE2 and / or DGA1, preferably wherein one or more of the said ACC1, MCE2 and / or DGA1 genes is overexpressed. The strain according to the first aspect is particularly suitable for production of the lipids on a large or industrial scale, during a short period of production time. Furthermore, the strain according to the invention is of a higher lipogenic capacity, for example the use of the strain can allow for a higher lipid titer, DCW and / or lipid yield compared to other strains known in the prior art. Lipogenic capacity, i.e. the capability of a yeast to increase lipid production regardless of the fermentation conditions, is an important feature of obese, oleaginous yeasts used for large-volume and / or industrial-scale lipid production.

[0061] In a preferred embodiment, Y. lipolytica strain CBS 8108 comprising three chimeric constructs is provided, wherein a first construct comprises the DNA region encoding the ACC1 protein, a second construct comprises the DNA region encoding the MCE2 protein, and a third construct comprises the DNA region encoding the DGA1 protein. In said embodiment, Y. lipolytica strain CBS 8108 is engineered by overexpression of three chimeric gene constructs comprising ACC1 DGA1 and MCE2 genes thereby featuring all 3 genes ACC1, DGA1 and MCE2 recombinantly expressed, preferably overexpressed. Said strain preferably shows higher lipid titer compared to known obese strains reported in the prior art, as shown in Fig. IB.

[0062] The Y. lipolytica CBS 8108 strain, comprising one or more of chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes can utilize variety of carbon sources, including, but not limited to fermentable sugars, for example sugars, such as glucose, galactose, arabinose, xylose, and / or organic acids, e.g., acetic acid, oleic acid and / or their salts, e.g., acetate, oleate and / or glycerol, methanol, and / or ethanol. In an embodiment, the strain according to invention is able of growing on lignocellulosic biomass, preferably biomass hydrolysate thereof, more preferably monomeric sugars via enzymatic hydrolysis of said lignocellulosic biomass. Said preferred strain is unexpectedly able to grow on alternative carbon sources such as, but not limited to glycerol, xylose, arabinose, and decane. Development of novel yeast strains capable to grow on the alternative substrates is particularly advantageous, as it allows for utilization of the biomass waste and utilization of available biomass sources.

[0063] The Y. lipolytica CBS 8108 strain, comprising one or more of chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes is shown to be more resistant to known and / or common industrial inhibitors of the yeast growth, such as, but not limited to acetic acid, coumaric acid, methanol, 5- hydroxymethylfurfural, coniferyl aldehyde and the like, as compared to the reference strain W29. Thus, said particularly preferred yeast is more robust to influences of common inhibitors, and thus it can be used to for the lipid production in not optimal, harsh conditions.

[0064] The yeast strain of the invention comprises one or more chimeric gene construct, which one or more constructs comprise yeast-expressible promoter. In some embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a translation elongation factor 1 alpha (TEF) promoter or a Yarrowia lipolytica glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter. In some embodiments, the nucleic acid construct further comprises an intron. In some embodiments, the intron is downstream of the transcription initiation site, optionally wherein the intron is within the nucleic acid sequence encoding the gene product.

[0065] In a preferred embodiment, the yeast expressible promoter is export protein promoter (EXP1) preferably Y. lipolytica EXP1 and / or GPD, preferably Y. lipolytica GPD. Said promoters are found to be particularly suited for achieving the overexpression of ACC1, DGA1, and MCE2 genes in the engineered Y. lipolytica strain of the invention, thereby allowing an increased lipid production of lipids.

[0066] The Y. lipolytica strain which recombinantly expresses native YL ACC1, DGA1 and MCE2 is also called herein an "obese" YL strain, as it is in particular suited for high lipid titer production. Hence, the term "obese" is used herein to refer to a genetically engineered Y. lipolytica strain, more specifically in this application the "obese" strains are defined as being engineered by introducing the following chimeric genes: EXP1:ACC1, GPD:DGA1 and GPD:MCE2 wherein EXP1 and GPD1 (the amino acid sequences of EXP1 and GPD genes shown in SEQ ID NO:1 and SEQ ID NO: 2, respectively) are the yeast expressible promoters shown in nucleic acid sequence SEQ ID NO: 44 and SEQ ID NO: 45, respectively and ACC1, DGA1 and MCE2 are the DNA regions encoding into the proteins ACC1, DGA1 and MCE2 with a sequence as shown in SEQ ID NOs: 3, 5, and 4, respectively.

[0067] So the "obese" yeast strain is one that is engineered and / or optimized for producing a higher titer of lipids when the yeast is cultured as compared to the "wild type" and / or non-engineered yeast strain with the same genetic background.

[0068] In some embodiments, and preferably in the embodiments wherein the utilisation of glycerol or other alternative carbon sources is preferred, the promoters such as pFBAlin, pEXPl, and pTEFlin or EXP1, TEF1, GPD, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPM1, and FBA1 or any of the hybrid promoters disclosed in Georgiadis et al. (Microorganisms, 2023, 11(5), 1152) can be used.

[0069] In another embodiment, the chimeric gene constructs comprises any yeast-expressible promoter, preferably Y. lipolytica expressible promoter. For example, said Y. lipolytica promoter can be constitutive TEF promoter, the inducible POX2 promoter, and the hybrid hp4d promoter, the promoters that couple various numbers of UAS1 tandem elements with the minimal LEU2 promoter or the TEF promoter for example 2UASl-pTEF, 3UASl-pTEF, 4UASl-pTEF, 8UASl-pTEF, hp8dpreLip2, preXpr2, and preSuc2, or any other suitable promoter disclosed in Dulermo et al. (Microbial Cell Factories, 2017, 16:31).

[0070] In a preferred embodiment, the yeast expressible promoter is EXP1 promoter, or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, of sequence identity to amino acid SEQ ID NO 1 or to the corresponding nucleic acid sequence SEQ ID NO: 44. SEQ. ID NO: 1

[0071] MVKVADLILRGLIFM FAAIIMGLAGSLASTHKKGHYNPQVSYAVFCGAWSALFGVFYPVLANFIEAIAFPIVILIIDFISW VLTLAGGAALATAIRCHSCGNM NYVNSNKVTQGSKGRCRKAQATVAFLFFANFSFLATM ILSAISVKQLGAFTLPGRS RRSAPRTGIPTMSQV

[0072] SEQ ID NO: 44

[0073] AAGGAGTTTGGCGCCCGTTTTTTCGAGCCCCACACGTTTCGGTGAGTATGAGCGGCGGCAGATTCGAGCGTTTCC

[0074] GGTTTCCGCGGCTGGACGAGAGCCCATGATGGGGGCTCCCACCACCAGCAATCAGGGCCCTGATTACACACCCA CCTGTAATGTCATGCTGTTCATCGTGGTTAATGCTGCTGTGTGCTGTGTGTGTGTGTTGTTTGGCGCTCATTGTTG CGTTATGCAGCGTACACCACAATATTGGAAGCTTATTAGCCTTTCTATTTTTTCGTTTGCAAGGCTTAACAACATTG CTGTGGAGAGGGATGGGGATATGGAGGCCGCTGGAGGGAGTCGGAGAGGCGTTTTGGAGCGGCTTGGCCTGG CGCCCAGCTCGCGAAACGCACCTAGGACCCTTTGGCACGCCGAAATGTGCCACTTTTCAGTCTAGTAACGCCTTA CCTACGTCATTCCATGCATGCATGTTTGCGCCTTTTTTCCCTTGCCCTTGATCGCCACACAGTACAGTGCACTGTAC AGTGGAGGTTTTGGGGGGGTCTTAGATGGGAGCTAAAAGCGGCCTAGCGGTACACTAGTGGGATTGTATGGAG TGGCATGGAGCCTAGGTGGAGCCTGACAGGACGCACGACCGGCTAGCCCGTGACAGACGATGGGTGGCTCCTG TTGTCCACCGCGTACAAATGTTTGGGCCAAAGTCTTGTCAGCCTTGCTTGCGAACCTAATTCCCAATTTTGTCACTT CGCACCCCCATTGATCGAGCCCTAACCCCTGCCCATCAGGCAATCCAATTAAGCTCGCATTGTCTGCCTTGTTTAG TTTGGCTCCTGCCCGTTTCGGCGTCCACTTGCACAAACACAAACAAGCATTATATATAAGGCTCGTCTCTCCCTCC CAACCACACTCACTTTTTTGCCCGTCTTCCCTTGCTAACACAAAAGTCAAGAACACAAACAACCACCCCAACCCCCT TACACACAAGACATATCTACAGCA

[0075] In a preferred embodiment, the yeast expressible promoter is GPD promoter, or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, of sequence identity to amino acid SEQ ID NO 2 or to the corresponding nucleic acid sequence SEQ ID NO: 45.

[0076] SEQ ID NO: 2

[0077] MAIKVGINGFGRIGRIVLRNALKNPEVEVVAVNDPFIDTEYAAYMFKYDSTHGRFKGKVEAKDGGLIIDGKHIQVFGER DPSNIPWGKAGADYVVESTGVFTGKEAASAHLKGGAKKVIISAPSGDAPM FVVGVNLDAYKPDMTVISNASCTTNCL APLAKVVNDKYGIIEGLMTTVHSITATQKTVDGPSHKDWRGGRTASGNIIPSSTGAAKAVGKVIPELNGKLTGMSLRV

[0078] PTVDVSVVDLTVRIKNGASYEDIKATM KAASESPELKGILGYTDEDVVSTDFIGDTHSSIFDAKAGIGLNDNFVKLISWY

[0079] DNEYGYSARVVDLIVAVAKKDASA SEQ. ID NO: 45

[0080] GTTGATGTGTGTTTAATTCAAGAATGAATATAGAGAAGAGAAGAAGAAAAAAGATTCAATTGAGCCGGCGATGC

[0081] AGACCCTTATATAAATGTTGCCTTGGACAGACGGAGCAAGCCCGCCCAAACCTACGTTCGGTATAATATGTTAAG CTTTTTAACACAAAGGTTTGGCTTGGGGTAACCTGATGTGGTGCAAAAGACCGGGCGTTGGCGAGCCATTGCGC GGGCGAATGGGGCCGTGACTCGTCTCAAATTCGAGGGCGTGCCTCAATTCGTGCCCCCGTGGCTTTTTCCCGCCG TTTCCGCCCCGTTTGCACCACTGCAGCCGCTTCTTTGGTTCGGACACCTTGCTGCGAGCTAGGTGCCTTGTGCTAC TTAAAAAGTGGCCTCCCAACACCAACATGACATGAGTGCGTGGGCCAAGACACGTTGGCGGGGTCGCAGTCGG CTCAATGGCCCGGAAAAAACGCTGCTGGAGCTGGTTCGGACGCAGTCCGCCGCGGCGTATCGATATCCGCAAGG TTCCATGGCGCCATTGCCCTCCGTCGGCGTCTATCCCGCAACCTCTAAATAGAGCGGGAATATAACCCAAGCTTCT TTTTTTTTCCTTTAACACGCACACCCCCAACTATCATGTTGCTGCTGCTGTTTGACTCTACTCTGTGGAGGGGTGCT CCCACCCAACCCAACCTACAGGTGGATCCGGCGCTGTGATTGGCTGATAAGTCTCCTATCCGGACTAATTCTGAC CAATGGGACATGCGCGCAGGACCCAAATGCCGCAATTACGTAACCCCAACGAAATGCCTACCCCTCTTTGGAGCC CAGCGGCCCCAAATCCCCCCAAGCAGCCCGGTTCTACCGGCTTCCATCTCCAAGCACCCCTTTCTCCACACCCCAC AAAAAGACCCGTGCAGGACATCCTACTGCGTC

[0082] In a preferred embodiment, the ACC1 protein is a native, Y. lipolytica protein or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, of sequence identity to the native ACC1 protein of SEQ ID NO: 3.

[0083] SEQ ID NO: 3

[0084] MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVASHGGHTVINKVLIANNGIA AVKEIRSVRKWAYETFGDERAISFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFGVDAV WAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKISSTIVAQHAKVPCIPWSGTGVDEVVVDKSTNLVSVSE EVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGIRKVEREEDFEAAYHQVEGEIPGSPIFIMQLAGNARHLEVQ LLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTFTAMEKAAVRLGKLVGYVSAGTVEYLYSHEDDKFYFLEL NPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPHTTTPIDFDFSGEDADKTQRRPVPRGHTTACR ITSEDPGEGFKPSGGTM HELNFRSSSNVWGYFSVGNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSIRGDF RTTVEYLIKLLETPDFEDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRASEDSIATYMASLEKGQVPARDILKT LFPVDFIYEGQRYKFTATRSSEDSYTLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTCLLEVE NDPTQLRSPSPGKLVKFLVENGDHVRANQPYAEIEVMKMYMTLTAQEDGIVQLMKQPGSTIEAGDILGILALDDPSK VKHAKPFEGQLPELGPPTLSGNKPHQRYEHCQNVLHNILLGFDNQVVM KSTLQEMVGLLRNPELPYLQWAHQVSSL HTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSGEVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLL QAYYDSEARFCGPNVRDEDVILKLREENRDSLRKVVMAQLSHSRVGAKNNLVLALLDEYKVADQAGTDSPASNVHVA KYLRPVLRKIVELESRASAKVSLKAREILIQCALPSLKERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVF DVLAQFFAHDDPWIVLAALELYIRRACKAYSILDINYHQDSDLPPVISWRFRLPTMSSALYNSVVSSGSKTPTSPSVSRA DSVSDFSYTVERDSAPARTGAIVAVPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASSVDTG LSNICNVM IGRVDESDDDDTLIARISQ.VIEDFKEDFEACSLRRITFSFGNSRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQ LELARLSNFDIKPVHTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADRLMSDILDALEVIGTTNS DLNHIFINFSAVFALKPEEVEAAFGGFLERFGRRLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVSGYVVQSELYA EAKNDKGQWIFKSLGKPGSMHMRSINTPYPTKEWLQPKRYKAHLMGTTYCYDFPELFRQSIESDWKKYDGKAPDDL MTCNELILDEDSGELQEVNREPGANNVGMVAWKFEAKTPEYPRGRSFIVVANDITFQIGSFGPAEDQFFFKVTELARK LGIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLATLKPDTVVTTEIEEEGPNGVEKRHVIDYIV GEKDGLGVECLRGSGLIAGATSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINKLLGREVYSSNLQ LGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGR TLEDGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDSIEMSTSEAGQVWYP NSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRDMYNEVLKYGSFIVDALVDYKQPIMVYIPPTGELRGGSWVV VDPTINSDMMEMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARLDPEYSSLKKQLEESPDSEELKVKLSVREKSLM PIYQQISVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRIRRRLVEEYLITKINSILPSCTRLECLARIKSWKPATLD QGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDRQGTLQGMKQALASLSEAERAELLKGL*

[0085] In a preferred embodiment, said native ACC1 gene is expressed, i.e., operably linked to a EXP1 promoter, allowing for overexpression of chimeric ACC1 gene in the yeast strain. In an embodiment, said native ACC1 gene is linked, preferably operably linked to another yeast-expressible promoter, preferably a Y. lipolytica promoter such as, for example TEF1, GPD, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPMl, and FBA1.

[0086] In a preferred embodiment, the MCE2 protein is Mucor circinelloides MCE2 protein or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the MCE2 protein of SEQ ID NO: 4.

[0087] SEQ ID NO:4.

[0088] MSPIIDFVRRQLSSTKLHEEQQTATTNDLVSRSGYLNEGKYEVRLNCINAGCLQKKLNYIGTAM DPAKRQRLGLNGLLP AGVETLEIQKARALRVLRSKHNLLEKYILMAQLRTTNVRLFYKIVIDELETVQLAPVIYTPTVGTACLEYSTIYPFLAAPGV PDGLYLTKAELPELCQTIRNYRPTDTEGFEPEIAVISDGSRILGLGDLGTNGMGIPMGKLQLYVAGAGIDPRRTLPIILDL GTNNEKLLNDEFYIGLRQKRPNDEEFYQTVDTVLTALHTVYPNLLIQFEDWSSEHAFGLLEKYQNQMLCFNDDIQGTG AVILSGVINAIRKVEKENQVSPRDHRIVFYGAGSAAIGVARQIQSYFQIEHNMTEEEAKHVFWIVDSKGLVTTTRGDKL AQHKVYYARGDNEGQQYKELIDIVNYNLYSLIGLSSTTGAFNTQVLERLASLNEQPIVFPLSNPATQAECTFEQAMEAT NNKVIFASGTAFPAYTIKSTGEVNTPGQGNNMYIFPGLGLGACLANPAHFDRMIYEASKALADSLTEEEISKAWLYPSL NYRSVSAIVAAAVCQETLNENLATSQAMMTQCKSHEDILDYVSAHMWSPDYGNNNSNQQAGKL*

[0089] In a preferred embodiment, said M. circinelloides MCE2 gene is expressed, i.e., operably linked to a GPD promoter, allowing for overexpression of chimeric MCE2 gene in the yeast strain. In an embodiment, said MCE2 gene is linked, preferably operably linked to another yeast-expressible promoter, preferably a Y. lipolytica promoter such as, for example TEF1, EXP1, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPM1, and FBA1.

[0090] In a preferred embodiment, the DGA1 protein is Y. lipolytica DGA1 protein or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the DGA1 protein of SEQ. ID NO: 5.

[0091] SEQ ID NO: 5.

[0092] MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPTFLTIFM LCCAIPLLWPFVIAYVVYAVKDDSPS NGGVVKRYSPISRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAIISTIEYFLPAFMKRSL SINEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSRYSRGESSGSNGHASGSELNGNGNNGTTNRRPLSSASAGSTAS DSTLLNGSLNSYANQIIGENDPQLSPTKLKPTGRKYIFGYHPHGIIGMGAFGGIATEGAGWSKLFPGIPVSLMTLTNNF RVPLYREYLMSLGVASVSKKSCKALLKRNQSICIVVGGAQESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAF GENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMHARGVFNYDVGLVPYRRPVNIVVGSPIDLPYLPHPTDEEVSEY HDRYIAELQRIYNEHKDEYFIDWTEEGKGAPEFRMIE*

[0093] In a preferred embodiment, said Y. lipolytica DGA1 gene is expressed, i.e., operably linked to a GPD promoter, allowing for overexpression of chimeric DGA1 gene in the yeast strain. In an embodiment, said DGA1 gene is linked, preferably operably linked to another yeast-expressible promoter, preferably a Y. lipolytica promoter such as, for example TEF1, EXP1, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPM1, and FBA1.

[0094] In an alternative embodiment, said ACC1, MCE2 or DGA1 genes are chosen from another microbial organism, preferably a yeast.

[0095] In another preferred embodiment, DGA1 gene is selected from an animal species, preferably animal species such as pig, bovine or chicken. This is advantageous for production of tailored lipids which are chemically more "animal-like" by the yeast strain of invention. In a preferred embodiment, the DGA is a pig or Sus scrofa DGA1 gene or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the DGA1 protein of SEQ ID NO: 6.

[0096] SEQ ID NO: 6

[0097] MGDRSGAGGSRRRRTGSRPSSQSGSGFAAAEEEVRDVGAGGDAPTPDKDKDGHDDVSSGHWDLRCHRLQDSLFSS

[0098] DSGFSNYRGILNWCVVMLVLSNARLFLENLIKYGILVDPIQVVSLFLKDPYSWPALCLVIVANVFAVTAFQVEKRLAVG ALTEQAGLLIHVANLATILCFPAAVAFLLESITPVGSLLALMVYAILFLKLFSYRDVNLWCRERRATAKAKAASAGKKAN GGAAQHSVSYPDNLTYRDLYYFLLAPTLCYELNFSRFPRIRKRFLLRRLLEMLFLIQLQVGLIQQWMVPTIQNSMKPFK DM DYSRIIERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWCLRH FYKPMLRRGSSKWVARMGVFLASAFFHEYLVSIPLRMFRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIG QPVAVLM YVH DYYVLH H EAPTAG A*

[0099] In a preferred embodiment, the DGA is a bovine or Bos taurus DGA1 gene, or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99%, of sequence identity to the DGA1 protein of SEQ ID NO: 7.

[0100] SEQ ID NO: 7

[0101] MGDRGGAGGSRRRRTGSRPSIQGGSGPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFS SDSGFSNYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFLKDPYSWPALCLVIVANIFAVAAFQVEKRLAVGA LTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMVYTILFLKLFSYRDVNLWCRERRAGAKAKAALAGKAANG GAAQRTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKD MDYSRIVERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESITYFWQNWNIPVHKWCIRHF YKPMLRRGSSKWAARTAVFLASAFFHEYLVSIPLRM FRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQ PVAVLM YVH DYYVLN REAPAAGT*

[0102] In a preferred embodiment, the DGA is a chicken or Gallus gallus DGA1 gene or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the DGA1 protein of SEQ ID NO: 8.

[0103] SEQ ID NO: 8

[0104] MAGEDCVRKRPSGSTTTYKSPENEEMQRRPDGDRSFQNSSNGRVDVDHVITRKMQLIAEAEQLKPVFM KEVDSHFT EFVNSLVAKSALLDSSSSASLFPASCSEKELHKAKALRAPPEHGKIFTARRSLLDELFEVSHIRTIYHM FIALLIVFILSTLLVD FIDEGRLVLGFDLLVYVFGKFPVVFCTWLCMFCATVIIPYSLFSQWAQGYCSSSHRVIYSLFYGTLFTLFQTVGLGIGPTY

[0105] VAISYALPPASRFIVILEQVRLVMKAHSFVRENVPRVLSSVKEKSSSVPIPRISQYLYFLFAPTLIYRDNYPRNPMVRWGY

[0106] VATKFAQVLGSLFYAYYIFVRLCIPQFRNSSQETFNLRGLVLCIFNSILPGVLILFLVFFAFLHCWLNAFAEMMRFADRM FYKDWWNSTSYANYYRTWNVVVHDWLYYYAYRDFLWFFGKKFKAAAMLSVFTVSAAVHEYVLSICFGFFYPVLFCLF MCFGMLFNFILNDRRKGPIWNVIMWTSLFLGQGVIICLYSQEWYARQYCPAENPAFLDYLKPRSWSCHVQM*

[0107] In a second aspect, the present invention relates to a method for producing lipids using the strain according to any embodiment of the first aspect, the method comprising the following steps:

[0108] -culturing the strain according to any one of embodiments according to the first aspect in a culture medium and in culturing conditions suitable for production of lipids, and

[0109] -optionally, extracting said lipids from said culture medium and / or said strain.

[0110] In the method of the second aspect, the lipids, such as fatty acids and triacylglycerols, can be synthetised by culturing of engineered Yarrowia lipolytica strain, preferably Y. lipolytica CBS 8108 strain comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes, from a variety of carbon sources, such as, for example, glucose, xylose, fructose or arabinose and / or glycerol, ethyl acetate, methanol, and the like.

[0111] Preferably said culturing is done in the culture medium which is a liquid medium. In some embodiments, the culture medium comprises a carbon source, for example, a fermentable carbohydrate source, or an organic acid or salt thereof. In some embodiments, the culture medium comprises a salt and / or buffer establishing conditions of salinity, osmolarity, dissolved oxygen (dO2), and pH, that are amenable to survival, growth, and / or carbohydrate to lipid, such as TAG or a fatty acid conversion by the yeast strain disclosed herein. In some embodiments, the culture comprises an additional component, for example, an additive. Non-limiting examples of additives are nutrients, enzymes, amino acids, albumin, growth factors, enzyme inhibitors (for example protease inhibitors), fatty acids, lipids, hormones (e.g., dexamethasone and gibberellic acid), trace elements, inorganic compounds (e.g., reducing agents, such as manganese), redox-regulators (e.g., antioxidants), stabilizing agents (e.g., dimethylsulfoxide), polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g., NaCI), chelating agents (e.g., EDTA, EGTA), and enzymes (e.g., cellulase, dispase, hyaluronidase, or DNase). In some embodiments, the culture medium may comprise a drug inducing or inhibiting transcription from a conditional or inducible promoter, for example doxicycline, tetracycline, tamoxifen, IPTG, hormones, or metal ions.

[0112] While the specific culture conditions, for example, the concentration of the carbon source, will depend upon the respective Y. lipolytica yeast strain to be cultured, general methods and culture conditions for the generation of microbial cultures are well known to those of skill in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (Jan. 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (Mar. 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (Jul. 2, 2002); and Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (Jul. 9, 2002), all of which are incorporated by reference herein. For the lipid production, the cultures of engineered Y. lipolytica yeast strains described herein are cultured under conditions suitable for lipid accumulation, as known in the art.

[0113] In some embodiments, the Y. lipolytica yeast strains of the invention exhibit a growth advantage over wild type microbes of the same kind and / or over other microbes, for example, microbes commonly found to contaminate microbial cultures for carbon source to biofuel or biofuel precursor conversion. In some embodiments, the growth and / or proliferation advantage of an engineered microbe provided by aspects of this invention translates into the possibility of using non-sterile culturing and fermentation conditions for lipid or lipid precursor production, because the problem of culture overgrowth by contaminating microbes is mitigated or completely abolished. In some embodiments, genetically modified Y. lipolytica yeast strains of the invention exhibit is cultured under non-sterile conditions for biofuel or biofuel precursor production. For example, in some embodiments, non-sterilized feedstock, non-sterilized culture media, non-sterilized supplements, or a non-sterilized bioreactor (e.g. an open reactor under non-sterile conditions) is used for lipid or lipid precursor production.

[0114] The method according to the second aspect allows an increased lipid synthesis and reaching the lipid titer of 50 g / L, preferably 60 g / L, more preferably 65 g / L, even more preferably 70 g / L, most preferably 75 g / L, during growth in a bioreactor for 80, 70, 65, 60, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46 or 45 hours. This is particularly advantageous as this bioreactor growth-time is shorter, with a maintenance of a high yield compared to methods known in the prior art. In a particularly preferred embodiment, the method allows for a lipid titer of at least 75 g / L in a bioreactor of at least 8 L, on a suitable medium, after 55 h, preferably after 50 h, more preferably after 45 h of the growth.

[0115] In some embodiments, the engineered Y. lipolytica strain CBS 8108, comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes, can achieve or does achieve a lipid titer of at least 10 g / L (grams of lipid per liter of microbial culture), at least 15 g / L, at least 20 g / L, at least 25 g / L, at least 30 g / L, at least 35 g / L, at least 40 g / L, at least 45 g / L, at least 50 g / L, at least 55 g / L, at least 60 g / L, at least 65 g / L, at least 70 g / L, at least 75 g / L, at least 80 g / L, at least 85 g / L, at least 90 g / L, at least 91 g / L, at least 92 g / L, at least 93 g / L, at least 94 g / L, at least 95 g / L, at least 96 g / L, at least 97 g / L, at least 98 g / L, at least 99 g / L, at least 100 g / L, at least 101 g / L, at least 102 g / L, at least 103 g / L, at least 104 g / L, at least 105 g / L, at least 106 g / L, at least 107 g / L, at least 108 g / L, at least 109 g / L, at least 110 g / L, at least 120 g / L, at least 130 g / L, at least 140 g / L, at least 150 g / L, at least 160 g / L, at least 170 g / L, at least 180 g / L, at least 190 g / L, at least 200 g / L, or at least 250 g / L. In some embodiments, the lipid titer is 40 g / L to 110 g / L, 50 g / L to 105 g / L, 50 g / L to 100 g / L, 50 g / L to 99 g / L, 60 g / L to 99 g / L, 70 g / L to 99 g / L, 80 g / L to 99 g / L, 90 g / L to 99 g / L, or 95 g / L to 99 g / L.

[0116] In some embodiments, the engineered Y. lipolytica strain CBS 8108, comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes exhibits an increased lipid titer during carbon to oil conversion. The term "increased lipid titer" as used herein in the context of yeast lipid synthesis, e.g., in the context of a fatty acid synthesis by an oil-producing microbe described herein, refers to an amount of lipid synthesized per volume of a microbial culture comprising the oil- producing microbe that is increased as compared to the corresponding lipid titer of a wild-type microbe of the same species and under the same conditions (e.g., in the same growth medium, with the same C / N ratio, the same amount of oxygen, the same pH, the same nutrients, and so forth). For example, an increased lipid titer achieved by an engineered the engineered Y. lipolytica strain CBS 8108, comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2 and / or DGA1 chimeric genes described herein refers to a lipid titer that is increased as compared to the lipid titer that can be achieved by a wild-type Y. lipolytica under identical conditions. In some embodiments, an increased lipid titer refers to a lipid titer of at least 1 g / L (grams of lipid per liter of microbial culture), at least 2 g / L, at least 3 g / L, at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, at least 10 g / L, at least 11 g / L, at least 12 g / L, at least 13 g / L, at least 14 g / L, at least 15 g / L, at least 20 g / L, at least 25 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, at least 80 g / L, at least 90 g / L, at least 100 g / L, at least 200 g / L, or at least 250 g / L. In some embodiments, an increased lipid titer is 1 g / L to 100 g / L, 2 g / L to 100 g / L, 5 g / L to 100 g / L, 5 g / L to 95 g / L, 5 g / L to 90 g / L, 5 g / L to 80 g / L, 5 g / L to 70 g / L, 5 g / L to 0 g / L, 5 g / L to 50 g / L, 10 g / L to 100 g / L, 10 g / L to 90 g / L, 10 g / L to 80 g / L, 10 g / L to 70 g / L, 10 g / L to 60 g / L, or 10 g / L to 50 g / L.

[0117] In a preferred embodiment, the method for producing lipids comprises the step of culturing executed by fed-batch fermentation, preferably in a bioreactor. Preferably, the yeast according to any embodiment of the first aspect allows for a method which utilizes various carbon sources, preferably sugars such as xylose, arabinose or polyols, such as glycerol, which does not compromise the lipid yield of the method of the invention. In some embodiments, the carbon source comprises a fermentable sugar. In some embodiments, the carbon source comprises glucose, dextrose, maltose, fructose, xylose, arabinose and the like. In some embodiments, the carbon source comprises a low-carbon substrates such as glycerol, methanol, ethanol, ethylene glycol, isopropanol and the like. In some embodiments, The method is shows advantageous robustness, as it concerns the yeast which is more tolerant and / or less affected to common industrial inhibitors of yeast growth.

[0118] In a third independent aspect, the invention concerns lipids obtainable by the method according to any embodiment of the second aspect, preferably said lipids comprising or essentially consisting of triacylglycerols. Triacylglycerols are preferred as highly concentrated stores of metabolic energy because of their reduced, anhydrous nature. TAGs obtainable by the method and / or in the yeast of the invention are suitable for various food, non-food, for example pharmaceutical, cosmetical and / or biofuel applications.

[0119] In some embodiments, the lipids comprise fatty acids and their derivatives, such as TAGs, as an ideal form to store metabolic energy. The energy contained in the C— C bonds can be efficiently released by P-oxidation, a reaction formally equivalent to the reverse of fatty acid biosynthesis, but mediated and regulated by different enzymes constituting a different molecular pathway. The mentioned fatty acids can be derived from external supply, endogenous turnover, and de novo synthesis in the yeast.

[0120] In some embodiments, the lipids provided by the method and / or in the yeast of the invention may be suitable for biofuel or biofuel precursor production based on the yeast's ability to synthesize and store fatty acids or fatty acid derivatives, such as TAGs, efficiently from an externally supplied carbon source. In some embodiments, the lipids comprise natural fatty acid molecules, having an unbranched, aliphatic chain, or tail, of 4 to 28 carbon atoms. In some embodiments, the fatty acids are "saturated", meaning that all carbon atoms of the aliphatic chain are connected via a C— C single bond. In some embodiments, the fatty acids are "unsaturated", if two or more carbon atoms are connected via a C=C double bond. Unsaturated fatty acids play important roles in the regulation of membrane fluidity, cellular activity, metabolism and nuclear events governing gene transcription.

[0121] In some embodiments, the lipids comprise at least 80%, preferably at least 85%, most preferably at least 90% of C16 and C18 fatty acids, for example palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), oleic acid (C18) and linoleic acid (C18). Palmitic acid is an unbranched, saturated fatty acid, with an aliphatic chain of 16 carbon atoms (carbon atoms / unsaturated bonds: 16.0). Stearic acid is an unbranched, saturated fatty acid with an aliphatic chain of 18 carbon atoms (18.0). Palmitoleic acid is a monounsaturated fatty acid with an aliphatic chain of 16 carbon atoms (16.1). Oleic acid is a monounsaturated fatty acid with an aliphatic chain of 18 carbon atoms (18.1). In some embodiments, the lipids comprise minor fatty acid species such as C14 and C26 fatty acids, which can play essential functions in protein modification or as components of sphingolipids and GPI anchors, respectively.

[0122] The immediate product of de novo fatty acid synthesis are saturated fatty acids. Saturated fatty acids are known to be the precursors of unsaturated fatty acids in eukaryotes, including yeast Y. lipolytica. Unsaturated fatty acids are generally produced by desaturation of C— C single bonds in saturated fatty acids by specialized enzymes, called desaturases.

[0123] In some embodiments, the lipids comprise at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94% or 95%, most preferably at least 96% of C16 and C18 fatty acids, said C16 and / or C18 fatty acids being for example palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), oleic acid (C18) and linoleic acid (C18). Palmitic acid is an unbranched, saturated fatty acid, with an aliphatic chain of 16 carbon atoms (carbon atoms / unsaturated bonds: 16.0). Stearic acid is an unbranched, saturated fatty acid with an aliphatic chain of 18 carbon atoms (18.0). Palmitoleic acid is a monounsaturated fatty acid with an aliphatic chain of 16 carbon atoms (16.1). Oleic acid is a monounsaturated omega-9 fatty acid, with an aliphatic chain of 18 carbon atoms (18.1). In some embodiments, the lipids comprise minor fatty acid species such as C14 and C26 fatty acids, which can play essential functions in protein modification or as components of sphingolipids and GPI anchors, respectively.

[0124] The immediate product of de novo fatty acid synthesis are saturated fatty acids. Saturated fatty acids are known to be the precursors of unsaturated fatty acids in eukaryotes, including yeast Y. lipolytica. Unsaturated fatty acids are generally produced by desaturation of C— C single bonds in saturated fatty acids by specialized enzymes, called desaturases.

[0125] In some embodiments, about 50%, 55%, 60%, 65% or 70% of fatty acids of the total fatty acid content (calculated as 100%) in the lipids of the invention is are monounsaturated, meaning that they contain one unsaturated bond in their aliphatic chain. In some embodiments, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40% or 45% of the fatty acids of the total fatty acid content (calculated as 100%) in the lipids of the invention are polyunsaturated fatty acid.

[0126] In a preferred embodiment, the lipids comprise at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of linoleic acid, in the total fatty acid content (calculated as 100%). Linoleic acid is an octadecadienoic acid in which the two double bonds are at positions 9 and 12 and have Z (cis) stereochemistry. Since linoleic acid is an essential fatty acid, and an omega-6 polyunsaturated fatty acid, and the consumption of linoleic acid is vital to proper health, the lipids comprising at least 15% of linoleic acid, preferably at least 20% of linoleic acid are advantageous for food and / or human consumption. In a preferred embodiment, the weight ratio of oleic acid, which is, to linoleic acid is from about 4:1 to about 1.5:1 in the lipid. Preferably, said weight ratio of oleic acid to linoleic acid is about 2:1, more preferably around 2.5:1 or 3:1.

[0127] In some embodiments, about 40%, 50%, 60%, 70% or 80% of fatty acids of the total fatty acid content (calculated as 100%) in the lipids of the invention is are saturated, meaning that they contain saturated bonds in their aliphatic chain. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% of the fatty acids of the total fatty acid content (calculated as 100%) in the lipids of the invention is stearic acid. In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% of the fatty acids of the total fatty acid content (calculated as 100%) in the lipids of the invention is palmitic acid.

[0128] In yet another aspect, the invention concerns composition comprising the lipids obtained from the method according to any embodiment of the second aspect, or the yeast strain according to any embodiment of the first aspect.

[0129] In another independent aspect, the invention concerns a food product, preferably a plant-based food product, comprising the lipids of the third aspect, or the composition according to the fourth aspect.

[0130] Some non-limiting examples of said food product are plant-based dairy products, plant based meat products, also known as vegan meat products, vegan chocolate components, and creams, wherein said product are preferably substantially free of any animal-derived ingredients (for example, milk fat, animal fat or the like).

[0131] In some embodiments, the lipids provided by the yeast strain according to any embodiment of the first aspect, or the method according to any embodiment of the second aspect of the invention may be used in products as replacement of traditional fats like butter and / or duck, pork or other fat or lard in baked products . Some non-limiting examples of such products are pastries, bread, cookies and the like.

[0132] Advantageously, the lipids according to the present invention can be used in replacement of oils for cooking and frying.

[0133] Particularly advantageously, the lipids of the present invention may be used in supplements, particularly in the supplements wherein lipids rich e.g., rich in omega-3 or omega-6 or omega - 9 fatty acids are desired.

[0134] Advantageously, the lipids of the present invention may be used in use in food products which aim to achieve a high content of omega-3 or omega-6 or omega - 9 fatty acids. The non limiting examples " plant based egg" products, or plant based spreads, margarine and the like.

[0135] The lipids according to the invention are suitable for cosmetical purposes, i.e. personal care and cosmetic compositions. The following non-limiting Examples describe methods and means according to the invention. Unless stated otherwise in the Examples, all techniques are carried out according to protocols standard in the art. The following examples are included to illustrate embodiments of the invention. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0136] EXAMPLES

[0137] Example 1: Obtaining a recombinant 'obese' Y. lipolytica strain.

[0138] Most of the engineering efforts in Y. lipolytica are focused on the use of a reference strain called ATCC 20460, herein further referred to as 'W29' and its auxotrophic version 'POlf' (MATA, LEU2-270, URA3- 302, XPR2-322, AXP-2). In this invention, a new wild type strain known as Yarrowia lipolytica CBS 8108, and also referred to herein to as 'yl51' or 'YL51' or 'YI51', has been identified to provide for a background strain or host with superior phenotypic characteristics, especially for its use in lipid production, as compared to W29. Specifically, YL51 showed higher ability to grow on alternative carbon sources, higher resistance against commonly found industrial inhibitors, and allowed up to 47 % higher lipid titers (shown in Figs. 1A-B).

[0139] To further increase lipid titers, the lipid metabolism of the yeast organism was engineered using a known strategy, namely "PUSH-PULL" (Tai and Stephanopoulos, 2013, Metab. Eng., 15, 1-9) which strategy was combined with the restoration of the redox imbalance caused by lipid biosynthesis (Qiao et al., 2017, Nat. Biotechnol., 35, 174-177). This engineering approach entails the overexpression of the Y. lipolitica native ACC1 gene (XM_501721) which encodes for an enzyme that converts the acetyl-CoA to malonyl- CoA, precursor of lipid biosynthesis (PUSH). In addition, overexpression of the native DGA1 (XM_504700), which catalyses the final step of triacylglycerols (TAGs) by acylating diacylglycerol using acyl-CoA as an acyl donor, helps to increase the ratio of fatty acids incorporation to TAGs, thus increasing lipid production (PULL).

[0140] It is known that the primary bottleneck of lipid biosynthesis is NADPH availability (Qiao et al., 2017, Nat. Biotechnol., 35, 174-177). Yarrowia lipolytica lacks the cytosolic POM (pyruvate-oxaloacetate-malate) pathway which is responsible for recycling the cytosolic NADPH in other oleaginous microorganisms. Previous research has described that overexpression of the heterologous MCE2 (DQ975377) from Mucor circinelloides, encoding a cytosolic NADP+-dependent malic enzyme, effectively restores the NADPH homeostasis during lipogenesis (Qiao et al., 2017, Nat. Biotechnol., 35, 174-177).

[0141] Based on what was described above we designed three different constructs to drive the overexpression of the aforementioned genes. We chose to control the expression of the ACC1 using the strong promoter EXP1 (XM_501745) (SEQ ID NO:1, i.e. the corresponding nucleic acid sequence SEQ ID NO: 44), while the expression of DGA1 and MCE2 was regulated from the strong promoter GPD (XM_501515) (SEQ ID NO:2, i.e. the corresponding nucleic acid sequence SEQ ID NO: 45). To engineer YI51 we used as a selection marker the hygromycin resistance cassette (HPH), flanked by different orthogonal LoxPSym sites2, to facilitate marker recycling between all the intermediate engineering steps (as shown in WQ2024 / 068845). To establish the same engineering strategy in the W29 background, we used the auxotrophic variant POlf and use as selectable markers the LEU2 and URA3 genes to successively restore the auxotrophies of this strain. We introduced each of these three overexpression constructs successively till we build the final engineered strains i.e., "obese" W29 and "obese" YI51. Hence, the term "obese" is used herein to refer to a genetically engineered Y. lipolitica strain, more specifically in this application the "obese" strains are defined as being engineered by introducing the following chimeric genes: EXP1:ACC1, GPD:DGA1 and GPD:MCE2 wherein EXPland GPD are the yeast expressible promoters shown in nucleic acid sequences SEQ ID NO: 44 and SEQ ID NO: 45, respectively, and ACC1, DGA1 and MCE2 are the DNA regions encoding into the proteins ACC1, DGA1 and MCE2 with a sequence as shown in SEQ ID NOs: 3, 5, and 4, respectively.

[0142] So the "obese" yeast strain is one that is engineered and / or optimized for producing a higher titer of lipids when the yeast is cultured as compared to the "wild type" and / or non-engineered yeast strain with the same genetic background.

[0143] We evaluated the performance of the strains in shake flasks fermentations in a low N medium to effectively induce lipogenesis. The engineering strategy introduced to the YI51 background (i.e. the "obese" yl51) leads to higher lipid accumulation compared to same strategy implemented in the W29 background (i.e. "obese" W29; Figure IB; Table 1). Specifically the "obese" YI51 strain exhibited a 51.3 % higher lipid titer and 50 % lipid content compared to the "obese" W29 lipid titer and content. Furthermore, when compared to their WT backgrounds the "obese" YI51 has a 136 % increase in lipid titer over wild type (non-engineered) YI51, while the "obese" W29 yielded a 129 % increase over wild type (non-engineered) W29 (Figure IB). Example 2. Generation of Yarrowia lipolytica recombinant strains with heterologously expressed DGA1 gene.

[0144] Using the same methodology, three new strains were generated wherein the engineering strategy for recombinant expression of ACC1 and MCE2 was identical to the "obese" strains, but wherein a DGA1 orthologue sequence was used to heterologously express DGA1 gene (diacylglycerol O-acyltransferase 1). DGA1 catalyses the final step of triacylglycerols (TAGs) by acylating diacylglycerol using acyl-CoA as an acyl donor, and thereby helps to increase the ratio of fatty acids incorporation to TAGs, thus increasing lipid production. So it would be interesting to optimize the enzyme for its production in the yl51 strain, and look for improved engineered versions.

[0145] So based on the results of Example 1, we investigated whether heterologous DGAs could further increase lipid titers if incorporated into our engineering strategy to replace the overexpression of the native DGA1. For that we used homologs from different organisms i.e., Sus scrofa (pig; NM_214051), Bos taurus (bovine; NM_174693), and Gallus gallus (chicken; JQ031642), as provided in SEQ. ID NOs: 6-8, resp. Although the native Y. lipolytica DGA1 overexpression provided the best result in the YI51 background in view of lipid titers (Table 1), still independent of the origin of the DGA1 protein, all of the "obese" strains with the heterologous DGAs showed increased lipid titers by 12.8 %-23.01 % as compared to the "obese" W29 containing the Y. lipolytica DGA1 (Figure 1C; Table 1).

[0146] Example 3. Fed-batch fermentation using "obese" Y. lipolytica.

[0147] Given that shake flasks underestimate strain performance we compared the "obese" W29 with "obese" YI51 through fed-batch fermentations in 8 L bioreactors. Lipid production in bioreactors typically occurs in 2 sequential phases, a first growth-phase where the yeast consumes the carbon source to produce biomass (around 40 h) and then once the nitrogen is depleted the lipogenic phase starts and the cells consume the carbon source to produce lipids. We started with 20 g / L of ammonium sulfate as nitrogen source and 100 g / L of glucose as the carbon source. After 5 h from the start of the process constant feeding of the fermentation was performed with a 75 % solution of glucose for 60 h in total. We performed a side-by-side comparison between the "obese" W29 and "obese" YI51 strains and we showed a 47.9 % increase in biomass production, 58.3 % increase in lipid titers (Figure 2A), and almost 100 % increase in productivity for the "obese" YI51 over the "obese" W29 (Figure 2B). We also noticed that pseudohyphae formation was drastically lower in the "obese" YI51 compared to "obese" W29 and that also reflected in the oxygen requirements of the strain during the process. Finally also the FA composition of the 2 "obese" strains was different with the "obese" YI51 showing higher levels of linoleic acid (C18:2; Figure 2B). Table 1. Phenotypic characteristics of the engineered strains presented in Figure 1.

[0148] The strains were grown in a low N medium supplemented with 6% glucose, at 30 °C (220 rpm) for 5 days.

[0149] The average values are presented (experiments were performed in triplicates).

[0150] Example 4. Decoupling growth from lipogenesis to better monitor the lipogenic ability of the 'obese' Y. lipolytica strains of Example 1 (also referred to as W29_ACCl_MCE2_ylDGAl and yl51_ACCl_MCE2_ylDGAl in Table 1) under conditions optimized to monitor lipogenic activity.

[0151] Further evaluation of differences between the two "obese" strains YI51 and W29 obtainable in Example 1, and mimicking the conditions into the bioreactors during lipogenic phase on a smaller scale, were performed in an experiment in shake flasks that separated yeast growth from lipogenesis. In lipid production fermentations, nitrogen (N) availability may be seen as the main distinction parameter between growth of yeasts and their lipogenesis. Upon N depletion the yeast growth phase stops and lipogenesis begins redirecting glucose from biomass production to lipid formation. To replicate this in shake flasks typically a low N medium with a high carbon-to-nitrogen (C / N) ratio is used (Figure 1B,C). However, this method can lead to inconclusive results about a strain's lipogenic activity, as strains may vary in the abilities to produce biomass or in the efficiency to utilize N and C sources. By uncoupling growth from lipogenesis , the aim was to focus on the differences of the strains only in their lipogenic activity. The "obese" strains of Example 1 were grown under conditions characterized by the N limitation, to study if the obese YI51 can more efficiently convert the excess of glucose to lipids compared to the obese W29. In this way it was possible to i) achieve and / or mimic, in a smaller scale, the conditions in the bioreactor during the lipogenic phase (N depletion and excess of glucose), and ii) validate that the differences in lipid titer shown in Example 1 are not the result of a growth defect of W29, but a result of the most efficient lipogenesis in YI51.

[0152] To get a more direct and accurate insight in lipogenesis of the newly obtained "obese" strains YI51 and by using two different media in shake flasks. For the growth phase, the bioreactor medium but with lower ammonium sulfate (5 g / L instead of 2O g / L) and glucose (5% instead of 10%) was used. To maintain a stable pH of 5.5, we used 20 mM citrate buffer. We inoculated the strains at a starting optical density (OD of 0.1) and we harvested the biomass after 48 hours. The DCW of "obese" YI51 was 7.96 g / L (± 0.09), compared to 7.07 g / L (± 0.12) for "obese" W29. Both strains have consumed similar amounts of ammonium sulfate (=2.7 g / L) and glucose (=22 g / L), suggesting that "obese" YI51 converts N and C sources into biomass more efficiently than W29.

[0153] To evaluate the differences in lipogenic capacity between the "obese" YI51 and W29, following the growth phase, the same amount of biomass of "obese" YI51 and W29 was inoculated in shaking flasks with 5% glucose and 20 mM citrate buffer (for pH stability). In said substantially N-free medium, the cells can shift their metabolism towards lipid accumulation. A time-course experiment was performed by collecting samples at 24h, 68h and 96h post-inoculation. At each time-point, we measured lipid titer, DCW (reflecting lipid accumulation), and glucose consumption to calculate lipid yield (grams of lipids produced per gram of glucose consumed). The "obese" YI51 accumulated more efficiently lipids than "obese" W29 (Figure 3 A-C). By the end of the experiment, lipid production in YI51 was 23% higher than that of the "obese" W29. In addition, the higher lipid yield of YI51 (compared to W29) indicates that it can more efficiently direct glucose towards lipid production, rather than producing byproducts such as citrate, etc.

[0154] These findings show that regardless of the conditions, scale, and medium "obese" YI51 is a significantly better lipid producer than "obese" W29. The differences between the lipogenic capacity of the two obese strains tested herein become even more pronounced in fed-batch fermentations (Figure 2), highlighting YI51's higher lipogenic capacity.

[0155] Materials and Methods

[0156] Strains and culture conditions.

[0157] The reference strain Y. lipolytica W29 (ATCC20460), its auxotrophic version POlf (ATCC MYA2613) and the YI51 (CBS 8108) were used in this study. The yeasts were grown in Yeast Extract-Peptone-Dextrose (YPD) at 30 °C / 220 r.p.m (when liquid cultures were used) for 2-3 days. The selective reagents were added at the following concentrations: nourseothricin (CloNAT), 220 pg / ml; hygromycin B 100 pg / ml.

[0158] Phenotypic characterization.

[0159] The strains W29 (ATCC20460) and YI51 (CBS 8108) were phenotypically characterized in robot-assisted, high-throughput spotting assays (ability to grow on different carbon sources, resistance to industrial inhibitors). The evaluation was performed on YP 2% agar plates (Yeast extract 1% w / v; Peptone 2% w / v) supplement with 2% w / v of the respective carbon source. For the industrial inhibitors we used YPGIu 2% agar plates supplemented with 3 g / l acetic acid; 0.2 g / l coumaric acid; 1% v / v methanol; 1.5 g / l 5- hydroxy-methyl-furfural (5-HMF); and 0.2 g / l coniferyl aldehyde. Growth on YPGIu 2% agar on 30 °C was used as a control condition.

[0160] After spotting, all plates were incubated at 30 0C for 4 days. All plates were scanned using a high- definition scanner (Seiko Epson, Japan). Scanned images were processed using an in-house Matlab script. Data were processed by calculating relative growth compared to the control condition.

[0161] Strain engineering.

[0162] All overexpression constructs were assembled though the Gibson assembly reaction (NEBuilder HiFi DNA Assembly Reaction Protocol). The coding sequence of the heterologous genes was codon optimized for Y. lipolytica and synthesized from BGL The codon optimized sequences were amplified with PCR and cloned to the overexpression constructs with Gibson assembly.

[0163] The engineered Y. lipolytica strains were constructed by transforming the respective plasmids by an already published protocol (Abdel-Mawgoud & Stephanopoulos, Metab. Eng., 2020, 62, 106-115).

[0164] For the recycling of the selectable marker we used a Y. lipolytica replicative plasmid containing an expression cassette for the Cre recombinase (codon optimized for S. cerevisiae) and the NAT selectable marker conferring resistance to nourseothricin. The LoxPsym sites we used were previously described for their efficacy in marker recycling in Y. lipolytica (WO2024 / 068845).

[0165] Shake flasks fermen tations.

[0166] For the evaluation of the performance of the strains we used shake flasks fermentations using a low nitrogen medium (6% glucose; 1.7 g / l Yeast Nitrogen Base without amino acids and ammonium sulfate; 0.6 g / l urea). A single colony of Y. lipolytica was grown in 2 mL of YPGIu at 200 r.p.m. for 20 h in 10 mL test tubes. The cells were harvested by centrifugation at 10000 r.p.m. and washed with the low-nitrogen medium. The washed cells were inoculated into 20 mL low-nitrogen medium (250 mL shake flask) at OD600 of 0.1 and grown at 30 °C / 220 r.p.m. for 5 days. At the end of the fermentation dried cell weight (DCW) and lipid content determination was performed.

[0167] DCW determination.

[0168] 1 ml of cell suspension from the fermentations was collected. The cells were harvested by centrifugation, washed with water and dried at 60 °C for 24 h.

[0169] Lipids extraction.

[0170] To determine the lipid content of the fermentations we used a modified protocol of the Folch method (Folch et al., J. Biol. Chem., 1957, 226, 497-509). Around 200 mg of wet biomass was lysed with TissueLyser II after the addition of acid-washed glass beads (425-600 pm), and a 2:1 methanolchloroform solution to extract lipids into the organic phase. We centrifuged the samples after the lysing and we collected the organic phase. This was washed with 0.2V of 0.3% NaCI solution, and then collected into pre-weighed tubes after which it was evaporated using a centrivap at 50 °C.

[0171] Bioreactor fermentations.

[0172] The 8-1 iter BioNet bioreactor was operated with 3-1 iter working volume for all the experiments. The seed cultures of Y. lipolytica were prepared by inoculating a single colony of the strain into YPD medium and growing at 30 °C / 220 r.p.m. for 20 h. The seed culture was harvested by centrifugation, washed using the bioreactor medium (3.8 g / L yeast nitrogen base without amino acids and ammonium sulfate, 3 g / L yeast extract, 17.5 g / L ammonium sulfate, 100 g / L glucose) and inoculated into a bioreactor containing 3 liters bioreactor medium. The starting OD600 of each bioreactor run was ~1.0. During the fermentation, oxygen was supplied at 3 v.v.m. (volume 02 per volume liquid / min) and agitation speed was cascaded (400 r.p.m. to 1000 r.p.m.) such that dissolved oxygen levels were maintained at 20% during growth phase (typically from 0 h to 40 h) and ~5% during lipid production phase (~till the end of the fermentation). The temperature was controlled at 30 °C, and pH was maintained at 5.5 during growth phase and at 3.5 during lipogenesis with the addition of 6 M sodium hydroxide solution. From 5h till the 72h the bioreactor was continuously supplemented with glucose from a 75% stock solution at 0.8 mL / h rate.

[0173] Fatty acid composition.

[0174] We determined the FA composition of the strains using a Gas Chromatograph equipped with a flame ionization detector (GC-FID). Samples preparation and GC-FID settings were conducted as previously described (Qiao et al., 2017, Nat. BiotechnoL, 35, 174-177).

[0175] Chimeric gene constructs.

[0176] Sequence of the construct used for the overexpression of the of Y. lipolytica ACC1 (XM_501721) under the expression of EXP1 promoter (XM_501745) is shown as SEQ ID NO: 9 in the sequence listing, and consists of the sequences in the following order: SEQ ID NO: 10 corresponding to the EXP1 promoter, SEQ ID NO: 11 corresponding to the region encoding ACC1, SEQ ID NO: 12 corresponding to CYC1 terminator, SEQ ID NO: 13 corresponding to LoxPsym site, SEQ ID NO:14 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 13 corresponding to LoxPsym site.

[0177] For the transformation of POlf strain (the auxotrophic variant) the same construct was used, but URA3 selection marker was used instead of the HPH resistance cassette. The sequence of the URA3 marker used is shown as SEQ ID NO: 15. Sequence of the construct used for the overexpression of M. circinelloides MCE2 (DQ975377) under the control of GPD promoter (XM_501515) is shown as SEQ ID NO: 16 in the sequence listing, and consists of the sequences in the following order: SEQ ID NO: 17 corresponding to the GPD promoter, SEQ ID NO: 18 corresponding to the region encoding MCE2; SEQ ID NO: 19 corresponding to TEF1 terminator, SEQ ID NO: 20 corresponding to LoxPsym site, SEQ ID NO: 21 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 20 corresponding to LoxPsym site.

[0178] Sequence of the construct used for the overexpression of the of Y. lipolytica DGAl(XM_504700) under the control of the GPD promoter (XM_501515) is shown as SEQ ID NO: 22 in the sequence listing, and consists of the sequences in the following order: SEQ ID NO: 23 corresponding to GPD promoter, SEQ ID NO: 24 corresponding to the region encoding DGA1, SEQ ID NO: 25 corresponding to PEX20 terminator, SEQ ID NO: 26 corresponding to LoxPsym site, SEQ ID NO: 27 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 26 corresponding to LoxPsym site.

[0179] For the transformation of POlf strain (the auxotrophic variant) the same construct was used, but LEU2 selection marker was used instead of the HPH resistance cassette. The sequence of the LEU2 marker used is shown as SEQ ID NO: 28.

[0180] Sequence of the construct used for the overexpression of G. gallus (chicken) DGA1 (JQ031642) under the control of the EXP1 promoter (XM_501745) is shown as SEQ ID NO: 29, and consists of the sequences in the following order: SEQ ID NO: 30 corresponding to EXP1 promoter, SEQ ID NO: 31 to the region encoding DGA1, SEQ ID NO: 32 corresponding to TEF1 terminator, SEQ ID NO: 26 corresponding to LoxPsym site, SEQ ID NO: 33 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 26 corresponding to LoxPsym site.

[0181] Sequence of the construct used for the overexpression of B. taurus (bovine) DGA1 (NM_174693) under the control of the FBA1 promoter (XM_504407) is shown as SEQ ID NO: 34, and consists of the sequences in the following order: SEQ ID NO: 35 corresponding to FBA1 promoter, SEQ ID NO: 36 corresponding to the region encoding DGA1, SEQ ID NO: 37 corresponding to PEX20 terminator, SEQ ID NO: 26 corresponding to LoxPsym site, SEQ ID NO: 38 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 26 corresponding to LoxPsym site.

[0182] Sequence of the construct used for the overexpression of S. scrofa (pig) DGA1(NM_214O5) under the control of the GPD promoter (XM_501515) is shown as SEQ ID NO: 39, and consists of the sequences in the following order: SEQ ID NO: 40 corresponding to GPD promoter, SEQ ID NO: 41 corresponding to the region encoding DGA1, SEQ ID NO: 42 corresponding to PEX20 terminator, SEQ ID NO: 26 corresponding to LoxPsym site, SEQ ID NO: 43 corresponding to HPH (hygromycin B) resistance cassette and SEQ ID NO: 26 corresponding to LoxPsym site.

Claims

Claims1. A Yarrowia lipolytica CBS 8108 strain for producing lipids, comprising one or more chimeric gene constructs, wherein each one of said chimeric gene constructs comprises: i) a yeast-expressible promoter, ii) a DNA region encoding an ACC1, MCE2 and / or DGA1 protein; and iii) a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

2. The strain according to claim 1, comprising three chimeric constructs, a first construct comprising the DNA region encoding the ACC1 protein, a second construct comprising the DNA region encoding the MCE2 protein, and a third construct comprising the DNA region encoding the DGA1 protein.

3. The strain according to any one of claims 1 to 2, wherein said DNA region encoding the ACC1 protein codes for the Y. lipolytica ACC1 protein.

4. The strain according to any one of claims 1 to 3, wherein said DNA region encoding the MCE2 protein codes for the Mucor circinelloides MCE2 protein.

5. The strain according to any one of claims 1 to 4, wherein said wherein said DNA region encoding the DGA1 protein codes for the Y. lipolytica DGA1 protein.

6. The strain according to any one of claims 1 to 5, wherein said ACC1, MCE2 or DGA1 protein comprises SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively or an orthologue characterized by ay least 90% amino acid sequence identity thereof.

7. The strain according to any one of claims 1 to 5, wherein said DGA1 protein is selected from any one of the species of Sus scrofa, Bos taurus, and Gallus gallus, preferably said DGA1 protein comprising the sequences SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, respectively, or an orthologue with at least 90 % amino acid sequence identity of any one thereof.

8. The strain according to any one of claims 1 to 7, wherein said yeast expressible promoter is chosen from an EXP1 and / or GPD promoter region.

9. A method for producing lipids using the strain according to any one of claims 1 to 8 comprising the steps of:• culturing the strain according to any one of claims 1 to 8 in a culture medium and in culturing conditions suitable for production of lipids, and• optionally, extracting said lipids from said culture medium and / or said strain.

10. The method for producing lipids according to claim 9, wherein said culturing is executed by fed-batch fermentation, preferably in a bioreactor.

11. Lipids obtainable by the method of claims 9 or 10, preferably said lipids comprising or essentially consisting of triacylglycerols.

12. A composition comprising the lipids obtained from the method of claims 9 or 10, or the yeast strain according to any one of claims 1 to 8.

13. A food product, preferably a plant-based food product, comprising the lipids of claim 11, or the composition of claim 12.

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