Yeast for increased lipid production

Abstract

The present invention relates to a recombinant oleaginous yeast cell having at least one CEX1 gene disruption, comprising chimeric gene constructs, each of said chimeric gene constructs comprising a yeast-expressible promoter and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell, wherein said chimeric gene constructs allow for overexpression of DGA1, ACC1, MCE2, and ACL genes, said ACL gene being ACL1 and / or ACL2 gene. In a second aspect, the invention relates to a method for producing lipids using said transformed oleaginous yeast cell. The invention further pertains to lipids obtainable by the method, a composition comprising said lipids, a composition comprising transformed oleaginous yeast cell, and a food product obtainable therefrom.

Description

[0001] KeVer / SLIM_5gene / 850

[0002] YEAST FOR INCREASED LIPID PRODUCTION

[0003] Field of the invention

[0004] The present invention relates to the field of molecular biology, more particularly the field of microbial engineering, even more particularly to field of improving yields of lipids produced by microbial cells. The invention relates to engineering methods and / or yeasts for increased and / or improved lipid production comprising chimeric genes and constructs. Furthermore, the invention relates to methods for lipid production using the oleaginous yeast cells of the invention.

[0005] Background

[0006] In the recent years, the use of microorganisms such as bacteria, yeasts, fungi and microalgae for production of microbial lipids 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. Lipids have multiple industrial applications, including in the cosmetic and food industries, as well as serving as precursors for biodiesel and biochemical production. Microbial lipids are produced by many oleaginous organisms, including the well-characterized yeast Yarrowia lipolytica. Lipid yield in oleaginous organisms can be increased by up-regulation or down-regulation or deletion of genes implicated in the lipid pathway. However, it remains a challenge to achieve a less costly and more sustainable way for a biomass transformation especially on a large- and / or industrial-scale level. Another challenge is to obtain highly versatile and / or tailored microbial lipids, which can be applied in various industries.

[0007] 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 said 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 KeVer / SLIM_5gene / 850 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.

[0008] 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, US20240043788 and US11692209 relate to a fungal cell capable of producing fatty acids, fatty acid derivatives, including triacylglycerols, by providing the fungal cell with at least one modification to the endogenous fatty acid metabolism. EP3110947, EP3149153 and EP3137616 relate to different engineering strategies applied to oleaginous yeast cells to increase the lipid yield and / or tailor the composition of lipids produced by said transformed cells.

[0009] There is a need to develop new engineering strategies for design of oleaginous, i.e., "obese" microorganisms, preferably yeasts for the sustainable and cost-effective production of lipids. There is a need for providing novel engineered yeasts which would be suitable for the industrial scaling-up and / or the production of the lipids in high quantities. There is a need for an improved method for a large-scale production of lipids.

[0010] Summary

[0011] The present invention aims to provide an alternative engineering strategy to obtain oleaginous yeast cells, preferably industrially applicable oleaginous yeast cells, which could be used for lipid production in a more sustainable manner. The recombinant oleaginous yeast cell of the invention can preferably produce a high lipid titer resulting in high lipid content also on a large- and / or industrial- scale, for example in industrial bioreactors.

[0012] In an independent aspect, the present invention aims to provide a method for producing lipids using the recombinant oleaginous yeast cell of the invention, which method would preferably allow obtaining a high yield of lipids during preferably shorter incubation time, than alternative oleaginous yeast cells known in the art. The method of the invention is particularly suitable for an industrial scale production, where a high lipid productivity and more economical production is a must. KeVer / SLIM_5gene / 850

[0013] In a first aspect, the invention relates to a recombinant oleaginous yeast cell having at least one CEX1 gene disruption, further comprising at least one chimeric gene construct, the chimeric gene construct comprising:

[0014] • a yeast-expressible promoter,

[0015] • a nucleotide sequence encoding DGA1, ACC1, MCE2, and ACL protein, said ACL protein being ACL1 and / or ACL2 protein; wherein said nucleotide sequence is operably linked to said yeast expressible promoter; and

[0016] • a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0017] Preferably, said recombinant oleaginous yeast cell having said at last one CEX1 gene disruption, comprises the following chimeric gene constructs:

[0018] • a first chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding of diacylglycerol acyltransferase 1 (DGA1) native to the yeast cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0019] • a second chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding Acetyl-CoA carboxylase 1 (ACC1) native to the yeast cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0020] • a third chimeric gene construct comprises a yeast expressible promoter operably linked to a nucleotide sequence encoding NADP+-dependent malic enzyme 2 (MCE2) native to the cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0021] • a fourth chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding ATP citrate lyase 1 (ACL1) native to the cell or from a different species, and / or a nucleotide sequence encoding ATP citrate lyase 2 (ACL2) native to the cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0022] The recombinant oleaginous yeast cell according to a first aspect of the invention is engineered to allow an increased lipid synthesis. Said oleaginous yeast cell has at least one CEX1 gene disruption as well an expression, preferably an overexpression of DGA1, ACC1, MCE2, and ACL genes, said ACL being ACL1 and / or ACL2 gene, which can lead to an increased lipid production compared to the known wild type KeVer / SLIM_5gene / 850 yeast cells. The recombinant oleaginous yeast cell of the invention allows higher lipid production compared to the preferred recombinant yeast cell alternative, for example the recombinant cell being modified by overexpression of genes such as DGA1, ACC1 and MCE2. The recombinant oleaginous yeast cell of the invention being modified with CEX1 disruption and having overexpressed genes DGA1, ACC1, MCE2, and ACL1 and / or ACL2, shows an increased yield of lipid and / or a higher lipid productivity compared to the wild type cell, especially on a large-scale, industrial setup, for example, when a fast production of high quantities of lipids, such as triacylglycerols (TAGs) is favoured.

[0023] The recombinant oleaginous yeast cell of the invention allows for the increased TAGs production compared to the wild type, reference strain cell, independent of the composition of culturing and / or growth medium. The recombinant oleaginous yeast cell of the invention can allow for an increased lipid yield and / or lipid titer regardless of the culturing and / or growth medium, for example, regardless of the use of glucose, glycerol, xylose and / or arabinose as the main ingredient of said medium. The lipid yield of the recombinant oleaginous yeast cell of the invention which can be expressed as grams of lipids produced per gram of the consumed carbon substrate (for example, glucose, glycerol, xylose), is increased compared to a wild type cell, as well as compared to the reference recombinant cell featuring overexpression of genes such as DGA1, ACC1 and MCE2.

[0024] The recombinant yeast cell of the invention allows increase in the lipid titer, i.e. reaching the high "plateau" of the lipid production which is particularly remarkable during the lipogenic phase of lipid production in the bioreactor, for example after 48 hours of the incubation. The term "lipogenic phase" should be understood by the person skilled in the art as the phase which begins once nitrogen is depleted, thus during which the cells consume the carbon source to produce lipids. The lipid titer obtainable by the recombinant oleaginous yeast cell of the invention preferably outperforms the titer obtainable by a reference recombinant oleaginous yeast cell featuring the overexpression of the genes such as DGA1, ACC1 and MCE2 as well as the titer of the wild-type cell under the same culturing conditions.

[0025] Preferably said recombinant oleaginous yeast cells are of high lipid productivity, which is both high biomass productivity and the lipid content. Hence, the oleaginous yeast cells of the invention can be used as a more sustainable and / or economically beneficial alternative for lipid production to known microbial oleaginous yeasts. Said recombinant oleaginous yeast can allow a more stable and / or high- volume lipid production preferably on a large-, industrial- scale. Preferably the recombinant oleaginous yeast cell of the invention could be used for production of tailored, more animal-like lipids, preferably triacylglycerols. Such tailored, more animal like lipids, could be used for various applications, for KeVer / SLIM_5gene / 850 example, as a versatile component for various applications in food products, as said lipids can mimic the taste of the animal fats.

[0026] In a second aspect, the invention relates to a method for producing lipids using said recombinant oleaginous yeast cell.

[0027] According to a second aspect, the invention relates to a method for producing lipids using the recombinant oleaginous yeast cell according to any embodiment of a first aspect of the invention, the method comprising the steps of:

[0028] -culturing the recombinant oleaginous yeast cell of the invention in a culture medium and in culturing conditions suitable for production of lipids, and

[0029] -optionally, extracting said lipids from said culture medium and / or said cell.

[0030] The method according to the second aspect is particularly suited for a large-scale TAGs production, for example bioreactor-scale production. The method of the invention which utilizes the recombinant oleaginous yeast cell according to any embodiment of the first aspect of the invention, allows reaching the high plateau of lipid production phase of lipid production faster than the known methods utilizing for example, a wild type cell or, for example, a reference recombinant oleaginous yeast cell featuring overexpressed DGA1, ACC1 and MCE2 genes. Said method is particularly suited for large-scale, industrial yeast production, wherein methods are to be robust, reproducible and fast.

[0031] The invention further pertains to lipids obtainable by the method, a composition comprising said lipids, a composition comprising the recombinant oleaginous yeast cell of the invention, and a food product obtainable therefrom.

[0032] Brief description of the Figures

[0033] 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.

[0034] Figure 1. Schematic representation of the main metabolic pathways involved in lipogenesis in Yarrrowia lipolytica. A Description of the "PUSH-PULL" (PP) strategy that was implemented to increase lipid production in Y. lipolytica. Briefly, it includes the overexpression of the native acetyl-CoA carboxylase (ACC1) to "push" acetyl-CoA to the main precursor of lipid biosynthesis (malonyl-CoA) and the overexpression of the native diacylglycerol acyltransferase (DGA1) to "pull" fatty acids to the TAGs pool. In parallel, the activation of the pyruvate / oxaloacetate / malate (POM) cycle, by the overexpression of an heterologous, cytoplasmic NADP+-dependent malic enzyme from Mucor circinelloides (MCE2), provides an excess of NADPH, which is the limiting factor in lipogenesis. B Description of the newly developed KeVer / SLIM_5gene / 850

[0035] "PUSH-PULL-TRAP" (PPT) engineering strategy for obtaining the recombinant oleaginous yeast cell of the present invention. The PPT engineering allows to boost lipid production and reduce byproduct formation in a yeast cell, for example Y. lipolytica. This strategy combines the already existing "PUSH-PULL" approach with the deletion of the cytoplasmic citrate exporter (CEX1), that "traps" inside the cell the citrate produced by the TCA cycle, and the simultaneous overexpression of the Mus musculus homologs of the ATP-citrate lyase 1 and 2 (ACL1 and ACL2) that convert citrate to acetyl-CoA, fluxing the excess of citrate to lipid production.

[0036] Figure 2. Deletion of the CEX1 gene diminishes citrate production in Y. lipolytica, herein shown in Y. lipolytica CBS 8108. The cells were grown in shake flasks in a low N medium supplemented with 6% glucose, at 30 °C (220 rpm) for 5 days. At the end of the fermentations the citrate production was determined by HPLC measurements. The experiment was performed in triplicate, and the bars indicate the SD between the replicates. Statistical significance of the difference between the wild-type (WT) strain and the mutant featuring CEX1 deletion (ACEX1) was evaluated using t-test (***: p < 0.001).

[0037] Figure 3. Comparison of the performance of the "obese", i.e. oleaginous strains engineered with the "PUSH-PULL" (PP strain) and the "PUSH-PULL-TRAP" (PPT strain) strategies, with the wild type and the deletion mutant of the citrate exporter (ACEX1). Comparison is herein shown for Y. lipolytica CBS 8108. The performance of the strains was evaluated in shake flask fermentations in 4 different conditions, at 30 °C (220 rpm) for 5 days. The effect of two different C sources (glucose and glycerol) was tested in two different N / C rations (1 / 100 and 1 / 140). At the end of the fermentations the dried cell weight (DCW) of the strains was evaluated and the lipids were extracted to determine the lipid content of each strain. The fermentations were performed in triplicate and the bars indicate the SD between the replicates. The black triangles represent the average DCW of each strain from the three replicates. Statistical significance of the differences between the wild-type (WT) strain and the three engineered strains (ACEX1, PP, and PPT) was evaluated using t-test (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0038] Figure 4. A Comparison of the performance of the oleaginous, i.e. "obese" strains engineered with the "PUSH-PULL" (PP strain) and the "PUSH-PULL-TRAP" (PPT strain) strategies, with the wild type (WT) Y. lipolytica strain in fed-batch fermentations, herein shown in Y. lipolytica CBS 8108. The strains were grown in 8 L bioreactors for 4 days. Lipid titers, dried cell weight (DCW), and citrate production were monitored at the indicated timepoints. B-C Comparison of the lipid productivity (B) and yield (C) of the same strains as calculated in the indicated timepoints.

[0039] Figure 5. Evaluation of the "PUSH-PULL-TRAP" engineering strategy on two different Y. lipolytica strains:

[0040] CBS 8108 (A) and CBS 10739 (B) in fed-batch fermentations. For Y. lipolytica strain CBS 8108 the wild type KeVer / SLIM_5gene / 850

[0041] (WT) and the 'obese' engineered strains with the 'PUSH-PULL' (PP) and the 'PUSH-PULL-TRAP' (PPT) strains were compared (A). For Y. lipolytica strain CBS 10739 the WT and PPT variant were compared (B). The strains were grown in 8 L bioreactors for 4 days. Lipid titers and dried cell weight (DCW) were monitored at the indicated timepoints. The tests for all strains shown in Fig. 5 were done in triplicates. Statistical significance of the differences between pairs of strains was evaluated using t-test (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0042] Detailed description

[0043] Definitions

[0044] 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.

[0045] 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. KeVer / SLIM_5gene / 850

[0046] 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", "fourth", and the like in the description and in the claims, are used for distinguishing between 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.

[0047] 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.

[0048] A "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.

[0049] 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.

[0050] The term "terminator" or "3' end region" 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 KeVer / SLIM_5gene / 850 polyadenylation of a primary transcript and termination of transcription in a cell. The terminator can be derived from the natural gene, from a variety of other known yeast genes, or from T-DN A. The terminator to be added may be derived from, for example, any yeast derived gene, or less preferably from any other eukaryotic gene.

[0051] "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.

[0052] "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.

[0053] "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.

[0054] "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.

[0055] 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 KeVer / SLIM_5gene / 850 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". The term "expression cassette" can also refer 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 self-replicate 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.

[0056] 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. KeVer / SLIM_5gene / 850

[0057] 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.

[0058] 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-recombinant , 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.

[0059] "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 KeVer / SLIM_5gene / 850

[0060] 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.

[0061] "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.

[0062] 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 engineered oleaginous yeast cell and / or 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.

[0063] The production of the lipids can be defined as "the lipid productivity", and can be, for example, at least 0.25 g / L / h, preferably at least 0.50 g / L / h, most preferably at least 0.80 g / L / h for the recombinant oleaginous yeast cell of the invention. In some embodiments, the lipid productivity of the recombinant cell of the invention is 0.25 g / L / h to 1.5 g / L / h. In some embodiments, the lipid productivity is 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.00, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45 or 1.50 g / L / h.

[0064] 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 can produce lipids to store energy, for example in the form of triacylglycerols in lipid bodies, lipid droplets, or vacuoles.

[0065] 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 KeVer / SLIM_5gene / 850 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 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 KeVer / SLIM_5gene / 850 mitochondria-associated NAD+-dependent enzyme, whose overexpression and knockout has little impact on lipid production.

[0072] 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.

[0073] 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.

[0074] The term CEX1 or CEX1 protein, as used herein, refers to a citrate exporter. Disruption, of said CEX1 protein can lead to abolished citrate production in a yeast cell, resulting in a lower citrate production in a recombinant yeast cell. Citrate is considered a main byproduct of lipid production, and citrate may lead to lower lipid yields (Erian, et al., 2020 FEMS Yeast Res. 20(7):foaa055). The new engineering strategy of the invention developed to eliminate citrate production while also partially decoupling lipid production from N availability is named "PUSH-PULL-TRAP".

[0075] The term ACL protein refers to ATP-citrate synthase protein. The types of ACL, in particular ACL1 and ACL2 or ACL1 / ACL2 proteins as used herein, refer to ATP-citrate synthase protein, subunit 1 and 2 (referred to as class EC 2.3.3.8) belong to the group of citrate-mediated acyl-CoA synthesis related genes. ATP citrate-lyase is the enzyme responsible for the synthesis of cytosolic acetyl-CoA. ATP citrate-lyase has a central role in de novo lipid synthesis, biosynthesis of fatty acids and sterols.

[0076] The terms "oleaginous" and "obese" are herein interchangeably used, and refer to a recombinant yeast cell 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 cell and / or strain with the same genetic background.

[0077] The terms "recombinant yeast cell of the invention" and "recombinant oleaginous yeast cell of the invention" are used interchangeably, and relate to the recombinant yeast cell having at least one CEX1 gene disruption, comprising at least one chimeric gene construct allowing for an overexpression of genes KeVer / SLIM_5gene / 850

[0078] DGA1, ACC1, MCE2, and ACL, according to any one embodiment of the first aspect of the invention discloses in the detailed description of embodiments below.

[0079] Detailed description of embodiments

[0080] In the present invention, a new engineering strategy directed to a recombinant oleaginous yeast cell is disclosed. The disclosed engineering strategy and a recombinant yeast cell obtainable thereby are particularly suited for a yeast lipid production, wherein high lipid titer and or high lipid productivity are required. The recombinant oleaginous yeast cell of the invention can allow production of large volume of lipids on an industrial scale, preferably regardless of the medium used.

[0081] In a first aspect, the present invention relates to a recombinant oleaginous yeast cell having at least one CEX1 gene disruption, comprising at least one chimeric gene construct, the chimeric gene construct comprising:

[0082] • a yeast-expressible promoter,

[0083] • a nucleotide sequence encoding DGA1, ACC1, MCE2, and ACL protein, wherein said ACL protein is ACL1 and / or ACL2 protein; wherein said nucleotide sequence is operably linked to said yeast expressible promoter; and

[0084] • a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0085] The recombinant cell according to a first aspect features at least one CEX1 gene disruption and comprises one or more chimeric gene constructs, which gene constructs allow overexpression of genes such as DGA1, ACC1, MCE2 and ACL, wherein said ACL gene is ACL1 and / or ACL2 gene. The recombinant oleaginous yeast cell of the invention shows decreased citrate production compared to the wild type strain cell and increased lipid production, especially during lipogenic phase, when the nitrogen from the culturing and / or growth medium is depleted.

[0086] In a preferred embodiment, a recombinant oleaginous, i.e., "obese" yeast cell having at least one CEX1 gene disruption, comprises the following chimeric gene constructs:

[0087] • a first chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding of diacylglycerol acyltransferase 1 (DGA1) native to the yeast cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0088] • a second chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding Acetyl-CoA carboxylase 1 (ACC1) native to the yeast cell or from a KeVer / SLIM_5gene / 850 different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0089] • a third chimeric gene construct comprises a yeast expressible promoter operably linked to a nucleotide sequence encoding NADP+-dependent malic enzyme 2 (MCE2) native to the cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell;

[0090] • a fourth chimeric gene construct comprising a yeast expressible promoter operably linked to a nucleotide sequence encoding ATP citrate lyase 1 (ACL1) native to the cell or from a different species, and / or a nucleotide sequence encoding ATP citrate lyase 2 (ACL2) native to the cell or from a different species, and a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

[0091] It should be understood by a skilled person that said first, second, third and fourth chimeric gene construct as used herein, may or may not be different chimeric gene constructs.

[0092] In a preferred embodiment, said first, second, third and fourth chimeric gene constructs are different gene constructs.

[0093] In a preferred embodiment, the recombinant yeast cell of the invention is engineered by overexpression of the chimeric gene constructs comprising ACC1 DGA1, MCE2, and ACL genes, the ACL gene being ACL1 and / or ACL2, thereby featuring all four genes ACC1, DGA1, MCE2 and ACL recombinantly expressed, preferably overexpressed. The recombinant yeast cell of the invention preferably shows higher lipid titer and / or lipid productivity compared to known obese strains reported in the prior art as shown in Figs 3, 4 and 5. The recombinant yeast cell of the invention shows increased lipid production compared to the recombinant cell obtainable by overexpression of genes of the push-pull phase, cush as ACC1, DGA1 and MCE2. It has been surprisingly found that a disruption of at least one CEX1 gene, which controls the metabolism of the citrate and / or the citric acid in the citric-acid producing yeast cell, for example Y. lipolytica can lead to increased lipid yield, particularly pronounced in a large-scale and / or industrial setup. The recombinant oleaginous yeast cell of the present invention comprises at least a disruption of CEX1 gene, with overexpressed genes such as DGA1, ACC1, MCE2, ACL1 and / or ACL2, and surprisingly shows pronounced lipid synthesis, which particularly increases during the early lipogenic phase, for example even before the nitrogen is completely depleted, and / or the metabolism of the oleaginous i.e., obese yeast shifts to lipid synthesis. Preferably, the recombinant yeast cell of the invention allows for the increased lipid synthesis which is not dependent on the yeast type and / or strain. The engineering strategy of the invention is preferably applicable in various yeast cells and / or strains. The citric acid is one of the metabolites obtainable by carbohydrates oxidation in Krebs cycle. Yarrowia lipolytica, but also some other yeast of the genera: Candida, Hansenula, Pichia, Debaryomyces, Torulopsis, Kloekera, KeVer / SLIM_5gene / 850

[0094] Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia, Nematospora, Saccharomyces, and Zygosaccharomyces are known producers for citric acid. It has been reported previously that disrupting CEX1 gene YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing Y. lipolytica wildtype (Erian et al., FEMS Yeast Res., 2020, 20(7) foaa055). However, the link between the decreased citrate production and effects onto lipid synthesis parameters such as lipid productivity and / or lipid yield, especially for yeast lipid production on a large scale and / or in industry, has not been shown in the prior art. Duman-Ozdamar et al., 2024, (https: / / www.biorxiv.org / content / 10.1101 / 2024.07.31.6060Q2yl) reported that a sole deletion of CEX1 did not affect lipid production in yeast cells.

[0095] Said recombinant oleaginous yeast cell is modified by at least one CEX1 gene disruption. The term "CEX1 gene disruption" means any disturbance of CEX 1 gene expression and / or decrease in overall CEX1 gene function. Some non-limiting examples of said CEX1 disruption are heterozygous or homozygous or CEX1 disruption, wherein said yeast cell lacks one, or both CEX1 alleles, for example functional CEX1 alleles, respectively, or a reduced CEX expression by using a weak and / or a non-functional promoter or no promoter at all, or fusion of CEX1 to a signal peptide, or CRISPRi reduced expression, knock-out mutation, or hemizygous disruption, RNAi, asRNA, riboswitches, RNA aptamers, fusing gene to no / weak / non- functional terminator, fuse to protein degradation tag, reduced CEX1 expression by knocking out some key transcription factor, any CEX1 gene modification allowing the loss of the affinity of the exporter to citrate, and the like. In some embodiments, said at least one CEX1 disruption means blocking actively the exporter either intracellularly or extracellularly by using a blocking peptide or some other kind of inhibitor. Said CEX1 gene disruption can be obtainable by using gene edition using any kind of programmable nucleases, gene homologous recombination, and the like. Any technique known in the art for disruption of a gene function can be used for said CEX1 gene disruption, without departing from the scope of the present invention.

[0096] Preferably, said recombinant oleaginous yeast cell is modified by said at least one CEX1 gene disruption obtainable by CEX1 knockout mutation. Said CEX1 knockout mutation is particularly favoured in diploid or haploid yeast cells. In a particularly preferred embodiment, said CEX1 knockout mutation is replaced by heterologous overexpression of homologs of ATP-citrate lyase 1 and 2 (ACL1 / 2) genes, which allows more efficient cleavage of citrate than the native proteins and / or to direct more citrate towards acetyl- CoA thus enhancing lipid production independently from the nitrogen availability.

[0097] In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1 and MCE2 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1 and ACC1 proteins within the same construct. In another embodiment, a KeVer / SLIM_5gene / 850 chimeric gene construct comprises nucleotides coding for MCE2 and ACC1 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1, MCE2 and ACC1 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1, MCE2, ALC1 and / or ACL2 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for ACC1, ALC1 and / or ACL2 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1, MCE2 and ACL1 or DGA1, MCE2 or ACL2 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for DGA1, ACC1 and ACL1 or DGA1, ACC1 or ACL2 proteins within the same construct. In another embodiment, a chimeric gene construct comprises nucleotides coding for MCE2, ACC1 and ACL1 or MCE2, ACC1 or ACL2 proteins within the same construct.

[0098] In an alternative embodiment, a chimeric gene construct comprises nucleotides coding for DGA1, MCE2, ACC1, ACL1 and ACL2 proteins within the same construct.

[0099] In a preferred embodiment, said yeast cell is Y. lipolytica cell. Yarrowia lipolytica is known oleaginous yeast which is capable of producing citrate in high amounts, when grown on glucose-based substrate, and / or n-alkane substrates. In another embodiment said yeast cell is of the genus Candida, Hansenula, Pichia, Debaryomyces, Torulopsis, Kloekera, Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia, Nematospora, Saccharomyces, or Zygosaccharomyces. In another embodiment, said yeast cell is cell form Candida species such as C. lipolytica, C. tropicalis, C. oleophila, C. intermedia, C. guilliermondii, C. paratropicalis, C. zeylanoides, C. catenulata, C. parapsilosis, C. citroformans, C.fibrae, C. subtropicalis, C. albicans, C. atmospherica, C. brumptii, C. chalmersii, C. hitachinica, C. krusei, C. melibiosa, C. mycoderma, C. pelliculosa, C. petrophilum, C. pulcherrima and C. rugosa. In an alternative embodiment, the yeast cell of the inventio is the cell of Lipomyces starkeyi. In another embodiment, the yeast cell of the invention is the cell of Rhodosporidium toruloides, Rhodotorula glutinis, R. toruloides.

[0100] In a preferred embodiment, said yeast cell is Y. lipolytica strain CBS 8108 cell or Y. lipolytica CBS 10739 cell. It has been surprisingly shown that engineering of Y. lipolytica strains such as CBS 8108 or CBS 10739 according to the present invention can lead to particularly pronounced increase in lipid synthesis, preferably a significantly increased lipid titer and / or lipid productivity.

[0101] In a preferred embodiment, said first chimeric gene construct codes for Y. lipolytica DGA1 protein.

[0102] 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: 1. KeVer / SLIM_5gene / 850

[0103] SEQ ID NO: 1

[0104] MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPTFLTIFM LCCAIPLLWPFVIAYVVYAVKDDSPS NGGVVKRYSPISRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAIISTIEYFLPAFMKRSL SINEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSRYSRGESSGSNGHASGSELNGNGNNGTTNRRPLSSASAGSTAS DSTLLNGSLNSYANQIIGENDPQLSPTKLKPTGRKYIFGYHPHGIIGMGAFGGIATEGAGWSKLFPGIPVSLMTLTNNF RVPLYREYLMSLGVASVSKKSCKALLKRNQSICIVVGGAQESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAF GENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMHARGVFNYDVGLVPYRRPVNIVVGSPIDLPYLPHPTDEEVSEY HDRYIAELQRIYNEHKDEYFIDWTEEGKGAPEFRMIE*

[0105] In an 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 oleaginous yeast cell. 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.

[0106] In a preferred embodiment, said second chimeric gene construct codes for Y. lipolytica ACC1 protein.

[0107] 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: 2.

[0108] SEQ ID NO: 2

[0109] MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVASHGGHTVINKVLIANNGIA AVKEIRSVRKWAYETFGDERAISFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFGVDAV WAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKISSTIVAQHAKVPCIPWSGTGVDEVVVDKSTNLVSVSE EVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGIRKVEREEDFEAAYHQVEGEIPGSPIFIMQLAGNARHLEVQ LLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTFTAMEKAAVRLGKLVGYVSAGTVEYLYSHEDDKFYFLEL NPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPHTTTPIDFDFSGEDADKTQRRPVPRGHTTACR ITSEDPGEGFKPSGGTM HELNFRSSSNVWGYFSVGNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSIRGDF RTTVEYLIKLLETPDFEDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRASEDSIATYMASLEKGQVPARDILKT LFPVDFIYEGQRYKFTATRSSEDSYTLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTCLLEVE NDPTQLRSPSPGKLVKFLVENGDHVRANQPYAEIEVMKMYMTLTAQEDGIVQLMKQPGSTIEAGDILGILALDDPSK VKHAKPFEGQLPELGPPTLSGNKPHQRYEHCQNVLHNILLGFDNQVVM KSTLQEMVGLLRNPELPYLQWAHQVSSL HTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSGEVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLL QAYYDSEARFCGPNVRDEDVILKLREENRDSLRKVVMAQLSHSRVGAKNNLVLALLDEYKVADQAGTDSPASNVHVA KYLRPVLRKIVELESRASAKVSLKAREILIQCALPSLKERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVF KeVer / SLIM_5gene / 850

[0110] DVLAQFFAHDDPWIVLAALELYIRRACKAYSILDINYHQDSDLPPVISWRFRLPTMSSALYNSVVSSGSKTPTSPSVSRA DSVSDFSYTVERDSAPARTGAIVAVPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASSVDTG LSNICNVM IGRVDESDDDDTLIARISQ.VIEDFKEDFEACSLRRITFSFGNSRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQ LELARLSNFDIKPVHTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADRLMSDILDALEVIGTTNS DLNHIFINFSAVFALKPEEVEAAFGGFLERFGRRLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVSGYVVQSELYA EAKNDKGQWIFKSLGKPGSMHMRSINTPYPTKEWLQPKRYKAHLMGTTYCYDFPELFRQSIESDWKKYDGKAPDDL MTCNELILDEDSGELQEVNREPGANNVGMVAWKFEAKTPEYPRGRSFIVVANDITFQIGSFGPAEDQFFFKVTELARK LGIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLATLKPDTVVTTEIEEEGPNGVEKRHVIDYIV GEKDGLGVECLRGSGLIAGATSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINKLLGREVYSSNLQ LGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGR TLEDGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDSIEMSTSEAGQVWYP NSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRDMYNEVLKYGSFIVDALVDYKQPIMVYIPPTGELRGGSWVV VDPTINSDMMEMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARLDPEYSSLKKQLEESPDSEELKVKLSVREKSLM PIYQQISVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRIRRRLVEEYLITKINSILPSCTRLECLARIKSWKPATLD QGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDRQGTLQGMKQALASLSEAERAELLKGL*

[0111] 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 oleaginous yeast cell. 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, GPM1, and FBA1.

[0112] In a preferred embodiment the recombinant oleaginous yeast cell of the invention comprises the third chimeric gene construct codes for Mucor circinelloides MCE2 protein.

[0113] In a preferred embodiment, the MCE2 protein is M. 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: 3.

[0114] SEQ ID NO: 3

[0115] MSPIIDFVRRQLSSTKLHEEQQTATTNDLVSRSGYLNEGKYEVRLNCINAGCLQKKLNYIGTAM DPAKRQRLGLNGLLP AGVETLEIQKARALRVLRSKHNLLEKYILMAQLRTTNVRLFYKIVIDELETVQLAPVIYTPTVGTACLEYSTIYPFLAAPGV PDGLYLTKAELPELCQTIRNYRPTDTEGFEPEIAVISDGSRILGLGDLGTNGMGIPMGKLQLYVAGAGIDPRRTLPIILDL GTNNEKLLNDEFYIGLRQKRPNDEEFYQTVDTVLTALHTVYPNLLIQFEDWSSEHAFGLLEKYQNQMLCFNDDIQGTG AVILSGVINAIRKVEKENQVSPRDHRIVFYGAGSAAIGVARQIQSYFQIEHNMTEEEAKHVFWIVDSKGLVTTTRGDKL AQHKVYYARGDNEGQQYKELIDIVNYNLYSLIGLSSTTGAFNTQVLERLASLNEQPIVFPLSNPATQAECTFEQAMEAT KeVer / SLIM_5gene / 850

[0116] NNKVIFASGTAFPAYTIKSTGEVNTPGQGNNMYIFPGLGLGACLANPAHFDRMIYEASKALADSLTEEEISKAWLYPSL NYRSVSAIVAAAVCQETLNENLATSQAMMTQCKSHEDILDYVSAHMWSPDYGNNNSNQQAGKL*

[0117] 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 oleaginous yeast cell. 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.

[0118] In a preferred embodiment, the recombinant oleaginous yeast cell comprises the fourth chimeric gene construct codes for M. musculus ACL1 protein and / or M. musculus ACL2 protein. It has been surprisingly found that overexpressing homologs of the ATP-citrate lyase 1 and 2 (ACL1,2), with more efficient cleavage of citrate than the native proteins (i.e., the ACL1,2 from M. musculus), aimed to direct more citrate towards acetyl-CoA thus enhancing lipid production independently from the N availability.

[0119] In a preferred embodiment, the ACL1 protein is M. musculus ACL1 protein or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the ACL1 protein of SEQ. ID NO: 4

[0120] SEQ ID NO: 4

[0121] MSAKAISEQTGKELLYKYICTTSAIQNRFKYARVTPDTDWAHLLQDHPWLLSQSLVVKPDQLIKRRGKLGLVGVNLSLD GVKSWLKPRLGHEATVGKAKGFLKNFLIEPFVPHSQAEEFYVCIYATREGDYVLFHHEGGVDVGDVDAKAQKLLVGV DEKLNTEDIKRHLLVHAPEDKKEVLASFISGLFNFYEDLYFTYLEINPLVVTKDGVYILDLAAKVDATADYICKVKWGDIEF PPPFGREAYPEEAYIADLDAKSGASLKLTLLNPKGRIWTMVAGGGASVVYSDTICDLGGVNELANYGEYSGAPSEQQT YDYAKTILSLMTREKHPEGKILIIGGSIANFTNVAATFKGIVRAIRDYQGPLKEHEVTIFVRRGGPNYQEGLRVMGEVGK TTGIPIHVFGTETHMTAIVGMALGHRPIPNQPPTAAHTANFLLNASGSTSTPAPSRTASFSESRADEVAPAKKAKPAM PQDSVPSPRSLQGKSATLFSRHTKAIVWGMQTRAVQGMLDFDYVCSRDEPSVAAMVYPFTGDHKQKFYWGHKEILI PVFKNMADAMKKHPEVDVLINFASLRSAYDSTMETMNYAQIRTIAIIAEGIPEALTRKLIKKADQKGVTIIGPATVGGIK PGCFKIGNTGGMLDNILASKLYRPGSVAYVSRSGGMSNELNNIISRTTDGVYEGVAIGGDRYPGSTFM DHVLRYQDT PGVKMIVVLGEIGGTEEYKICRGIKEGRLTKPVVCWCIGTCATMFSSEVQFGHAGACANQASETAVAKNQALKEAGV FVPRSFDELGEIIQSVYEDLVAKGAIVPAQEVPPPTVPMDYSWARELGLIRKPASFMTSICDERGQELIYAGMPITEVFK EEMGIGGVLGLLWFQRRLPKYSCQFIEMCLMVTADHGPAVSGAHNTIICARAGKDLVSSLTSGLLTIGDRFGGALDAA AKM FSKAFDSGIIPMEFVNKM KKEGKLIMGIGHRVKSINNPDMRVQILKDFVKQHFPATPLLDYALEVEKITTSKKPNL ILNVDGFIGVAFVDMLRNCGSFTREEADEYVDIGALNGIFVLGRSMGFIGHYLDQKRLKQGLYRHPWDDISYVLPEHM SM* KeVer / SLIM_5gene / 850

[0122] In a preferred embodiment, said M. musculus ACL1 gene is expressed, i.e., operably linked to a TEF1 promoter, allowing for overexpression of chimeric ACL1 gene in the oleaginous yeast cell. In an embodiment, said native ACL1 gene is linked, preferably operably linked to another yeast-expressible promoter, preferably a Y. lipolytica promoter such as, for example EXP1, GPD, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPM1, and FBA1.

[0123] In a preferred embodiment, the ACL2 protein is M. musculus ACL2 protein or an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the ACL2 protein of SEQ. ID NO: 5.

[0124] SEQ ID NO: 5

[0125] MSAKAISEQTGKELLYKYICTTSAIQNRFKYARVTPDTDWAHLLQDHPWLLSQSLVVKPDQLIKRRGKLGLVGVNLSLD GVKSWLKPRLGHEATVGKAKGFLKNFLIEPFVPHSQAEEFYVCIYATREGDYVLFHHEGGVDVGDVDAKAQKLLVGV DEKLNTEDIKRHLLVHAPEDKKEVLASFISGLFNFYEDLYFTYLEINPLVVTKDGVYILDLAAKVDATADYICKVKWGDIEF PPPFGREAYPEEAYIADLDAKSGASLKLTLLNPKGRIWTMVAGGGASVVYSDTICDLGGVNELANYGEYSGAPSEQQT YDYAKTILSLMTREKHPEGKILIIGGSIANFTNVAATFKGIVRAIRDYQGPLKEHEVTIFVRRGGPNYQEGLRVMGEVGK TTGIPIHVFGTETHMTAIVGMALGHRPIPNQPPTAAHTANFLLNASGSTSTPAPSRTASFSESRADEVAPAKKAKPAM PQGKSATLFSRHTKAIVWGMQTRAVQGM LDFDYVCSRDEPSVAAMVYPFTGDHKQKFYWGHKEILIPVFKNMADA MKKHPEVDVLINFASLRSAYDSTMETMNYAQIRTIAIIAEGIPEALTRKLIKKADQKGVTIIGPATVGGIKPGCFKIGNTG GMLDNILASKLYRPGSVAYVSRSGGMSNELNNIISRTTDGVYEGVAIGGDRYPGSTFMDHVLRYQDTPGVKMIVVLG EIGGTEEYKICRGIKEGRLTKPVVCWCIGTCATM FSSEVQFGHAGACANQASETAVAKNQALKEAGVFVPRSFDELGE IIQSVYEDLVAKGAIVPAQEVPPPTVPMDYSWARELGLIRKPASFMTSICDERGQELIYAGM PITEVFKEEMGIGGVLG LLWFQRRLPKYSCQFIEMCLMVTADHGPAVSGAHNTIICARAGKDLVSSLTSGLLTIGDRFGGALDAAAKMFSKAFDS GIIPMEFVNKMKKEGKLIMGIGHRVKSINNPDM RVQILKDFVKQHFPATPLLDYALEVEKITTSKKPNLILNVDGFIGVA FVDM LRNCGSFTREEADEYVDIGALNGIFVLGRSMGFIGHYLDQKRLKQGLYRHPWDDISYVLPEHMSM*

[0126] In a preferred embodiment, said M. musculus ACL2 gene is expressed, i.e., operably linked to a TEF1 promoter, allowing for overexpression of chimeric ACL2 gene in the oleaginous yeast cell. In an embodiment, said M. musculus ACL2 gene is linked, preferably operably linked to another yeast- expressible promoter, preferably a Y. lipolytica promoter such as, for example EXP1, GPD, GPAT, YAT1, XPR2, FBA1, LV5, ICL, DGA1, GPM1, and FBA1.

[0127] In a preferred embodiment the recombinant oleaginous yeast cell comprises the fourth chimeric gene construct, said construct coding for M. musculus ACL1 and M. musculus ACL2 proteins. KeVer / SLIM_5gene / 850

[0128] The recombinant oleaginous yeast cell 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 Y. 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.

[0129] 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, MCE2 and ACL1 and / or ACL2 genes in the recombinant oleaginous yeast cell of the invention, thereby allowing an increased lipid production.

[0130] 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.

[0131] 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 TEF1 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).

[0132] In one embodiment, the genes are under control of the TEF1 promoter, for example Y. lipolytica TEF1 promoter (XM_501628), or an orthologue an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the sequence of TEF1 promoter showed in SEQ ID NO: 6.

[0133] SEQ ID NO: 6

[0134] MGKEKTHVNLVVIGHVDAGKSTTTGHLIYKCGGIDKRTIEKFEKEADELGKGSFKYAWVLDKLKAERERGITIDIALWKF QTPKYYVTVIDAPGHRDFIKNMITGTSQADCAILIIAGGVGEFEAGISKDGQTREHALLAFTLGVKQLIVAINKMDSVK WSQDRYNEICKETANFVKKVGYNPKAVPFVPISGWNGDNM IEASTNCDWYKGWTKETKAGEVKGKTLLEAIDAIEP PVRPSDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKAGMVVTFAPANVTTEVKSVEM HHEILPDGGFPGDNVGFNVK KeVer / SLIM_5gene / 850

[0135] NVSVKDIRRGNVAGDSKNDPPKGCDSFNAQVIVLNHPGQIGAGYAPVLDCHTAHIACKFDTLIEKIDRRTGKKM EDSP KFIKSGDAAIVKMVPSKPMCVEAFTEYPPLGRFAVRDM RQTVAVGVIKSVEKSDKAGGKVTKAAQKAAKK*

[0136] 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 SEQ ID NO: 7.

[0137] SEQ ID NO: 7

[0138] MVKVADLILRGLIFM FAAIIMGLAGSLASTHKKGHYNPQVSYAVFCGAWSALFGVFYPVLANFIEAIAFPIVILIIDFISW VLTLAGGAALATAIRCHSCGNM NYVNSNKVTQGSKGRCRKAQATVAFLFFANFSFLATM ILSAISVKQLGAFTLPGRS RRSAPRTGIPTMSQV*

[0139] 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 SEQ ID NO: 8.

[0140] SEQ ID NO: 8

[0141] MAIKVGINGFGRIGRIVLRNALKNPEVEVVAVNDPFIDTEYAAYMFKYDSTHGRFKGKVEAKDGGLIIDGKHIQVFGER DPSNIPWGKAGADYVVESTGVFTGKEAASAHLKGGAKKVIISAPSGDAPM FVVGVNLDAYKPDMTVISNASCTTNCL APLAKVVNDKYGIIEGLMTTVHSITATQKTVDGPSHKDWRGGRTASGNIIPSSTGAAKAVGKVIPELNGKLTGMSLRV

[0142] PTVDVSVVDLTVRIKNGASYEDIKATMKAASESPELKGILGYTDEDVVSTDFIGDTHSSIFDAKAGIGLNDNFVKLISWY DNEYGYSARVVDLIVAVAKKDASA*

[0143] In a preferred embodiment, said at least one CEX1 gene disruption concerns a disruption of a citrate exporter CEX1 corresponding to gene YALI0D20196g, or an orthologue an orthologue with at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of sequence identity to the CEX1 protein of SEQ ID NO: 9.

[0144] SEQ ID NO: 9

[0145] MFNLNTKSSKEPEVTEVAVDSTPSSPVTRESSPESSPDNSAVDLEKGKKSKFEM HDQTNLLPRSQLFLVFGAMAFCM L VSFMDQNGISVALPDIAKDLNATDTISWAGTSGLIANTVFQVLYGRLSDIFGRKLVFMIVVCTLVCADIGCACAQTSTQ LYIFRAFSGIANGGMSCLTMIVVSDIVTLKERGKYQGILGACVGLGNTIGPFLGAAFTENVSWRAIFYLLAPMGGLCAV VIFFILPSKKPQGSAIQKAKAIDYPGLFCSSVALVFLLVPIAGGGSYYEWDSPMVISMLCIGGVFFIAFFVIEGFFAKLPM MPLRIFGTKALFALMM HSVLLGIAYYGDLYYLPMYMRNIRGWSSMKAAGMSCALVTTQAVTTVISGQYLSRMGRYL

[0146] EVIYFGFGIWTVGAIMKCFWKRDSNMALLIFSLLFEGAGVGCCFQPTLVAAQALSRKEDRSVVISSRNFLRSFGGAVGL AVCSAILANSLKADLKTKNLPSELYELIVKAPFSLPDLSHYPEYRDQVLDAYM NGSHTVFVFLCPIVGACLLVTVFVKDH KeVer / SLIM_5gene / 850

[0147] GLQTHEKKAAEAKTVEDKDKDESGTDCEDMTKGEVLVSEKEGKLSRNSSSQSMHFGDHTAVNTPAPTGYNTPVVGT LCHNSPNFPPMDHNDVITPLEDFDESPLPPHSPNSSGKRVTIQEE*

[0148] In a particularly preferred embodiment, said CEX1 gene, for example the CEX1 gene corresponding to YALI0D20196g, is replaced by a knock-out mutation and replaced by two constructs of the M. musculus ACL1 (SEQ ID No:4) and ACL2 (SEQ IN NO:5) genes, which said two constructs are overexpressed under the control of TEF1 promoter.

[0149] In an alternative 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 recombinant oleaginous yeast cell of the present invention.

[0150] In an 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: 10.

[0151] SEQ ID NO: 10

[0152] MGDRSGAGGSRRRRTGSRPSSQSGSGFAAAEEEVRDVGAGGDAPTPDKDKDGHDDVSSGHWDLRCHRLQDSLFSS DSGFSNYRGILNWCVVMLVLSNARLFLENLIKYGILVDPIQVVSLFLKDPYSWPALCLVIVANVFAVTAFQVEKRLAVG ALTEQAGLLIHVANLATILCFPAAVAFLLESITPVGSLLALMVYAILFLKLFSYRDVNLWCRERRATAKAKAASAGKKAN GGAAQHSVSYPDNLTYRDLYYFLLAPTLCYELNFSRFPRIRKRFLLRRLLEMLFLIQLQVGLIQQWMVPTIQNSMKPFK DMDYSRIIERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWCLRH FYKPMLRRGSSKWVARMGVFLASAFFHEYLVSIPLRMFRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIG QPVAVLM YVH DYYVLH H EAPTAG A*

[0153] 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: 11.

[0154] SEQ ID NO: 11

[0155] MGDRGGAGGSRRRRTGSRPSIQGGSGPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFS SDSGFSNYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFLKDPYSWPALCLVIVANIFAVAAFQVEKRLAVGA LTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMVYTILFLKLFSYRDVNLWCRERRAGAKAKAALAGKAANG GAAQRTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKD MDYSRIVERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESITYFWQNWNIPVHKWCIRHF KeVer / SLIM_5gene / 850

[0156] YKPMLRRGSSKWAARTAVFLASAFFHEYLVSIPLRM FRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQ PVAVLM YVH DYYVLN REAPAAGT*

[0157] 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: 12.

[0158] SEQ ID NO: 12

[0159] MAGEDCVRKRPSGSTTTYKSPENEEMQRRPDGDRSFQNSSNGRVDVDHVITRKMQLIAEAEQLKPVFM KEVDSHFT EFVNSLVAKSALLDSSSSASLFPASCSEKELHKAKALRAPPEHGKIFTARRSLLDELFEVSHIRTIYHM FIALLIVFILSTLLVD FIDEGRLVLGFDLLVYVFGKFPVVFCTWLCMFCATVIIPYSLFSQWAQGYCSSSHRVIYSLFYGTLFTLFQTVGLGIGPTY

[0160] VAISYALPPASRFIVILEQVRLVMKAHSFVRENVPRVLSSVKEKSSSVPIPRISQYLYFLFAPTLIYRDNYPRNPMVRWGY VATKFAQVLGSLFYAYYIFVRLCIPQFRNSSQETFNLRGLVLCIFNSILPGVLILFLVFFAFLHCWLNAFAEM MRFADRM FYKDWWNSTSYANYYRTWNVVVHDWLYYYAYRDFLWFFGKKFKAAAMLSVFTVSAAVHEYVLSICFGFFYPVLFCLF MCFGMLFNFILNDRRKGPIWNVIMWTSLFLGQGVIICLYSQEWYARQYCPAENPAFLDYLKPRSWSCHVQM*

[0161] In a second independent aspect, the invention concerns a method for producing lipids using the recombinant oleaginous yeast cell according to any embodiment described above, the method comprising the steps:

[0162] • culturing the recombinant oleaginous yeast cell according to any embodiment of a first aspect of the invention in a culture medium and in culturing conditions suitable for production of lipids, and

[0163] • optionally, extracting said lipids from said culture medium and / or said recombinant oleaginous yeast cell.

[0164] In a preferred embodiment said culturing is executed by fed-batch fermentation, preferably in a bioreactor. The method of the invention is particularly suited for the lipid production on large scale. For example, the method of the invention may allow for lipid titers of at least 20 g / L, 25 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 70 g / L, 75 g / L, or 80 g / L. In some embodiments , the method of the invention allows for obtaining lipid titers as high as 85 g / L, 90 g / L or 100 g / L.

[0165] In the method of the second aspect, the lipids, such as fatty acids and triacylglycerols, can be synthetised by culturing of the recombinant oleaginous yeast cell according to any one of the above described KeVer / SLIM_5gene / 850 embodiments of the first aspect from a variety of carbon sources, such as, for example, fermentable sugars, such as glucose and maltose, monosaccharides, such as xylose, fructose or arabinose and / or other carbon sources, such as glycerol, ethyl acetate, methanol, and the like. In an embodiment the method of the invention utilizes the by-product of cellulose and / or lignin production, agricultural waste streams, such as pentose sugars or sugar acids, or molasses, i.e. the products obtainable by sugar refining and / or production. In a particular example, in the method of the invention viscous byproducts obtainable from the refining of sugarcane or sugar beet juice into sugar are used as a carbon source.

[0166] 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 recombinant oleaginous yeast cell 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.

[0167] While the specific culture conditions, for example, the concentration of the carbon source, will depend upon the respective recombinant yeast cell 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 1 KeVer / SLIM_5gene / 850 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 recombinant oleaginous yeast cells of the invention described herein are cultured under conditions suitable for lipid accumulation, as known in the art.

[0168] Preferably, said medium used in a method of a second aspect is glucose- or glycerol- based.

[0169] The recombinant yeast cell of the invention can be grown on different culturing media, as it 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 recombinant yeast cell according to the invention is able of growing on lignocellulosic biomass, preferably biomass hydrolysate thereof, more preferably monomeric sugars via enzymatic hydrolysis of said lignocellulosic biomass. The recombinant oleaginous yeast cell of the invention is unexpectedly able to grow on alternative carbon sources such as, but not limited to glycerol, xylose, arabinose, and decane. Development of novel recombinant yeast cells 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.

[0170] The recombinant oleaginous yeast cell of the invention can 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 wild type cell. Thus, the recombinant yeast cell of the invention is more robust to influences of common inhibitors, and thus it can be used to for the lipid production in not optimal, harsh conditions.

[0171] In some embodiments, the recombinant oleaginous yeast cells 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, the recombinant oleaginous yeast cell 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. KeVer / SLIM_5gene / 850

[0172] 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 75 g / L, most preferably 80 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 80 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, 46 h, 47 h, 48 h, 49 h, 50 h, 55 h or 60 h of the growth.

[0173] In some embodiments, the recombinant yeast cell of the invention, having at least one CEX1 gene disruption and comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2, DGA1 and ACL chimeric genes, wherein said ACL gene is ACL1 or ACL2 gene, 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 111 g / L, at least 112 g / L, at least 113 g / L, at least 114 g / L, at least 115 g / L, at least 116 g / L, at least 117 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.

[0174] In some embodiments, the recombinant yeast cell of the invention, having at least one CEX1 gene disruption and comprising one or more chimeric gene constructs, for overexpression of ACC1, MCE2, DGA1 and ACL 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 the recombinant oleaginous yeast cell according to any one of the above described embodiments herein refers to a lipid titer that is increased as compared to the lipid titer that can be achieved by a wild-type yeast cell under KeVer / SLIM_5gene / 850 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 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 95 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. In some embodiments, the lipid titer obtainable by bioreactor scale culturing of the recombinant yeast cell of the invention after 48 h of culturing is at least 35 g / L, 40 g / L, 45 g / L or 50 g / L. In some embodiments, the lipid titer obtainable by bioreactor scale culturing of the recombinant yeast cell of the invention after 70 h of bioreactor culturing is at least 35 g / L, 40 g / L, 45 g / L or 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L or 100 g / L.

[0175] 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. The method of the invention shows advantageous robustness, as it concerns the utilization of an recombinant yeast cell which is more tolerant to and / or less affected by common industrial inhibitors of yeast growth.

[0176] 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. KeVer / SLIM_5gene / 850

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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 KeVer / SLIM_5gene / 850 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.

[0182] 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 for example, 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.

[0183] In some embodiments, about 40%, 45%, 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.

[0184] In some embodiments, about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%? 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65% of the total fatty acids is oleic acid. 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.

[0185] 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. In some embodiments, said weight ratio of oleic acid to linoleic acid is about 3.5:1 or 4:1.

[0186] 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. KeVer / SLIM_5gene / 850

[0187] In yet another aspect, the invention concerns composition comprising the lipids according to any one of embodiments of the third aspect and / or lipids obtainable by the method according to any embodiment of the second aspect, or the recombinant oleaginous yeast cell according to any embodiment of the first aspect of the present invention.

[0188] In another independent aspect, the invention concerns a food product, preferably a plant-based food product, comprising the lipids of the third aspect and / or the composition according to the fourth aspect, and or recombinant oleaginous yeast cell according to any embodiment of the first aspect of the present invention.

[0189] 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).

[0190] In some embodiments, the lipids provided by the recombinant oleaginous yeast cell 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.

[0191] Advantageously, the lipids according to the present invention can be used in replacement of oils for cooking and frying.

[0192] 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.

[0193] 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.

[0194] The lipids according to the invention are suitable for cosmetical purposes, i.e. personal care and cosmetic compositions.

[0195] 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 KeVer / SLIM_5gene / 850 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.

[0196] EXAMPLES

[0197] Example 1: Obtaining a recombinant oleaginous Yarrowia lipolytica cell of the invention.

[0198] Lipid production in Y. lipolytica could be increased using the "PUSH-PULL" strategy, which involves the overexpression of two native enzymes ACC1 and DGA1 along with the simultaneous restoration of the cytoplasmic POM cycle (pyruvate- oxaloacetate-malate)(Tai and Stephanopoulos, Metab. Eng., 2013, 15, 1-9; Qiao et al., Nat. Biotechnol., 2017, 35, 173-177), which is shown in Fig. 1A. Although this approach has been proved very efficient, it still may have some disadvantages: i) lipid production is dependent on nitrogen availability in the bioreactor, and ii) the main byproduct of lipid production is citrate, which results in lower lipid yields and necessitates modifications to the process to reduce its production (e.g., lower oxygen supply during lipogenesis) (Qiao et al., Nat. Biotechnol., 2017, 35, 173-177). However, in Duman-Ozdamar et al., 2024, (https: / / www.biorxiv.org / content / 10.1101 / 2024.07.31.6060Q2yl), it was demonstrated that the disruption of MFE1 and deletion of CEX1 (Amfe_Acex) but not the sole deletion of CEX1 lead to an increased lipid production in the oleaginous Y. lipolytica strain CBS 8108. The new engineering strategy of the present invention, referred to as "PUSH-PULL-TRAP", was developed to eliminate citrate production while also partially decoupling lipid production from N availability. It was surprisingly shown that deletion of the cytoplasmic exporter (CEX1) would "trap" the produced citrate inside the cell. In addition to said deletion of CEX1 exporter, the overexpressing of the homologs of the ATP-citrate lyase 1 and 2 (ACL1,2), with more efficient cleavage of citrate than the native proteins (i.e., the ACL1,2 from Mus musculus (Zhang et al., J. Biotechnol., 2014, 192, 78-84.), more citrate was directed towards acetyl-CoA thereby enhancing lipid production independently from the N availability (Fig. IB).

[0199] To validate that the citrate exporter indeed plays a role in citrate production we deleted the respective gene , YALI0D20196g (Erian et al., FEMS Yeast Res., 2020, 20, foaa055), in the chosen oleaginous background strain of Y. lipolytica CBS 8108 (herein further referred to as "ACEX1" strain). The yeast growth and the citrate production was compared between the ACEX1 strain with the wild type (WT) strain in shake flask fermentations using a low N medium. After 5 days the deletion mutant did not show any growth defects compared to the WT and citrate production was almost diminished (Fig. 2). KeVer / SLIM_5gene / 850

[0200] To assess the effect of the citrate exporter deletion in combination with the overexpression of the ACL1,2 enzymes which convert excess citrate into acetyl-CoA, we first engineered an "obese" variant of the chosen oleaginous Y. lipolytica strain CBS 8108 by implementing the "PUSH-PULL" strategy. This was achieved by overexpressing the native gene ACC1 (XM_501721) which encodes for an enzyme that converts the acetyl-CoA to malonyl-CoA and the native DGA1 (XM_504700), which catalyses the final step of triacylglycerols (TAGs) by acylating diacylglycerol using acyl-CoA as an acyl donor. In addition, we overexpressed the heterologous MCE2 (DQ975377) from Mucor circinelloides, encoding a cytosolic NADP+-dependent malic enzyme that effectively restores the NADPH homeostasis during lipogenesis (Qiao et al., Nat. Biotechnol., 2017, 35, 173-177) to obtain a reference recombinant Y. lipolytica strain, herein referred to as "PP" strain. On top of the PP background we replaced the CEX1 gene with two overexpression constructs of the Mus musculus homologs of ACL1 (NM_001199296) and ACL2 (NM_134037) genes. Both heterologous genes in these overexpression constructs were under the control of Y. lipolytica strong intronic promoter TEF1 in (XM_501628) (to obtain the strain which is herein further referred to as a "PPT" strain).

[0201] Example 2. The comparison of the growth and lipid content parameters of reference strain to newly obtained, recombinant Y. lipolytica cells.

[0202] To evaluate the performance of the engineering strategies the growth and the lipid production parameters such as lipid content, DCW and / or lipid titer, of the generated recombinant strains (ACEX1, PP, and PPT) were compared to the WT background Y. lipolytica strain CBS 8108 (Fig. 3). The growth and the lipid production parameters were tested in four different conditions: i) a "high" N condition with a lower N:C ratio (1:100) and glucose as the sole carbon source; ii) a "high" N condition with a lower N:C ratio (1:100) and glycerol as the sole carbon source; iii) a "low" N condition with a higher N:C ratio (1:140) and glucose as the sole carbon source, and iv) a "low" N condition with a higher N:C ratio (1:140) and glycerol as the sole carbon source. None of the strains exhibited significantly different growth, under any of the conditions, indicating that the observed differences in lipid production are due to the strains' abilities to produce lipids rather than fitness-related reasons. The difference in biomass production between the "high" and the "low" N conditions can be explained by the fact that N is the limiting nutrient in these types of fermentations, leading to more pronounced effects on growth and less variation in lipid production between the different strains. The best performing strain in all conditions was the PPT strain with a lipid content ranging from 33.3% to 52.6% and an increase in lipid production ranging from 12.8% to 108.5% compared to the WT (Fig. 3). When compared to the PP strain, PPT shows an increase in lipid production from 4.8% to 16.0%. The deletion of the citrate exporter alone (ACEX1 strain) results in an KeVer / SLIM 5gene / 850 increase in lipid production ranging from 11.0% to 16.7% compared to the WT in most of the tested conditions (Fig. 3).

[0203] Example 3. Comparative results for the bioreactor experiments.

[0204] To further assess strain performance and the effects of the different engineering strategies used to generate various oleaginous Y. lipolytica strains, we performed fed-batch fermentations in 8-liter bioreactors. Lipid production in bioreactors typically occurs in 2 phases, a first growth-phase where the yeast consumes the carbon source to produce biomass (lasting around 30 h) followed by a lipogenic phase that begins once nitrogen is depleted, during which the cells consume the carbon source to produce lipids. In the experiment, it was started with 25 g / L of ammonium sulfate as the nitrogen source and 100 g / L of glucose as the carbon source. After 5 h, a continuous feed of 75% glucose solution for a total of 65 hours was initiated. The performance of the oleaginous, i.e., "obese" PP and PPT strains was compared with the WT Y. lipolytica CBS 8108 strain. Consistent with the observations in the shake flasks experiments, the PPT strain outperformed the others, reaching a maximum lipid titer of 63.3 g / l after 90 h and a lipid content of 58.9%. This represents a 23.9% increase in lipid titer compared to the PP strain and a 103.5% increase compared to the WT strain, while citrate production was below 1 g / L (Fig. 4A). In addition, the PPT strain exhibits faster lipid accumulation, even from 48 hours, due to the enhanced activity of the ACL proteins that supply acetyl-CoA independently of N availability. At 48 hours the PPT strain reaches its maximum lipid productivity (0.98 g / L / h) which is 1.6 times higher than that of PP and WT strains at the same timepoint (Fig. 4B). By the end of the fermentation PPT engineered Y. lipolytica CBS 8108 has a lipid productivity (0.7 g / L / h), more than twice that of the WT strain and a 22.8% increase compared to the PP strain (Fig. 4B). The reduced production of byproducts in the PPT strain leads to a 90% increase in lipid yield from the WT and to a 18.8% from the PP strain (with the lipid yield of the PP strain being 60% higher than that of the WT; Fig. 4C).

[0205] Example 4. Composition (i.e. fatty acid content) in the lipids obtainable by the recombinant oleaginous yeast Y. lipolytica engineered according to the invention.

[0206] Fatty acid content in the lipids obtained from the recombinant Y. lipolytica of the Fatty acid invention (strain PPT), expressed as wt.% C14:0 0,18

[0207] C16:0 11,90

[0208] C16:l 12,66

[0209] C18:0 4,45

[0210] C18:l 52,80

[0211] C18:2 15,15

[0212] C20:0 0,23 KeVer / SLIM_5gene / 850

[0213] Fatty acid content in the lipids obtained from the recombinant Y. lipolytica of the Fatty acid invention (strain PPT), expressed as wt.%

[0214] C22:0 0,30

[0215] C24:0 1,82

[0216] Example 5. A preferred embodiments (nucleotide sequences) of a chimeric construct used for overexpression of Y. lipolytica DGA1 (XM_504700) under the control of the GPD promoter (XM_501515), shown in the following order (letter-type): GPD PROMOTER, Y. LIPOLYTICA DGA1 ORF, pex20 terminator, loxp site, hph (hygromycin B) resistance cassette, loxp site.

[0217] GTTGATGTGTGTTTAATTCAAGAATGAATATAGAGAAGAGAAGAAGAAAAAAGATTCAATTGAGCCGGCGATGCAGACCCTTAT ATAAATGTTGCCTTGGACAGACGGAGCAAGCCCGCCCAAACCTACGTTCGGTATAATATGTTAAGCTTTTTAACACAAAGGTTT GGCTTGGGGTAACCTGATGTGGTGCAAAAGACCGGGCGTTGGCGAGCCATTGCGCGGGCGAATGGGGCCGTGACTCGTCT

[0218] CAAA TTCGAGGGCGTGCCTCAATTCGTGCCCCCGTGGCTTTTTCCCGCCGTTTCCGCCCCGTTTGCACCACTGCAGCCGCTT CTTTGGTTCGGACACCTTGCTGCGAGCTAGGTGCCTTGTGCTACTTAAAAAGTGGCCTCCCAACACCAACATGACATGAGTGC GTGGGCCAAGACACGTTGGCGGGGTCGCAGTCGGCTCAATGGCCCGGAAAAAACGCTGCTGGAGCTGGTTCGGACGCAGT CCGCCGCGGCGTATCGATATCCGCAAGGTTCCATGGCGCCATTGCCCTCCGTCGGCGTCTATCCCGCAACCTCTAAATAGAG CGGGAATATAACCCAAGCTTCTTTTTTTTTCCTTTAACACGCACACCCCCAACTATCATGTTGCTGCTGCTGTTTGACTCTACTC TGTGGAGGGGTGCTCCCACCCAACCCAACCTACAGGTGGATCCGGCGCTGTGATTGGCTGATAAGTCTCCTATCCGGACTAA TTCTGACCAATGGGACATGCGCGCAGGACCCAAATGCCGCAATTACGTAACCCCAACGAAATGCCTACCCCTCTTTGGAGCC CAGCGGCCCCAAATCCCCCCAAGCAGCCCGGTTCTACCGGCTTCCATCTCCAAGCACCCCTTTCTCCACACCCCACAAAAAG

[0219] ACCCGTGCAGGACATCCTTICTGCGTCATGACTATCGACTCACAATACTACAAGTCGCGAGACAAAAACGACACGGCACCCA AAATCGCGGGAATCCGATATGCCCCGCTATCGACACCATTACTCAACCGATGTGAGACCTTCTCTCTGGTCTGGCACATTTT CAGCATTCCCACTTTCCTCACAATTTTCATGCTATGCTGCGCAATTCCACTGCTCTGGCCATTTGTGATTGCGTATGTAGTGT ACGCTGTTAAAGACGACTCCCCGTCCAACGGAGGAGTGGTCAAGCGATACTCGCCTATTTCAAGAAACTTCTTCATCTGGA AGCTCTTTGGCCGCTACTTCCCCATAACTCTGCACAAGACGGTGGATCTGGAGCCCACGCACACATACTACCCTCTGGACG TCCAGGAGTATCACCTGATTGCTGAGAGATACTGGCCGCAGAACAAGTACCTCCGAGCAATCATCTCCACCATCGAGTACT TTCTGCCCGCCTTCATGAAACGGTCTCTTTCTATCAACGAGCAGGAGCAGCCTGCCGAGCGAGATCCTCTCCTGTCTCCCG TTTCTCCCAGCTCTCCGGGTTCTCAACCTGACAAGTGGATTAACCACGACAGCAGATATAGCCGTGGAGAATCATCTGGCT CCAACGGCCACGCCTCGGGCTCCGAACTTAACGGCAACGGCAACAATGGCACCACTAACCGACGACCTTTGTCGTCCGCC TCTGCTGGCTCCACTGCATCTGATTCCACGCTTCTTAACGGGTCCCTCAACTCCTACGCCAACCAGATCATTGGCGAAAAC GACCCACAGCTGTCGCCCACAAAACTCAAGCCCACTGGCAGAAAATACATCTTCGGCTACCACCCCCACGGCATTATCGG CATGGGAGCCTTTGGTGGAATTGCCACCGAGGGAGCTGGATGGTCCAAGCTCTTTCCGGGCATCCCTGTTTCTCTTATGAC TCTCACCAACAACTTCCGAGTGCCTCTCTACAGAGAGTACCTCATGAGTCTGGGAGTCGCTTCTGTCTCCAAGAAGTCCTG CAAGGCCCTCCTCAAGCGAAACCAGTCTATCTGCATTGTCGTTGGTGGAGCACAGGAAAGTCTTCTGGCCAGACCCGGTGT CATGGACCTGGTGCTACTCAAGCGAAAGGGTTTTGTTCGACTTGGTATGGAGGTCGGAAATGTCGCCCTTGTTCCCATCAT GGCCTTTGGTGAGAACGACCTCTATGACCAGGTTAGCAACGACAAGTCGTCCAAGCTGTACCGATTCCAGCAGTTTGTCAA GAACTTCCTTGGATTCACCCTTCCTTTGATGCATGCCCGAGGCGTCTTCAACTACGATGTCGGTCTTGTCCCCTACAGGCGA CCCGTCAACATTGTGGTTGGTTCCCCCATTGACTTGCCTTATCTCCCACACCCCACCGACGAAGAAGTGTCCGAATACCAC

[0220] GACCGATACATCGCCGAGCTGCAGCGAATCTACAACGAGCACAAGGATGAATATTTCATCGATTGGACCGAGGAGGGCAA AGGAGCCCCAGAGTTCCGAATGATTGAGTAAacgcaacfaacafgaafgaafacgafafacafcaaagacfafgafacgcagfatgcacacfgfacgagfaag ggcactagccactgcactcaagtgaaaccgttgcccgggtacgagtatgagtatgtacagtatgtttagtattgtacttggacaatgcttgtatcgtacattctcaagtgtcaaacataaatat ccgttgctatatcctcgcaccaccacgtagctcgctatatccctgtgttgaatccatccatcttggattgccaattgtgcacacagaaccgggcactcacttccccatccacacttataacttc gtataatgtatgctatacgaagttataaggagtttggcgcccgttttttcgagccccacacgtttcggtgagtatgagcggcggcagattcgagcgtttccggtttccgcggctggacgag agcccatgatgggggctcccaccaccagcaatcagggccctgattacacacccacctgtaatgtcatgctgttcatcgtggttaatgctgctgtgtgctgtgtgtgtgtgttgtttggcgctcatt gttgcgttatgcagcgtacaccacaatattggaagcttattagcctttctattttttcgtttgcaaggcttaacaacattgctgtggagagggatggggatatggaggccgctggagggagtcg gagaggcgttttggagcggcttggcctggcgcccagctcgcgaaacgcacctaggaccctttggcacgccgaaatgtgccacttttcagtctagtaacgccttacctacgtcattccatgc atgcatgtttgcgccttttttcccttgcccttgatcgccacacagtacagtgcactgtacagtggaggttttgggggggtcttagatgggagctaaaagcggcctagcggtacactagtgggat tgtatggagtggcatggagcctaggtggagcctgacaggacgcacgaccggctagcccgtgacagacgatgggtggctcctgttgtccaccgcgtacaaatgtttgggccaaagtcttg tcagccttgcttgcgaacctaattcccaattttgtcacttcgcacccccattgatcgagccctaacccctgcccatcaggcaatccaattaagctcgcattgtctgccttgtttagtttggctcctg cccgtttcggcgtccacttgcacaaacacaaacaagcattatatataaggctcgtctctccctcccaaccacactcacttttttgcccgtcttcccttgctaacacaaaagtcaagaacacaa acaaccaccccaacccccttacacacaagacatatctacagcaatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgac ctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcgg cactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaa ccgaactgcccgctgttctgcagccggtcgcggaggcaatggatgccattgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacac tacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccga ggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcc caatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgta KeVer / SLIM_5gene / 850 tatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcggg cgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataatcat gtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaaga acgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgATAACTTCGTATAgcgTAcgc TATACGAAGTTATcgtaatagcctccaagagattgatcatcactcataacttcgtataatgtatgctatacgaagttat

[0221] Example 6. A preferred embodiments (nucleotide sequences) of a chimeric construct used for overexpression of Y. lipolytica ACC1 (XM_501721) under the expression of the EXP1 promoter

[0222] (XM_501745) shown in the following order (letter-type): EXP1 PROMOTER, Y. LIPOLYTICA ACC1 ORF, cycl terminator, loxp site, hph (hygromycin B) resistance cassette, loxp site.

[0223] AAGGAGTTTGGCGCCCGTTTTTTCGAGCCCCACACGTTTCGGTGAGTATGAGCGGCGGCAGATTCGAGCGTTTCCGGTTTCC GCGGCTGGACGAGAGCCCATGATGGGGGCTCCCACCACCAGCAATCAGGGCCCTGATTACACACCCACCTGTAATGTCATG CTGTTCATCGTGGTTAATGCTGCTGTGTGCTGTGTGTGTGTGTTGTTTGGCGCTCATTGTTGCGTTATGCAGCGTACACCACA ATATTGGAAGCTTATTAGCCTTTCTATTTTTTCGTTTGCAAGGCTTAACAACATTGCTGTGGAGAGGGATGGGGATATGGAGGC CGCTGGAGGGAGTCGGAGAGGCGTTTTGGAGCGGCTTGGCCTGGCGCCCAGCTCGCGAAACGCACCTAGGACCCTTTGGC ACGCCGAAATGTGCCACTTTTCAGTCTAGTAACGCCTTACCTACGTCATTCCATGCATGCATGTTTGCGCCTTTTTTCCCTTGC

[0224] CCTTGATCGCCACACAGTACAGTGCACTGTACAGTGGAGGTTTTGGGGGGGTCTTAGATGGGAGCTAAAAGCGGCCTAGCG GTACACTAGTGGGATTGTATGGAGTGGCATGGAGCCTAGGTGGAGCCTGACAGGACGCACGACCGGCTAGCCCGTGACAGA CGATGGGTGGCTCCTGTTGTCCACCGCGTACAAATGTTTGGGCCAAAGTCTTGTCAGCCTTGCTTGCGAACCTAATTCCCAAT TTTGTCACTTCGCACCCCCATTGATCGAGCCCTAACCCCTGCCCATCAGGCAATCCAATTAAGCTCGCATTGTCTGCCTTGTTT AGTTTGGCTCCTGCCCGTTTCGGCGTCCACTTGCACAAACACAAACAAGCATTATATATAAGGCTCGTCTCTCCCTCCCAACC ACACTCACTTTTTTGCCCGTCTTCCCTTGCTAACACAAAAGTCAAGAACACAAACAACCACCCCAACCCCCTTACACACAAGAC

[0225] ATATCTACAGCAATGCGACTGCAATTGAGGACACTAACACGTCGGTTTTTCAGGTGAGTAAACGACGGTGGCCGTGGCCAC GACAGCCGAGGCGTCACGATGGGCCAGACGAGCACATTCTCGCCGCCACAACCTCGCCAGCACAAGAAACTAACCCAGT ATGGCTTCAGGATCTTCAACGCCAGATGTGGCTCCCTTGGTGGACCCCAACATTCACAAAGGTCTCGCCTCTCATTTCTTTG GACTCAATTCTGTCCACACAGCCAAGCCCTCAAAAGTCAAGGAGTTTGTGGCTTCTCACGGAGGTCATACAGTTATCAACA AGGTGAGTATTTGACGTTTAGACTGTATAACAGGCGGCCGCAGTGCAACAACGACCAAAAAGGGTCGAAAAAGGGTCGAA AACGGACACAAAAGCTGGAAAACAAGAGTGTAATACATTCTTACACGTCCAATTGTTAGACAAACACGGCTGTTCGGTCCC

[0226] AAAACCACCAGTATCACCTATTTTCCACTTGTGTCTCGGATCTGATCATAATCTGATCTCAAGATGAAATTTACGCCACCGA CATGATATTGTGATTTTCGGATTCTCCAGACCGAGCAGATTCCAGCAATACCACCACTTGCCCACCTTCAGCGGCCTCTCG GCGCGATTCGCCACTTTCCCCAACGAGTGTTACTAACCCAGGTCCTCATCGCTAACAACGGTATTGCCGCAGTAAAGGAGA TCCGTTCAGTACGAAAATGGGCCTACGAGACCTTTGGCGACGAGCGAGCAATCTCGTTCACCGTCATGGCCACCCCCGAA GATCTCGCTGCCAACGCCGACTACATTAGAATGGCCGATCAGTACGTCGAGGTGCCCGGAGGAACCAACAACAACAACTA CGCCAACGTCGAGCTGATTGTCGACGTGGCTGAGCGATTCGGCGTCGATGCCGTGTGGGCCGGATGGGGCCATGCCAGT

[0227] GAAAATCCCCTGCTCCCCGAGTCGCTAGCGGCCTCTCCCCGCAAGATTGTCTTCATCGGCCCTCCCGGAGCTGCCATGAG ATCTCTGGGAGACAAAATTTCTTCTACCATTGTGGCCCAGCACGCAAAGGTCCCGTGTATCCCGTGGTCTGGAACCGGAGT

[0228] GGACGAGGTTGTGGTTGACAAGAGCACCAACCTCGTGTCCGTGTCCGAGGAGGTGTACACCAAGGGCTGCACCACCGGTC CCAAGCAGGGTCTGGAGAAGGCTAAGCAGATTGGATTCCCCGTGATGATCAAGGCTTCCGAGGGAGGAGGAGGAAAGGG TATTCGAAAGGTTGAGCGAGAGGAGGACTTCGAGGCTGCTTACCACCAGGTCGAGGGAGAGATCCCCGGCTCGCCCATCT TCATTATGCAGCTTGCAGGCAATGCCCGGCATTTGGAGGTGCAGCTTCTGGCTGATCAGTACGGCAACAATATTTCACTGTT TGGTCGAGATTGTTCGGTTCAGCGACGGCATCAAAAGATTATTGAGGAGGCTCCTGTGACTGTGGCTGGCCAGCAGACCTT CACTGCCATGGAGAAGGCTGCCGTGCGACTCGGTAAGCTTGTCGGATATGTCTCTGCAGGTACCGTTGAATATCTGTATTC

[0229] CCATGAGGACGACAAGTTCTACTTCTTGGAGCTGAATCCTCGTCTTCAGGTCGAACATCCTACCACCGAGATGGTCACCGG TGTCAACCTGCCCGCTGCCCAGCTTCAGATCGCCATGGGTATCCCCCTCGATCGAATCAAGGACATTCGTCTCTTTTACGGT GTTAACCCTCACACCACCACTCCAATTGATTTCGACTTCTCGGGCGAGGATGCTGATAAGACACAGCGACGTCCCGTCCCC CGAGGTCACACCACTGCTTGCCGAATCACATCCGAGGACCCTGGAGAGGGTTTCAAGCCCTCCGGAGGTACTATGCACGA GCTCAACTTCCGATCCTCGTCCAACGTGTGGGGTTACTTCTCCGTTGGTAACCAGGGAGGTATCCATTCGTTCTCGGATTCG CAGTTTGGTCACATCTTCGCCTTCGGTGAGAACCGAAGTGCGTCTCGAAAGCACATGGTTGTTGCTTTGAAGGAACTATCTA

[0230] TTCGAGGTGACTTCCGAACCACCGTCGAGTACCTCATCAAGCTGCTGGAGACACCGGACTTCGAGGACAACACCATCACC ACCGGCTGGCTGGATGAGCTTATCTCCAACAAGCTGACTGCCGAGCGACCCGACTCGTTCCTCGCTGTTGTTTGTGGTGCT GCTACCAAGGCCCATCGAGCTTCCGAGGACTCTATTGCCACCTACATGGCTTCGCTAGAGAAGGGCCAGGTCCCTGCTCG AGACATTCTCAAGACCCTTTTCCCCGTTGACTTCATCTACGAGGGCCAGCGGTACAAGTTCACCGCCACCCGGTCGTCTGA GGACTCTTACACGCTGTTCATCAACGGTTCTCGATGCGACATTGGAGTTAGACCTCTTTCTGACGGTGGTATTCTGTGTCTT GTAGGTGGGAGATCCCACAATGTCTACTGGAAGGAGGAGGTTGGAGCCACGCGACTGTCTGTTGACTCCAAGACCTGCCT

[0231] TCTCGAGGTGGAGAACGACCCCACTCAGCTTCGATCTCCCTCTCCCGGTAAGCTGGTTAAGTTCCTGGTCGAGAACGGCGA CCACGTGCGAGCCAACCAGCCCTATGCCGAGATTGAGGTCATGAAGATGTACATGACTCTCACTGCTCAGGAGGACGGTA TTGTCCAGCTGATGAAGCAGCCCGGTTCCACCATCGAGGCTGGCGACATCCTCGGTATCTTGGCCCTTGATGATCCTTCCA AGGTCAAGCATGCCAAGCCCTTTGAGGGCCAGCTTCCCGAGCTTGGACCCCCCACTCTCAGCGGTAACAAGCCTCATCAG CGATACGAGCACTGCCAGAACGTGCTCCATAACATTCTGCTTGGTTTCGATAACCAGGTGGTGATGAAGTCCACTCTTCAG GAGATGGTTGGTCTGCTCCGAAACCCTGAGCTTCCTTATCTCCAGTGGGCTCATCAGGTGTCTTCTCTGCACACCCGAATG

[0232] AGCGCCAAGCTGGATGCTACTCTTGCTGGTCTCATTGACAAGGCCAAGCAGCGAGGTGGCGAGTTTCCTGCCAAGCAGCT TCTGCGAGCCCTTGAGAAGGAGGCGAGCTCTGGCGAGGTCGATGCGCTCTTCCAGCAAACTCTTGCTCCTCTGTTTGACCT KeVer / SLIM_5gene / 850

[0233] TGCTCGAGAGTACCAGGACGGTCTTGCTATCCACGAGCTTCAGGTTGCTGCAGGCCTTCTGCAGGCCTACTACGACTCTGA GGCCCGGTTCTGCGGACCCAACGTACGTGACGAGGATGTCATTCTCAAGCTTCGAGAGGAGAACCGAGATTCTCTTCGAA AGGTTGTGATGGCCCAGCTGTCTCATTCTCGAGTCGGAGCCAAGAACAACCTTGTGCTGGCCCTTCTCGATGAATACAAGG TGGCCGACCAGGCTGGCACCGACTCTCCTGCCTCCAACGTGCACGTTGCAAAGTACTTGCGACCTGTGCTGCGAAAGATT GTGGAGCTGGAATCTCGAGCTTCTGCCAAGGTATCTCTGAAAGCCCGAGAGATTCTCATCCAGTGCGCTCTGCCCTCTCTA AAGGAGCGAACTGACCAGCTTGAGCACATTCTGCGATCTTCTGTCGTCGAGTCTCGATACGGAGAGGTTGGTCTGGAGCA CCGAACTCCCCGAGCCGATATTCTCAAGGAGGTTGTCGACTCCAAGTACATTGTCTTTGATGTGCTTGCCCAGTTCTTTGCC CACGATGATCCCTGGATCGTCCTTGCTGCCCTGGAGCTGTACATCCGACGAGCTTGCAAGGCCTACTCCATCCTGGACATC AACTACCACCAGGACTCGGACCTGCCTCCCGTCATCTCGTGGCGATTTAGACTGCCTACCATGTCGTCTGCTTTGTACAACT CAGTAGTGTCTTCTGGCTCCAAAACCCCCACTTCCCCCTCGGTGTCTCGAGCTGATTCCGTCTCCGACTTTTCGTACACCGT TGAGCGAGACTCTGCTCCCGCTCGAACCGGAGCGATTGTTGCCGTGCCTCATCTGGATGATCTGGAGGATGCTCTGACTCG TGTTCTGGAGAACCTGCCCAAACGGGGCGCTGGTCTTGCCATCTCTGTTGGTGCTAGCAACAAGAGTGCCGCTGCTTCTGC TCGTGACGCTGCTGCTGCTGCCGCTTCATCCGTTGACACTGGCCTGTCCAACATTTGCAACGTTATGATTGGTCGGGTTGAT GAGTCTGATGACGACGACACTCTGATTGCCCGAATCTCCCAGGTCATTGAGGACTTTAAGGAGGACTTTGAGGCCTGTTCT CTGCGACGAATCACCTTCTCCTTCGGCAACTCCCGAGGTACTTATCCCAAGTATTTCACGTTCCGAGGCCCCGCATACGAG GAGGACCCCACTATCCGACACATTGAGCCTGCTCTGGCCTTCCAGCTGGAGCTCGCCCGTCTGTCCAACTTCGACATCAAG CCTGTCCACACCGACAACCGAAACATCCACGTGTACGAGGCTACTGGCAAGAACGCTGCTTCCGACAAGCGGTTCTTCAC CCGAGGTATCGTACGACCTGGTCGTCTTCGAGAGAACATCCCCACCTCGGAGTATCTCATTTCCGAGGCTGACCGGCTCAT GAGCGATATTTTGGACGCTCTAGAGGTGATTGGAACCACCAACTCGGATCTCAACCACATTTTCATCAACTTCTCAGCCGTC TTTGCTCTGAAGCCCGAGGAGGTTGAAGCTGCCTTTGGCGGTTTCCTGGAGCGATTTGGCCGACGTCTGTGGCGACTTCGA GTCACCGGTGCCGAGATCCGAATGATGGTATCCGACCCCGAAACTGGCTCTGCTTTCCCTCTGCGAGCAATGATCAACAAC GTCTCTGGTTACGTTGTGCAGTCTGAGCTGTACGCTGAGGCCAAGAACGACAAGGGCCAGTGGATTTTCAAGTCTCTGGGC AAGCCCGGCTCCATGCACATGCGGTCTATCAACACTCCCTACCCCACCAAGGAGTGGCTGCAGCCCAAGCGGTACAAGGC CCATCTGATGGGTACCACCTACTGCTATGACTTCCCCGAGCTGTTCCGACAGTCCATTGAGTCGGACTGGAAGAAGTATGA CGGCAAGGCTCCCGACGATCTCATGACTTGCAACGAGCTGATTCTCGATGAGGACTCTGGCGAGCTGCAGGAGGTGAACC GAGAGCCCGGCGCCAACAACGTCGGTATGGTTGCGTGGAAGTTTGAGGCCAAGACCCCCGAGTACCCTCGAGGCCGATC TTTCATCGTGGTGGCCAACGATATCACCTTCCAGATTGGTTCGTTTGGCCCTGCTGAGGACCAGTTCTTCTTCAAGGTGACG GAGCTGGCTCGAAAGCTCGGTATTCCTCGAATCTATCTGTCTGCCAACTCTGGTGCTCGAATCGGCATTGCTGACGAGCTC GTTGGCAAGTACAAGGTTGCGTGGAACGACGAGACTGACCCCTCCAAGGGCTTCAAGTACCTTTACTTCACCCCTGAGTCT CTTGCCACCCTCAAGCCCGACACTGTTGTCACCACTGAGATTGAGGAGGAGGGTCCCAACGGCGTGGAGAAGCGTCATGT GATCGACTACATTGTCGGAGAGAAGGACGGTCTCGGAGTCGAGTGTCTGCGGGGCTCTGGTCTCATTGCAGGCGCCACTT CTCGAGCCTACAAGGATATCTTCACTCTCACTCTTGTCACCTGTCGATCCGTTGGTATCGGTGCTTACCTTGTTCGTCTTGGT CAACGAGCCATCCAGATTGAGGGCCAGCCCATCATTCTCACTGGTGCCCCCGCCATCAACAAGCTGCTTGGTCGAGAGGT CTACTCTTCCAACTTGCAGCTTGGTGGTACTCAGATCATGTACAACAACGGTGTGTCTCATCTGACTGCCCGAGATGATCTC AACGGTGTCCACAAGATCATGCAGTGGCTGTCATACATCCCTGCTTCTCGAGGTCTTCCAGTGCCTGTTCTCCCTCACAAGA CCGATGTGTGGGATCGAGACGTGACGTTCCAGCCTGTCCGAGGCGAGCAGTACGATGTTAGATGGCTTATTTCTGGCCGA ACTCTCGAGGATGGTGCTTTCGAGTCTGGTCTCTTTGACAAGGACTCTTTCCAGGAGACTCTGTCTGGCTGGGCCAAGGGT GTTGTTGTTGGTCGAGCTCGTCTTGGCGGCATTCCCTTCGGTGTCATTGGTGTCGAGACTGCGACCGTCGACAATACTACC CCTGCCGATCCCGCCAACCCGGACTCTATTGAGATGAGCACCTCTGAAGCCGGCCAGGTTTGGTACCCCAACTCGGCCTT CAAGACCTCTCAGGCCATCAACGACTTCAACCATGGTGAGGCGCTTCCTCTCATGATTCTTGCTAACTGGCGAGGCTTTTCT GGTGGTCAGCGAGACATGTACAATGAGGTTCTCAAGTACGGATCTTTCATTGTTGATGCTCTGGTTGACTACAAGCAGCCC ATCATGGTGTACATCCCTCCCACCGGTGAGCTGCGAGGTGGTTCTTGGGTTGTGGTTGACCCCACCATCAACTCGGACATG ATGGAGATGTACGCTGACGTCGAGTCTCGAGGTGGTGTGCTGGAGCCCGAGGGAATGGTCGGTATCAAGTACCGACGAGA CAAGCTACTGGACACCATGGCTCGTCTGGATCCCGAGTACTCCTCTCTCAAGAAGCAGCTTGAGGAGTCTCCCGATTCTGA GGAGCTCAAGGTCAAGCTCAGCGTGCGAGAGAAGTCTCTCATGCCCATCTACCAGCAGATCTCCGTGCAGTTTGCCGACTT GCATGACCGAGCTGGCCGAATGGAGGCCAAGGGTGTCATTCGTGAGGCTCTTGTGTGGAAGGATGCTCGTCGATTCTTCTT CTGGCGAATCCGACGACGATTAGTCGAGGAGTACCTCATTACCAAGATCAATAGCATTCTGCCCTCTTGCACTCGGCTTGA GTGTCTGGCTCGAATCAAGTCGTGGAAGCCTGCCACTCTTGATCAGGGCTCTGACCGGGGTGTTGCCGAGTGGTTTGACGA GAACTCTGATGCCGTCTCTGCTCGACTCAGCGAGCTCAAGAAGGACGCTTCTGCCCAGTCGTTTGCTTCTCAACTGAGAAA GGACCGACAGGGTACTCTCCAGGGCATGAAGCAGGCTCTCGCTTCTCTTTCTGAGGCTGAGCGGGCTGAGCTGCTCAAGG GGJJGJGAtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatag ttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttg cataacttcgtatagcctaggctatacgaagttataaggagtttggcgcccgttttttcgagccccacacgtttcggtgagtatgagcggcggcagattcgagcgtttccggtttccgcgg ctggacgagagcccatgatgggggctcccaccaccagcaatcagggccctgattacacacccacctgtaatgtcatgctgttcatcgtggttaatgctgctgtgtgctgtgtgtgtgtgttgttt ggcgctcattgttgcgttatgcagcgtacaccacaatattggaagcttattagcctttctattttttcgtttgcaaggcttaacaacattgctgtggagagggatggggatatggaggccgctgg agggagtcggagaggcgttttggagcggcttggcctggcgcccagctcgcgaaacgcacctaggaccctttggcacgccgaaatgtgccacttttcagtctagtaacgccttacctacgt cattccatgcatgcatgtttgcgccttttttcccttgcccttgatcgccacacagtacagtgcactgtacagtggaggttttgggggggtcttagatgggagctaaaagcggcctagcggtaca ctagtgggattgtatggagtggcatggagcctaggtggagcctgacaggacgcacgaccggctagcccgtgacagacgatgggtggctcctgttgtccaccgcgtacaaatgtttgggc caaagtcttgtcagccttgcttgcgaacctaattcccaattttgtcacttcgcacccccattgatcgagccctaacccctgcccatcaggcaatccaattaagctcgcattgtctgccttgtttagt ttggctcctgcccgtttcggcgtccacttgcacaaacacaaacaagcattatatataaggctcgtctctccctcccaaccacactcacttttttgcccgtcttcccttgctaacacaaaagtcaa gaacacaaacaaccaccccaacccccttacacacaagacatatctacagcaatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacag cgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgtt atgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagac ctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggcaatggatgccattgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcg gtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatg KeVer / SLIM_5gene / 850 ctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgt tcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcgg ctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccg ggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaa ggaataatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatg ttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgataacttcgtatagcc taggctatacgaagttat

[0234] Example 7. A preferred embodiment (nucleotide sequences) of a chimeric construct used for overexpression of M. circinelloides MCE2 (DQ975377) under the control of the GPD promoter (XM_501515), shown in the following order (letter-type): GPD PROMOTER, M. CIRCINELLOIDES MCE2 ORF, tefl terminator, loxp site, hph (hygromycin B) resistance cassette, loxp site.

[0235] GTTGATGTGTGTTTAATTCAAGAATGAATATAGAGAAGAGAAGAAGAAAAAAGATTCAATTGAGCCGGCGATGCAGACCCTTAT ATAAATGTTGCCTTGGACAGACGGAGCAAGCCCGCCCAAACCTACGTTCGGTATAATATGTTAAGCTTTTTAACACAAAGGTTT GGCTTGGGGTAACCTGATGTGGTGCAAAAGACCGGGCGTTGGCGAGCCATTGCGCGGGCGAATGGGGCCGTGACTCGTCT CAAA TTCGAGGGCGTGCCTCAATTCGTGCCCCCGTGGCTTTTTCCCGCCGTTTCCGCCCCGTTTGCACCACTGCAGCCGCTT CTTTGGTTCGGACACCTTGCTGCGAGCTAGGTGCCTTGTGCTACTTAAAAAGTGGCCTCCCAACACCAACATGACATGAGTGC GTGGGCCAAGACACGTTGGCGGGGTCGCAGTCGGCTCAATGGCCCGGAAAAAACGCTGCTGGAGCTGGTTCGGACGCAGT CCGCCGCGGCGTATCGATATCCGCAAGGTTCCATGGCGCCATTGCCCTCCGTCGGCGTCTATCCCGCAACCTCTAAATAGAG CGGGAATATAACCCAAGCTTCTTTTTTTTTCCTTTAACACGCACACCCCCAACTATCATGTTGCTGCTGCTGTTTGACTCTACTC TGTGGAGGGGTGCTCCCACCCAACCCAACCTACAGGTGGATCCGGCGCTGTGATTGGCTGATAAGTCTCCTATCCGGACTAA TTCTGACCAATGGGACATGCGCGCAGGACCCAAATGCCGCAATTACGTAACCCCAACGAAATGCCTACCCCTCTTTGGAGCC CAGCGGCCCCAAATCCCCCCAAGCAGCCCGGTTCTACCGGCTTCCATCTCCAAGCACCCCTTTCTCCACACCCCACAAAAAG ACCCGTGCAGGACATCCTACTGCGTCATGTCGCCTATTATTGATTTTGTTCGTCGCCAATTGTCCTCTACAAAGTTGCATGAA GAGCAGCAAACAGCAACTACAAATGATTTGGTCTCTAGATCAGGCTATCTAAATGAAGGCAAGTATGAGGTCCGCTTGAAT TGTATCAATGCTGGCTGCTTACAAAAAAAACTAAACTATATAGGTACTGCCATGGATCCTGCTAAACGTCAAAGACTTGGAT TGAACGGTCTTTTACCTGCTGGTGTAGAGACATTGGAAATTCAAAAAGCTCGCGCCCTCAGAGTGCTTCGTTCAAAACACA ATTTATTAGAAAAATACATTTTAATGGCTCAACTTCGTACCACCAACGTCCGCTTATTTTACAAGATTGTCATTGATGAATTA GAGACCGTTCAATTGGCTCCTGTTATCTATACCCCGACTGTTGGTACCGCATGCTTGGAATACTCTACCATCTATCCCTTCTT GGCTGCCCCTGGTGTGCCGGATGGTCTTTACCTCACCAAAGCCGAATTACCGGAACTGTGTCAAACCATTCGTAACTATCG TCCTACGGATACTGAGGGTTTTGAGCCAGAGATTGCTGTGATTTCTGATGGGTCTCGAATTTTGGGTCTGGGTGATTTGGGA ACAAATGGCATGGGTATTCCAATGGGTAAACTTCAGCTCTATGTTGCTGGTGCTGGTATTGATCCTCGTCGTACGTTACCCA TCATTTTGGATTTGGGTACAAACAATGAAAAGTTGCTCAATGATGAGTTTTATATTGGTCTTCGTCAAAAGCGACCCAATGA TGAGGAGTTTTATCAAACAGTTGATACAGTCTTGACAGCATTACATACCGTGTACCCCAACCTACTCATCCAGTTTGAAGAT TGGTCTTCTGAACACGCATTTGGCCTCTTGGAAAAGTACCAAAATCAAATGCTTTGTTTTAACGACGACATACAGGGCACAG GTGCTGTCATATTATCTGGTGTCATTAATGCTATTCGCAAGGTTGAGAAAGAGAATCAAGTGTCTCCTCGTGATCATCGTAT CGTGTTCTACGGTGCTGGTTCTGCTGCTATCGGTGTTGCTCGTCAAATTCAAAGCTACTTCCAAATTGAACACAACATGACT GAGGAAGAAGCTAAGCATGTGTTCTGGATTGTTGATTCCAAGGGTCTTGTTACTACTACACGAGGCGATAAATTAGCTCAA CACAAGGTGTATTACGCACGAGGCGATAATGAAGGCCAACAGTACAAGGAATTGATTGATATTGTCAACTATAATCTCTAC AGTTTGATTGGTTTATCATCTACTACAGGTGCCTTTAATACTCAAGTCCTTGAGCGTCTTGCCTCACTCAATGAGCAACCTAT TGTTTTCCCTCTTTCCAATCCAGCCACACAAGCAGAATGTACATTTGAGCAAGCCATGGAAGCTACCAACAACAAGGTTATT TTTGCATCTGGTACTGCTTTCCCTGCATATACCATCAAATCCACTGGCGAAGTAAATACCCCTGGTCAAGGCAACAACATGT ACATCTTCCCTGGTTTGGGTCTGGGTGCTTGTCTGGCTAACCCAGCACATTTCGATCGCATGATCTACGAAGCATCCAAAGC ACTTGCTGACTCACTTACAGAGGAAGAAATCAGTAAGGCCTGGTTATATCCATCTTTAAACTATCGTAGCGTATCAGCCATC GTTGCAGCAGCTGTATGTCAAGAGACTTTGAATGAAAACCTAGCAACGTCTCAAGCTATGATGACGCAGTGTAAATCACAT GAAGATATTCTAGATTATGTTAGTGCTCATATGTGGTCTCCCGACTATGGAAACAACAACAGCAATCAGCAAGCTGGTAAAT TGtAccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacc tcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaa ctgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttatt ttccatacatatgcaagtgagatgcccgtgtccgaattcataacttcgtatagcgtacgctatacgaagttataaggagtttggcgcccgttttttcgagccccacacgtttcggtgagtat gagcggcggcagattcgagcgtttccggtttccgcggctggacgagagcccatgatgggggctcccaccaccagcaatcagggccctgattacacacccacctgtaatgtcatgctgtt catcgtggttaatgctgctgtgtgctgtgtgtgtgtgttgtttggcgctcattgttgcgttatgcagcgtacaccacaatattggaagcttattagcctttctattttttcgtttgcaaggcttaacaacat tgctgtggagagggatggggatatggaggccgctggagggagtcggagaggcgttttggagcggcttggcctggcgcccagctcgcgaaacgcacctaggaccctttggcacgccg aaatgtgccacttttcagtctagtaacgccttacctacgtcattccatgcatgcatgtttgcgccttttttcccttgcccttgatcgccacacagtacagtgcactgtacagtggaggttttggggg ggtcttagatgggagctaaaagcggcctagcggtacactagtgggattgtatggagtggcatggagcctaggtggagcctgacaggacgcacgaccggctagcccgtgacagacga tgggtggctcctgttgtccaccgcgtacaaatgtttgggccaaagtcttgtcagccttgcttgcgaacctaattcccaattttgtcacttcgcacccccattgatcgagccctaacccctgcccat caggcaatccaattaagctcgcattgtctgccttgtttagtttggctcctgcccgtttcggcgtccacttgcacaaacacaaacaagcattatatataaggctcgtctctccctcccaaccacac tcacttttttgcccgtcttcccttgctaacacaaaagtcaagaacacaaacaaccaccccaacccccttacacacaagacatatctacagcaatgggtaaaaagcctgaactcaccgcg acgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcg ggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattg catctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggcaatggatgccattgctgcggccgatcttagccaga cgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgt KeVer / SLIM_5gene / 850 cagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggcc gcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcg agcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgca gggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtg gaaaccgacgccccagcactcgtccgagggcaaaggaataatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttaga caacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgctt gagaaggttttgggacgctcgataacttcgtatagcgtacgctatacgaagttatcgtaatagcctccaagagattgatcatcactcataacttcgtatagcgtacgctatacgaagttat

[0236] Example 8. A preferred embodiment (nucleotide sequences) of a chimeric construct used for the replacement of the CEX1 (YALI0D20196g) by two overexpression constructs of the Mus Musculus ACL1 (NM_001199296) and ACL2 (NM_134037) genes under the control of the TEF1 promoter (XM_501628):

[0237] HOMOLOGY REGION UPSTREAM OF THE CEX1 GENE, TEF1 PROMOTER, M. MUSCULUS ACL2 GENE, tefl terminator, TEF1 PROMOTER, M. MUSCULUS ACL1 GENE, tefl terminator, loxp site, hph (hygromycin B) resistance cassette, loxp site, HOMOLOGY REGION DOWNSTREAM OF THE CEX1 GENE

[0238] CAAGTGGCTGGATGTCTCAAACAAGGCACTATGATGTACTCGTAGATGGTACAGGTGGGTGAGACCTGAAACCGTTGAAG GGTCTAA CTTA TCCAA GAA GCA TA TGAA CGAAA GCGTCTCCAAA TA CA CCCA GCAAA CA GAAA CTCCCA CA GA CA CGGA C GCCCA CA GCGGTGCA GA CA CA CGTA CGGCAGGTA CCA GGGCCGA CTGCCCCTTCCTTA TCA GAA CGCCAA GCCCGGA GG GCGGAAACGTGCTCACGATAAGTCCAAGAGACGACAGTGTGACTACAGGTCTATCGTGCCAAGACAACCCTATCCATCCG GCTTGCGAAACGGTGGGCCGGATTAAATTGGAGGTTTATTTTCGGTCAGCCCTGTGCGGCATATTGTGGCTTGGGCAACTG GA CGGTTGAAA CCTGGGTGTA CA GA CCTGCCCGAAA TA TGAA TCGAA TCA GCA GA GCGA GA CGAGTA CA GCGGCGAA GT CCCCACGGCTGTATCTCCCCTTTTTTGACCTCCGGTCGCCTTCCCAAATAAACTCTGGCAGTGCAGTCTGGGTGCTACACTA TCGCA GCGTGTGCAAAA CA CA TGGCGGA CTGTTCA GGCGGTGCTGTCTGA CGGAA TAAAA TTCAAA GCA GA TAAA TCGA G GGGCAGAGTCGTGGTGAGGAAGTCCGCCAAAGTCGGATTACATTTGTGACATCTGAAGGATGTAAACTGGCGCTTACAAA TGGCTGAATGTTAGCGTCCCTTAAACTCTGGCAGTGTCAAAAGGGTTCAGACGCCGAAAGCCCAAAGACACTTGGGCCTTC CCGGAGCGATAGCACTGCCCCCTTTGTCTGTGTGGAAGACCCGGTGAATGTCGAGTACCTGCTTATGCAGCGACACATGTT TCGCTGGTGGTAATGGCGGCAAAGTTGGGTGTTTGACACTGCGACCCGAAATCTCGTGCCAGATTGTACTGAGACGTATAT CGAGGTGTGCGTTCGAAAATCACTCTATAGCACCGTGGACGGCGCAAACCATCCTTGTCGTGCGACCGGTACCGCCCACT CAACCCACAGATGGTCGGATTGTCAGATTGGCCAGGTACTGCGTTCCTGACCAGCAGCGTTTAAAATAGTGTTTGGACACT CAGGGTGGGCAGCCAGATAGGTTATCAGTGTGCCTCGAAGTCTCTCTAAAATGTGCGGCTTGAGGTCTGGGCACGTCTACT GTGCGAGGGTCTATGTCAGTGTGAAAAAAAGACGCCAAACAAGCGTTTAGGTCTGTGTGGTGGGGGAGGGGGAGATGAGT AGTGGGGCTGTGTGTGGA TGGGGTGGA TA TA TGGA TA TA TCGGCA TGTGTCGCTTTGTGTCTTCCGGGGGTTAGGCTGTTG AATTACATTGCGTTGTGCCTGTCACATCGGCTTTTTTGTTTCTTACTCTCTCTCTCTCTCTCTCTCTCTTTTTTTTTTTTTGCCCT TGGA GCCA TCCTCA CGTTTTGCCA TTTTCAA CTTTTGTCA TTTTCCA CTTTTGCCTTTTTCCAA CTTTTCA TTTCCA CCA TCAA G TCGA CTTTGGTTAA GCTCCGTTA TGGCTTCTAA CCTCAA CCA CGCTTCCA CCA CCA TA TCTCTGCA CAA GGTCGGGTGCA GA AGCCCTGCATGGTGGGCAGAATGATAGTTCTGAGGAGCCGCGAGTCGTCAGCAAATCACAGAGACCGGGTTGGCGGCGCA

[0239] TTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCC CTTCTCCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTG TTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCT TTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAA CTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAAAATGGTGAGTTTCAGAGGCAGCAGCAATTGCCA CGGGCTTTGAGCACACGGCCGGGTGTGGTCCCATTCCCATCGACACAAGACGCCACGTCATCCGACCAGCACTTTTTGCAGT / IC77WCCGC / IGAGTGCGAAGGCTATAAGCGAACAGACTGGAAAAGAACTGTTATACAAATATATATGCACCACATCAGCTA TTCAAAATCGGTTCAAGTACGCTAGAGTAACCCCCGATACCGACTGGGCTCACTTGCTGCAAGATCACCCTTGGTTACTGA GTCAGAGCCTGGTCGTGAAGCCTGACCAACTTATCAAAAGACGAGGGAAACTTGGCTTAGTAGGTGTGAACCTGAGTTTG GACGGAGTCAAGAGTTGGTTGAAACCGCGTCTTGGCCATGAGGCCACCGTAGGTAAAGCCAAAGGGTTCTTGAAGAACTT CCTGATTGAACCCTTCGTCCCCCACTCTCAAGCAGAGGAGTTCTATGTGTGCATCTATGCGACGCGGGAGGGGGATTACGT ATTATTTCACCATGAAGGCGGCGTGGATGTTGGCGACGTAGACGCGAAAGCTCAAAAGTTACTTGTAGGTGTGGATGAAAA GCTGAACACCGAGGATATTAAGCGGCACCTGCTTGTCCATGCACCAGAAGATAAAAAGGAGGTGCTGGCTTCTTTTATAAG TGGATTGTTTAACTTTTACGAAGATCTTTATTTTACCTACCTGGAAATTAATCCATTGGTGGTAACAAAGGACGGTGTGTACA TCCTGGATCTGGCCGCAAAAGTAGACGCAACTGCGGACTACATTTGTAAAGTTAAATGGGGCGATATAGAGTTCCCTCCGC CGTTTGGTCGCGAAGCATATCCCGAAGAAGCGTACATCGCCGATTTAGATGCTAAATCTGGGGCGTCATTAAAGCTGACTT TATTAAATCCTAAAGGACGCATATGGACGATGGTAGCTGGGGGAGGAGCAAGTGTTGTCTACAGTGACACTATATGCGACT TGGGGGGGGTCAACGAATTAGCGAACTATGGAGAGTACAGTGGAGCACCTAGTGAACAACAAACGTACGACTATGCAAAG ACAATTTTATCACTTATGACCAGAGAAAAGCATCCTGAGGGAAAGATTCTTATAATCGGTGGGTCTATTGCCAATTTCACAA ACGTAGCAGCGACCTTTAAGGGAATCGTTCGGGCGATACGAGATTACCAGGGCCCGTTAAAAGAACATGAAGTAACAATC TTCGTCCGACGTGGCGGTCCAAATTATCAGGAAGGATTGCGAGTTATGGGTGAAGTAGGGAAAACTACTGGGATACCCATT CATGTCTTTGGGACGGAGACACACATGACAGCTATTGTCGGAATGGCTCTGGGGCATAGACCTATTCCTAATCAGCCCCCC ACCGCAGCCCATACCGCAAACTTCTTGCTTAACGCCAGCGGAAGTACGAGTACGCCGGCCCCCTCTCGTACTGCGTCATTT TCAGAAAGTCGAGCAGACGAGGTCGCTCCCGCTAAAAAAGCGAAGCCCGCCATGCCGCAGGGAAAAAGCGCAACTTTGT

[0240] TCTCTCGCCACACGAAAGCAATCGTGTGGGGGATGCAAACTCGAGCCGTACAAGGAATGCTTGACTTTGACTACGTGTGCT KeVer / SLIM_5gene / 850

[0241] CACGCGATGAGCCTAGCGTTGCTGCAATGGTGTACCCTTTCACGGGGGATCATAAGCAGAAATTTTATTGGGGCCACAAGG AGATTCTTATCCCAGTCTTCAAGAATATGGCGGATGCAATGAAAAAACACCCTGAAGTTGATGTACTGATAAACTTCGCATC ATTGCGATCAGCATACGACTCAACGATGGAGACTATGAACTACGCACAAATCCGTACCATAGCGATAATCGCGGAAGGCA TTCCCGAAGCCTTGACCCGTAAACTGATCAAAAAAGCTGACCAGAAAGGGGTTACTATTATCGGGCCAGCAACTGTCGGA GGGATAAAGCCGGGGTGTTTCAAGATAGGAAATACTGGTGGTATGTTGGATAATATTCTTGCCAGCAAGCTGTATAGACCA GGATCAGTCGCATATGTAAGTCGGTCAGGCGGTATGTCTAACGAACTTAATAATATCATTTCTCGTACCACGGATGGTGTAT ACGAAGGTGTAGCGATTGGCGGGGATCGATATCCGGGGTCTACATTCATGGATCATGTTCTGCGCTATCAAGACACGCCG GGTGTAAAAATGATTGTAGTATTGGGAGAAATCGGTGGGACTGAAGAGTACAAAATTTGTCGCGGCATTAAGGAGGGACG TCTTACTAAACCCGTTGTTTGCTGGTGCATCGGTACCTGCGCAACTATGTTTTCAAGTGAGGTCCAATTTGGGCATGCAGGA GCTTGCGCAAACCAAGCTTCAGAAACTGCGGTCGCCAAAAACCAAGCCTTAAAGGAAGCAGGCGTATTCGTCCCACGCAG TTTTGACGAATTAGGAGAAATTATCCAGTCTGTTTACGAGGACTTAGTTGCGAAGGGTGCTATTGTCCCTGCGCAGGAAGTT CCGCCACCCACCGTGCCAATGGATTATAGCTGGGCGCGTGAGTTAGGCTTAATTCGTAAACCTGCGTCTTTCATGACCAGC ATATGCGATGAACGGGGGCAAGAGTTAATATACGCGGGGATGCCTATTACAGAAGTATTTAAAGAGGAAATGGGGATAGG AGGCGTGCTGGGTCTGTTATGGTTTCAACGCCGTCTTCCAAAATACTCTTGCCAGTTCATCGAAATGTGCCTGATGGTAACG GCGGACCACGGTCCGGCGGTATCAGGCGCTCACAACACAATAATCTGTGCGCGGGCCGGAAAAGATCTGGTCTCAAGTTT AACAAGCGGCCTTTTGACCATTGGAGATAGATTTGGGGGTGCCTTAGACGCAGCGGCAAAAATGTTCAGTAAAGCATTTGA

[0242] TTCTGGGATAATACCTATGGAGTTTGTGAACAAAATGAAGAAAGAAGGCAAATTGATAATGGGGATAGGACATCGAGTAAA ATCAATAAATAATCCGGATATGCGCGTTCAAATATTGAAAGATTTTGTAAAGCAACACTTCCCCGCAACTCCCTTGTTGGAC TACGCCCTTGAGGTTGAGAAAATAACTACTTCAAAGAAGCCCAACCTTATATTGAACGTCGACGGCTTCATTGGAGTTGCG TTTGTTGATATGTTGAGAAACTGTGGTAGTTTCACACGAGAAGAGGCGGACGAGTACGTAGATATAGGGGCATTAAACGGT ATATTTGTCTTGGGGCGATCTATGGGATTCATAGGTCATTACTTAGACCAGAAACGACTTAAGCAAGGGCTGTATCGGCATC CCTGGGATGACATTAGTTACGTTTTACCTGAGCATATGAGTATGTAAacgcaacfaacafgaafgaafacgafafacafcaaagacfafgafacgca gtattgcacactgtacgagtaagggcactagccactgcactcaagtgaaaccgttgcccgggtacgagtatgagtatgtacagtatgtttagtattgtacttggacaatgcttgtatcgtacat tctcaagtgtcaaacataaatatccgttgctatatcctcgcaccaccacgtagctcgctatatccctgtgttgaatccatccatcttggattgccaattgtgcacacagaaccgggcactcactt ccccafccacactAGAGACCGGGTTGGCGGCGCATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCC AGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCTCCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGG CGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCG GACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTA CCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAAA ATGGTGAGTTTCAGAGGCAGCAGCAATTGCCACGGGCTTTGAGCACACGGCCGGGTGTGGTCCCATTCCCATCGACACAAG ACGCCACGTCATCCGACCAGCACTTTTTGCAGTACTAACCGCAGTCAGCCAAGGCAATTTCAGAGCAGACCGGCAAAGAAC TCCTGTACAAGTACATCTGCACCACCTCGGCCATCCAGAACCGGTTCAAGTACGCCCGGGTGACTCCCGACACAGACTGG

[0243] GCCCATCTGCTGCAGGACCACCCGTGGCTGCTCAGCCAGAGCTTGGTTGTCAAGCCAGACCAGTTAATCAAACGTCGAGG AAAGCTTGGCCTCGTCGGGGTCAATCTCTCTCTGGATGGAGTCAAATCCTGGCTAAAACCTCGCCTGGGACATGAAGCCAC TGTCGGCAAGGCCAAAGGCTTCCTCAAGAACTTTCTCATTGAACCCTTCGTCCCCCACAGTCAGGCGGAGGAGTTCTACGT GTGCATCTATGCTACCCGGGAAGGAGACTACGTCCTGTTCCACCATGAAGGGGGTGTGGATGTGGGCGATGTGGATGCCA AAGCCCAGAAGCTGCTTGTGGGTGTGGACGAAAAGCTGAATACCGAGGACATTAAGAGACACCTGTTGGTCCATGCACCT GAGGACAAGAAAGAAGTCCTGGCCAGCTTCATCTCTGGTCTATTCAATTTCTACGAGGATCTGTACTTCACCTACCTTGAGA TCAACCCCCTTGTGGTGACCAAAGATGGTGTCTACATCCTTGACTTGGCGGCCAAGGTGGATGCCACAGCTGACTACATCT GTAAAGTCAAGTGGGGTGATATAGAGTTCCCTCCCCCCTTTGGGCGTGAGGCGTACCCCGAGGAAGCCTACATTGCAGAC CTGGATGCCAAAAGTGGAGCAAGCTTGAAGCTGACCTTGCTGAACCCCAAGGGGCGGATCTGGACCATGGTTGCTGGGGG TGGCGCCTCCGTCGTGTACAGTGACACCATCTGTGATCTTGGAGGTGTCAATGAACTGGCGAATTACGGGGAATACTCGGG TGCCCCCAGTGAACAACAGACCTATGACTATGCCAAGACCATCCTCTCACTTATGACTCGAGAGAAGCACCCAGAAGGCA AGATCCTCATCATTGGAGGCAGCATTGCAAACTTCACCAATGTGGCCGCCACCTTCAAGGGCATTGTGAGAGCGATTCGAG ATTACCAGGGTCCCCTGAAGGAGCATGAGGTCACCATCTTTGTCCGAAGAGGTGGCCCCAACTATCAAGAGGGATTACGA GTGATGGGAGAAGTTGGGAAGACCACTGGGATCCCCATCCATGTCTTTGGCACAGAAACTCACATGACGGCCATTGTGGG CATGGCCCTGGGCCACCGGCCCATTCCCAACCAGCCACCCACAGCAGCTCACACTGCCAACTTCCTCCTTAATGCCAGCG GGAGCACATCGACTCCAGCACCCAGTAGGACAGCATCTTTTTCTGAGTCCCGAGCTGATGAAGTGGCACCTGCAAAGAAA

[0244] GCCAAGCCAGCTATGCCCCAAGATTCAGTCCCAAGTCCAAGATCCCTGCAAGGAAAGAGTGCCACCCTCTTCAGCCGACA TACCAAGGCCATCGTGTGGGGCATGCAGACCCGGGCTGTGCAGGGCATGCTGGACTTTGACTACGTGTGTTCCCGAGACG AGCCCTCAGTGGCTGCCATGGTCTACCCTTTCACTGGGGATCACAAGCAGAAGTTTTACTGGGGACACAAGGAAATCCTGA TCCCTGTCTTCAAGAACATGGCTGACGCCATGAAGAAGCACCCGGAGGTAGACGTGCTGATCAACTTTGCGTCTCTGCGGT CCGCTTACGACAGCACCATGGAGACCATGAACTATGCCCAGATCCGCACCATAGCCATCATAGCAGAAGGTATCCCTGAG GCCCTCACACGGAAGCTCATCAAGAAGGCCGACCAGAAGGGAGTGACCATCATTGGGCCAGCTACGGTTGGGGGCATTA AGCCTGGATGCTTTAAGATCGGGAATACTGGTGGAATGCTGGACAACATCCTGGCCTCCAAACTGTACCGCCCAGGCAGC GTGGCCTACGTCTCACGTTCAGGAGGCATGTCTAATGAACTCAATAACATCATCTCTCGGACCACAGATGGTGTCTATGAG GGCGTGGCCATCGGCGGGGACAGGTACCCTGGGTCCACATTCATGGATCACGTGCTGCGCTACCAGGACACTCCAGGAGT CAAGATGATCGTAGTTCTTGGGGAGATAGGGGGCACAGAGGAATATAAGATCTGCCGGGGCATCAAGGAGGGCCGCCTC ACCAAGCCAGTGGTCTGCTGGTGTATCGGGACCTGTGCCACCATGTTCTCCTCCGAGGTCCAGTTTGGCCATGCTGGAGCT TGTGCCAACCAGGCTTCTGAAACTGCAGTAGCCAAGAACCAGGCCTTGAAGGAAGCAGGAGTGTTTGTGCCCCGAAGCTT CGATGAGCTTGGAGAAATCATTCAGTCTGTGTATGAAGATCTGGTGGCCAAAGGAGCCATTGTACCTGCCCAGGAAGTGCC ACCTCCAACAGTGCCCATGGACTACTCTTGGGCCAGAGAGCTGGGTTTGATCCGAAAACCTGCCTCATTCATGACCAGCAT CTGTGATGAGCGAGGGCAGGAGCTCATTTATGCGGGCATGCCCATCACCGAGGTCTTCAAGGAGGAGATGGGCATCGGTG GTGTCCTCGGCCTCCTCTGGTTCCAGAGAAGGTTGCCCAAGTATTCCTGCCAGTTCATTGAGATGTGTCTGATGGTCACAG

[0245] CTGATCACGGGCCAGCTGTCTCTGGAGCCCATAACACCATCATCTGTGCTCGGGCTGGGAAGGACCTGGTCTCCAGCCTC KeVer / SLIM_5gene / 850

[0246] ACCTCAGGGCTGCTCACCATTGGAGACCGGTTTGGGGGTGCCTTGGATGCCGCAGCAAAGATGTTCAGTAAAGCCTTTGA CAGCGGCATCATTCCCATGGAGTTCGTCAACAAGATGAAGAAGGAGGGGAAGCTGATCATGGGCATCGGCCATCGAGTAA AATCGATAAACAACCCAGACATGCGAGTGCAGATCCTCAAGGACTTCGTCAAACAGCACTTCCCCGCCACCCCGCTGCTC GACTATGCCCTGGAAGTGGAGAAGATTACCACCTCCAAGAAGCCAAATCTTATCCTGAATGTGGACGGCTTCATCGGCGTT GCGTTTGTGGACATGCTCAGGAACTGTGGCTCCTTCACCCGGGAGGAAGCTGATGAATATGTTGACATTGGAGCCCTCAAT GGCATCTTTGTGCTAGGAAGGAGTATGGGCTTCATTGGGCACTACCTTGACCAGAAGAGGCTGAAGCAAGGGCTGTATCG TCACCCCTGGGATGACATTTCCTATGTTCTTCCAGAACACATGAGCATGTAAccccacgtgccggfctgccfccfacfaccfgfccafcaafgacg aggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaa ttaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggc aaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgaattcataacttcgtat attatataatatacgaagttataaggagtttggcgcccgttttttcgagccccacacgtttcggtgagtatgagcggcggcagattcgagcgtttccggtttccgcggctggacgagagccc atgatgggggctcccaccaccagcaatcagggccctgattacacacccacctgtaatgtcatgctgttcatcgtggttaatgctgctgtgtgctgtgtgtgtgtgttgtttggcgctcattgttgc gttatgcagcgtacaccacaatattggaagcttattagcctttctattttttcgtttgcaaggcttaacaacattgctgtggagagggatggggatatggaggccgctggagggagtcggaga ggcgttttggagcggcttggcctggcgcccagctcgcgaaacgcacctaggaccctttggcacgccgaaatgtgccacttttcagtctagtaacgccttacctacgtcattccatgcatgca tgtttgcgccttttttcccttgcccttgatcgccacacagtacagtgcactgtacagtggaggttttgggggggtcttagatgggagctaaaagcggcctagcggtacactagtgggattgtat ggagtggcatggagcctaggtggagcctgacaggacgcacgaccggctagcccgtgacagacgatgggtggctcctgttgtccaccgcgtacaaatgtttgggccaaagtcttgtcag ccttgcttgcgaacctaattcccaattttgtcacttcgcacccccattgatcgagccctaacccctgcccatcaggcaatccaattaagctcgcattgtctgccttgtttagtttggctcctgcccg tttcggcgtccacttgcacaaacacaaacaagcattatatataaggctcgtctctccctcccaaccacactcacttttttgcccgtcttcccttgctaacacaaaagtcaagaacacaaacaa ccaccccaacccccttacacacaagacatatctacagcaatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgat gcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcacttt gcatcggccgcgctcccgattccggaagtgcttgacattggggaattcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccga actgcccgctgttctgcagccggtcgcggaggcaatggatgccattgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactaca tggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgagga ctgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaa tacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatg ctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgta cacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataatcatgtaat tagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgtt atttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgataacttcgtatattatataatatacgaagtt atTTCTTCCGGGCCAAAA TTGCTCA CCA TAA CGCCA CGGA CCGTGTCA CA TA TCTCA GTCA CCCTGGCCA CGTA CTGTA CCA

[0247] GTCCAGATCTACAGTTGCACACTCTGGTAACCGCAGTGTACCTGATCGTCTAGGTTCAAGACAAGGGTTGAGAGGGCTGTG AGACAATTCTTGGCGCAAGTTACTGTAGAGAAAAGTGATGCGGCCGGAATGAGTACAGTCAAGGCAATACTGGGGCTGTT TCTGGAAA GGAAAA GCA GA CCA TCA GCCGGGTTGGCCCCA CA TGCAGTTCTTTTTTGTGCTGTTTCA CTTTTCAAA TTCAA T TTGGCCAA TCTCGTA CA TCTCCTTCTCTCA CCAA GA CCCA CGA TA GTGTGCTTCCTCCA CCTCCCA GCTCCCTGCA TA TTA T CAGTGA CA CGGA CCA TTTGTCTCA CCAA TTA CA GAAAA GA CGCTGGTTGCCCAA CCGCCTAA CCTTGTGCCCCTGCA TGA T ACATTGTTATCTTTGCTGTTGGCGGCCGGTCTCTTGCTCTGGGACGGTGACGATCGCCCACGTGCTTGAGGACGAGCGGAC GCTGCACTTTGGAGCAGTGTGGAGCAGTGTGGAGCACTTTGGGCACCGGTCTGGAGGATGGACTCTGGGGAGTTGAATGA AACGGGGAGTCGTGGCGGTGGGCAAGTTGCACTTTTTTTACACAAGCCATTCTTGGCGGAGGAACCACACTCAGTCTTGCT GAGTCGAGTCTGTCTTTCTTTCTTACCCAGATTTGTCCGACTCTGAGTTGGTCCCTCTACAAGTAGCTGACTCTGGGTCTCA GTCTCTCCA CGGGGCTGTGTGA TTGA TAA CTCGA GA CA CGAGTTTCAA GA CGA CTGGA TTCA CTGA TTGGGCTGCTGAAA G TTGA CAAA GTTCTCGGAAAA CA GGCTTCGCGCTAA CAA GTCA A A GA CA GA CA GA CA GCA TGCTTCCA CA GCTTA CA CA GG A GCCAAAA TAA GA CAAAAA GAAA GGCCCCGA GCGTGA CGGA GA CA TTCGGA TTAA TGGGTCTGGGTTA GGGTGA GGCTTT TTA CCCA GA CGGTCGAAA TCTCGGTCAAA TA CGGCTTCCCA CAA GGTCGTCCTGA CA CA GCA TTGTGGGTGGA GGAA TTTG CTCA CGA TAA GAA TA GGTCTGA GA GCGGA CGTTTTTTCA CAAAGTTGAAA GCCGA TGCGGA CAGCTA CTTGGA GCA GAA G AGATCCTCCTCACAGCCCCGTTAAAGGGCCGGTTTTAAGTAGAGCTCCCGAGATCACACGTCTATCAAACGCAGAGGTTGA GTCTTGCACACATACAGTTGCGACAATGGAGATCAGTGGCAATCTCCGTTGGTTGACATGACTAAAGACAGGAGGCTGAGT AACATCCACAGTGAGAGCGAAAGTTCACCTTGCTCGGTTAGGGATCGCAGTGAGAGAACAAGTCACCCCCGGTTAATGTT GCA GA CTGTCCTGGCTA GGGGCAA CTTGA GCCA GGAA CGTGCCTGCCA GTA GCTGGCAA TA TCTCCTTCCTGTTA GCTGA T AA TAA CA GTGCCGGGCTA CTGTA GCTTAA GCAA CGA CTCTA TCTA CCGCTTA GCCA TTCTCTTTGCGGAA CAA GGCGCTAA ACAGACGCTAGTGGCCCATTGTTTGGAGTCATGACGTGCTAGGCTGGGAATGACTAGAGTAGCTGTCTCGTTAGATCTGTC ATAGACGACGTTGGTTCTTTATCAAGTTGATGACGAAGTATTAACAACGACCTTGGCAGCGGTTG

[0248] Example 9. Comparative results of Y. lipolytica CBS 8108 and CBS 10739 in bioreactor experiments.

[0249] To examine whether the effect of 'PUSH-PULL-TRAP', herein referred to as 'PPT' engineering of the invention is applicable to other Y. lipolytica strains, thus preferably strain-independent, the engineering of another Y. lipolytica strain (CBS 10739) was performed. The performance of the two wild type (WT) strains (Y. lipolytica CBS 8108 and CBS 10739) was monitored and compared to the performance of their corresponding 'obese' variants engineered with either the 'PUSH-PULL' and / or the 'PUSH-PULL-TRAP' strategies in fed-batch fermentations (shown in Figure 5A, B wherein the strains engineered using the KeVer / SLIM_5gene / 850

[0250] PPT method of the invention are marked as "PPT"). To monitor the performance of wild type (shown as "WT" in Figures 5A and B) and genetically engineered strains, the parameters for example dry cell weight (DCW) and lipid titer ('lipids") were measured at 16, 24, 48, 72 and 90h after inoculation. In both tested strains, the 'PUSH-PULL-TRAP' strategy of the present invention led to a significant increase in lipid titers compared to their wild-type counterparts (on average 98.28% increase for Y. lipolytica CBS 8108 and 48.57% for Y. lipolytica CBS 10739; Figure 5A, B). For Y. lipolytica CBS 8108 strain, an engineered yeast strain was additionally prepared according to "PUSH-PULL" strategy explained in Example 1, and referred herein to as "PP" strain of Y. lipolytica CBS 8108. Statistical significance of the differences between the dry cell weight and lipid titers of the "WT", "PP", and "PPT" strains of Y. lipolytica CBS 8108 and "WT" and "PPT" strains of Y. lipolytica CBS 10793 are shown in Figs. 5A and 5B, respectively. The statistical significance was evaluated using t-test (*: p < 0.05; **: p < 0.01; ***: p < 0.001).

[0251] The engineered strains obtained using the 'PUSH-PULL-TRAP' strategy of the invention were all characterized by an early onset of lipids accumulation, as determined already at 48h time point. At this time point both engineered oleaginous yeasts using the PPT strategy of the present invention reached similar lipid titers (on avg. 44.17 g / l for Y. lipolytica CBS 8108 and 41.91 g / l for Y. lipolytica CBS 10739; Figure 5A, B, respectively). This increase in lipid titer represented statistically significant increases of 78.61% and 34.49%, respectively, in comparison to the corresponding wild types. The increase in the lipid titer is a particularly important parameter for a metabolic and / of synthetic capacity of an oleaginous yeast strain. The recombinant yeast strains engineered using the engineering method of the invention are particularly suitable for a large scale lipid production, where shorter production times are favored. Furthermore, said early lipid production and / or accumulation led to increased maximum productivities of recombinant yeast strains of the invention (i.e. "PPT" strains as indicated in Figs. 5A and B), averaging 0.92 g / l / h for Y. lipolytica CBS 8108 and 0.87 g / l / h for Y. lipolytica CBS 10739, while the wild type counterparts showed their maximum productivity only after 72h (0.36 g / l / h and 0.44 g / l / h, respectively).

[0252] Materials and Methods

[0253] Strains and culture conditions.

[0254] Two different wild type, reference strains Y. lipolytica CBS 8108 and CBS10739 were used in this study, together with two recombinant strains, the reference recombinant yeast herein referred to as "PP" which corresponds to Y. lipolytica recombinant cell featuring overexpression of genes such as DGA1, ACC1 and MCE2 and the recombinant oleaginous yeast cell of the invention, herein referred to as "PPT", which features replaced the CEX1 gene with two overexpression constructs of the M. musculus homologs of ACL1 and ACL2 genes, besides the overexpression of genes such as DGA1, ACC1 and MCE2 featured in the reference recombinant strain "PP". The yeasts were grown in Yeast Extract-Peptone-Dextrose KeVer / SLIM_5gene / 850

[0255] (YPD) agar plates or liquid cultures containing 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar, 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.

[0256] Strain engineering.

[0257] 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 BGI. The codon optimized sequences were amplified with PCR (PrimeSTAR GXL DNA Polymerase, Takara) and cloned to the overexpression constructs with Gibson assembly.

[0258] 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). Integration of the overexpression constructs and deletion of the CEX1 gene was achieved through double homologous recombination.

[0259] 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).

[0260] Shake flasks fermen tations.

[0261] For the evaluation of the performance of the strains we used shake flasks fermentations using minimal media (Duman-Ozdamar et al., Microb. Cell Factories, 2022, 21, 228) consisting of urea as the nitrogen source and glucose or glycerol as the carbon source. Two different ratios of N / C (g / g) were tested. In the condition of N / C 1 / 100 0.6 g / L of urea and 60 g of either glucose or glycerol were added in the minimal medium while in N / C 1 / 140 condition 0.13 g / L of urea and 18.2 g / L of glucose or glycerol. A single colony of Y. lipolytica was grown in 2 mL of YPD 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 minimal medium. The washed cells were inoculated into 20 mL minimal 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 determination of the dried cell weight (DCW) and lipid content determination was performed.

[0262] DCW determination.

[0263] 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. KeVer / SLIM_5gene / 850

[0264] Lipids extraction.

[0265] 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.

[0266] Bioreactor fermentations.

[0267] 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, 25 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 0.7 mL / h rate.

[0268] Fatty acid composition.

[0269] We determined the fatty acid 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).

[0270] Analytical quantification of metabolites.

[0271] Quantification of the citrate and glucose concentrations was performed with high-performance liquid chromatography (Acquity Arc HPLC (Waters) equipped 2414 Rl detector). At the selected time points 1 mL of the fermentation broth was sampled and centrifuged at 10000 rpm for 1 min. The supernatant was filtered with a syringe filter with 0.2 pm membrane, before loaded to the HPLC. The mobile phase (Sulphuric acid 5 mM) was used to flow through a separation column (Aminex HPX-87H 300 x 7.8 mm, Biorad) at a rate of 0.6 mL / min.

Claims

1. KeVer / SLIM_5gene / 850Claims1. A recombinant oleaginous yeast cell having at least one CEX1 gene disruption, comprising at least one chimeric gene construct, said chimeric gene construct comprising:• a yeast-expressible promoter,• a nucleotide sequence encoding DGA1, ACC1, MCE2, and ACL protein, wherein said ACL protein is ACL1 and / or ACL2 protein; wherein said nucleotide sequence is operably linked to said yeast expressible promoter; and• a 3' end region comprising transcription termination and polyadenylation signals functioning in a yeast cell.

2. A recombinant oleaginous yeast according to claim 1, comprising the following chimeric gene constructs:• a chimeric gene construct comprising a nucleotide sequence encoding DGA1 protein being native to the yeast cell or from a different species;• a chimeric gene construct comprising a nucleotide sequence encoding ACC1 protein being native to the yeast cell or from a different species;• a chimeric gene construct comprising a nucleotide sequence encoding MCE2 protein being native to the yeast cell or from a different species;• a chimeric gene construct comprising a nucleotide sequence encoding ACL1 protein and / or a nucleotide sequence encoding ACL2 protein, said ACL1 and / or ACL2 protein being native to the yeast cell or from a different species.

3. The recombinant oleaginous yeast cell according to any one of claims 1 or 2, wherein said at least one CEX1 gene disruption is obtainable by CEX1 knockout mutation.

4. The recombinant oleaginous yeast cell according to any one of the preceding claims, wherein said yeast cell is Yarrowia lipolytica cell.

5. The recombinant oleaginous yeast cell according to any one of the preceding claims, wherein said DGA1 protein is Y. lipolytica DGA1 protein.

6. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said ACC1 protein is Y. lipolytica ACC1 protein.

7. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said MCE2 protein is Mucor circinelloides MCE2 protein.

8. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said ACL1 protein is Mus musculus ACL1 protein.

9. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said MCL2 protein is M. musculus ACL2 protein.47KeVer / SLIM_5gene / 85010. The recombinant oleaginous yeast cell according any one of the preceding claims, comprising the chimeric gene construct, said construct coding for M. musculus ACL1 and M. musculus ACL2 proteins.

11. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said DGA1, ACC1, MCE2, ACL1 and / or ACL2 protein comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4 and / or SEQ ID NO:5, respectively, or an orthologue characterized by ay least 90% amino acid sequence identity thereof.

12. The recombinant oleaginous yeast cell according any one of the preceding claims, wherein said yeast expressible promoter is chosen from an EXP1, GPD and / or TEF1 promoter region.

13. A method for producing lipids using the recombinant oleaginous yeast cell according to any one of claims 1 to 12 comprising the steps of:• culturing the recombinant oleaginous yeast cell according to any one of claims 1 to 12 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 cell.

14. The method for producing lipids according to claim 13 wherein said culturing is executed by fed- batch fermentation, preferably in a bioreactor.

15. The method for producing lipids according to any one of claims 13 or 14, wherein said medium is glucose- or glycerol- based.

16. Lipids obtainable by the method according to any one of claims 13 to 15, preferably said lipids comprising or essentially consisting of triacylglycerols.

17. A composition comprising the lipids obtained from the method of any one of claims 13 to 15, or the recombinant oleaginous yeast cell according to any one of claims 1 to 12.

18. A food product, preferably a plant-based food product, comprising the lipids of claim 16, or the composition of claim 17.48

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