Methods and compositions

Abstract

Provided herein is an oleaginous yeast cell that has been engineered to increase the lipid content with minimal engineering steps. The engineered yeast cells are able to produce yields of lipids that are improved over known yeast cells from a single insertion of an expression cassette. The invention also provides accompanying methods, such as methods of culturing said yeast cells, methods of producing lipids, and accompanying nucleic acids, such as the various nucleic acid constructs, cassettes and genes and proteins for putting the invention into practice.

Description

[0001]Methods and Compositions Background Global production of vegetable oils reached 210 million tonnes per year in 2022 / 23. Global demand for vegetable oils is predicted to rise by over 30% between 2024 and 2034, with little improvement in the crop yields. These oils are used across food, cosmetics, oleochemicals and biofuels. Fats and oils are composed mostly of triacylglycerols (which are composites of fatty acids and glycerol molecules) and are produced by all organisms as a way to store energy and carbon. Palm oil accounts for 40% of this production. Palm oil is in approximately 50% of packaged supermarket products in the UK. It can only be grown in the tropics, causing widespread deforestation and peatland loss, particularly in Malaysia and Indonesia who produce 85% of the world’s palm oil. Palm oil contains a high proportion of saturated C16 (palmitic) and C18 (stearic) fatty acids (approximately 50%). This gives it a high oxidative stability and melting point, making it particularly ideal for frying and baking, as well as giving a desired consistency not only to food products but also to cosmetic ones. Oil palms are the highest yielding vegetable oil crop. They can achieve 4 tonnes / hectare / year, which is significantly higher than other staple oil crops such as sunflower oil (0.69 tonnes / hectare / year) and soybean oil (0.44 tonnes / hectare / year). Replacing palm oil with an existing animal or vegetable oil would therefore significantly increase the land footprint of oil production, which is infeasible at scale and generates unwanted environmental impact. Even though palm oil is the most efficient vegetable oil crop, the increase in demand is leading to an increase in plantations, which is linked to deforestation in the producer countries, even if the exact amount of damage to forests is debated in scientific literature. Since palm trees grow in tropical regions, the biodiversity affected by its associated deforestation is exacerbated compared to other vegetable oil crops, due to the diverse ecosystems found in the tropics. Nevertheless, one of the main impacts of palm oil plantations is the destruction of peatlands, which are ecosystems characterised by low degradation rates, which allow them to be the main carbon storage ecosystem in the world. The drainage of these ecosystems to generate land for palm oil plantations leads to the release of the CO2 stored, contributing to global climate change. The climate effect is extended at local levels, since palm oil plantations are hotter, drier and more prone to wildfires than endogenous ecosystems. Furthermore, the destruction of peatlands can have wide effects in the hydrological cycles or the neighbouring ecosystems as well as the access of the population to clean water. A rising alternative to animal and vegetable oils is the use of microorganisms. Some microorganisms are naturally able to accumulate or produce large quantities of oil, which is usually termed single-cell oil (SCO). These organisms are termed oleaginous, and they are defined as those able to produce 20% or more of their cell dry weight as lipids. There is a wide diversity in the microbial world in terms of lipid production and bacteria, yeasts, filamentous fungi and microalgae have been studied and used as lipid producers for industrial purposes. Microalgae offer the advantage of being photosynthetic organisms able to produce lipids from atmospheric CO2. However, their need for light makes the scale up of their lipid production challenging. While most bacteria do not produce large amounts of oil, there are some species able to do so. However, oleaginous bacteria produce oil with different compositions than that of vegetable oils and therefore they are not of interest for the food sector. Yeasts and filamentous fungi are the best microorganisms in terms of productivity, but they require different growth conditions. While yeasts grow in liquid environments, filamentous fungi grow best in solid surfaces. While this means that yeasts require more water, it also implies that the scale up to industrial levels is easier. The process of growing microorganisms for the production of a specific product is known as precision fermentation, which has commonly relied on fermentable sugars from first generation crops as the carbon source, such as glucose from sugar cane. While providing a standardised media input, utilising such crops for microbial oil feedstock results in land use shifted from oil crop growth towards fermentation feedstock growth. Utilisation of second-generation feedstocks that are derived from industrial or agricultural side-streams removes the environmental burden of feedstock growth, while allowing access to lower-cost inputs for the fermentation process. Various yeasts have been genetically engineered to improve lipid production, for example including numerous gene knockouts and / or overexpressions. However, the regulatory landscape governing genetically modified microorganisms (GMMs), particularly those used in food production, is highly sensitive, and the nature and extent of genetic modifications play a critical role in determining the pathway to market approval. In many jurisdictions—including the United States, European Union, Singapore, Australia, and others—the number, type, and method of gene edits directly impact the regulatory classification of an engineered strain. Strains with extensive or complex modifications may face more stringent safety evaluations, including full novel food dossiers, toxicology studies, and mandatory premarket approval. In contrast, strains with fewer, well-characterized edits, may qualify for simplified review processes or exemptions under emerging regulatory frameworks for precision gene editing. Therefore, there is a clear need for production strains that can deliver high oil yields while minimizing the number and complexity of genetic alterations. Such strains offer a more favourable regulatory profile, reducing the time, cost, and uncertainty associated with bringing food ingredients to market. Summary of invention The present invention relates to engineered yeast cells that have been engineered so as to improve the ability of the yeast to produce lipids such as triacylglycerols. In particular, the engineered yeast cells provided herein are considered to have high lipid production capabilities whilst requiring just one, or a small number of gene edits. Detailed description of the invention As set out herein, the invention provides engineered yeast cells that are able to produce yields of lipids that are improved over known yeast cells. The invention also provides engineered yeast cells that are able to produce yields of lipids that are improved over, or similar to known yeast cells, but where the engineered yeast cell comprises just one or a small number of gene edits. The invention also provides accompanying methods, such as methods of culturing said yeast cells, methods of producing lipids, and accompanying nucleic acids, such as the various nucleic acid constructs, cassettes and genes and proteins for putting the invention into practice. The invention provides an oleaginous yeast cell that has been engineered to increase the lipid content. The oleaginous yeast cell may be engineered using any means, and may be engineered to overexpress one or more proteins or non-coding RNAs, to reduce the expression of or totally knockout the expression of one more proteins or non-coding RNAs, or may be engineered to introduce heterologous proteins or non-coding RNAs, or may be engineered to mutate various residues within endogenous proteins or to express mutated heterologous proteins. The yeast cell may be engineered to increase production of lipids and / or reduce degradation of lipids and / or improve secretion of lipids. The yeast cell may be of any species. Preferably the yeast is an oleaginous yeast. Oleaginous yeasts are found across different taxonomic groups, and new strains are being constantly discovered. Preferably the yeast is of the genus Yarrowia. In specific embodiments the yeast is Yarrowia lipolytica. Yarrowia lipolytica holds GRAS (generally recognised as safe) status and it was the first genetically modified yeast harnessed industrially for oil production. This obligate- aerobe ascomycete yeast thrives in diverse environments, notably those abundant in lipids and proteins such as meat and dairy products. Y. lipolytica exhibits dimorphic growth, adopting either individual cellular forms or pseudohyphae / hyphae structures, influenced by various factors including strain type and environmental conditions. Y. lipolytica has been utilised as a single-cell protein (SCP) source, biomass for feed production using crude oil substrates, and even as a bioremediation agent for oil spills. Although Y. lipolytica has been used for metabolite production such as erythritol and citric acid, it is primarily recognized as a lipid producer and it accumulates triacylglycerides (TAG) and sterol esters within its lipid bodies, with some strains able to amass up to 50% of their dry cell weight as oil. Apart from Saccharomyces cerevisiae, the lipid metabolism of Yarrowia lipolytica is one of the best characterised among yeasts. The synthesis of lipids occurs in the cytosol and it requires cytosolic input of acetyl-CoA as a source of carbon, ATP as a source of energy, and NADPH as a reducing agent. ATP is obtained from respiration and consumption of sugar or other organic compounds. NADPH may be obtained from different sources in yeasts, such as cytosolic malic enzymes, but the only source of NADPH in Y. lipolytica is the pentose phosphate pathway. Cytosolic acetyl-CoA can be obtained from two different routes. The one common to S. cerevisiae is the action of the acetyl-CoA synthetase (ACS), which is able to add CoA directly on acetate with the expense of energy in the form of ATP. The acetate can come from the extracellular medium, or it can be derived from glucose or other sugar through the glycolysis pathway. However, most of the acetyl-CoA formed in the cell is located inside the mitochondria. In the case of Y. lipolytica, as well as other oleaginous yeasts, mitochondrial acetyl-CoA is converted into citrate which is then exported to the cytosol through a shuttle mechanism involving the cytosolic ATP:citrate lyase (ACL). In the cytosol, this citrate is broken down into oxaloacetate and acetyl-CoA. Furthermore, the decrease in nitrogen availability during the yeast growth leads to the accumulation of citrate in the mitochondria, which translates into an increase of the cytosolic levels of acetyl-CoA thanks to the ATP:citrate lyase. Before acetyl-CoA molecules are used for the synthesis of fatty acids, they need to be converted into malonyl-CoA by the acetyl-CoA carboxylase (ACC). The malonyl-CoA molecules are fed to the fatty acid synthase (FAS) complex, which in a cyclical manner keeps adding them as building blocks to generate fatty acids with 16 atoms of carbon in length. Later, these fatty acids in the form of acyl-CoA molecules can undergo elongation to increase their length, or desaturation to add double bonds in its structure. Afterwards, three acyl-CoA molecules will be added to glycerol molecules to produce triacylglycerides in a stepwise manner, of which the last step is catalysed by diacylglycerol transferases DGA. Various strategies have been used to increase the lipid content of oleaginous yeast such a Yarrowia lipolytica. These approaches share the key step of overexpressing DGA1, with additional engineering steps such as further knockouts of overexpressions building on this base modification, resulting in strains with several modifications relative to the wild type strain. The inventors have surprisingly found that it is possible to generate a high-lipid producing yeast strain such as Y. lipolytica with just a single modification – to overexpress DGA2, in particularly to overexpress DGA2 from Claviceps purpurea, and in particular to express the DGA2 from Claviceps purpurea, from the 4UAS-pTEF promoter, and more in particular to express DGA2 from a cassette comprising just one copy of DGA2 from Claviceps purpurea under control of the 4UAS- pTEF promoter – the inventors found that adding extra copies of the DGA2 gene to the expression cassette did not increase lipid yield further. The inventors have created a minimally edited, high-lipid-producing oleaginous yeast. Accordingly the invention provides an oleaginous yeast cell that expresses a heterologous diacylglycerol acyltransferase 2 (DGA2). The yeast cell can be any cell and it is considered that the benefits on lipid production achieved by the present invention is independent of specific strain background. In preferred embodiments the oleaginous yeast cell is a Yarrowia lipolytica cell. The engineerings and deletions and overexpressions described herein aimed at improving lipid production and lipid yield are suitable for incorporation into any strain of Y. lipolytica. For example the Y. lipolytica cell may be a wild type undomesticated strain, or may be a wild type domesticated strain, or may be a domesticated or undomesticated strain that has been engineered to have improved characteristics in aspects other than lipid production, for example genetic tractability. Exemplary strains are set out below: ● W29 (CBS7504): A conventional, domesticated laboratory strain widely used as a reference for lipid production and genetic engineering. ● CLIB122: A domesticated strain derived from a cross between CBS7504 (W29) and CBS6124-2, commonly used in research. ● CBS6124-2: One of the parental strains involved in creating CLIB122. ● YB392, YB419, YB420, YB566, YB567: Undomesticated strains isolated from corn milling plants and other environments, notable for robust growth, lipid accumulation, and superior xylose utilization, especially in biomass hydrolysates. ● Po1g and derivatives (e.g., FKP355): Engineered strains with mutations such as ku70 deletion to improve genetic tractability and homologous recombination efficiency. ● Y9481, Y9497, Y9502: Optimized strains developed for high eicosapentaenoic acid (EPA) production. ● PSA02004PP-ACS: A strain domesticated for hypertolerance and improved industrial performance. Preferably the Y. lipolytic cell is a wildtype cell such as W29 – for example is a cell that comprises no further nucleic acid additions, deletions or substitutions relative to a wild- type strain of Y. lipolytica. In some embodiments the only gene edits relative to a wild-type strain are the edits set out herein that are required to overexpress or knockout the specific genes. However as set out above a strain may comprise one or more gene edits that allow for improved properties other than lipid production, such as to improve genetic tractability and / or homologous recombination as set out above for the Polg strains. Accordingly in some embodiments the yeast cell is not a true wildtype cell, but is a cell that may comprise one or more other gene edits, but has not been edited for improved lipid production. There are certain regulatory advantages where the yeast cell is a wildtype cell. By wildtype we include the meaning of a naturally occurring strain that has not been edited at all. In some instances a wildtype strain is a strain that may have one or more gene edits, but does not have the one or more gene edits set out herein. The heterologous DGA2 gene may be expressed within the cell by any means. It is typical practice when expressing a gene to create a gene expression cassette that comprises the gene operably linked to a promoter which is able to drive transcription from that gene. The expression cassettes are inserted into the relevant cell and may integrate into the genome, or the expression cassette may be incorporated into a vector such as a plasmid and be maintained in the cell episomally. Accordingly in some embodiments the oleaginous yeast cell which is preferably a Y. lipolytica cell comprises a DGA2 expression cassette comprising a gene encoding the heterologous diacylglycerol acyltransferase 2 (DGA2) operably linked to a promoter. The DGA2 expression cassette may comprise one or two or more copies of the DGA2 gene. However as set out in the Examples, the inventors found no benefit to increasing the number of DGA2 genes in the cassette. Accordingly in some embodiments the DGA2 expression cassette comprises a single copy of the DGA2 gene operably linked to a single promoter. In preferred embodiments the DGA2 expression cassette is integrated into the genome of the oleaginous yeast cell. In some embodiments, the expression cassette is integrated into the genome, and the expression cassette comprises only one copy of the DGA2 gene. In some embodiments the oleaginous yeast cell comprises only one copy of the DGA2 expression cassette integrated into the genome. In some embodiments the oleaginous yeast cell comprises only one copy of a DGA2 expression cassette that comprises only one copy of the DGA2 gene. In other instances the DGA2 expression cassette may be maintained episomally within the oleaginous yeast cell, for example the heterologous DGA2 is expressed from a plasmid, for example a multicopy plasmid, for example a high copy plasmid. Preferably the expression cassette is integrated into the genome. The inventors have surprisingly found a specific benefit associated with expressing the DGA2 gene from a promoter that comprises upstream activating sequences (UAS) such as UAS from XRP1. The benefit was not observed with promoters that lack the UAS sequences. The UAS from XRP2 has the sequence of gccggatcgaggtgggcgggaacaaccctctcgataatgtacgtacactacacacaaagagaagcaagtgggggcgg gactgcacggcacttgtgagacacctcagcatg [SEQ ID NO: 40]. In some embodiments the promoter that drives expression of the heterologous DGA2 is a TEF promoter that comprises 1 or more UAS sequences from XRP2. For example in some embodiments the promoter is a pTEF promoter with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more UAS sequences from XRP2 arranged upstream of the pTEF promoter, preferably arranged in a tandem array. In some embodiments the promoter is selected from the group comprising or consisting of the pTEF-4UAS promoter (SEQ ID NO: 16); the 2UAS1-pTEF promoter; the 3UAS1- pTEF promoter; the 4UAS1-pTEF promoter; the 8UAS1-pTEF promoter; and the hp8d promoter. In some preferred embodiments the promoter is the pTEF-4UAS promoter (SEQ ID NO: 16). The 4UAS tandem repeat is set out in SEQ ID NO: 41. The DGA2 may be any DGA2. However particular advantages are associated with expression of the DGA2 from Claviceps purpurea. Accordingly in preferred embodiments the DGA2 is from Claviceps purpurea. DGA2 from Claviceps purpurea has the sequence set out in SEQ ID NO: 5. In some embodiments the DGA2 has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In some embodiments the DGA2 has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2. It will be appreciated that the gene that encodes the DGA2, for example that encodes the DGA2 from Claviceps purpurea and which has the amino acid sequence set out in SEQ ID NO: 5 may be codon optimised for expression in a specific sequence, for example for expression in Yarrowia lipolytica. In some embodiments the gene that is in the DGA2 expression cassette has a nucleic acid sequence of for SEQ ID NO: 25 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. The skilled person will readily be able to determine if a given DGA2 or variant thereof retains the activity of a diacylglycerol acyltransferase 2. For example a DGA2 polypeptide's activity can be assessed by its capacity to convert diacylglycerol (DAG) and fatty acyl-CoA into triacylglycerol (TAG). This is typically measured in vitro by incubating a DGA2-containing cell lysate or purified enzyme with exogenously supplied DAG and a radioactively labeled fatty acyl-CoA, followed by lipid extraction and separation (e.g., via thin-layer chromatography) to quantify the incorporation of the label into TAG. Successful conversion of substrates to TAG indicates retention of the desired DGA2 enzymatic activity. As set out above, the inventors have surprisingly found that it is not necessary to overexpress DGA1 alongside expression of the heterologous DGA2 to achieve high levels of lipid production. Strains that are minimally edited have advantages such as reduced regulatory hurdles. The strains provided herein are considered to be minimally edited relative to other engineered lipid producing strains, whilst producing as much as, or more, lipid. In some embodiments then the oleaginous yeast cell of the invention has not been engineered to overexpress DGA1. The yeast cell of the invention comprises the native DGA1, but does not comprise any additional copies of DGA1, and / or does not comprise any modifications to the DGA1 that would increase expression. For example the yeast cell of the invention has not been engineered to overexpress the native DGA1 or engineered to introduce a heterologous DGA1, or engineered to add an additional copy of the native DGA1, or engineered to modify the promoter or other regulatory regions of the native DGA1 to increase expression. In preferred embodiments the oleaginous yeast cell of the invention comprises the native DGA1 gene at the native locus under the control of the native DGA1 promoter and does not comprise an additional DGA1 gene elsewhere in the cell. In some embodiments where the oleaginous yeast cell is in a haploid state it comprises only one copy of the native DGA1 gene; and where the oleaginous yeast cell is in a diploid state the yeast cell comprises only two copies the native DGA1 gene. In some embodiments the oleaginous yeast cell does not comprise a heterologous DGA1 gene. In some embodiments the oleaginous yeast cell does not comprise a native or heterologous DGA1 gene operably linked to: a TEF promoter; or to a GPD1 promoter; a EXP1 promoter; pTEF-4UAS promoter (SEQ ID NO: 16); the 2UAS1-pTEF promoter; the 3UAS1-pTEF promoter; the 4UAS1- pTEF promoter; the 8UAS1-pTEF promoter; or the hp8d promoter. In some embodiments the oleaginous yeast cell does not comprise: a) an expression cassette comprising the DGA1 gene from Rhodotorula toruloides (rtDGA1) operably linked to a promoter; and / or b) an expression cassette comprising the DGA1 gene from L. starkeyi operably linked to a promoter. In some embodiments the oleaginous yeast cell does not comprise an overexpression of: a) native GPD1, SLC1 and / or LRO1; or b) heterologous GPD1, SLC1 and / or LRO1. In some embodiments the oleaginous yeast cell is a Yarrowia lipolytica cell and does not have a deletion or disruption of the TGL3 gene. In some embodiments the oleaginous yeast cell does not comprise a DGA2 expression cassette that comprises a DGA2 gene operably linked to a promoter that either i) consists of a pTEF promoter; or ii) is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2. For example, the inventors have found that expression of DGA2 from the standard pTEF promoter does not confer the necessary advantages and so expression of the heterologous DGA2 from the standard pTEF promoter, which is a pTEF promoter that is not associated with one or more UAS sequences from XRP2, is not considered to be useful. As set out above, preferably the DGA2 is: a) is from Claviceps purpurea; and / or b) has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2. In some embodiments the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a 4UAS-pTEF; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2. In some embodiments the oleaginous yeast cell comprises: a) a DGA2 expression cassette that comprises one copy of the DGA2 gene from Claviceps purpurea operably linked to a 4UAS-pTEF; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2. In some embodiments the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter. In some embodiments the oleaginous yeast cell comprises: a) a DGA2 expression cassette that comprises one copy of the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter. In some embodiments the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter; and d) the native DGA1 gene in its native locus under the control of the native DGA1 promoter. In some embodiments the oleaginous yeast cell comprises: a) a DGA2 expression cassette that comprises one copy of the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter; and d) the native DGA1 gene in its native locus under the control of the native DGA1 promoter. As set out above preferably the DGA2 gene from Claviceps purpurea has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2. In some embodiments the oleaginous yeast cell comprises the native Δ9 desaturase, for example wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and comprises the native Yarrowia lipolytica Δ9 desaturase. In some embodiments the oleaginous yeast cell comprises the native Δ12 desaturase, for example wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and comprises the native Yarrowia lipolytica Δ12 desaturase. In some embodiments the oleaginous yeast cell does not comprise an A. adeninivorans Δ9 desaturase, optionally wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and does not comprise an A. adeninivorans Δ9 desaturase. In some embodiments the oleaginous yeast cell does not comprise E. coli phosphotransferase, for example wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and does not comprise E. coli phosphotransferase. In some embodiments the oleaginous yeast cell does not comprise Herpes simplex thymidine kinase. The oleaginous yeast cell of the invention does not have any one or more or all of or any combination of the above features. Some oleaginous yeast such as Y. lipolytica have a yeast and a hyphal phenotype. The hyphal phenotype can interfere with efforts to grow the yeast at scale and can interfere with lipid production / extraction. In some preferred embodiments the oleaginous yeast cell of the invention does not have a hyphal phenotype. In some embodiments the oleaginous yeast cell natively has a hyphal growth phenotype and wherein the oleaginous yeast cell comprises a gene deletion or disruption which prevents or reduces hyphal growth. In some embodiments the oleaginous yeast cell is a Yarrowia lipolytica cell and comprises a knockout or disruption of the MHY1 gene, wherein the disruption affects the function of the Mhy1p protein and prevents or reduces hyphal growth. It should be clear to the skilled person that, amongst other embodiments, the oleaginous yeast cell of the invention is: a Yarrowia lipolytica cell that comprises a single genome integrated DGA2 expression cassette wherein the DGA2 gene is the Claviceps purpurea DGA2 gene under the control of the 4UAS-pTEF promoter and wherein the yeast cell does not comprise: A) Any copy of DGA1 other than the native Y. lipolytica DGA1 at the native locus under the control of the native promoter; and B) A copy of DGA2 other than i) the Y. lipolytica DGA2 at the native locus and under control of the native DGA2 promoter; and ii) the Claviceps purpurea DGA2 present in the genomically integrated DGA2 expression cassette; and C) a knockout or disruption of the MHY1 gene so as to reduce or prevent hyphal growth; And may also: D) comprise only the native Δ9 desaturase and / or the native Δ12 desaturase (i.e. does not comprise a heterologous Δ9 desaturase and / or heterologous native Δ12 desaturase); E) not comprise any one or more or all of: i) an A. adeninivorans Δ9 desaturase; ii) E. coli phosphotransferase; iii) Herpes simplex thymidine kinase. As set out elsewhere there are benefits in reducing the number of gene edits a cell comprises, whilst maintaining the useful phenotype - in the present case high lipid production. Accordingly in some embodiments the oleaginous yeast cell comprises just one modification relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome. However it is appreciated that the yeast cell may comprise just one modification that aims to increase lipid production but the yeast cell may comprise one or more further modifications aimed at addressing different issues, such as the hyphal growth issue mentioned above. Accordingly in some embodiments the oleaginous yeast cell comprises just one modification that results in increased lipid production relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome, but the yeast cell may comprise one or more further modifications for purposes other than lipid production. For example in some embodiments the oleaginous yeast cell comprises just one modification that results in increased lipid production relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome, and wherein the yeast cell also comprises a deletion or disruption of the MHY1 gene. The oleaginous yeast cell of the invention is considered to produce high levels of lipids – levels that are equivalent to or higher than lipid levels produced by known strains that comprise more modifications than the strains of the current invention. In some embodiments a population of the oleaginous yeast cells of the invention has a lipid content (% cell dry weight) of at least 50%, or at least 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 80% or more after culture in BL05M lipogenic medium for 96 hours. In some embodiments a population of the oleaginous yeast cells has a lipid titre of at least 12g / L, or at least 12.2, 12.4, 12.6, 12.8, 13.0, 13.2, 13.4, 13.6, 13.8, 14.0, 14.2, 14.4, 14.6, 14.8, 15.0, 15.2, 15.4, 15.6, 15.8, 16.0, 16.2, 16.4, 16.6, 16.8, 17.0, 17.2, 17.4, 17.6, 17.8, 18.0, 18.2, 18.4, 18.6, 18.8, 19.0, 19.2, 19.4, 19.6, 19.8, or at least 20.0 g / L when cultured in BL05M lipogenic medium for 96 hours. In some embodiments a population of the oleaginous yeast cells of the invention has a lipid content (% cell dry weight) of at least 50%, or at least 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 80% or more after a culture duration of at least 96 hours, 100 hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 142 hours, 144 hours or at least 145 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells has a lipid titre of at least 12g / L, or at least 12.2, 12.4, 12.6, 12.8, 13.0, 13.2, 13.4, 13.6, 13.8, 14.0, 14.2, 14.4, 14.6, 14.8, 15.0, 15.2, 15.4, 15.6, 15.8, 16.0, 16.2, 16.4, 16.6, 16.8, 17.0, 17.2, 17.4, 17.6, 17.8, 18.0, 18.2, 18.4, 18.6, 18.8, 19.0, 19.2, 19.4, 19.6, 19.8, or at least 20.0 g / L after a culture duration of at least 96 hours, 100 hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 142 hours, 144 hours or at least 145 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells of the invention has a lipid content (% cell dry weight) of at least 70%, 72%, 74%, 76%, 80% or more after a culture duration of at least 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 142 hours, 144 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells has a lipid titre of at least 12g / L, or at least 12.2, 12.4, 12.6, 12.8, 13.0, 13.2, 13.4, 13.6, 13.8, 14.0, 14.2, 14.4, 14.6, 14.8, 15.0, 15.2, 15.4, 15.6, 15.8, 16.0, 16.2, 16.4, 16.6, 16.8, 17.0, 17.2, 17.4, 17.6, 17.8, 18.0, 18.2, 18.4, 18.6, 18.8, 19.0, 19.2, 19.4, 19.6, 19.8, or at least 20.0 g / L after a culture duration of at 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 142 hours, 144 hours or at least 145 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells of the invention has a lipid content (% cell dry weight) of at least 80% or more after a culture duration of at least 144 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells has a lipid titre of at least 20.0 g / L after a culture duration of at least 144 hours, optionally when cultured in BL05M lipogenic medium. In some embodiments a population of the oleaginous yeast cells has a lipid content (% cell dry weight) of at least 74%, 76%, 80% or more after culture in BL05M lipogenic medium for 168 hours. In some embodiments a population of the oleaginous yeast cells has a lipid titre of at least 25.0 g / L or at least 25.2, 25.4, 25.6, 25.8, 26.0, 26.2, 26.4, 26.6, 26.8, 27.0, 27.2, 27.4, 27.6, 27.8, 28.0, 28.2, 28.4, 28.6, 28.8, 29.0, 29.2, 29.4, 29.6, 29.8, 30.0, 30.2, 30.4, 30.6, 30.8, 31.0, 31.2, 31.4, 31.6, 31.8, or at least 32.0 g / L when cultured in BL05M lipogenic medium for 168 hours. In preferred embodiments of all embodiments of the oleaginous yeast cell the DGA is DGA2 from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In some preferred embodiments of all embodiments of the oleaginous yeast cell, the yeast cell is a Yarrowia lipolytica yeast cell and has been engineered to overexpress DGA2, for example overexpress DGA2 from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In some preferred embodiments of all embodiments of the oleaginous yeast cell the yeast cell is a Yarrowia lipolytica yeast cell and the only modifications relative to a wildtype Yarrowia lipolytica yeast cell is that it has been engineered to overexpress DGA2, for example overexpress DGA2 from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5, and has been modified to knockout or disrupt the MHY1 gene. In some preferred embodiments the oleaginous yeast cell is a Yarrowia lipolytica yeast cell and has been engineered to overexpress DGA2, for example overexpress DGA2 from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; and the Yarrowia lipolytica yeast cell and has not been engineered to overexpress DGA1. In a different aspect, the invention provides other engineered oleaginous yeast cells that are considered to have improved lipid content and that do have additional or different modifications. For example in some of the embodiments below the yeast cell does not express a heterologous DGA2. In preferred embodiments the yeast cell of the invention does express a heterologous DGA2 as set out above, but may also comprise one or more further modification as set out below. In some embodiments the oleaginous yeast cell is engineered so as to result in overexpression of diacylglycerol acyltransferase (DGA) (for example the yeast cell may not be engineered to express a heterologous DGA2). DGA1 codes for a diacylglycerol acyltransferase that catalyses the last step in the production of triacylglycerols (TAGs). Yarrowia contains two diacylglycerol acyltransferases: DGA1 and DGA2. DGA2 is a type 1 diacylglycerol acyltransferase and it is sometimes termed DGAT1. The DGA may be any DGA. In some embodiments the DGA is DGA1 or DGA2 or DGA3 or any combination thereof. In some embodiments the DGA1 or DGA2 is from Yarrowia lipolytica, for example DGA1 having the sequence of SEQ ID NO: 1 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1. As set out above, in preferred embodiments the DGA is DGA2 (or DGAT1) from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5. In other embodiments the DGA is soluble DGA3 from Rhodotorula glutinis. In some embodiments the oleaginous yeast cell, including the yeast cell set out above that expresses a heterologous DGA2, is engineered so as to result in the increased production of NADPH. This cofactor is necessary for the activity of the fatty acid synthase and it provides the redox difference to reduce the acetyl-CoA molecules into fatty acid chains. In some embodiments this is achieved by overexpression of glyceraldehyde-3-phosphate dehydrogenase (GapC). The GapC may be any GapC. In some embodiments the GapC is from Clostridium acetobutylicum. In some embodiments where the yeast is a Yarrowia lipolytica yeast, the GapC is from Clostridium acetobutylicum and has a sequence that has been codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3. In other embodiments the increase in NADPH production is achieved by the overexpression of NADP-dependant malic enzyme (MCE2). The MCE2 may be any MCE2. In some embodiments the MCE2 is from Mucor circinelloides. In embodiments where the yeast is Yarrowia lipolytica, the MCE2 is from Mucor circinelloides and has a sequence that has been codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. In preferred embodiments the increase in NADPH production is achieved by overexpression of both GapC and MCE2, for example GapC and MCE2 as set out above. Before acetyl-CoA is used to elongate fatty acid changes, it needs to be activated to malonyl-CoA through the addition of a CO2 group. This is done by the acetyl-CoA carboxylase (ACC1), which makes use of the cofactor biotin to catalyse the reaction. While in bacteria this activity is catalysed by three different separate enzymes, in eukaryotes ACC1 is a large multi-domain enzyme with a central non-catalytic domain. This domain, called central domain, contains several residues that can be phosphorylated to regulate the activity of the enzyme (Hunkeler et al 2016 Nature Communications 7: 11196). These features make ACC1 the central enzyme in lipid biosynthesis, whose regulation allows control of the whole metabolic pathway. Accordingly in some embodiments the yeast cell is engineered so as to overexpress acetyl-coA-carboxylase (ACC1). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to overexpress ACC1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to overexpress ACC1. In some embodiments the ACC1 is from Yarrowia lipolytica, for example is an ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. In some embodiments it is considered to be beneficial of the ACC1 is unable to be inactivated by phosphorylation. Accordingly in some embodiments the yeast cell expresses or overexpresses an ACC1 (for example the Yarrowia lipolytica ACC1 as set out above) that has been engineered to be insensitive to deactivation via phosphorylation. In some embodiments the ACC1 comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2. In some embodiments the ACC1 has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine. When the nitrogen source is exhausted in the medium, the key intermediate, citrate, is converted to acetyl-CoA by ATP:citrate lyase (ACL) for lipid accumulation. ACL is a heterodimeric enzyme responsible for cleaving citrate into acetyl-CoA. In some embodiments it is considered to be beneficial to express or overexpress ACL in the yeast cell. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress ACL. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress ACL. The ACL can be any ACL. In some embodiments the yeast cell has been engineered to overexpress the ATP:citrate lyase (ACL). In some embodiments the ACL1 and / or ACL2, for example the ACL is ACL1 and / or ACL2 from Yarrowia lipolytica, for example: i) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or ii) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1) produces glycerol-3-phosphate from dihydroxyacetone, which derives from the glycolysis pathway. Glycerol-3-phosphate is essential for the production of TAGs, as it forms the backbone that binds the three fatty acids together. Inside the glycolytic pathway there is bifurcation that can lead to a competition. On one hand, the metabolites can be directed toward the production of pyruvate, with the consequent production of acetyl- CoA essential for the production of fatty acids. On the other hand, the metabolites can be directed toward the production of glycerol-3-phosphate through the action of GPD1. Since both molecules are essential for the production of TAGs, it is necessary to achieve a proper balance between these alternative pathways. In some embodiments it is considered that overexpression of GPD1 has a beneficial effect on lipid production. Accordingly in some embodiments the yeast cell of the invention is engineered to express or overexpress NAD-dependent cytosolic glycerol- 3-phosphate dehydrogenase (GPD1). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress GDP1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress GDP1. The GPD1 may be any GPD1. In some embodiments the GPD1 is from Yarrowia lipolytica, for example a GPD1 from Yarrowia with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13. In addition to storing carbon as triacylglycerols, carbon can alternatively be stored by yeast as glycogen. In some embodiments it is considered to be advantageous if the ability of the yeast cell to store glycogen is removed. Accordingly in some embodiments the yeast cell is engineered so as to have a reduced ability to store carbon, for example to store carbon as glycogen. For example in some embodiments the yeast is engineered to as to have a disrupted glycogen synthesis pathway. For example in some embodiments the yeast is engineered so as to have an underexpression or knockout of glycogen synthase (GSY1). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to underexpress or have a knockout of overexpress GSY1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to underexpress or have a knockout of GSY1. In addition to engineering the yeast to have an improved ability to produce lipids and triacylglycerols, it is considered to be beneficial if the yeast is additionally or instead engineered to have a reduced ability to degrade lipids. For example in some embodiments the yeast is engineered to have an underexpression or knockout of triacylglycerol lipase (TGL4). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to underexpress or have a knockout of TGL4. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to underexpress or have a knockout of TGL4. As set out above it is also considered to be beneficial in some embodiments if the yeast is engineered so as to remove the ability of the yeast to produce hyphae. In some embodiments then the yeast is engineered to as to have a reduced expression of or a knockout of the Mhy1p gene or equivalent. In some embodiments where the yeast is Yarrowia lipolytica, the yeast as a knockout or reduced expression of Mhy1p. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to have a reduced expression of a knockout of Mhy1p. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to have a reduced expression or a knockout of Mhy1p. In some embodiments it is considered to be advantageous if the yeast has an overexpression of fatty acid synthase (FAS). Accordingly in one embodiment the yeast of the invention has been engineered so as to have an overexpression of FAS, for example FAS from Yarrowia lipolytica, and / or overexpression of FAS from Rhodosporidium toruloides. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress FAS. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress FAS. In some embodiments it is considered to be advantageous if the yeast has been engineered to express or overexpress pyruvate dehydrogenase, for example pyruvate dehydrogenase from a bacterial species, for example from E.coli. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress pyruvate dehydrogenase from a bacterial species such as E. coli. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress pyruvate dehydrogenase from a bacterial species, such as E. coli. In some embodiments it is considered to be advantageous if the yeast has been engineered to have an overexpression of acyl-CoA-binding protein ACB1. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress ACB1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress ACB1. In some embodiments it is considered to be advantageous if the yeast has been engineered so as to have a reduced expression or a knockout of URE2. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to reduced expression of or a knockout of URE2. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to have reduced expression or a knockout of URE2. In some embodiments it is considered to be advantageous if the yeast has been engineered so as to have an expression or overexpression of 6- phosphogluconolactonase (SOL3). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress SOL3. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress SOL3. In some embodiments it is considered to be advantageous if the yeast has been engineered to overexpress SLC1 (1-acylglycerol-3-phosphate O-acyltransferase), which is involved in the synthesis of TAGs. For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to express or overexpress SLC1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to express or overexpress SLC1. In the same or different embodiments, it is considered to be advantageous if the expression of particular endogenous genes is reduced, or knocked out completely. For example in some embodiments the yeast has been engineered to as to have reduced expression, or a gene knockout, of PEX10 (Peroxisomal membrane E3 ubiquitin ligase). In the same or different embodiments it is considered to be advantageous if the yeast has been engineered to as to have reduced expression or a gene knockout of MFE1 (a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family). For example the yeast cell set out above that expresses a heterologous DGA2 may be engineered to have a reduced expression or a knockout of PEX10 and / or MFE1. Alternatively the yeast cell may not have been engineered to express a heterologous DGA2 but has been engineered to have reduced expression of or a knockout of PEX10 and / or MFE1. The skilled person is well equipped to generate knockdowns or knockouts, and a reduction in expression of the relevant genes can be achieved by any appropriate means. For example a one or more of the relevant genes could be knocked out by physically disrupting the DNA that encodes for the protein, for example via homologous recombination. Other suitable means include RNAi based methods, or CRISPR based methods. The skilled person will appreciate what is meant by expression or overexpression. In some instances the yeast may not have its own endogenous version of the protein that is to be expressed. In those cases, the yeast can be engineered to express a particular gene and protein. The yeast can also be engineered to express high quantities of said protein. In other embodiments the yeast may already have its own endogenous version of the relevant gene and protein, and so inherently expresses said gene and protein. However in many instances set out herein, it is considered to be beneficial if the expression of said gene and protein is a high or very high expression. In these instances it is appropriate to refer to an overexpression of said gene or protein. By overexpression we include the meaning that said gene or protein is expressed to a higher level than the expression of said gene or protein in a corresponding wildtype strain, for example strain W29. In other embodiments said gene or protein is expressed to a higher level in an engineered strain than the expression of said gene or protein in a corresponding unengineered strain – i.e. the strain absent the specific engineering modifications that result in the overexpression. For example in some embodiments a strain may already be engineered to increase the expression of protein X to produce a first engineered strain. However, and as set out herein, the expression level of protein X in the first engineered strain may not be sufficient to exert the desired effect. In those instances a second strain may be generated in which the expression level of protein X is greater than the expression level of protein X in the first engineered strain. The first engineered strain and the second engineered strain may be related to one another, or may be unrelated strains. In some particular embodiments, said overexpression of DGA is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of DGA under the control of the TEFin promoter. In some particular embodiments, said overexpression of GapC is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of GapC under the control of the GPDp promoter. In some particular embodiments, said overexpression of MCE2 is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of MCE2 under the control of the TEFin promoter. The skilled person will appreciate that the expression of genes and proteins can be via episomally maintained nucleic acid constructs, or genomically integrated nucleic acid constructs. In some embodiments the yeast of the invention comprises one or more genomically integrated copies of said corresponding gene so as to result in expression or overexpression of said gene, e.g. DGA, MCE2 for example. In some embodiments, the yeast cell of the invention comprises at least one genomically integrated expression cassette. In some embodiments said expression cassette comprises a promoter operably linked to one or more gene encoding any one or more of: DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1,FAS, pyruvate dehydrogenase, ACB1, 6- phosphogluconolactonase, wherein said DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1, FAS, pyruvate dehydrogenase, ACB1, 6-phosphogluconolactonase, Preferences for each of these proteins and enzymes is as set out elsewhere herein. In some embodiments the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genomically integrated cassettes, for example wherein at least two of the genomically integrated cassettes encode the same gene. Preferences for the oleaginous yeast cell of the invention are set out elsewhere, for example in preferred embodiments the oleaginous yeast cell of the invention expresses the heterologous DGA2 gene as described herein, and does not comprise any further edits that relate to increasing lipid production. However in some instances the yeast cell that expresses the heterologous DGA2 may comprise one or more other overexpressions, underexpressions or knockouts as set out herein. In some embodiments each expression cassette, for example each genomically integrated expression cassette encodes a different protein. In other embodiments at least two of the expression cassettes, for example at least two genomically integrated expression cassette encodes the same protein. In this way it is considered that a higher expression level of said protein can be achieved. For example, in some embodiments the yeast cell of the invention comprises at least two genomically integrated cassettes, wherein: a) the at least two cassettes comprise a DGA gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a DGA gene; b) the at least two cassettes comprise a GAPC gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a GAPC gene; and / or the at least two cassettes comprise a MCE2 gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a MCE2 gene. Preferably the oleaginous yeast cell of the invention comprises only one DGA expression cassette that is a DGA2 expression cassette, preferably where the DGA2 is a DGA2 is from Claviceps purpurea, and preferably has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2; and preferably where the promoter in the cassette is the 4UAS-pTEF promoter. As set out above, in some instances the DGA2 expression cassette comprises only one copy of the DGA2 gene. As described above, in some embodiments said overexpression is episomal, for example wherein said corresponding gene is expressed from a plasmid, for example a multicopy plasmid, for example a high copy plasmid. The skilled person will appreciate that in order for a protein to be expressed or overexpressed, the coding region must be operably linked to a promoter. The promoters used in the invention can be any promoter. Preferably in the context of DGA2 expression the promoter is the 4UAS-pTEF promoter. In some embodiments the expression or overexpression is driven by any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) In some embodiments said expression or overexpression is not driven by the TEFin promoter. In some particular embodiments: a) The diacylglycerol acyltransferase (DGA) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) b) the acetyl-coA-carboxylase (ACC1) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) c) the glyceraldehyde-3-phosphate dehydrogenase (GapC) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) d) the NADP-dependant malic enzyme (MCE2) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). and / or e) the NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). Preferably the DGA is DGA2 and is operably linked to the 4UAS-pTEF promoter. In one particular embodiment the yeast cell of the invention comprises an overexpression of all of DGA, ACC1, GapC and MCE2, for example where each of DGA, ACC1, GapC and MCE2 is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). Preferably the DGA is DGA2 and is operably linked to the 4UAS-pTEF promoter. In some embodiments the yeast cell comprises an overexpression of all of DGA, ACC1, GapC and MCE2, and wherein: the DGA is operably linked to the pTEF promoter, the ACC1 is operably linked to the pMnDH2 promoter, the GapC is operably linked to the pGPD1 promoter, and the MCE2 is operably linked to the pTEF promoter. However in preferred embodiments the DGA is DGA2 and is operably linked to the 4UAS-pTEF promoter. In further embodiments, the yeast cell comprises: a genomically integrated DGA cassette comprising a pTEF promoter operably linked to a DGA gene and wherein said DGA cassette is genomically integrated into a least two loci within the yeast genome and wherein said DGA is as described herein; a genomically integrated ACC1 cassette comprising a pMnDH2 promoter operably linked to an ACC1 gene and wherein said ACC1 is defined as described herein; a genomically integrated GapC cassette comprising a pGPD1 promoter operably linked to a GapC gene and wherein said GapC gene is as described herein; and a genomically integrated MCE2 cassette comprising a pTEF promoter operably linked to a MCE2 gene and wherein said MCE2 gene is as described herein. Preferably the DGA is DGA2 and is operably linked to the 4UAS-pTEF promoter. In other embodiments, the yeast cell comprises: a genomically integrated DGA cassette comprising a pTEF promoter operably linked to a DGA gene and wherein said DGA is defined in claim 1; a genomically integrated ACC1 cassette comprising a pMnDH2 promoter operably linked to an ACC1 gene and wherein said ACC1 is defined in claim 1; a genomically integrated GapC cassette comprising a pGPD1 promoter operably linked to a GapC gene and wherein said GapC cassette is genomically integrated into a least two loci within the yeast genome and wherein said GapC gene is defined in claim 1; and a genomically integrated MCE2 cassette comprising a pTEF promoter operably linked to a MCE2 gene and wherein said MCE2 cassette is genomically integrated into a least two loci within the yeast genome and wherein said MCE2 gene is defined in claim 1. Preferably the DGA is DGA2 and is operably linked to the 4UAS-pTEF promoter. The skilled person will appreciate that in order to express a gene, other various standard features are required, for example a transcriptional terminator. In some embodiments the expression cassette(s) comprise a transcription termination, for example a transcription terminator selected from the group comprising or consisting of the following terminators: i) tLip2 [SEQ ID NO: 20]; or ii) tXPR2. In some specific embodiments the yeast cell does not compromise or consist of: i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and / or iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:17. In other embodiments, the overexpression is not an overexpression of DGA1 from Lipomyces starkeyi; and / or the overexpression is not an overexpression of DGA1 from Rhodosporidum toruloides. In some embodiments, the yeast cell does not comprise an overexpression of any one or more of ACC1, GapC and / or MCE2. In other embodiments, the yeast cell does not comprise: i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:17. The yeast cells of the invention are able to produce high levels of lipids such as triacylglycerols. In some embodiments a culture of the yeast cells of the invention produces a lipid titre of at least 20 g / L, for example at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L or more. In some embodiments the yeast cells of the invention are able to produce a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more, optionally wherein the wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more when the culture conditions comprise active agitation in an aerated fermenter. In some embodiments a culture of the yeast cells produces a lipid or triacylglycerol content of at least 0.2 g / g CDW, for example at least 0.225, 0.25, 0.275, 0.30, 0.325, 0.35, 0.375, 0.40, 0.425, 0.45, 0.475, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00. The skilled person will appreciate that the invention also provides accompanying methods that utilise the yeast cells of the invention to produce lipids or triacylglycerols. Accordingly, the invention provides a method of producing lipids or triacylglycerols wherein the method comprises culturing a population of yeast cells according to the invention. In some embodiments it is considered to be useful if the culture media comprises urea. For example where the yeast is a Yarrowia species, for example a Yarrowia lipolytica yeast, in some embodiments the culture media comprises urea. It is also considered to be useful in some embodiments if the population of yeast cells are cultured under conditions of nitrogen starvation. The method of producing lipids or triaclyglycerols in some embodiments further comprises purifying or isolating the lipids or triacylglycerols from the yeast cells or from the culture media. The invention also provides a composition comprising lipids prepared by the method according to the invention. The skilled person will appreciate that the invention also provides the various nucleic acids and expression cassettes for putting the various overexpressions / knockouts etc into practice. For example the invention provides a nucleic acid that is a DGA2 expression cassette, wherein the expression cassette comprises or consists of the DGA2 gene from Claviceps purpurea operably linked to a promoter selected from the group consisting of: pTEF- 4UAS promoter (SEQ ID NO: 16); the 2UAS1-pTEF promoter; the 3UAS1-pTEF promoter; the 4UAS1-pTEF promoter; the 8UAS1-pTEF promoter; and the hp8d promoter. In preferred embodiments the nucleic acid is a DGA2 expression cassette comprising or consisting of the DGA2 gene from Claviceps purpurea operably linked to the 4UAS-pTEF promoter. The DGA2 gene may have the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2. The cassette may comprise one or more additional features such as a terminator. In some embodiments the invention provides a nucleic acid that is an expression cassette, wherein the expression cassette comprises or consists of: a) a DGA expression cassette, wherein the DGA expression cassette comprises a DGA-driving promoter operably linked to a sequence that encodes a diacylglycerol acyltransferase (DGA); b) an ACC1 expression cassette, wherein the ACC1 expression cassette comprises an ACC1-driving promoter operably linked to a sequence that encodes an acetyl-coA- carboxylase (ACC1); c) a GapC expression cassette, wherein the GapC expression cassette comprises a GapC-driving promoter operably linked to a sequence that encodes a glyceraldehyde- 3-phosphate dehydrogenase (GapC); d) a MCE2 expression cassette, wherein the MCE2 expression cassette comprises a MCE2-driving promoter operably linked to a sequence that encodes a NADP-dependant malic enzyme (MCE2); and / or e) a ATP:citrate lyase (ACL) expression cassette, wherein the ACL expression cassette comprises a ACL-driving promoter operably linked to a sequence that encodes an ACL enzyme. Preferences for the various genes, proteins and promoters are as set out elsewhere herein. For example in some embodiments: a) DGA is selected from the group comprising or consisting of: i) DGA1 or DGA2 from Yarrowia lipolytica, for example DGA1 having the sequence of SEQ ID NO: 1 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; or ii) DGA2 (or DGAT1) from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; and / or iii) soluble DGA3 from Rhodotorula glutinis; b) the ACC1 is selected from the group comprising or consisting of: i) ACC1 from Yarrowia lipolytica, for example ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; ii) ACC1 that has been engineered to be insensitive to deactivation via phosphorylation, for example ACC1 that comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2 or that has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine; c) the glyceraldehyde-3-phosphate dehydrogenase (GapC) is from Clostridium acetobutylicum, for example a GapC from Clostridium acetobutylicum having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; d) the NADP-dependant malic enzyme (MCE2) is from Mucor circinelloides, for example a MCE2 from Mucor circinelloides having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4; and / or e) the ATP:citrate lyase (ACL) is ACL1 and / or ACL2 from Yarrowia lipolytica, for example: i) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or ii) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. As described in relation to the yeast cell, in the context of the nucleic acids and expression cassettes of the invention, any one or more or all of the DGA-driving promoter, the ACC1-driving promoter, the GapC-driving promoter the MCE2-driving promoter and / or the ACL-driving promoter may be selected from group of promoters comprising or consisting of: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). Preferably where the DGA is DGA2 the promoter is the 4-UAS pTEF promoter. More particularly where the DGA is DGA2 from Claviceps purpurea the promoter is 4-UAS pTEF. The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the invention provides: a Yarrowia lipolytica cell that comprises a single genome integrated DGA2 expression cassette wherein the DGA2 gene is the Claviceps purpurea DGA2 gene under the control of the 4UAS-pTEF promoter and where the DGA2 expression cassette comprises only one copy of the DGA2 gene, and wherein the yeast cell does not comprise: A) Any copy of DGA1 other than the native Y. lipolytica DGA1 at the native locus under the control of the native promoter; and B) A copy of DGA2 other than i) the Y. lipolytica DGA2 at the native locus and under control of the native DGA2 promoter; and ii) the Claviceps purpurea DGA2 present in the genomically integrated DGA2 expression cassette; and C) a knockout or disruption of the MHY1 gene so as to reduce or prevent hyphal growth; And may also: D) comprise only the native Δ9 desaturase and / or the native Δ12 desaturase (i.e. does not comprise a heterologous Δ9 desaturase and / or heterologous native Δ12 desaturase); E) not comprise any one or more or all of: i) an A. adeninivorans Δ9 desaturase; ii) E. coli phosphotransferase; iii) Herpes simplex thymidine kinase. The invention also provides: a Yarrowia lipolytica cell that comprises an overexpression of DGA1 wherein DGA1 is under the control of pTEF-4UAS promoter and an overexpression of GPD1; and a yeast cell that comprises an overexpression of DGA1 and ACL2; and a yeast cell that comprises an overexpression of DGA1 and a gene knockout of PEX10 and / or MFE1. The invention also provides: a Yarrowia lipolytica cell that comprises a genome-integrated DGA2 expression cassette wherein the DGA2 gene is the Claviceps purpurea DGA2 gene under the control of the 4UAS-pTEF promoter and where the DGA2 expression cassette comprises only one copy of the DGA2 gene, and wherein the yeast cell does not comprise: A) Any copy of DGA1 other than the native Y. lipolytica DGA1 at the native locus under the control of the native promoter; and B) A copy of DGA2 other than i) the Y. lipolytica DGA2 at the native locus and under control of the native DGA2 promoter; and ii) the Claviceps purpurea DGA2 present in the genomically integrated DGA2 expression cassette; and C) a knockout or disruption of the MHY1 gene so as to reduce or prevent hyphal growth; And may also: D) comprise only the native Δ9 desaturase and / or the native Δ12 desaturase (i.e. does not comprise a heterologous Δ9 desaturase and / or heterologous native Δ12 desaturase); E) not comprise any one or more or all of: i) an A. adeninivorans Δ9 desaturase; ii) E. coli phosphotransferase; iii) Herpes simplex thymidine kinase. The invention also provides the following numbered embodiment paragraphs: Embodiments 1. An oleaginous yeast cell that has been engineered to have any one or more of the following: a) Overexpression of diacylglycerol acyltransferase (DGA), for example DGA1 or DGA2 or DGA3 or any combination thereof, for example: i) DGA1 or DGA2 from Yarrowia lipolytica, for example DGA1 having the sequence of SEQ ID NO: 1 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; ii) DGA2 (or DGAT1) from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; and / or iii) soluble DGA3 from Rhodotorula glutinis; b) increased production of NADPH, for example: i) Overexpression of NADP-dependant glyceraldehyde-3-phosphate dehydrogenase (GapC), for example GapC from Clostridium acetobutylicum, for example a GapC from Clostridium acetobutylicum having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; and / or ii) Overexpression of NADP-dependant malic enzyme (MCE2), for example MCE2 from Mucor circinelloides, for example a MCE2 from Mucor circinelloides having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4 c) Overexpression of acetyl-CoA-carboxylase (ACC1), for example ACC1 from Yarrowia lipolytica, for example ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; d) express or overexpress ACC1 that has been engineered to be insensitive to deactivation via phosphorylation, for example ACC1 that comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2 or that has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine; e) Overexpression of the ATP:citrate lyase (ACL), for example overexpression of ACL1 and / or ACL2, for example the ACL1 and / or ACL2 from Yarrowia lipolytica, for example: i) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or ii) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7; f) Overexpression of NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), for example GPD1 from Yarrowia lipolytica, for example a GPD1 from Yarrowia with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13; g) a reduced ability to store carbon for example to store glycogen for example engineered so as to have an underexpression or knockout of glycogen synthase (GSY1); h) a reduced ability to degrade lipids, for example engineered to have an underexpression or knockout of triacyglyceroal lipase (TGL); i) an inability to produce hyphae, for example where the yeast is an oleaginous yeast for example where the yeast is Yarrowia lipolytica the knockout is of the Mhy1p gene; j) overexpression of fatty acid synthase (FAS), for example FAS from Yarrowia lipolytica, and / or overexpression of FAS from Rhodosporidium toruloides; k) express or overexpress pyruvate dehydrogenase from a bacterial species, for example from E.coli; l) overexpression of acyl-CoA-binding protein ACB1; m) reduced expression or a knockout of URE2; n) overexpression of 6-phosphogluconolactonase ; o) overexpression of SLC1 (1-acylglycerol-3-phosphate O-acyltransferase); p) a reduced expression, or a gene knockout, of PEX10 (Peroxisomal membrane E3 ubiquitin ligase); and / or q) a reduced expression or a gene knockout of MFE1 (a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family). 2. The oleaginous yeast cell according to embodiment 1 wherein the yeast is of the genus Yarrowia, for example is a Yarrowia lipolytica yeast cell. 3. The yeast cell according to embodiment 1 or 2 wherein said overexpression is an overexpression with respect to a wild type strain, for example W29. 4. The yeast cell according to any of the preceding embodiments wherein: said overexpression of DGA is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of DGA under the control of the TEFin promoter; said overexpression of GapC is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of GapC under the control of the GPDp promoter; and / or said overexpression of MCE2 is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of MCE2 under the control of the TEFin promoter. 5. The yeast cell of any of the preceding embodiments wherein said overexpression is via one or more genomically integrated copies of said corresponding gene. 6. The yeast cell of any of the preceding embodiments wherein said yeast cell comprises at least one genomically integrated expression cassette, optionally wherein said expression cassette comprises a promoter operably linked to one or more gene encoding any one or more of: DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1, FAS, pyruvate dehydrogenase, ACB1, and / or 6-phosphogluconolactonase, wherein said DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1, FAS, pyruvate dehydrogenase, ACB1, and / or 6- phosphogluconolactonase, is as set out in embodiment 1, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genomically integrated cassettes, for example wherein at least two of the genomically integrated cassettes encode the same gene. 7. The yeast cell of embodiment 6 wherein the yeast cell comprises at least two genomically integrated cassettes, wherein: a) the at least two cassettes comprise a DGA gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a DGA gene; b) the at least two cassettes comprise a GAPC gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a GAPC gene; and / or the at least two cassettes comprise a MCE2 gene, for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes wherein said at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more genomically integrated cassettes comprises a MCE2 gene. 8. The yeast cell of any of the preceding embodiments wherein said overexpression is episomal, for example wherein said corresponding gene is expressed from a plasmid, for example a multicopy plasmid, for example a high copy plasmid. 8. The yeast cell according to any of the preceding embodiments wherein said overexpression is driven by any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) 10. The yeast cell according to any of the preceding embodiments wherein said overexpression is not driven by the TEFin promoter. 11. The yeast cell according to any of the preceding embodiments wherein: a) The diacylglycerol acyltransferase (DGA) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) b) the acetyl-coA-carboxylase (ACC1) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) c) the glyceraldehyde-3-phosphate dehydrogenase (GapC) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) d) the NADP-dependant malic enzyme (MCE2) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18); and / or e) the NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). 10. The yeast cell of any of the preceding embodiments wherein the yeast cell comprises an overexpression of all of DGA, ACC1, GapC and MCE2, for example where each of DGA, ACC1, GapC and MCE2 is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). 12. The yeast cell of any of the preceding embodiments wherein the yeast cell comprises an overexpression of all of DGA, ACC1, GapC and MCE2, and wherein: the DGA is operably linked to the pTEF promoter, the ACC1 is operably linked to the pMnDH2 promoter, the GapC is operably linked to the pGPD1 promoter, and the MCE2 is operably linked to the pTEF promoter. 13. The yeast cell of embodiment 12 wherein the yeast cell comprises: a genomically integrated DGA cassette comprising a pTEF promoter operably linked to a DGA gene and wherein said DGA cassette is genomically integrated into a least two loci within the yeast genome and wherein said DGA is defined in embodiment 1; a genomically integrated ACC1 cassette comprising a pMnDH2 promoter operably linked to an ACC1 gene and wherein said ACC1 is defined in embodiment 1; a genomically integrated GapC cassette comprising a pGPD1 promoter operably linked to a GapC gene and wherein said GapC gene is defined in embodiment 1; and a genomically integrated MCE2 cassette comprising a pTEF promoter operably linked to a MCE2 gene and wherein said MCE2 gene is defined in embodiment 1. 14. The yeast cell of embodiment 12 wherein the yeast cell comprises: a genomically integrated DGA cassette comprising a pTEF promoter operably linked to a DGA gene and wherein said DGA is defined in embodiment 1; a genomically integrated ACC1 cassette comprising a pMnDH2 promoter operably linked to an ACC1 gene and wherein said ACC1 is defined in embodiment 1; a genomically integrated GapC cassette comprising a pGPD1 promoter operably linked to a GapC gene and wherein said GapC cassette is genomically integrated into a least two loci within the yeast genome and wherein said GapC gene is defined in embodiment 1; and a genomically integrated MCE2 cassette comprising a pTEF promoter operably linked to a MCE2 gene and wherein said MCE2 cassette is genomically integrated into a least two loci within the yeast genome and wherein said MCE2 gene is defined in embodiment 1. 15. The yeast cell according to any of the preceding embodiments wherein the expression cassette(s) comprise a transcription termination, for example a transcription terminator selected from the group comprising or consisting of the following terminators: i) tLip2 [SEQ ID NO: 20]; or ii) tXPR2. 16. The cell of any of the preceding embodiments wherein the cell does not compromise: i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and / or iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:17. 17. The yeast cell of any of the preceding embodiments where: the overexpression is not an overexpression of DGA1 from Lipomyces starkeyi; the overexpression is not an overexpression of DGA1 from Rhodosporidum toruloides. 18. The yeast cell of any of the preceding embodiments where: The cell does not comprise an overexpression of any one or more of ACC1, GapC and / or MCE2. 19. The cell of any of the preceding embodiments wherein the cell does not comprise: i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:17. 20. The yeast cell according to any of the preceding embodiments wherein a culture of the yeast cells produces a lipid titre of at least 20 g / L, for example at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L or more.0 21. The yeast cell according to any of the preceding embodiments wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more, optionally wherein the wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more when the culture conditions comprise active agitation in an aerated fermenter. 21. The yeast cell according to any of the preceding embodiments wherein a culture of the yeast cells produces a lipid content of at least 0.2 g / g CDW, for example at least 0.225, 0.25, 0.275, 0.30, 0.325, 0.35, 0.375, 0.40, 0.425, 0.45, 0.475, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95 or 1.00. 22. A method of producing lipids wherein the method comprises culturing a population of yeast cells according to any of the preceding embodiments. 23. The method of embodiment 22 wherein the culture media comprises urea, for example where the yeast is a Yarrowia species, for example a Yarrowia lipolytica yeast, the culture media comprises urea. 24. The method of any of embodiments 22 or 23 wherein the method comprises culturing said population of yeast cells under conditions of nitrogen starvation. 25. A composition comprising lipids prepared by the method according to any of embodiments 22-24. 26. A nucleic acid that is an expression cassette, wherein the expression cassette comprises or consists of: a) a DGA expression cassette, wherein the DGA expression cassette comprises a DGA- driving promoter operably linked to a sequence that encodes a diacylglycerol acyltransferase (DGA); b) an ACC1 expression cassette, wherein the ACC1 expression cassette comprises an ACC1-driving promoter operably linked to a sequence that encodes an acetyl-coA- carboxylase (ACC1); c) a GapC expression cassette, wherein the GapC expression cassette comprises a GapC-driving promoter operably linked to a sequence that encodes a glyceraldehyde- 3-phosphate dehydrogenase (GapC); d) a MCE2 expression cassette, wherein the MCE2 expression cassette comprises a MCE2-driving promoter operably linked to a sequence that encodes a NADP-dependant malic enzyme (MCE2); and / or e) a ATP:citrate lyase (ACL) expression cassette, wherein the ACL expression cassette comprises a ACL-driving promoter operably linked to a sequence that encodes an ACL enzyme. 27. The expression cassette according to embodiment 26 wherein: a) DGA is selected from the group comprising or consisting of: i) DGA1 or DGA2 from Yarrowia lipolytica, for example DGA1 having the sequence of SEQ ID NO: 1 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; or ii) DGA2 (or DGAT1) from Claviceps purpurea for example DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; and / or iii) soluble DGA3 from Rhodotorula glutinis; b) the ACC1 is selected from the group comprising or consisting of: i) ACC1 from Yarrowia lipolytica, for example ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; ii) ACC1 that has been engineered to be insensitive to deactivation via phosphorylation, for example ACC1 that comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2 or that has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine; c) the glyceraldehyde-3-phosphate dehydrogenase (GapC) is from Clostridium acetobutylicum, for example a GapC from Clostridium acetobutylicum having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; d) the NADP-dependant malic enzyme (MCE2) is from Mucor circinelloides, for example a MCE2 from Mucor circinelloides having a sequence that is codon optimised for expression in Yarrowia lipolytic, for example having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4; and / or e) the ATP:citrate lyase (ACL) is ACL1 and / or ACL2 from Yarrowia lipolytica, for example: i) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or ii) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. 28. The nucleic acid according to any of embodiments 26 or 27 wherein any one or more or all of the DGA-driving promoter, the ACC1-driving promoter, the GapC-driving promoter the MCE2-driving promoter, and / or the ACL-driving promoter are selected from group of promoters comprising or consisting of: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). Disclosures The invention also provides the following numbered disclosures: 1. An oleaginous yeast cell that has been engineered to have any one or more of the following: a) Overexpression of diacylglycerol acyltransferase (DGA), optionally DGA1 or DGA2 or DGA3 or any combination thereof, optionally: i) DGA1 or DGA2 from Yarrowia lipolytica, optionally DGA1 having the sequence of SEQ ID NO: 1 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1; ii) DGA2 (or DGAT1) from Claviceps purpurea optionally DGA2 having the sequence of SEQ ID NO: 5 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5; and / or iii) soluble DGA3 from Rhodotorula glutinis. 2. The oleaginous yeast cell of disclosure 1 wherein the yeast cell has been further engineered so as to have increased production of NADPH, optionally: i) Overexpression of NADP-dependant glyceraldehyde-3-phosphate dehydrogenase (GapC), optionally GapC from Clostridium acetobutylicum, optionally a GapC from Clostridium acetobutylicum having a sequence that is codon optimised for expression in Yarrowia lipolytica, optionally having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; and / or ii) Overexpression of NADP-dependant malic enzyme (MCE2), optionally MCE2 from Mucor circinelloides, optionally a MCE2 from Mucor circinelloides having a sequence that is codon optimised for expression in Yarrowia lipolytica, optionally having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4. 3. The oleaginous yeast cell of any of disclosures 1 or 2 wherein the yeast cell has been engineered so as to have an overexpression of acetyl-CoA-carboxylase (ACC1), optionally ACC1 from Yarrowia lipolytica, optionally ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. 4. The oleaginous yeast cell of any one or more of disclosures 1-3 wherein the yeast cell has been engineered so a to have an overexpression of ACC1 that has been engineered to be insensitive to deactivation via phosphorylation, optionally ACC1 that comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2 or that has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine. 5. The oleaginous yeast cell of any one or more of disclosures 1-4 wherein the yeast cell has been engineered so as to have an overexpression of the ATP:citrate lyase (ACL), optionally overexpression of ACL1 and / or ACL2, optionally the ACL1 and / or ACL2 from Yarrowia lipolytica, optionally: i) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or ii) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7. 6. The oleaginous yeast cell of any one or more of disclosures 1-5 wherein the yeast cell has been engineered so as to have an overexpression of NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), optionally GPD1 from Yarrowia lipolytica, optionally a GPD1 from Yarrowia with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13. 7. The oleaginous yeast cell of any one or more of disclosures 1-6 wherein the yeast cell has been engineered so as to have a reduced ability to store carbon optionally to store glycogen optionally engineered so as to have an underexpression or knockout of glycogen synthase (GSY1). 8. The oleaginous yeast cell of any one or more of disclosures 1-7 wherein the yeast cell has been engineered so as to have a reduced ability to degrade lipids, optionally engineered to have an underexpression or knockout of triacyglyceroal lipase (TGL). 9. The oleaginous yeast cell of any one or more of disclosures 1-8 wherein the yeast cell has been engineered so as to have an inability to produce hyphae, optionally where the yeast is an oleaginous yeast optionally where the yeast is Yarrowia lipolytica the knockout is of the Mhy1p gene. 10. The oleaginous yeast cell of any one or more of disclosures 1-9 wherein the yeast cell has been engineered so as to have an overexpression of fatty acid synthase (FAS), optionally FAS from Yarrowia lipolytica, and / or overexpression of FAS from Rhodosporidium toruloides. 11. The oleaginous yeast cell of any one or more of disclosures 1-10 wherein the yeast cell has been engineered so as to express or overexpress pyruvate dehydrogenase from a bacterial species, optionally from E.coli. 12. The oleaginous yeast cell of any one or more of disclosures 1-11 wherein the yeast cell has been engineered so as to have an overexpression of acyl-CoA-binding protein ACB1. 13. The oleaginous yeast cell of any one or more of disclosures 1-12 wherein the yeast cell has been engineered so as to have reduced expression or a knockout of URE2. 14. The oleaginous yeast cell of any one or more of disclosures 1-13 wherein the yeast cell has been engineered so as to have an overexpression of 6- phosphogluconolactonase. 15. The oleaginous yeast cell of any one or more of disclosures 1-14 wherein the yeast cell has been engineered so as to have an overexpression of of SLC1 (1- acylglycerol-3-phosphate O-acyltransferase). 16 The oleaginous yeast cell of any one or more of disclosures 1-15 wherein the yeast cell has been engineered so as to have a reduced expression, or a gene knockout, of PEX10 (Peroxisomal membrane E3 ubiquitin ligase). 17. The oleaginous yeast cell of any one or more of disclosures 1-16 wherein the yeast cell has been engineered so as to have a reduced expression or a gene knockout of MFE1 (a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family). 18. The oleaginous yeast cell according to any one of disclosures 1-17 wherein the yeast is of the genus Yarrowia, optionally is a Yarrowia lipolytica yeast cell. 19. The oleaginous yeast cell of any one or more of disclosures 1-18 wherein a) said overexpression is an overexpression with respect to a wild type strain, optionally W29; and b) said reduced expression is a reduced expression with with respect to a wild type strain, optionally W29. 20. The yeast cell of any of the preceding disclosures wherein said yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genomically integrated cassettes, wherein at least two of the genomically integrated cassettes encode the same gene selected from the group comprising or consisting of: DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1, FAS, pyruvate dehydrogenase, ACB1, and / or 6-phosphogluconolactonase, optionally wherein said DGA, GapC, MCE2, ACC1, ACL1, ACL2, GPD1, FAS, pyruvate dehydrogenase, ACB1, and / or 6- phosphogluconolactonase, is as set out in any of the preceding disclosures. 21. The yeast cell according to any of the preceding disclosures wherein said overexpression is driven by any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). 22. The yeast cell according to any of the preceding disclosures wherein said overexpression is not driven by the TEFin promoter. 23. The yeast cell of any of the preceding disclosures wherein the yeast cell comprises an overexpression of all of DGA, ACC1, GapC and MCE2, and wherein each of DGA, ACC1, GapC and MCE2 is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18). 24. The yeast cell of any of the preceding disclosures wherein the yeast cell comprises an overexpression of all of DGA, ACC1, GapC and MCE2, and wherein: the DGA is operably linked to the pTEF promoter, the ACC1 is operably linked to the pMnDH2 promoter, the GapC is operably linked to the pGPD1 promoter, and the MCE2 is operably linked to the pTEF promoter. 25. The yeast cell according to any of the preceding disclosures wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more, optionally wherein the wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more when the culture conditions comprise active agitation in an aerated fermenter. 26. A method of producing lipids wherein the method comprises culturing a population of yeast cells according to any of the preceding disclosures, optionally where the culture media comprises urea, optionally where the yeast is a Yarrowia species, optionally a Yarrowia lipolytica yeast, the culture media comprises urea. Sequences Protein sequences >SEQ_ID_NO:_1_-_DGA1_from_Yarrowia_lipolytica: MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPTFLTIFMLCCAIPLLWPFVIAYVVYAVKDDSPSNGGVVKRY SPISRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAIISTIEYFLPAFMKRSLSINEQEQPAERDPLLS PVSPSSPGSQPDKWINHDSRYSRGESSGSNGHASGSELNGNGNNGTTNRRPLSSASAGSTASDSTLLNGSLNSYANQIIGENDPQLSPTK LKPTGRKYIFGYHPHGIIGMGAFGGIATEGAGWSKLFPGIPVSLMTLTNNFRVPLYREYLMSLGVASVSKKSCKALLKRNQSICIVVGGA QESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAFGENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMHARGVFNYDVGLVPYR RPVNIVVGSPIDLPYLPHPTDEEVSEYHDRYIAELQRIYNEHKDEYFIDWTEEGKGAPEFRMIE >SEQ_ID_NO:_2_ACC1_from_Yarrowia_lipolytica: MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVASHGGHTVINKVLIANNGIAAVKEIRSVRKWA YETFGDERAISFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFGVDAVWAGWGHASENPLLPESLAASPRKIVF IGPPGAAMRSLGDKISSTIVAQHAKVPCIPWSGTGVDEVVVDKSTNLVSVSEEVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGI RKVEREEDFEAAYHQVEGEIPGSPIFIMQLAGNARHLEVQLLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTFTAMEKAAVRL GKLVGYVSAGTVEYLYSHEDDKFYFLELNPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPHTTTPIDFDFSGEDAD KTQRRPVPRGHTTACRITSEDPGEGFKPSGGTMHELNFRSSSNVWGYFSVGNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSI RGDFRTTVEYLIKLLETPDFEDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRASEDSIATYMASLEKGQVPARDILKTLFPVD FIYEGQRYKFTATRSSEDSYTLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTCLLEVENDPTQLRSPSPGKLV KFLVENGDHVRANQPYAEIEVMKMYMTLTAQEDGIVQLMKQPGSTIEAGDILGILALDDPSKVKHAKPFEGQLPELGPPTLSGNKPHQRY EHCQNVLHNILLGFDNQVVMKSTLQEMVGLLRNPELPYLQWAHQVSSLHTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSG EVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLLQAYYDSEARFCGPNVRDEDVILKLREENRDSLRKVVMAQLSHSRVGAKNNLVLA LLDEYKVADQAGTDSPASNVHVAKYLRPVLRKIVELESRASAKVSLKAREILIQCALPSLKERTDQLEHILRSSVVESRYGEVGLEHRTP RADILKEVVDSKYIVFDVLAQFFAHDDPWIVLAALELYIRRACKAYSILDINYHQDSDLPPVISWRFRLPTMSSALYNSVVSSGSKTPTS PSVSRADSVSDFSYTVERDSAPARTGAIVAVPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASSVDTGLSNICN VMIGRVDESDDDDTLIARISQVIEDFKEDFEACSLRRITFSFGNSRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQLELARLSNFDIKPV HTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADRLMSDILDALEVIGTTNSDLNHIFINFSAVFALKPEEVEAA FGGFLERFGRRLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVSGYVVQSELYAEAKNDKGQWIFKSLGKPGSMHMRSINTPYPTKEWL QPKRYKAHLMGTTYCYDFPELFRQSIESDWKKYDGKAPDDLMTCNELILDEDSGELQEVNREPGANNVGMVAWKFEAKTPEYPRGRSFIV VANDITFQIGSFGPAEDQFFFKVTELARKLGIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLATLKPDTVVTT EIEEEGPNGVEKRHVIDYIVGEKDGLGVECLRGSGLIAGATSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINK LLGREVYSSNLQLGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGRTLE DGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDSIEMSTSEAGQVWYPNSAFKTSQAINDFNHG EALPLMILANWRGFSGGQRDMYNEVLKYGSFIVDALVDYKQPIMVYIPPTGELRGGSWVVVDPTINSDMMEMYADVESRGGVLEPEGMVG IKYRRDKLLDTMARLDPEYSSLKKQLEESPDSEELKVKLSVREKSLMPIYQQISVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRI RRRLVEEYLITKINSILPSCTRLECLARIKSWKPATLDQGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDRQGTLQGMKQA LASLSEAERAELLKGL* MAKIAINGFGRIGRLALRRILEVPGLEVVAINDLTDAKMLAHLFKYDSSQGRFNGEIEVKEGAFVVNGKEVKVFAEADPEKLPWGDLGID VVLECTGFFTKKEKAEAHVRAGAKKVVISAPAGNDLKTIVFNVNNEDLDGTETVISGASCTTNCLAPMAKVLNDKFGIEKGFMTTIHAFT NDQNTLDGPHRKGDLRRARAAAVSIIPNSTGAAKAISQVIPDLAGKLDGNAQRVPVPTGSITELVSVLKKKVTVEEINAAMKEAADESFG YTEDPIVSADVVGINYGSLFDATLTKIVDVNGSQLVKTAAWYDNEMSYTSQLVRTLAYFAKIAK* >SEQ_ID_NO:_4_MCE2_from_Mucor_circinelloides MSPIIEFVRRQLSSTKLHEEQQTATTNDLVSRSGYLNECKYEVRLNCINAGCLQKKLNYIGTAMDPAKRQRLGLNGLLPAGVETLEIQKA RALRVLRSKHNLLEKYILMAQLRTTNVRLFYKIVIDELETVQLAPVIYTPTVGTACLEYSTIYPFLAAPGVPDGLYLTKAELPELCQTIR NYRPTDTEGFEPEIAVISDGSRILGLGDLGTNGMGIPMGKLQLYVAGAGIDPRRTLPIILDLGTNNEKLLNDEFYIGLRQKRPNDEEFYQ TVDTVLTALHTVYPNLLIQFEDWSSEHAFGLLEKYQNQMLCFNDDIQGTGAVILSGVINAIRKVEKENQVSPRDHRIVFYGAGSAAIGVA RQIQSYFQIEHNMTEEEAKHVFWIVDSKGLVTTTRGDKLAQHKVYYARGDNEGQQYKELIDIVNYNLYSLIGLSSTTGAFNTQVLERLAS LNEQPIVFPLSNPATQAECTFEQAMEATNNKVIFASGTAFPAYTIKSTGEVNTPGQGNNMYIFPGLGLGACLANPAHFDRMIYEASKALA DSLTEEEISKAWLYPSLNYRSVSAIVAAAVCQETLNENLATSQAMMTQCKSHEDILDYVSAHMWSPDYGNNNSNQQAGKL* >SEQ_ID_NO:_5_DGA2_from_Claviceps_purpurea MSATGVDVANGRSGARRRNDTAVDETISAVTAEMRSSSHPTYRHVSAVHSTSRPSCLSHDSDAAPSFIGFRNLMVIVLVVGNVRLMIENL KKYGVLICLRCHSYKNEDIIIGGLLYFLIPCHLLVAYGIELAAARQARESRTRPPGQSDTASKSTEDDNKHFHSTWVLAAWAHIINMTLS FILTTFVVYYYVHHPLVGTLTEMHAVIVSLKTASYAFTNRDLRHAYLHPDKRKHIPELYLECPYPQNLTFGNLVYFWWAPTLVYQPVYPR TDKIRWVFVFKRLGEVCCLSAFIWFASFQYAAPVLRNSLDKIASLDFIMIFERLLKLSTISLVIWLAGFFALFQSFLNALAEVLRFGDRC FYDDWWNSESLGAYWRTWNRPVYTYFKRHVYVPMIGRGWSPWTASCTVFFVSAVLHEVLVGVPTHNIIGVAFVGMFLQLPLIALTAPMEK KKWGHTGRVMGNVIFWVSFTIFGQPFAALMYFYAWQAKYGSVSRQIVLVNPVEEAS* >SEQ_ID_NO:_6_ATP:citrate_lyase_(ACL1)_from_Yarrowia_lipolytica MSANENISRFDAPVGKEHPAYELFHNHTRSFVYGLQPRACQGMLDFDFICKRENPSVAGVIYPFGGQFVTKMYWGTKETLLPVYQQVEKA AAKHPEVDVVVNFASSRSVYSSTMELLEYPQFRTIAIIAEGVPERRAREILHKAQKKGVTIIGPATVGGIKPGCFKVGNTGGMMDNIVAS KLYRPGSVAYVSKSGGMSNELNNIISHTTDGVYEGIAIGGDRYPGTTFIDHILRYEADPKCKIIVLLGEVGGVEEYRVIEAVKNGQIKKP IVAWAIGTCASMFKTEVQFGHAGSMANSDLETAKAKNAAMKSAGFYVPDTFEDMPEVLAELYEKMVAKGELSRISEPEVPKIPIDYSWAQ ELGLIRKPAAFISTISDDRGQELLYAGMPISEVFKEDIGIGGVMSLLWFRRRLPDYASKFLEMVLMLTADHGPAVSGAMNTIITTRAGKD LISSLVAGLLTIGTRFGGALDGAATEFTTAYDKGLSPRQFVDTMRKQNKLIPGIGHRVKSRNNPDFRVELVKDFVKKNFPSTQLLDYALA VEEVTTSKKDNLILNVDGAIAVSFVDLMRSCGAFTVEETEDYLKNGVLNGLFVLGRSIGLIAHHLDQKRLKTGLYRHPWDDITYLVGQEA IQKKRVEISAGDVSKAKTRS* >SEQ_ID_NO:_7_ATP:citrate_lyase_(ACL2)_from_Yarrowia_lipolytica MSAKSIHEADGKALLAHFLSKAPVWAEQQPINTFEMGTPKLASLTFEDGVAPEQIFAAAEKTYPWLLESGAKFVAKPDQLIKRRGKAGLL VLNKSWEECKPWIAERAAKPINVEGIDGVLRTFLVEPFVPHDQKHEYYINIHSVREGDWILFYHEGGVDVGDVDAKAAKILIPVDIENEY PSNATLTKELLAHVPEDQHQTLLDFINRLYAVYVDLQFTYLEINPLVVIPTAQGVEVHYLDLAGKLDQTAEFECGPKWAAARSPAALGQV VTIDAGSTKVSIDAGPAMVFPAPFGRELSKEEAYIAELDSKTGASLKLTVLNAKGRIWTLVAGGGASVVYADAIASAGFADELANYGEYS GAPNETQTYEYAKTVLDLMTRGDAHPEGKVLFIGGGIANFTQVGSTFKGIIRAFRDYQSSLHNHKVKIYVRRGGPNWQEGLRLIKSAGDE LNLPMEIYGPDMHVSGIVPLALLGKRPKNVKPFGTGPSTEASTPLGV* >SEQ_ID_NO:_13:_NAD-dependent_cytosolic_glycerol-3- phosphate_dehydrogenase_(GPD1)_from_Yarrowia_lipolytica MSALLRSSLRFKHMSAVNRLTQQLRLLTASAPLSAANTAGKAPFKVAVVGSGNWGTTVAKIVAENCTAHPELFEPEVRVWVREEKVNGKN LTDIFNAEHENVRYLPKIKLPHNLIAEPDLLKAVEGANIIVFNLPHQFLAGVCKQLKGHVNPKARAISCLKGLDVTPQGVYLLSDVIENE TGLHCGVLSGANLATEIALEKYSETTVAYNRPKDFFGEGDVTNDVLKALFHRPYFHVRCVQDVAGVSIGGALKNVVALCAGFVEGKNWGD NAKAAIMRRGMLEMINFSKRFFPETDINTLTVESAGVADLITSCAGGRNFKVGRAFGKESGSGKTIQDVEKELLNGQSAQGVITCNEVHE LLKNKNMQKDFPLFESTWGIIHGELKIDDLPEILYHAN >SEQ_ID_NO:_30_FAS-alpha_subunit MSSKMPGGFSLTSARSYLDSRWGLAAGRQDSVLLVALMNEPKNRLGSEAEAKAYLDEQTQKYAASAGLNLSAPAGGAEGGNGGGAVIDSA AFDALTKDQRYLVQQQLELFANYLKQDLRQGSKVAAAQKEAMDILQAELDLWNSEHGEVYAEGIKPAFSALKARVYDSYWNWARQDSLSM YFDIVFGRLSTVDREIMAKCIHLMNRTNHNLIDYMQYHMDHVPVHKGATYELAKQLGLQLLENCKETLTEAPVYKDVSYPTGPQTTIDVK GNIVYNEVPRPNVRKLEQYVHEMACGGELTKDPSFVGEGVQGELKKLYSQISALAKTQTGSTLDIEALYSDLVAKISQAEDASKPVVENK AVSASITPGTLPFLHIKKKTELGAWNYDSETTATYLDGLEVAARDGLTFQGKTALITGAGAGSIGASILQGLISGGCKVIVTTSRYSRKV TEYYQSLYTKFGAKGSTLIVVPFNQGSKKDVDELVSFIYNDPKNGGLGWDLDFVVPFAALPENGIELEHIDSKSELAHRIMLTNLLRLLG NVKKQKVAHSYETRPAQVMLPLSPNHGNFGSDGLYSESKISLETLFNRWHTESWGSYLTIVGVVIGWTRGTGLMSANNITAEGLEQLGVR TFSQTEMAFSIMGLMTKDIVRLAQNSPVWADLNGGFQYIPDLKGVVGKIRRDIVETSEIRRAVAQETAIEQKVVNGPHADLPYQKVEVKP RANLKFDFPTLKSYAEVKELSPAGDALEGLLDLSSVIVVTGFAEVGPWGNARTRWDMEANGVFSLEGAIEMAWIMGLIKHHNGPLPGMPQ YSGWIDTKTKQPVDDRDIKTKYEDYLLEHAGIRLIEPELFHGYNPKKKTFLQEVIVEHDLEPFEASKESAEQFALEQGANVEIFAVPESD QWTVRLLKGAKLLIPKALKFDRLVAGQIPTGWDARRYGIPEDICDQVDPITLYALVSTVEALLASGITDPYEFYKYVHVSEVGNCSGSGM GGITALRGMFKDRFMDKPVQNDILQESFINTMSAWVNMLLLSSSGPIKTPVGACATAVESVDIGCETILSGKARICLVGGYDDFQEESSQ EFANMNATSNAETEITHGRTPAEMSRPITSTRAGFMEAQGAGTQVLMAADLAIAMGVPIYCIVGYVNTATDKIGRSVPAPGKGILTTARE HQTLKHANPLLNIKYRKRQLDSRLRDIKRWAEGEMEAIDIELDDVSDADKESFIQERSAHIQSQSDRMIREAKNSWGNAFFKQDARISPI RGALATYGLTIDDISVASFHGTSTKANEKNETTTVNAMLEHLGRTRGNPVYGIFQKYLTGHPKGAAGAWMLNGAIQCLNSGIIPGNRNAD NVDAYFEQCQHVVFPSRSLQTDGLKAASVTSFGFGQKGAQAIVIHPDYLYAALTPSEYSEYTTRVAQRYKKAYRYYHNAIAEESMFQAKD KAPYSAELEQEVYLDPLVRVHQNEDTEQYSFNAKDLAASAFVKNSHKDTAKVLANLTSQVSGSGKNVGVDVEAISAINIDNDTFLDRNFT ANEQAYCFKAPSPQSSFAGTWSAKEAVFKSLGVKSQGGGAELKSIEITRDGNGAPVVVLHGAAKDAAASKGISTVKVSISHDDSQAVAVA VAE* >SEQ_ID_NO_31_FAS-beta_subunit MYPTTGVNTPQSAASLRPLVLSHGQTEHSLLVPTSLYINCTTLRDQFYASLPPATEDKADDDEPSSSTELLAAFLGFTAKTVEEEPGPYD DVLSLVLNEFETRYLRGNDIHAVASSLLQDEDVPTTVGKIKRVIRAYYAARIACNRPIKAHSSALFRAASEDSDNVSLYAIFGGQGNTED YFEELREIYDIYQGLVGDFIRECGAQLLALSRDHIAAEKIYTKGFDIVKWLEHPETIPDFEYLISAPISVPIIGVIQLAHYAVTCRVLGL NPGQVRDNLKGATGHSQGLITAIAISASDSWDEFYNSASRILKIFFFIGVRVQQAYPSTFLPPSTLEDSVKQGEGKPTPMLSIRDLSLNQ VQEFVDATNLHLPEDKQIVVSLINGPRNVVVTGPPQSLYGLCLVLRKQKAETGLDQSRVPHSQRKLKFTHRFLPITSPFHSYLLEKSTDL IINDLESSGVEFVSSELKVPVYDTFDGSVLSQLPKGIVSRLVNLITHLPVKWEKATQFQASHIVDFGPGGASGLGLLTHKNKDGTGVRTI LAGVIDQPLEFGFKQELFDRQESSIVFAQNWAKEFSPKLVKISSTNEVYVDTKFSRLTGRAPIMVAGMTPTTVNPKFVAATMNSGYHIEL GGGGYFAPGMMTKALEHIEKNTPPGSGITINLIYVNPRLIQWGIPLIQELRQKGFPIEGLTIGAGVPSLEVANEWIQDLGVKHIAFKPGS IEAISSVIRIAKANPDFPIILQWTGGRGGGHHSFEDFHAPILQMYSKIRRCSNIVLIAGSGFGASTDSYPYLTGSWSRDFDYPPMPFDGI LVGSRVMVAKEAFTSLGAKQLIVDSPGVEDSEWEKTYDKPTGGVITVLSEMGEPIHKLATRGVLFWHEMDKTVFSLPKKKRLEVLKSKRA YIIKRLNDDFQKTWFAKNAQGQVCDLEDLTYAEVIQRLVDLMYVKKESRWIDVTLRNLAGTFIRRVEERFSTETGASSVLQSFSELDSEP EKVVERVFELFPASTTQIINAQDKDHFLMLCLNPMQKPVPFIPVLDDNFEFFFKKDSLWQCEDLAAVVDEDVGRICILQGPVAVKHSKIV NEPVKEILDSMHEGHIKQLLEDGEYAGNMANIPQVECFGGKPAQNFGDVALDSVMVLDDLNKTVFKIETGTSALPSAADWFSLLAGDKNS WRQVFLSTDTIVQTTKMISNPLHRLLEPIAGLQVEIEHPDEPENTVISAFEPINGKVTKVLELRKGAGDVISLQLIEARGVDRVPVALPL EFKYQPQIGYAPIVEVMTDRNTRIKEFYWKLWFGQDSKFEIDTDITEEIIGDDVTISGKAIADFVHAVGNKGEAFVGRSTSAGTVFAPMD FAIVLGWKAIIKAIFPRAIDADILRLVHLSNGFKMMPGADPLQMGDVVSATAKIDTVKNSATGKTVAVRGLLTRDGKPVMEVVSEFFYRG EFSDFQNTFERREEVPMQLTLKDAKAVAILCSKEWFEYNGDDTKDLEGKTIVFRNSSFIKYKNETVFSSVHTTGKVLMELPSKEVIEIAT VNYQAGESHGNPVIDYLERNGTTIEQPVEFEKPIPLSKADDLLSFKAPSSNEPYAGVSGDYNPIHVSRAFASYASLPGTITHGMYSSAAV RSLIEVWAAENNVSRVRAFSCQFQGMVLPNDEIVTRLEHVGMINGRKIIKVTSTNRETEAVVLSGEAEVEQPISTFVFTGQGSQEQGMGM DLYASSEVAKKVWDKADEHFLQNYGFSIIKIVVENPKELDIHFGGPKGKKIRDNYISMMFETIDEKTGNLISEKIFKEIDETTDSFTFKS PTGLLSATQFTQPALTLMEKASFEDMKAKGLVPVDATFAGHSLGEYSALASLGDVMPIESLVDVVFYRGMTMQVAVPRDAQGRSNYGMCA VNPSRISTTFNDAALRFVVDHISEQTKWLLEIVNYNVENSQYVTAGDLRALDTLTNVLNVLKLEKINIDKLLESLPLEKVKEHLSEIVTE VAKKSVAKPQPIELERGFAVIPLKGISVPFHSSYLRNGVKPFQNFLVKKVPKNAVKPANLIGKYIPNLTAKPFEITKEYFEEVYKLTGSE KVKSIINNWESYESKQ* >SEQ_ID_NO:_32_RtFAS-alpha_subunit MVAAQDLPLALSISFAPESSTISMTLFNQPEASKPALPLELKYKYDPSTPYAPIHEITEDRNQRIKQHYWDLWGLGNKADQGISQLKITD EFQGDLVTISADEIEAFCRVVGIEGEAYKRNHKAGMQVPLDFAIKLGWKAIMKPIFPSTIDGDLLKLVHLSNGFRVLPDTPTLQVGDVVT TTSRIESITNSDTGKTVSVRGVISLVSSADSKGKDASTEDRIPLIEVTSSFFYRGKFSDYAQTFSRVAHPTYSVPITTPEAVAVLQSKEW FQWDDDSKPLEVGTKLQFKVESNYVYADKSSYAMATVTGGAYVITPELKLAVKVATVDYTSEGEGVIQGDPVIEYLKRHGSALDQPIMLE NGGYSLTKAGQCTFTTPASNLDYSLTSGDTNPIHTNPYFASLAYLPGTITHGMHSSARTRKFVEQVAADNVGARVRKYEVGFTAMCLPSR KMEVRLKHVGMTADGNRLIKVETVDVEGGNVVLSGTAEVAQAPTAYVFTGQGSQEPGMGMELYANSPVARAVWDEADRHLGEVYGFSILE IVRTNPKEKTVHFGGLKGQATRQKYMDMSYTTTDHEGNVKTLPLFGDIDLRTSRYTFSSPTGLLYATQFAQIALVVTEKAAFEDMRAKGL VQKDCVFAGHSLGEYSALASIADILPISALVDVVFYRGITMQRAVERDHLNRSSYGMVAVNPSRIGKSFGDAALREVVDTIARRGNILIE VVNYNVEGQQYVVAGHLVALQSLTNVLNFLKIQKIDLAKLTETMSIEQVKEHLCEIVDECVQKARDLQAKTGFITLERGFATIPLPGIDV PFHSRYLWAGVMPFRTYLSKKVNPAHFNADLLVGRYIPNLTAVHYEVSKEYAERIHTQTSSPRLNKILKAWDEERWGAPENRNKLGYAIL IELLAYQFASPVRWIETQDILFRDFKFERLVELGPSPTLTGMATRTQKLKYDAHDSSVGIKRSIYCIAKHQKEIYYQFDDVAGEEAPAPA AVAPSAPAPKAAPVAAAPPPPAPVAAAPAAAVADEPLKAVDTLRIIIAQKLKKPVGEVPLTKSIKELVGGKSTLQNEILGDLQGEFSSAP EKGEEMPLQELGAALQQGYSGKLGKYTTGVISRMIGAKMPGGFGLSAVQGHLGKTYGLGAGRIDGVLLFAVTQEPAKRLANEGEAKAWVD SVAQGYASMAGISLAAGGGAAAAAPAMAFAAPAAAGGGAPAAVDEPLKATDTLRAIIAQKLKKQIPDVPLTKSIKDLVGGKSTLQNEILG DLQGEFSSAPEKGEEMPLQELGAALNQGYSGTLGKHTSGLVARMMGAKMPGGFGLSAAKAHLSKAHGLGPGRTDGALLVALTKEPEKRLG SEADAKAWLDGVAQAYASQAGITLGAGGGGGGAAVGGAGFMINTEQLDKMQEKQDNFVSQQVELFLRYLGKDSREGHRLADMQKAEVANL QEKLDSIAREHGDAYVQGIQPVFDPLKARHFNSSWNWVRQDALMMWMDILFGRLTTVDRDITARCLVIMNRADPSLIDYMQYTIDNTPVE RGEHYVLAKQFGQQLLDNCREMIGQAPLYKDVTFPTAPKTTVNAKGDIITEEVNRPGVSRLEKYVAEMAAGSKVTVASVNLDKVQEQVEK LYKLVKSQPQISKQHMTSIKSLYAEVVRGLGKDAGPPPVHKAGTRARRPSSQFLRPAAVSEATFLPEDKVPLLHLKRKIGNDWQYSSKLT SLYLDILKEIATSGVTFEHKNALMTGVGKGSIGIEIVKGLLAGGARVVITTSRYSRSTVEYYQAIYQEVGSKGSSLTVVPFNQGSKQDVE ALVDFIYSKDKGLGMDLDYILPFAALPENGREIDGIDDRSELAHRIMLTNLLRLLGAVKSKKAALKLTTRPTEVVLPLSPNHGLFGNDGL YSESKISLETLFNRWSSESWGEYLCLAGAVIGWTRGTGLMSATNSVAEGIEAQGCRTFSAKEMAFNILGLMHPLVFDVAQIEPVWADLNG GMDKLPDLANLTTEIRKKLNLTASTRRAIAKDNSFDYKVAHGPAMEQIHQRINVAPRANFSLPFPELKPIDAKSELAKLRGLIDLEKVVV MTGYAEVGPFGSSRTRWEMEANGTFSIQGTLELAYVMGLIKHFEGRLKDGTLYVGWVDAKTNEPLDDKDVKAAYEKHILAHTGIRLIEPE IFNGYDPKRKGFTQEIEIQHDLEPIEASEEDAARFKREHGALVDVYTEDGSKFFVKFKKGAKLHIPKAVAFDRLVAGQIPTGWSHKAFGI PDDIASQVDRTSLWALVSVAEALMMAGITDPYELYKWIHPSEVGSSLGSGMGGITSISKMFRDRREEKDVQKDILQETFINTVAGWVNLL LLSSSGPIKIPVGACATALQSVEIACDTILSGKAKIMVSGGYDDFSEEGSYEFANMKATSNSETEFAAGREPNEMSRPTTSTRAGFMESM GCGAQVLMSAKTAIEMGATIYGIVAYTATATDKAGRSIPAPGRGVMGTAREITSKYPSPILDVTYRRRQLEFRRKQISQWLENETELLKF EVSSHGQATKLPDDYVSERLASIEREAKRQEAEALATYGMLAGQDPTIAPLRRALAVWGLTIDDVGVASFHGTSTVANDKNESNAYNEQF RHLGRAKGNACPVIAQKWLTGHPKGGAAAWMLNGLAQVIQSGLVPGNRNADNIGEELRAFEYLLYPSKSIQTDGIKAGLLTSFGFGQVGG QALIVHPSLLIGALEPAQFEAYKKLNDQRKKWSYRRFNDFFTNGKLVIIKDGTPFTPEQENTTLLNPLVRAVPDKTGSYSMPKEFPATVP RSNNAEVANKLVSAAVGGAFGVGTDVELISAVPTSESFLERNFTQDEIAYCKAAPDFRASLAARWSAKEATFKALKTESKGAAASMQDIE VVSTSQGPTIKLHGEVEKIAQAAGITAFEVSLSHSEDVACAVVIAQK >SEQ_ID_NO:_33_RtFAS-beta_subunit MNGRATRSVTGTSTPVHTATTRPLVLLHPSTQTRISLHVPSTSQEWIAAEVARDTFQDWLHAAEKSGNLVGFEAAELDDEQAGEGDDEKE LVLTAYFLKHVAGLLPFPSTATSPATAAVLLAAFNHFASVYLSGTDVHTLTASLAAPVRALVISSFFLAKTKLEVEGLGKVLPKQSESAL LQKAATGQAEVFALFGGQGMNEVYFDELQTLHDLYTPLLTPFLARASEHLVSLAAAEQHTLLYDHSLDALAWLQDPSTRPEVPYLATCAV SLPLIGLTQLCQYVVYGKGSSLGPAELGAKFKGATGHSQGVVSALVIAHEYPPASKDGSDAWEPFYEQALRGLTVLFQIGLQGTLAFPSI AISPALESSSVENGEGVPTAMLAVTGLDLKSLEKKIAEVNGHVKSEGRDETVSISLYNGARAFVVTGAPKDLVGLADGLRKNRAPAGKDQ HATNFPSSATHAIDFGTGGASGIGSLCARNWEGRGIRTIMLGNRGEGVGAGKEAWGKKVPTEEKWNERFHPRLVRTSDGKIHLDTPFSRL LSKPPLMVGGMTPTTVKAGFVSAVLRAGYHIELAGGGHYNEKAVRAKVAEIQKLVNKPGMGITLNSLYINQRQWTFQFPLWAKMKQEGEP VEGLCVAAGIPSTEKAKEIIDTLREAGIKHVSFKPGSVDGIRQVVNIASANPDFPIILQWTGGRAGGHHSCEDFHAPILATYASIRQHPN IKLVAGSGFGSAEGCYPYLSGEWSEKQYGVARMPFDGFMFASWVMVAKEAHTSESVKQLIVDAPGVEDGQWEQTYDKPTGGILTVNSELG EPIHKVATRGVKLWAEFDKKVFSLSKEKQLAWLADNKKYVIDRLNADFQKPWFPAKADGSPCDLADMTYAEVNARLVRLMYVAHEKRWID PSLRNLVGDWIRRVEERLSNVNDSGIKISALQSYSELNEPEAFLKQFLAQYPQAEDQILASADVSYFLAISQRPGQKPVPFIPVLDANFS IWFKKDSLWQAEDIEAVFDQDPQRVCILQGPVAAKHCTSTQTPIAEMLGNIEHQLVKNVLDDYYGGDESQIPTIDYLAPPPKPVDAGAIL AENNIAHSVEELADGGKKHVYSINGVLPPTGDWHAALAGPKLDWLQAFLSNVSIQAGEQSIPNPVKKVLAPRHGQRVELTLNKDGQPLKL DVFGGL >SEQ_ID_NO:_34_EcPHD_(AceE,_E1) MSERFPNDVDPIETRDWLQAIESVIREEGVERAQYLIDQLLAEARKGGVNVAAGTGISNYINTIPVEEQPEYPGNLELERRIRSAIRWNA IMTVLRASKKDLELGGHMASFQSSATIYDVCFNHFFRARNEQDGGDLVYFQGHISPGVYARAFLEGRLTQEQLDNFRQEVHGNGLSSYPH PKLMPEFWQFPTVSMGLGPIGAIYQAKFLKYLEHRGLKDTSKQTVYAFLGDGEMDEPESKGAITIATREKLDNLVFVINCNLQRLDGPVT GNGKIINELEGIFEGAGWNVIKVMWGSRWDELLRKDTSGKLIQLMNETVDGDYQTFKSKDGAYVREHFFGKYPETAALVADWTDEQIWAL NRGGHDPKKIYAAFKKAQETKGKATVILAHTIKGYGMGDAAEGKNIAHQVKKMNMDGVRHIRDRFNVPVSDADIEKLPYITFPEGSEEHT YLHAQRQKLHGYLPSRQPNFTEKLELPSLQDFGALLEEQSKEISTTIAFVRALNVMLKNKSIKDRLVPIIADEARTFGMEGLFRQIGIYS PNGQQYTPQDREQVAYYKEDEKGQILQEGINELGAGCSWLAAATSYSTNNLPMIPFYIYYSMFGFQRIGDLCWAAGDQQARGFLIGGTSG RTTLNGEGLQHEDGHSHIQSLTIPNCISYDPAYAYEVAVIMHDGLERMYGEKQENVYYYITTLNENYHMPAMPEGAEEGIRKGIYKLETI EGSKGKVQLLGSGSILRHVREAAEILAKDYGVGSDVYSVTSFTELARDGQDCERWNMLHPLETPRVPYIAQVMNDAPAVASTDYMKLFAE QVRTYVPADDYRVLGTDGFGRSDSRENLRHHFEVDASYVVVAALGELAKRGEIDKKVVADAIAKFNIDADKVNPRLA >SEQ_ID_NO:_35_EcPHD_(AceF,_E2) MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVPSPQAGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEE KKEAAPAAAPAAAAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVEGDKASMEVPAPFAGTVKEIKVNVGDKVSTGSLIMVFEVA GEAGAAAPAAKQEAAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKVGDKVAAEQSLITVEGDKASMEVPAPFAGVVKELKVNVGDKVK TGSLIMIFEVEGAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHATPLIRRLAREFGVNLAKVKGTGRKGRILRED VQAYVKEAIKRAEAAPAATGGGIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLSRNWVMIPHVTHFDKTDITELEAFRKQQNEEAAK RKLDVKITPVVFIMKAVAAALEQMPRFNSSLSEDGQRLTLKKYINIGVAVDTPNGLVVPVFKDVNKKGIIELSRELMTISKKARDGKLTA GEMQGGCFTISSIGGLGTTHFAPIVNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADGARFITIINNTLSDIRRLVM >SEQ_ID_NO:_36_EcPHD_(LpdA,_E3) MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLNVGCIPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWK EKVINQLTGGLAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPIQLPFIPHEDPRIWDSTDALELKEVPERLLV MGGGIIGLEMGTVYHALGSQIDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLETKVTAVEAKEDGIYVTMEGKKAPAEPQRYDAVLVA IGRVPNGKNLDAGKAGVEVDDRGFIRVDKQLRTNVPHIFAIGDIVGQPMLAHKGVHEGHVAAEVIAGKKHYFDPKVIPSIAYTEPEVAWV GLTEKEAKEKGISYETATFPWAASGRAIASDCADGMTKLIFDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHE SVGLAAEVFEGSITDLPNPKAKKK >SEQ_ID_NO:_37_ACB1 MPSAEFTAAADSVQKLPKTPSDDELLELYGLYKQATVGDNNTDRPGAFNFKAKYKWDAWDKLKGKSQEEAEQEYIALVQTLSDKYN* >SEQ_ID_NO:_38_SOL3 MPKVISKNESQLVAEAAAAEIIRLQNESIAATGAFHVAVSGGSLVSALRKGLVNNSETKFPKWKIFFSDERLVKLDDADSNYGLLKKDLL DHIPKDQQPQVFTVKESLLNDSDAVSKDYQEQIVKNVPLNGQGVPVFDLILLGCGPDGHTCSLFPGHALLKEETKFVATIEDSPKPPPRR ITITFPVLKAAKAIAFVAEGAGKAPVLKQIFEEPEPTLPSAIVNKVATGPVFWFVSDSAVEGVNLSKI* >SEQ_ID_NO:_39_SLC1 MNSWIYVAVIAVAAVLIARMSVASKLVFYVRAAIAVVIFAACATYGVLASTILTAIGKQGLAQWTVARAFYYSVRIFLGISIKLRSRQVT GTAGLDASKIQVANTTKPIDDITKHLPRPCILISNHQNEMDILVLGRIFPQYCSVTAKKALKWYPLLGQFMALSGTIFLDRKDRTKSVQT LGGAVKTIQSGNGGKGQSVFMFPEGTRSYSKDVGIMPFKKGCFHLAVQSGAPIVPVVVQNTSRMFSFGRGKLDAGEILVDVLSPIETKGL DASNVDALMATTYKAMCETADQIGYAGQKTQ* PROMOTERS >SEQ ID NO: 14 pTEF [the standard strong constitutive promoter] accgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggc gagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgct tgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgt ggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccg aaatcgttaagcatttccttctgagtataagaatcattcaa >SEQ ID NO: 15 pTEF-Kozak, with 3 copies of CACA in tandem immediately before the initiation codon AUG GGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAG AGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGT TCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGT TGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAC ACACACACACA >SEQ ID NO: 16 pTEF-4UAS4UAS, with 4x additional upstream activation sequences cgatacgcgtatcgatacgcgtgcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgta cgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgct tctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgc agtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgagg tgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacc tcgatccggcatgcactgatcacgggcaaaagtgcgttcgatagagagggggttggcggcgcatttgtgtcccaaaaaacagccccaatt gccccaattgaccccaaattgacccagtagcggacccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttccca cacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaa cccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaa ttacctttcctcttcttttctctctctccttgtcaactcacacccgaa >SEQ_ID_NO:_17_pGPD1 GACGCAGTAGGATGTCCTGCACGGGTCTTTTTGTGGGGTGTGGAGAAAGGGGTGCTTGGAGATGGAAGCCGGTAGAACCGGGCTGCTTGG GGGGATTTGGGGCCGCTGGGCTCCAAAGAGGGGTAGGCATTTCGTTGGGGTTACGTAATTGCGGCATTTGGGTCCTGCGCGCATGTCCCA TTGGTCAGAATTAGTCCGGATAGGAGACTTATCAGCCAATCACAGCGCCGGATCCACCTGTAGGTTGGGTTGGGTGGGAGCACCCCTCCA CAGAGTAGAGTCAAACAGCAGCAGCAACATGATAGTTGGGGGTGTGCGTGTTAAAGGAAAAAAAAAGAAGCTTGGGTTATATTCCCGCTC TATTTAGAGGTTGCGGGATAGACGCCGACGGAGGGCAATGGCGCCATGGAACCTTGCGGATATCGATACGCCGCGGCGGACTGCGTCCGA ACCAGCTCCAGCAGCGTTTTTTCCGGGCCATTGAGCCGACTGCGACCCCGCCAACGTGTCTTGGCCCACGCACTCATGTCATGTTGGTGT TGGGAGGCCACTTTTTAAGTAGCACAAGGCACCTAGCTCGCAGCAAGGTGTCCGAACCAAAGAAGCGGCTGCAGTGGTGCAAACGGGGCG GAAACGGCGGGAAAAAGCCACGGGGGCACGAATTGAGGCACGCCCTCGAATTTGAGACGAGTCACGGCCCCATTCGCCCGCGCAATGGCT CGCCAACGCCCGGTCTTTTGCACCACATCAGGTTACCCCAAGCCAAACCTTTGTGTTAAAAAGCTTAACATATTATACCGAACGTAGGTT TGGGCGGGCTTGCTCCGTCTGTCCAAGGCAACATTTATATAAGGGTCTGCATCGCCGGCTCAATTGAATCTTTTTTCTTCTTCTCTTCTC TATATTCATTCTTGAATTAAACACACATCAAC >SEQ_ID_NO:_18_pMnDH2 CTTGTATTGGCCAAGTTGAAGACGTTCTTGTTGCCACTTCCACCCAAATGATCCCCAAACGAACTACTGCATGGCGTCCGGGTAACCGCC GAGGGAGGGTCTTTGTCTGTTAATTAGTACTTTTGAGAAATACACCGGAGTATTATTATGGTGGATTTCCGGGCTCCTCTGTGACCCGAG TGTTGCTGGACGTTCGATGTTCGATGCTCGGCACATGCCGGTTCGAACAGGAATTATAGCGTTCATCTGGAGTTGGACGCAAGCAAAAAA GCAAATGAGGGAGTTATGGGAGGGTTCCGAGAAGTGAAAAATCGGTCAATGGGTTAGTTTGAAGTCTCGTTTTGTTTGTGTTGGCGAGAC AAGAAGAATGGTATAATTTTCGCACCAAAAAGAGACCATTTATCGTGGATTATGGGGGTGTGATGTGGGGGGAGGGGGGAGATGCCCCAT CTCTGGCAACCCTATTTGACGATAGTTGCTGGAGGCTTGACAGGACTTGGTGACGAGGGGTGTTTGGGCGCTGGAAGCGTAATTTTCGTC TTGAATGGGCCGTCGAGACTTGGGGTTCGACCCCGACTAAATGGCGCACCGCTAGATTCTCTTTTGGCGACTTTCTCGGGATTCTAGTCA CCCCCGCAATGTTCCAGCTTACGGTTTGAGACAGTATACGACTGGTTAGGCGAGTGTTGAAGTCGTAGCGTAGAGTGGGAGGCATGACGT CACGGGACAGCTGCGTGCACCACGCGAGCAGGTCAATTGACCTCATTTGAGTGGTGTGGCTTGGCGTTCTAGCGGTGGCGGCGTTGTCGA GCTCCCTCTACTTGTAGTGAGATTATGTCGACGAGCGGGGGGGGACTTCCATTGTGCTTGCCACTGCTAGTGCAGTACAACTGAAAGCTA AACCGCAATCAATCCCAAACTGCATGTCCGCCTTAACTCTGATATGTTATCAAGAGAGTGGTGTGGTGAGGTGAGGTGAGGTGACGTGGA CAAGTTGATGGGGAGTTGGGGCATTGACAAAAGGGAAATTGCAGGGGGATTCCGCCGGCTATATATATCTTATGTCTGCTCAATTCCCAG ACGGCTCCACACAAAACCAAGATACCACACCATCATGGTCACACCGGGTACATAACTCCCATCCATCTCATCCCACTTGCATGGCGACCG GAGAGAGAAAGCCCGGGGAGAGCACGTCGGCGCGGTCCCCAGGGCGACAACCAAAACAAAATCACCGAGTGACTCCGAAAGCCGCGTTCC AACACCCCCCCAAAATCCCCCCCTCAAACACGTCAGCCACCTGTCCCCCGAAAATTAACTTCACTGACATGGCGCAGCTATTAAGGCTAA AGTGAATGCATGGCTCATCTTTGTTTGCTGGTTGCTACTGTGACTGAGGTAAAAACCCTCGCTCCCAAGTCTATATATACCTGGGTGTGC TCCCTCGAACAGACCCGTCACAGTAAAACTACTACCTCCATACACAGCACCACCTTGATC >SEQ ID NO: 19 hp4d promoter atcgatacgcgtgcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattat cgagaccgttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtg tgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgcagtcccgccc ccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccggcatgctgaggtgtctcacaa gtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccggc atgcactgatcacgggcaaaagtgcgtatatatacaagagcgtttgccagccacagattttcactccacacaccacatcacacatacaac cacacacatccaca Terminators >SEQ ID NO: 20 tLIP2 (long version) CTTCTGTTCGGAATCAACCTCAAGGTTAACGGCCACGATCCCCTCGTTGTTACTCTTGGTCAGCCCATTGTCGGTAACGCTGGCTTTGCT AACTGGGTCGATAAACTCTTCTTTGGCCAGGAGAACCCCGATGTCTCCAAGGTGTCCAAAGACCGAAAGCTCTACCGAATCACCCACCGA GGAGATATCGTCCCTCAAGTGCCCTTCTGGGACGGTTACCAGCACTGCTCTGGTGAGGTCTTTATTGACTGGCCCCTGATCCACCCTCCT CTCTCCAACGTTGTCATGTGCCAGGGCCAGAGCAATAAACAGTGCTCTGCCGGTAACACTCTGCTCCAGCAGGTCAATGTGATTGGAAAC CATCTGCAGTACTTCGTCACCGAGGGTGTCTGTGGTATCTAAGCTATTTATCACTCTTTACAACTTCTACCTCAACTATCTACTTTAATA AATGAATATCGTTTATTCTCTATGATTACTGTATATGCGTTCCTCTAAGACAAATCGAAACCAGCATGCGATCGAATGGCATACAAAAGT TTCTTCCGAAGTTGATCAATGTCCTGATAGTCAGGCAGCTTGAGAAGATTGACACAGGTGGAGGCCGTAGGGAACCGATCAACCTGTCTA CCAGCGTTACGAATGGCAAATGACGGGTTCAAAGCCTTGAATCCTTGCAATGGTGCCTTGGATACTGATGTCACAAACTTAAGAAGCAGC CGCTTGTCCTCTTCCTCGAAACTCTCAAACACAGTCCAGAAGTCCTTTATAGTTTGATCTGTATCCAGATAGCCTCCGTAATTGGTGTGT GTCTTCAAATCCCAGACGTCCACATTGGCATGTCCTCCACTGATAAGCATTTGAAGTTCATCTGCGTTGAACATTGAGACCCACGAAGGG TCAATGAGCTGGTATAGACCGCCCAAGAATGCATCTG >SEQ_ID_NO:_29_tXPR2 sequence AGGCAATTAACAGATAGTTTGCCGGTGATAATTCTCTTAACCTCCCACACTCCTTTGACATAACGATTTATGTAACGAAACTGAAATTTG ACCAGATATTGTTGTAAATAGAAAATCTGGCTTGTAGGTGGCAAAATCCCGTCTTTGTTCATCAATTCCCTCTGTGACTACTCGTCATCC CTTTATGTTCGACTGTCGTATTTTTATTTTCCATACATACGCAAGTGAGATGCCCGTGTCCG Nucleic acid sequences >SEQ_ID_NO:_21_Nucleic_acid_sequence_encoding_DGA1_from_Yarrowia_lipolytica ATGACTATCGACTCACAATACTACAAGTCGCGAGACAAAAACGACACGGCACCCAAAATCGCGGGAATCCGATATGCCCCGCTATCGACA CCATTACTCAACCGATGTGAGACCTTCTCTCTGGTCTGGCACATTTTCAGCATTCCCACTTTCCTCACAATTTTCATGCTATGCTGCGCA ATTCCACTGCTCTGGCCATTTGTGATTGCGTATGTAGTGTACGCTGTTAAAGACGACTCCCCGTCCAACGGAGGAGTGGTCAAGCGATAC TCGCCTATTTCAAGAAACTTCTTCATCTGGAAGCTCTTTGGCCGCTACTTCCCCATAACTCTGCACAAGACGGTGGATCTGGAGCCCACG CACACATACTACCCTCTGGACGTCCAGGAGTATCACCTGATTGCTGAGAGATACTGGCCGCAGAACAAGTACCTCCGAGCAATCATCTCC ACCATCGAGTACTTTCTGCCCGCCTTCATGAAACGGTCTCTTTCTATCAACGAGCAGGAGCAGCCTGCCGAGCGAGATCCTCTCCTGTCT CCCGTTTCTCCCAGCTCTCCGGGTTCTCAACCTGACAAGTGGATTAACCACGACAGCAGATATAGCCGTGGAGAATCATCTGGCTCCAAC GGCCACGCCTCGGGCTCCGAACTTAACGGCAACGGCAACAATGGCACCACTAACCGACGACCTTTGTCGTCCGCCTCTGCTGGCTCCACT GCATCTGATTCCACGCTTCTTAACGGGTCCCTCAACTCCTACGCCAACCAGATCATTGGCGAAAACGACCCACAGCTGTCGCCCACAAAA CTCAAGCCCACTGGCAGAAAATACATCTTCGGCTACCACCCCCACGGCATTATCGGCATGGGAGCCTTTGGTGGAATTGCCACCGAGGGA GCTGGATGGTCCAAGCTCTTTCCGGGCATCCCTGTTTCTCTTATGACTCTCACCAACAACTTCCGAGTGCCTCTCTACAGAGAGTACCTC ATGAGTCTGGGAGTCGCTTCTGTCTCCAAGAAGTCCTGCAAGGCACTCCTCAAGCGAAACCAGTCTATCTGCATTGTCGTTGGTGGAGCA CAGGAAAGTCTTCTGGCCAGACCCGGTGTCATGGACCTGGTGCTACTCAAGCGAAAGGGTTTTGTTCGACTTGGTATGGAGGTCGGAAAT GTCGCCCTTGTTCCCATCATGGCCTTTGGTGAGAACGACCTCTATGACCAGGTTAGCAACGACAAGTCGTCCAAGCTGTACCGATTCCAG CAGTTTGTCAAGAACTTCCTTGGATTCACCCTTCCTTTGATGCATGCCCGAGGCGTCTTCAACTACGATGTCGGTCTTGTCCCCTACAGG CGACCCGTCAACATTGTGGTTGGTTCCCCCATTGACTTGCCTTATCTCCCACACCCCACCGACGAAGAAGTGTCCGAATACCACGACCGA TACATCGCCGAGCTGCAGCGAATCTACAACGAGCACAAGGATGAATATTTCATCGATTGGACCGAGGAGGGCAAAGGAGCCCCAGAGTTC CGAATGATTGAGTAA >SEQ_ID_NO:_22_Nucleic_acid_sequence_encoding_ACC1_from_Yarrowia_lipolytica ATGCGACTGCAATTGAGGACACTAACACGTCGGTTTTTCAGTATGGCTTCAGGATCTTCAACGCCAGATGTGGCTCCCTTGGTGGACCCC AACATTCACAAAGGTCTCGCCTCTCATTTCTTTGGACTCAATTCTGTCCACACAGCCAAGCCCTCAAAAGTCAAGGAGTTTGTGGCTTCT CACGGAGGTCATACAGTTATCAACAAGGTCCTCATCGCTAACAACGGTATTGCCGCAGTAAAGGAGATCCGTTCAGTACGAAAATGGGCC TACGAGACCTTTGGCGACGAGCGAGCAATCTCGTTCACCGTCATGGCCACCCCCGAAGATCTCGCTGCCAACGCCGACTACATTAGAATG GCCGATCAGTACGTCGAGGTGCCCGGAGGAACCAACAACAACAACTACGCCAACGTCGAGCTGATTGTCGACGTGGCTGAGCGATTCGGC GTCGATGCCGTGTGGGCCGGATGGGGCCATGCCAGTGAAAATCCCCTGCTCCCCGAGTCGCTAGCGGCCTCTCCCCGCAAGATTGTCTTC ATCGGCCCTCCCGGAGCTGCCATGAGATCTCTGGGAGACAAAATTTCTTCTACCATTGTGGCCCAGCACGCAAAGGTCCCGTGTATCCCG TGGTCTGGAACCGGAGTGGACGAGGTTGTGGTTGACAAGAGCACCAACCTCGTGTCCGTGTCCGAGGAGGTGTACACCAAGGGCTGCACC ACCGGTCCCAAGCAGGGTCTGGAGAAGGCTAAGCAGATTGGATTCCCCGTGATGATCAAGGCTTCCGAGGGAGGAGGAGGAAAGGGTATT CGAAAGGTTGAGCGAGAGGAGGACTTCGAGGCTGCTTACCACCAGGTCGAGGGAGAGATCCCCGGCTCGCCCATCTTCATTATGCAGCTT GCAGGCAATGCCCGGCATTTGGAGGTGCAGCTTCTGGCTGATCAGTACGGCAACAATATTTCACTGTTTGGTCGAGATTGTTCGGTTCAG CGACGGCATCAAAAGATTATTGAGGAGGCTCCTGTGACTGTGGCTGGCCAGCAGACCTTCACTGCCATGGAGAAGGCTGCCGTGCGACTC GGTAAGCTTGTCGGATATGTCTCTGCAGGTACCGTTGAATATCTGTATTCCCATGAGGACGACAAGTTCTACTTCTTGGAGCTGAATCCT CGTCTTCAGGTCGAACATCCTACCACCGAGATGGTCACCGGTGTCAACCTGCCCGCTGCCCAGCTTCAGATCGCCATGGGTATCCCCCTC GATCGAATCAAGGACATTCGTCTCTTTTACGGTGTTAACCCTCACACCACCACTCCAATTGATTTCGACTTCTCGGGCGAGGATGCTGAT AAGACACAGCGACGTCCCGTCCCCCGAGGTCACACCACTGCTTGCCGAATCACATCCGAGGACCCTGGAGAGGGTTTCAAGCCCTCCGGA GGTACTATGCACGAGCTCAACTTCCGATCCTCGTCCAACGTGTGGGGTTACTTCTCCGTTGGTAACCAGGGAGGTATCCATTCGTTCTCG GATTCGCAGTTTGGTCACATCTTCGCCTTCGGTGAGAACCGAAGTGCGTCTCGAAAGCACATGGTTGTTGCTTTGAAGGAACTATCTATT CGAGGTGACTTCCGAACCACCGTCGAGTACCTCATCAAGCTGCTGGAGACACCGGACTTCGAGGACAACACCATCACCACCGGCTGGCTG GATGAGCTTATCTCCAACAAGCTGACTGCCGAGCGACCCGACTCGTTCCTCGCTGTTGTTTGTGGTGCTGCTACCAAGGCCCATCGAGCT TCCGAGGACTCTATTGCCACCTACATGGCTTCGCTAGAGAAGGGCCAGGTCCCTGCTCGAGACATTCTCAAGACCCTTTTCCCCGTTGAC TTCATCTACGAGGGCCAGCGGTACAAGTTCACCGCCACCCGGTCGTCTGAGGACTCTTACACGCTGTTCATCAACGGTTCTCGATGCGAC ATTGGAGTTAGACCTCTTTCTGACGGTGGTATTCTGTGTCTTGTAGGTGGGAGATCCCACAATGTCTACTGGAAGGAGGAGGTTGGAGCC ACGCGACTGTCTGTTGACTCCAAGACCTGCCTTCTCGAGGTGGAGAACGACCCCACTCAGCTTCGATCTCCCTCTCCCGGTAAGCTGGTT AAGTTCCTGGTCGAGAACGGCGACCACGTGCGAGCCAACCAGCCCTATGCCGAGATTGAGGTCATGAAGATGTACATGACTCTCACTGCT CAGGAGGACGGTATTGTCCAGCTGATGAAGCAGCCCGGTTCCACCATCGAGGCTGGCGACATCCTCGGTATCTTGGCCCTTGATGATCCT TCCAAGGTCAAGCATGCCAAGCCCTTTGAGGGCCAGCTTCCCGAGCTTGGACCCCCCACTCTCAGCGGTAACAAGCCTCATCAGCGATAC GAGCACTGCCAGAACGTGCTCCATAACATTCTGCTTGGTTTCGATAACCAGGTGGTGATGAAGTCCACTCTTCAGGAGATGGTTGGTCTG CTCCGAAACCCTGAGCTTCCTTATCTCCAGTGGGCTCATCAGGTGTCTTCTCTGCACACCCGAATGAGCGCCAAGCTGGATGCTACTCTT GCTGGTCTCATTGACAAGGCCAAGCAGCGAGGTGGCGAGTTTCCTGCCAAGCAGCTTCTGCGAGCCCTTGAGAAGGAGGCGAGCTCTGGC GAGGTCGATGCGCTCTTCCAGCAAACTCTTGCTCCTCTGTTTGACCTTGCTCGAGAGTACCAGGACGGTCTTGCTATCCACGAGCTTCAG GTTGCTGCAGGCCTTCTGCAGGCCTACTACGACTCTGAGGCCCGGTTCTGCGGACCCAACGTACGTGACGAGGATGTCATTCTCAAGCTT CGAGAGGAGAACCGAGATTCTCTTCGAAAGGTTGTGATGGCCCAGCTGTCTCATTCTCGAGTCGGAGCCAAGAACAACCTTGTGCTGGCC CTTCTCGATGAATACAAGGTGGCCGACCAGGCTGGCACCGACTCTCCTGCCTCCAACGTGCACGTTGCAAAGTACTTGCGACCTGTGCTG CGAAAGATTGTGGAGCTGGAATCTCGAGCTTCTGCCAAGGTATCTCTGAAAGCCCGAGAGATTCTCATCCAGTGCGCTCTGCCCTCTCTA AAGGAGCGAACTGACCAGCTTGAGCACATTCTGCGATCTTCTGTCGTCGAGTCTCGATACGGAGAGGTTGGTCTGGAGCACCGAACTCCC CGAGCCGATATTCTCAAGGAGGTTGTCGACTCCAAGTACATTGTCTTTGATGTGCTTGCCCAGTTCTTTGCCCACGATGATCCCTGGATC GTCCTTGCTGCCCTGGAGCTGTACATCCGACGAGCTTGCAAGGCCTACTCCATCCTGGACATCAACTACCACCAGGACTCGGACCTGCCT CCCGTCATCTCGTGGCGATTTAGACTGCCTACCATGTCGTCTGCTTTGTACAACTCAGTAGTGTCTTCTGGCTCCAAAACCCCCACTTCC CCCTCGGTGTCTCGAGCTGATTCCGTCTCCGACTTTTCGTACACCGTTGAGCGAGACTCTGCTCCCGCTCGAACCGGAGCGATTGTTGCC GTGCCTCATCTGGATGATCTGGAGGATGCTCTGACTCGTGTTCTGGAGAACCTGCCCAAACGGGGCGCTGGTCTTGCCATCTCTGTTGGT GCTAGCAACAAGAGTGCCGCTGCTTCTGCTCGTGACGCTGCTGCTGCTGCCGCTTCATCCGTTGACACTGGCCTGTCCAACATTTGCAAC GTTATGATTGGTCGGGTTGATGAGTCTGATGACGACGACACTCTGATTGCCCGAATCTCCCAGGTCATTGAGGACTTTAAGGAGGACTTT GAGGCCTGTTCTCTGCGACGAATCACCTTCTCCTTCGGCAACTCCCGAGGTACTTATCCCAAGTATTTCACGTTCCGAGGCCCCGCATAC GAGGAGGACCCCACTATCCGACACATTGAGCCTGCTCTGGCCTTCCAGCTGGAGCTCGCCCGTCTGTCCAACTTCGACATCAAGCCTGTC CACACCGACAACCGAAACATCCACGTGTACGAGGCTACTGGCAAGAACGCTGCTTCCGACAAGCGGTTCTTCACCCGAGGTATCGTACGA CCTGGTCGTCTTCGAGAGAACATCCCCACCTCGGAGTATCTCATTTCCGAGGCTGACCGGCTCATGAGCGATATTTTGGACGCTCTAGAG GTGATTGGAACCACCAACTCGGATCTCAACCACATTTTCATCAACTTCTCAGCCGTCTTTGCTCTGAAGCCCGAGGAGGTTGAAGCTGCC TTTGGCGGTTTCCTGGAGCGATTTGGCCGACGTCTGTGGCGACTTCGAGTCACCGGTGCCGAGATCCGAATGATGGTATCCGACCCCGAA ACTGGCTCTGCTTTCCCTCTGCGAGCAATGATCAACAACGTCTCTGGTTACGTTGTGCAGTCTGAGCTGTACGCTGAGGCCAAGAACGAC AAGGGCCAGTGGATTTTCAAGTCTCTGGGCAAGCCCGGCTCCATGCACATGCGGTCTATCAACACTCCCTACCCCACCAAGGAGTGGCTG CAGCCCAAGCGGTACAAGGCCCATCTGATGGGTACCACCTACTGCTATGACTTCCCCGAGCTGTTCCGACAGTCCATTGAGTCGGACTGG AAGAAGTATGACGGCAAGGCTCCCGACGATCTCATGACTTGCAACGAGCTGATTCTCGATGAGGACTCTGGCGAGCTGCAGGAGGTGAAC CGAGAGCCCGGCGCCAACAACGTCGGTATGGTTGCGTGGAAGTTTGAGGCCAAGACCCCCGAGTACCCTCGAGGCCGATCTTTCATCGTG GTGGCCAACGATATCACCTTCCAGATTGGTTCGTTTGGCCCTGCTGAGGACCAGTTCTTCTTCAAGGTGACGGAGCTGGCTCGAAAGCTC GGTATTCCTCGAATCTATCTGTCTGCCAACTCTGGTGCTCGAATCGGCATTGCTGACGAGCTCGTTGGCAAGTACAAGGTTGCGTGGAAC GACGAGACTGACCCCTCCAAGGGCTTCAAGTACCTTTACTTCACCCCTGAGTCTCTTGCCACCCTCAAGCCCGACACTGTTGTCACCACT GAGATTGAGGAGGAGGGTCCCAACGGCGTGGAGAAGCGTCATGTGATCGACTACATTGTCGGAGAGAAGGACGGTCTCGGAGTCGAGTGT CTGCGGGGCTCTGGTCTCATTGCAGGCGCCACTTCTCGAGCCTACAAGGATATCTTCACTCTCACTCTTGTCACCTGTCGATCCGTTGGT ATCGGTGCTTACCTTGTTCGTCTTGGTCAACGAGCCATCCAGATTGAGGGCCAGCCCATCATTCTCACTGGTGCCCCCGCCATCAACAAG CTGCTTGGTCGAGAGGTCTACTCTTCCAACTTGCAGCTTGGTGGTACTCAGATCATGTACAACAACGGTGTGTCTCATCTGACTGCCCGA GATGATCTCAACGGTGTCCACAAGATCATGCAGTGGCTGTCATACATCCCTGCTTCTCGAGGTCTTCCAGTGCCTGTTCTCCCTCACAAG ACCGATGTGTGGGATCGAGACGTGACGTTCCAGCCTGTCCGAGGCGAGCAGTACGATGTTAGATGGCTTATTTCTGGCCGAACTCTCGAG GATGGTGCTTTCGAGTCTGGTCTCTTTGACAAGGACTCTTTCCAGGAGACTCTGTCTGGCTGGGCCAAGGGTGTTGTTGTTGGTCGAGCT CGTCTTGGCGGCATTCCCTTCGGTGTCATTGGTGTCGAGACTGCGACCGTCGACAATACTACCCCTGCCGATCCCGCCAACCCGGACTCT ATTGAGATGAGCACCTCTGAAGCCGGCCAGGTTTGGTACCCCAACTCGGCCTTCAAGACCTCTCAGGCCATCAACGACTTCAACCATGGT GAGGCGCTTCCTCTCATGATTCTTGCTAACTGGCGAGGCTTTTCTGGTGGTCAGCGAGACATGTACAATGAGGTTCTCAAGTACGGATCT TTCATTGTTGATGCTCTGGTTGACTACAAGCAGCCCATCATGGTGTACATCCCTCCCACCGGTGAGCTGCGAGGTGGTTCTTGGGTTGTG GTTGACCCCACCATCAACTCGGACATGATGGAGATGTACGCTGACGTCGAGTCTCGAGGTGGTGTGCTGGAGCCCGAGGGAATGGTCGGT ATCAAGTACCGACGAGACAAGCTACTGGACACCATGGCTCGTCTGGATCCCGAGTACTCCTCTCTCAAGAAGCAGCTTGAGGAGTCTCCC GATTCTGAGGAGCTCAAGGTCAAGCTCAGCGTGCGAGAGAAGTCTCTCATGCCCATCTACCAGCAGATCTCCGTGCAGTTTGCCGACTTG CATGACCGAGCTGGCCGAATGGAGGCCAAGGGTGTCATTCGTGAGGCTCTTGTGTGGAAGGATGCTCGTCGATTCTTCTTCTGGCGAATC CGACGACGATTAGTCGAGGAGTACCTCATTACCAAGATCAATAGCATTCTGCCCTCTTGCACTCGGCTTGAGTGTCTGGCTCGAATCAAG TCGTGGAAGCCTGCCACTCTTGATCAGGGCTCTGACCGGGGTGTTGCCGAGTGGTTTGACGAGAACTCTGATGCCGTCTCTGCTCGACTC AGCGAGCTCAAGAAGGACGCTTCTGCCCAGTCGTTTGCTTCTCAACTGAGAAAGGACCGACAGGGTACTCTCCAGGGCATGAAGCAGGCT CTCGCTTCTCTTTCTGAGGCTGAGCGGGCTGAGCTGCTCAAGGGGTTGTGA ATGGCCAAGATCGCCATCAACGGCTTCGGACGAATCGGCCGACTGGCCCTGCGACGAATCCTCGAGGTGCCCGGACTCGAGGTGGTGGCC ATTAACGACCTGACCGACGCCAAGATGCTGGCCCACCTGTTCAAGTACGACTCTTCTCAGGGCCGATTCAACGGCGAGATCGAGGTGAAG GAAGGCGCTTTCGTCGTCAACGGCAAGGAAGTGAAGGTTTTCGCCGAGGCTGACCCCGAGAAGCTGCCCTGGGGCGACCTGGGCATCGAC GTGGTGCTCGAGTGTACCGGCTTCTTCACCAAGAAGGAAAAGGCCGAGGCTCACGTGCGAGCCGGCGCTAAGAAGGTTGTCATCTCTGCT CCCGCCGGAAACGACCTCAAGACCATCGTGTTCAACGTGAACAACGAGGACCTGGACGGCACCGAGACTGTGATCTCTGGCGCCTCTTGC ACCACCAACTGTCTGGCCCCTATGGCCAAGGTGCTGAACGACAAGTTCGGCATCGAGAAGGGCTTCATGACCACCATCCACGCCTTCACC AACGACCAGAACACCCTGGACGGACCCCACCGAAAGGGCGACCTCCGACGAGCCCGAGCCGCCGCTGTGTCTATCATCCCCAACTCTACC GGCGCTGCCAAGGCCATCTCTCAGGTGATCCCCGACCTGGCCGGCAAGCTGGACGGAAACGCCCAGCGAGTGCCCGTGCCTACCGGCTCT ATCACCGAGCTGGTGTCTGTGCTGAAGAAGAAGGTCACCGTCGAAGAGATCAACGCCGCCATGAAGGAAGCCGCCGACGAGTCTTTCGGC TACACTGAGGACCCCATCGTGTCTGCCGACGTGGTCGGCATCAACTACGGATCTCTGTTCGACGCTACCCTGACCAAGATCGTGGACGTG AACGGCTCTCAGCTGGTCAAGACCGCCGCCTGGTACGACAACGAGATGTCTTACACCTCGCAGCTGGTGCGAACCCTGGCCTACTTCGCC AAGATTGCCAAGTAA >SEQ_ID_NO:_24_Nucleotide_sequence_for MCE2 from _Mucor_circinelloides_codon_optimised_for_expression_in_Yarrowia_lipolytica ATGTCGCCTATTATTGAATTTGTTCGTCGCCAATTGTCCTCTACAAAGTTGCATGAAGAGCAGCAAACAGCAACTACAAATGATTTGGTC TCTAGATCAGGCTATCTAAATGAATGCAAGTATGAGGTCCGCTTGAATTGTATCAATGCTGGCTGCTTACAAAAAAAACTAAACTATATA GGTACTGCCATGGATCCTGCTAAACGTCAAAGACTTGGATTGAACGGTCTTTTACCTGCTGGTGTAGAGACATTGGAAATTCAAAAAGCT CGCGCCCTCAGAGTGCTTCGTTCAAAACACAATTTATTAGAAAAATACATTTTAATGGCTCAACTTCGTACCACCAACGTCCGCTTATTT TACAAGATTGTCATTGATGAATTAGAGACCGTTCAATTGGCTCCTGTTATCTATACCCCGACTGTTGGTACCGCATGCTTGGAATACTCT ACCATCTATCCCTTCTTGGCTGCCCCTGGTGTGCCGGATGGTCTTTACCTCACCAAAGCCGAATTACCGGAACTGTGTCAAACCATTCGT AACTATCGTCCTACGGATACTGAGGGTTTTGAGCCAGAGATTGCTGTGATTTCTGATGGGTCTCGAATTTTGGGTCTGGGTGATTTGGGA ACAAATGGCATGGGTATTCCAATGGGTAAACTTCAGCTCTATGTTGCTGGTGCTGGTATTGATCCTCGTCGTACGTTACCCATCATTTTG GATTTGGGTACAAACAATGAAAAGTTGCTCAATGATGAGTTTTATATTGGTCTTCGTCAAAAGCGACCCAATGATGAGGAGTTTTATCAA ACAGTTGATACAGTCTTGACAGCATTACATACCGTGTACCCCAACCTACTCATCCAGTTTGAAGATTGGTCTTCTGAACACGCATTTGGC CTCTTGGAAAAGTACCAAAATCAAATGCTTTGTTTTAACGACGACATACAGGGCACAGGTGCTGTCATATTATCTGGTGTCATTAATGCT ATTCGCAAGGTTGAGAAAGAGAATCAAGTGTCTCCTCGTGATCATCGTATCGTGTTCTACGGTGCTGGTTCTGCTGCTATCGGTGTTGCT CGTCAAATTCAAAGCTACTTCCAAATTGAACACAACATGACTGAGGAAGAAGCTAAGCATGTGTTCTGGATTGTTGATTCCAAGGGTCTT GTTACTACTACACGAGGCGATAAATTAGCTCAACACAAGGTGTATTACGCACGAGGCGATAATGAAGGCCAACAGTACAAGGAATTGATT GATATTGTCAACTATAATCTCTACAGTTTGATTGGTTTATCATCTACTACAGGTGCCTTTAATACTCAAGTCCTTGAGCGTCTTGCCTCA CTCAATGAGCAACCTATTGTTTTCCCTCTTTCCAATCCAGCCACACAAGCAGAATGTACATTTGAGCAAGCCATGGAAGCTACCAACAAC AAGGTTATTTTTGCATCTGGTACTGCTTTCCCTGCATATACCATCAAATCCACTGGCGAAGTAAATACCCCTGGTCAAGGCAACAACATG TACATCTTCCCTGGTTTGGGTCTGGGTGCTTGTCTGGCTAACCCAGCACATTTCGATCGCATGATCTACGAAGCATCCAAAGCACTTGCT GACTCACTTACAGAGGAAGAAATCAGTAAGGCCTGGTTATATCCATCTTTAAACTATCGTAGCGTATCAGCCATCGTTGCAGCAGCTGTA TGTCAAGAGACTTTGAATGAAAACCTAGCAACGTCTCAAGCTATGATGACGCAGTGTAAATCACATGAAGATATTCTAGATTATGTTAGT GCTCATATGTGGTCTCCCGACTATGGAAACAACAACAGCAATCAGCAAGCTGGTAAATTGTAG >SEQ_ID_NO:_25_Nucleotide_sequence_for_DGA2 from Claviceps_purpurea_codon_optimised_for_expression_in_Yarrowia_lipolytica ATGTCTGCCACCGGCGTGGACGTGGCCAACGGCCGATCTGGCGCCCGACGACGAAACGACACCGCCGTCGACGAGACTATCTCTGCCGTG ACCGCCGAGATGCGATCTTCTTCTCACCCCACCTACCGACATGTGTCTGCCGTGCACTCTACCTCTCGACCCTCTTGCCTGTCTCACGAC TCTGACGCTGCTCCCTCTTTCATCGGCTTCCGAAACCTGATGGTGATCGTGCTGGTGGTGGGCAACGTGCGACTGATGATCGAGAACCTG AAGAAGTACGGCGTCCTGATCTGCCTCCGATGCCACTCTTACAAGAACGAGGACATCATCATCGGCGGCCTGCTGTACTTTCTGATCCCC TGCCACCTCCTGGTGGCCTACGGCATCGAGCTGGCCGCTGCTCGACAGGCCCGAGAGTCTCGAACCCGACCTCCTGGCCAGTCTGACACC GCCTCTAAGTCTACCGAGGACGACAACAAGCACTTCCACTCCACCTGGGTGCTCGCCGCCTGGGCTCACATCATCAACATGACCCTGTCT TTCATCCTGACCACCTTCGTGGTGTACTACTACGTGCACCATCCTCTGGTGGGCACCCTGACCGAGATGCACGCCGTGATCGTGTCTCTC AAGACCGCCTCTTACGCCTTCACCAACCGAGATCTGCGACACGCCTACCTGCATCCTGACAAGCGAAAGCACATCCCCGAGCTGTACCTC GAGTGCCCCTATCCTCAGAACCTGACCTTCGGCAACCTGGTGTACTTTTGGTGGGCTCCCACTCTGGTGTACCAGCCTGTGTACCCTCGA ACCGACAAGATCCGATGGGTGTTCGTGTTCAAGCGACTCGGCGAGGTGTGCTGCCTGTCTGCCTTTATCTGGTTCGCCTCTTTCCAGTAC GCCGCTCCTGTGCTGCGAAACTCTCTGGACAAGATCGCTTCTCTGGACTTCATCATGATCTTCGAGCGACTGCTGAAGCTGTCTACCATT TCTCTGGTGATCTGGCTGGCCGGCTTCTTCGCCCTGTTCCAGTCTTTCCTGAACGCCCTGGCCGAGGTGCTGCGATTCGGCGACCGATGC TTCTACGACGACTGGTGGAACTCTGAGTCTCTGGGCGCCTACTGGCGAACCTGGAACCGACCTGTGTACACCTACTTTAAGCGACACGTG TACGTGCCCATGATCGGCCGAGGCTGGTCGCCCTGGACCGCCTCCTGCACCGTGTTCTTCGTGTCCGCCGTGCTGCACGAGGTCCTGGTG GGAGTGCCCACTCACAACATCATCGGAGTGGCCTTCGTGGGCATGTTCCTGCAGCTGCCCCTGATCGCCCTGACTGCTCCCATGGAAAAG AAGAAGTGGGGCCACACCGGCCGAGTGATGGGAAACGTGATCTTCTGGGTGTCTTTCACCATCTTCGGACAGCCCTTCGCCGCTCTGATG TACTTCTACGCCTGGCAGGCCAAGTACGGCTCTGTGTCTCGACAGATTGTGCTGGTGAACCCCGTGGAAGAGGCCTCCTAA ATGTCTGCCAACGAGAACATCTCCCGATTCGACGCCCCTGTGGGCAAGGAGCACCCCGCCTACGAGCTCTTCCATAACCACACACGATCT TTCGTCTATGGCCTCCAGCCTCGAGCCTGCCAGGGTATGCTGGACTTCGACTTCATCTGTAAGCGAGAGAACCCCTCCGTGGCCGGTGTC ATCTATCCCTTCGGCGGCCAGTTCGTCACCAAGATGTACTGGGGCACCAAGGAGACTCTTCTCCCTGTCTACCAGCAGGTCGAGAAGGCC GCTGCCAAGCACCCCGAGGTCGATGTCGTGGTCAACTTTGCCTCCTCTCGATCCGTCTACTCCTCTACCATGGAGCTGCTCGAGTACCCC CAGTTCCGAACCATCGCCATTATTGCCGAGGGTGTCCCCGAGCGACGAGCCCGAGAGATCCTCCACAAGGCCCAGAAGAAGGGTGTGACC ATCATTGGTCCCGCTACCGTCGGAGGTATCAAGCCCGGTTGCTTCAAGGTTGGAAACACCGGAGGTATGATGGACAACATTGTCGCCTCC AAGCTCTACCGACCCGGCTCCGTTGCCTACGTCTCCAAGTCCGGAGGAATGTCCAACGAGCTGAACAACATTATCTCTCACACCACCGAC GGTGTCTACGAGGGTATTGCTATTGGTGGTGACCGATACCCTGGTACTACCTTCATTGACCATATCCTGCGATACGAGGCCGACCCCAAG TGTAAGATCATCGTCCTCCTTGGTGAGGTTGGTGGTGTTGAGGAGTACCGAGTCATCGAGGCTGTTAAGAACGGCCAGATCAAGAAGCCC ATCGTCGCTTGGGCCATTGGTACTTGTGCCTCCATGTTCAAGACTGAGGTTCAGTTCGGCCACGCCGGCTCCATGGCCAACTCCGACCTG GAGACTGCCAAGGCTAAGAACGCCGCCATGAAGTCTGCTGGCTTCTACGTCCCCGATACCTTCGAGGACATGCCCGAGGTCCTTGCCGAG CTCTACGAGAAGATGGTCGCCAAGGGCGAGCTGTCTCGAATCTCTGAGCCTGAGGTCCCCAAGATCCCCATTGACTACTCTTGGGCCCAG GAGCTTGGTCTTATCCGAAAGCCCGCTGCTTTCATCTCCACTATTTCCGATGACCGAGGCCAGGAGCTTCTGTACGCTGGCATGCCCATT TCCGAGGTTTTCAAGGAGGACATTGGTATCGGCGGTGTCATGTCTCTGCTGTGGTTCCGACGACGACTCCCCGACTACGCCTCCAAGTTT CTTGAGATGGTTCTCATGCTTACTGCTGACCACGGTCCCGCCGTATCCGGTGCCATGAACACCATTATCACCACCCGAGCTGGTAAGGAT CTCATTTCTTCCCTGGTTGCTGGCCTCCTGACCATTGGTACCCGATTCGGAGGTGCTCTTGACGGTGCTGCCACCGAGTTCACCACTGCC TACGACAAGGGTCTGTCCCCCCGACAGTTCGTTGATACCATGCGAAAGCAGAACAAGCTGATTCCTGGTATTGGCCATCGAGTCAAGTCT CGAAACAACCCCGATTTCCGAGTCGAGCTTGTCAAGGACTTTGTTAAGAAGAACTTCCCCTCCACCCAGCTGCTCGACTACGCCCTTGCT GTCGAGGAGGTCACCACCTCCAAGAAGGACAACCTGATTCTGAACGTTGACGGTGCTATTGCTGTTTCTTTTGTCGATCTCATGCGATCT TGCGGTGCCTTTACTGTGGAGGAGACTGAGGACTACCTCAAGAACGGTGTTCTCAACGGTCTGTTCGTTCTCGGTCGATCCATTGGCCTC ATTGCCCACCATCTCGATCAGAAGCGACTCAAGACCGGTCTGTACCGACATCCTTGGGACGATATCACCTACCTGGTTGGCCAGGAGGCT ATCCAGAAGAAGCGAGTCGAGATCAGCGCCGGCGACGTTTCCAAGGCCAAGACTCGATCATAG >SEQ_ID_NO:_27_Nucleotide_sequence_for_ATP:citrate_lyase_(ACL2)_from_Yarrowia_lipolytica ATGTCAGCGAAATCCATTCACGAGGCCGACGGCAAGGCCCTGCTCGCACACTTTCTGTCCAAGGCGCCCGTGTGGGCCGAGCAGCAGCCC ATCAACACGTTTGAAATGGGCACACCCAAGCTGGCGTCTCTGACGTTCGAGGACGGCGTGGCCCCCGAGCAGATCTTCGCCGCCGCTGAA AAGACCTACCCCTGGCTGCTGGAGTCCGGCGCCAAGTTTGTGGCCAAGCCCGACCAGCTCATCAAGCGACGAGGCAAGGCCGGCCTGCTG GTACTCAACAAGTCGTGGGAGGAGTGCAAGCCCTGGATCGCCGAGCGGGCCGCCAAGCCCATCAACGTGGAGGGCATTGACGGAGTGCTG CGAACGTTCCTGGTCGAGCCCTTTGTGCCCCACGACCAGAAGCACGAGTACTACATCAACATCCACTCCGTGCGAGAGGGCGACTGGATC CTCTTCTACCACGAGGGAGGAGTCGACGTCGGCGACGTGGACGCCAAGGCCGCCAAGATCCTCATCCCCGTTGACATTGAGAACGAGTAC CCCTCCAACGCCACGCTCACCAAGGAGCTGCTGGCACACGTGCCCGAGGACCAGCACCAGACCCTGCTCGACTTCATCAACCGGCTCTAC GCCGTCTACGTCGATCTGCAGTTTACGTATCTGGAGATCAACCCCCTGGTCGTGATCCCCACCGCCCAGGGCGTCGAGGTCCACTACCTG GATCTTGCCGGCAAGCTCGACCAGACCGCAGAGTTTGAGTGCGGCCCCAAGTGGGCTGCTGCGCGGTCCCCCGCCGCTCTGGGCCAGGTC GTCACCATTGACGCCGGCTCCACCAAGGTGTCCATCGACGCCGGCCCCGCCATGGTCTTCCCCGCTCCTTTCGGTCGAGAGCTGTCCAAG GAGGAGGCGTACATTGCGGAGCTCGATTCCAAGACCGGAGCTTCTCTGAAGCTGACTGTTCTCAATGCCAAGGGCCGAATCTGGACCCTT GTGGCTGGTGGAGGAGCCTCCGTCGTCTACGCCGACGCCATTGCGTCTGCCGGCTTTGCTGACGAGCTCGCCAACTACGGCGAGTACTCT GGCGCTCCCAACGAAACCCAGACCTACGAGTACGCCAAAACCGTACTGGATCTCATGACCCGGGGCGACGCTCACCCCGAGGGCAAGGTA CTGTTCATTGGCGGAGGAATCGCCAACTTCACCCAGGTTGGATCCACCTTCAAGGGCATCATCCGGGCCTTCCGGGACTACCAGTCTTCT CTGCACAACCACAAGGTGAAGATTTACGTGCGACGAGGCGGTCCCAACTGGCAGGAGGGTCTGCGGTTGATCAAGTCGGCTGGCGACGAG CTGAATCTGCCCATGGAGATTTACGGCCCCGACATGCACGTGTCGGGTATTGTTCCTTTGGCTCTGCTTGGAAAGCGGCCCAAGAATGTC AAGCCTTTTGGCACCGGACCTTCTACTGAGGCTTCCACTCCTCTCGGAGTTTAA ctgctcaagaacaagaacatgcagaaggacttccctctgttcgagtccacctggggcattatccacggtgagctcaagattgatgatctc cccgagattctttaccacgccaactag >SEQ_ID_NO:_40_core_UAS_sequence: gccggatcgaggtgggcgggaacaaccctctcgataatgtacgtacactacacacaaagagaagcaagtgggggcgggactgcacggcac ttgtgagacacctcagcatg >SEQ_ID_NO:_41:_Sequence_of_the_XRP2_UAS_4x_UAS: cgatacgcgtatcgatacgcgtgcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgta cgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgct tctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgaggtgtctcacaagtgccgtgc agtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacctcgatccggcatgctgagg tgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagagggttgttcccgcccacc tcgatccggcatgcactgatcacgggcaaaagtgcgttcgatagagagg Figure Legends – Schematic representation of the lipid metabolism in Yarrowia lipolytica. Metabolites are written in blue, enzymes in orange, and heterologous enzymes in purple. – Effect of DGA1 overexpression on lipid production. A) effect of DGA1, ACC1, GAPC and MCE2; B) effect of DGA1; C) effect of extra DGA1. – Study on the effect of GM on lipid production and yield. – Study of the effect of ACC1 in lipid production. content of genetic engineered Yarrowia lipolytica strains. Bar graph showing lipid content as a percentage of cell dry weight (%CDW) in wild- type (W29 WT) and engineered Y. lipolytica strains expressing either ylDGA1 or cpDGA2 under the control of the pTEF or 4UASpTEF promoters. Strains were cultivated in BL05M lipogenic medium for 96 hours prior to gravimetric lipid extraction. Values represent the mean ± standard deviation of biological replicates. Enhanced lipid accumulation was observed in strains expressing cpDGA2 under the 4UASpTEF promoter, reaching up to 57.89% CDW Figure 6 - Lipid titre of genetically engineered Yarrowia lipolytica strains. Bar graph showing lipid titre (g / L) in wild-type (W29 WT) and engineered Y. lipolytica strains expressing either ylDGA1 or cpDGA2 under the control of the pTEF or 4UASpTEF promoters. Strains were grown in BL05M lipogenic medium for 96 hours, and total lipid was quantified by gravimetric extraction. Values represent the mean ± standard deviation of biological replicates. The highest lipid titre was observed in the 4UASpTEF- cpDGA2-tLIP2 strain, achieving 14.76 g / L. Figure 7 - Lipid content (%CDW) of genetic engineered yeast genotypes (DGA combinatory overexpression). Bar chart showing the lipid content, expressed as percentage of cell dry weight (%CDW), for various engineered yeast genotypes. The x-axis indicates the different genotypes: (1) 4UASpTEF-ylDGA1 + 4UASpTEF-cpDGA2, (2) 4UASpTEF-ylDGA1 + pTEF-cpDGA2, (3) 4UASpTEF-ylDGA1 + pTEF-cpDGA2 + pTEF-rtDGA1, (4) pTEF-ylDGA1 + 4UASpTEF-cpDGA2, (5) pTEF-ylDGA1 + pTEF- cpDGA2, (6) pTEF-ylDGA1 + pTEF-cpDGA2 + pTEF-rtDGA1, (7) 2x 4UASpTEF-cpDGA2, (8) 4UASpTEF-cpDGA2 + pTEF-cpDGA2, (9) 4UASpTEF-cpDGA2 + pTEF-cpDGA2 + pTEF-rtDGA1, and Control: 4UASpTEF-cpDGA2. Bars represent mean values with error bars indicating standard deviation from biological replicates. Figure 8 - Lipid titre (g / L) of engineered yeast genotypes (DGA combinatorial overexpression). Bar chart presenting the lipid titre in grams per liter (g / L) for various engineered yeast genotypes. Genotypes are identical to those described in Figure 1: (1) 4UASpTEF-ylDGA1 + 4UASpTEF-cpDGA2, (2) 4UASpTEF-ylDGA1 + pTEF-cpDGA2, (3) 4UASpTEF-ylDGA1 + pTEF-cpDGA2 + pTEF-rtDGA1, (4) pTEF-ylDGA1 + 4UASpTEF-cpDGA2, (5) pTEF-ylDGA1 + pTEF-cpDGA2, (6) pTEF-ylDGA1 + pTEF- cpDGA2 + pTEF-rtDGA1, (7) 2x 4UASpTEF-cpDGA2, (8) 4UASpTEF-cpDGA2 + pTEF- cpDGA2, (9) 4UASpTEF-cpDGA2 + pTEF-cpDGA2 + pTEF-rtDGA1, and Control: 4UASpTEF-cpDGA2. Error bars denote standard deviation from biological replicates. Figure 9 - Comparison of lipid content (%CDW) and lipid titre (g / L) in Yarrowia strains expressing either one (strain pVS5 which is Yarrowia lipolytica, W29 strain, mhy1Δ, 4UAS-pTEF-cpDGA2-tLip2) or two (pVS5 with 2x 4UASpTEF-cpDGA2) copies of the cpDGA2 gene from Claviceps purpurea after 168 hours of fermentation. Bars represent mean values, with error bars indicating the range from technical replicates, measured by gravimetry from replicate cell harvests. Increasing gene copy number did not yield a notable improvement in lipid titre or content, demonstrating that more copies do not necessarily lead to better lipid accumulation under these conditions. Figure 10 - Comparison of lipid content (%CDW) and lipid titre (g / L) in Yarrowia strains expressing either pTEF-cpDGA2 + pTEF-rtDGA1 (from Rhodotorula toruloides) or pVS5 4UASpTEF-cpDGA2 after 144 hours of fermentation. Bars represent mean values, with error bars indicating the range from technical replicates, measured by gravimetry from replicate cell harvests. The pVS5 4UASpTEF-cpDGA2 strain exhibited slightly higher lipid content and titre compared to the pTEF-cpDGA2 + pTEF-rtDGA1 strain. These results demonstrate that combining cpDGA2 and rtDGA1 does not substantially outperform a single strong cpDGA2 expression cassette under these conditions. Figure 11 – microscopy images of (A) strain pVS5; and (B) strain pVS5 with the MHY1 gene knocked out. Figure 12 - Oil content, lipid titre, and biomass production in a bioreactor (strain pVS5 with MHY1 knocked out) at 120 h and 142 h. Bar chart showing oil content (as % cell dry weight, %CDW), lipid titre (g / L), and total cell dry weight (tCDW, g / L) in a bioreactor run (strain pVS5 with MHY1 knocked out) at two timepoints. Values represent mean and standard deviation from biological replicates. Oil content and lipid titre were determined gravimetrically following chloroform extraction, as described in Methods. EXAMPLES Example 1 Strains with the following genotypes were created in a wild-type W29 background: pTEF-ylDGA1; 4UASpTEF-ylDGA1; pTEF-cpDGA2 4UASpTEF-cpDGA2 ylDGA1 is the Yarrowia lipolytica DGA1; cpDGA2 is the Claviceps purpurea DGA2. Sequences were codon optimised for expression in Y. lipolytica with cpDGA2 having the nucleic acid sequence of SEQ ID NO: 25 and ylDGA1 having the sequence of SEQ ID NO: 21. Deep well plate screening of 21 individual colonies of each genotype was carried out, using bodipy (lipid-specific fluorescent stain) staining to estimate lipid content after 3 days of fermentation. The clones which produced the highest amount of lipids from each genotype was selected to take forwards into a five day deep well plate fermentation. The data is shown in Figure 5 and 6. W29 is the wild type. Overexpression of a diacylglycerol acyltransferase (DGA) enzyme using the pTEF promoter in Yarrowia lipolytica W29 increases lipid content compared to wild-type (W29). Further enhancement of lipid accumulation is achieved by expressing the DGA enzyme under the 4UAS-pTEF promoter, demonstrating the benefit of engineered promoter architecture. Remarkably, overexpression of Claviceps purpurea DGA2 (CpDGA2) under the 4UAS-pTEF promoter (4UAS-pTEF-CpDGA2) results in a significant and unique increase in lipid content and titre, surpassing all other tested strains. This was achieved in a background that had only the native DGA1 in the native locus, demonstrating that overexpression of DGA2 positively impacts lipid yield, independently of DGA1 expression levels. The particular combination of cpDGA2 and the 4UAS-pTEF promoter in Yarrowia lipolytica represents a minimally-edited approach for maximising lipid accumulation. Example 2 – additional copies of DGA do not further improve lipid yield The following strains were generated to determine whether additional copy number of DGA1 and / or DGA2 imparted additional effects over that achieved by expressing cpDGA2 from the 4UAS-pTEF promoter. 20 Gen N Full genotype 1 4UASpTEF-ylDGA1-tLip2, 4UASpTEF-cpDGA2-tLip2 2 4UASpTEF-ylDGA1-tLip2, pTEF-cpDGA2-tLip2 3 4UASpTEF-ylDGA1-tLip2, pTEF-cpDGA2-tLip2, pTEF- rtDGA1-tLip2 4 pTEF-ylDGA1-tLip2, 4UASpTEF-cpDGA2-tLip2 5 pTEF-ylDGA1-tLip2, pTEF-cpDGA2-tLip2 6 pTEF-ylDGA1-tLip2, pTEF-cpDGA2-tLip2, pTEF-rtDGA1- tLip2 7 2x 4UASpTEF-cpDGA2 8 4UASpTEF-cpDGA2-tLip2, pTEF-cpDGA2-tLip2 9 4UASpTEF-cpDGA2-tLip2, pTEF-cpDGA2-tLip2, pTEF- rtDGA1-tLip2 Control 4UASpTEF-cpDGA2-tLip2 Data is shown in Figure 7 and 8. None of the various combinations of additional DGA1 or DGA2 with the various different promoters enhanced lipid production over that achieved by simply expressing one copy of cpDGA2 from the 4UAS-pTEF promoter (strain pVS5 – genotype Yarrowia lipolytica, W29 strain, mhy1Δ, 4UAS-pTEF-cpDGA2-tLip2). Y. lipolytica expressing one copy of cpDGA2 from the 4UAS-pTEF promoter represents a minimally edited cell capable of achieving high lipid yields, high or higher than those achieved by yeast cells that comprise more gene edits. Example 3 The potential additive effects of increasing the copy number of 4UAS-pTEF-cpDGA2 was explored further in a 168 hour fermentation. See Figure 9. Comparison of lipid content (%CDW) and lipid titre (g / L) in Yarrowia strains expressing either one (pVS5 4UASpTEF-cpDGA2) or two (2x 4UASpTEF-cpDGA2) copies of the cpDGA2 gene from Claviceps purpurea after 168 hours of fermentation. Bars represent mean values, with error bars indicating the range from technical replicates, measured by gravimetry from replicate cell harvests. Surprisingly, increasing gene copy number did not yield a notable improvement in lipid titre or content, demonstrating that more copies do not lead to better lipid accumulation under these conditions. Example 4 The single modification to express cpDGA2 from 4UAS-pTEF produces cells that are at least as good as, if not better than, cells that comprise two modifications to overexpress both rtDGA1 and cpDGA2 Strains used: 1x 4UAS-pTEF-cpDGA2 vs pTEF-cpDGA2 + pTEF-rtDGA1 Yarrowia strains expressing either pTEF-cpDGA2 + pTEF-rtDGA1 (from Rhodotorula toruloides) or pVS54UASpTEF-cpDGA2 were cultured for 144 hours. Data is shown in Figure 10. Biomass and lipid content were measured after 4 days and 5 days of a 5 day 2-stage fermentation (with additional glucose added after 3 days). The pVS5 4UASpTEF- cpDGA2 strain achieves lipid content comparable with any other published Yarrowia lipolytica strain, but with one modification rather than 3 or 4, demonstrating that the rtDGA1 is not required to achieve the high lipid titre that can be obtained with expression of cpDGA2 from the 4UASpTEF promoter alone – i.e. one overexpression rather than two.vs5 Bars represent mean values, with error bars indicating the range from technical replicates, measured by gravimetry from replicate cell harvests. The pVS54UASpTEF- cpDGA2 strain exhibited slightly higher lipid content (73% after 120 hours) and titre compared to the pTEF-cpDGA2 + pTEF-rtDGA1 strain. These results demonstrate that combining cpDGA2 and rtDGA1 does not outperform a single cpDGA2 expression from 4UAS-pTEF. Example 5 The MHY1 gene was knocked out in the pVS5 background (4UASpTEF-cpDGA2 strain) to remove hyphal growth. Figure 11 B shows the lack of hyphal growth in the knockout strain with cells remaining in the yeast-like morphology. A 5 day 2-stage fermentation was performed, with additional glucose added after 3 days, and the lipid content was measured after 4 and 5 days. Data is shown in Figure 12. A lipid content of 71% lipid was achieved after 120h, 74% lipid content after 144h and 78% lipid content after 168h, demonstrating high lipid content in a strain modified to prevent hyphal growth. Example 6 – Methods for Examples 1-5 Cloning pipeline DNA fragments with BsaI sites were synthesized by GeneArt. Assembly was performed in-house using the NEB BsaI-HFv2 Golden Gate Assembly Kit (NEB #E1601). Reactions (20 µL) contained a vector (75 ng), inserts at 2:1 molar ratio, 2 µL 10X T4 DNA Ligase buffer, 1–2 µL enzyme mix, and water. Thermocycling: 25 cycles of 37°C for 3 min and 16°C for 4 min, followed by 50°C for 5 min. 2 µL of the assembly mix was transformed into 50 µL SURE2 Competent cells (Agilent) (42°C, 30 s), recovered in SOC for 1 h at 37°C, then plated on selective LB agar. Colonies were screened, and positive clones were grown overnight for plasmid miniprep. Yeast transformations For each transformation, 100 µL of the prepared transformation mix was aliquoted into sterile microcentrifuge tubes. A loopful of Yarrowia biomass from a fresh YPD plate was added, followed by 5 µL of denatured salmon sperm ssDNA carrier and 10 µL (2–3 µg) of linearised plasmid DNA. The mixture was briefly vortexed and incubated at 30°C with shaking for 30 minutes, then vortexed again. Cells were heat-shocked at either 42°C for 10 minutes or 39°C for 30 minutes to enhance transformation efficiency. Following heat shock, cells were washed twice by centrifugation, resuspended in sterile water, and plated onto selective media. Plates were incubated at 30°C for 48 hours, after which colonies were re-streaked onto fresh selective plates. Genomic DNA extraction and colony PCR were performed the next day to confirm successful transformants. Strain Revival and Plate Preparation Frozen (-80°C) yeast stocks were briefly thawed and streaked onto YPD agar plates using sterile loops. The plates were inverted and incubated at 30°C for 48 to 72 hours, until colonies reached approximately 1–2 mm in diameter. Prior to inoculation into pre- culture or transformation, strains were re-streaked once more on agar to ensure cells were in the exponential growth phase. Pre-culture Preparation Single colonies were picked and inoculated into 2.4mL of YPD medium in 24-deep-well plates (24dwp). The cultures were incubated at 30°C with shaking at 300 rpm for 12 to 16 hours to reach late logarithmic phase. Lipogenic Culture Setup The overnight cultures were diluted 1:40 into nitrogen-limited BL05M lipogenic medium supplemented with 1µg / mL BODIPY 493 / 503 dye. The cultures were incubated under the same conditions, protected from light to prevent photobleaching of the dye. Media used The YPD medium (Sigma-Aldrich) was used in both liquid and solid forms, with 2% agar added for solid plates, serving as a rich medium for yeast pre-culture and general propagation. Selective growth of transformants was achieved using SC–URA plates, prepared by supplementing Yeast Uracil Synthetic Drop-out Medium without uracil with 6.7 g / L Yeast Nitrogen Base and solidified with 2% bacteriological agar, providing a defined medium that selects for uracil prototrophy. Lipogenic conditions were established using BL05M medium, composed of 3.4 g / L Yeast Nitrogen Base, 1 g / L ammonium sulphate as the nitrogen source, 100 g / L glucose, 2.5 g / L yeast extract, and 23.2 g / L maleic acid, with the pH adjusted to 5, designed to promote lipid accumulation under nitrogen-limited conditions. Deep well plate (genotypic and clonal screening) The purpose of this screening was to perform genotypic and clonal evaluation of the best expression variants of ylDGA1 and cpDGA2 in Yarrowia lipolytica W29 base strain, aiming to identify clones with optimal lipid accumulation phenotypes. For a primary screening, a single colony per clone was inoculated into individual wells of a 24-deep-well plate containing 2.4 mL of YPD medium, serving as a starter pre- culture. Each genotype was represented by 21 clones, with the remaining wells allocated to controls including blank wells, a DGA1 overexpression strain as a positive control, and the W29 wild-type strain. Cultures were incubated at 30°C with shaking for 16 to 20 hours to reach late exponential phase. Following pre-culture, cells were inoculated into lipogenic medium BL05M supplemented with 1 µg / mL BODIPY 493 / 503 dye to stain intracellular lipid droplets. Cultures were then incubated under lipogenic conditions for 96 hours. At 96 hours post-inoculation, 100 µL of each culture was transferred in technical duplicate (two wells per clone) into black, flat-bottom 96-well plates suitable for fluorescence measurement. Fluorescence intensity was measured using a PHERAstar microplate reader with excitation / emission filters set at 493 / 520 nm, corresponding to BODIPY fluorescence. The average fluorescence of the technical replicates was calculated for each clone. Clones and genotypes exhibiting the highest fluorescence intensities, indicative of enhanced lipid accumulation, were selected for subsequent validation screening. Deep well plate (validation) The validation experiment was conducted using biological replicates of the three most promising clones per genotype identified during primary screening. These biological replicates consisted of distinct single colonies (labelled A, B, and C) that were individually inoculated into separate wells of 24-deep-well plates containing 2.4 mL YPD medium for pre-culture. Following growth, each biological replicate (A, B, and C) was used to inoculate a full column of wells (eight wells per column) in a new 24-deep- well plate, with 2.4 mL of lipogenic medium BL05M per well. This setup ensured consistent biological replication across the plate. To obtain sufficient biomass for direct lipid extraction and gravimetric quantification, the cultures from all wells within each replicate column were pooled. This approach provided adequate material for accurate lipid analysis, enabling robust validation of lipid accumulation in the selected clones. Gravimetry - Chloroform extraction protocol – see Example 8 Golden gate protocol - see Example 8 Transformation protocol - see Example 8 Knockout transformation protocol - see Example 8 Example 7 The following strains were generated: Strain Genotype W29 Wildtype SBB060 pGPD1-GAPC-tLip2, pTEF-MCE2-tXPR2, pMnDH2-ACC1-tLip2, pTEF-DGA1-tLip2 SBB065 pGPD1-GAPC-tLip2, pTEF-MCE2-tXPR2, pMnDH2-ACC1-tLip2, pTEF-DGA1-tLip2 (2x) SBB055 pGPD1-GAPC-tLip2, pTEF-MCE2-tXPR2 SBB074 pGPD1-GAPC-tLip2, pTEF-MCE2-tXPR2, pTEF-DGA1-tLip2 SBB071 pGPD1-GAPC-tLip2 (2x), pTEF-MCE2-tXPR2 (2x), pMnDH2-ACC1-tLip2, pTEF- DGA1-tLip2 Strain SBB060 showed an increased lipid content and titre compared to that of the wildtype (Figure 2A, media YNB02). We also tested the effect of overexpressing DGA1 in the wildtype genetic background, observing an increase in production with this single overexpression (Figure 2B, media YNB02). Furthermore, we studied the effect of adding a second copy of DGA1 on the SBB060 strain. This gave place to strain SBB065, which showed increased, and more robust, lipid production compared to the parental strain when compared directly, under the same conditions (Figure 2C, media YNB02). The strain SBB065 was used in further experiments using both benchtop bioreactors, as well as large-scale bioreactors. The best values observed in bioreactors were for the strain SBB060, which reached a titre of 24.2 g / L, a lipid content of 48% and a productivity of 0.33 g / L / h. This was achieved by using urea as the nitrogen source at the beginning of the fermentation. It was decided to test the effect of GAPC and MCE2 on their own (strain SBB055), as well as in combination with DGA1 (strain SBB074), and the best producer SBB065. The results indicate that the expression of GAPC and MCE2 does not have any effect on either lipid production or yield (Figure 3). Nevertheless, extra copies of GAPC and MCE2 were added to SBB060 to generate strain SBB071, which showed an increase in lipid production. The ACL1 and ACL2 genes code for the heterodimeric enzyme ATP:citrate lyase (ACL), which is regarded as the key enzyme for acetyl-CoA generation. Acetyl-CoA is the initial building block for fatty acids in oleaginous yeasts and fungi, In fact, the presence of ACL in Y. lipolytica is a major distinction between its metabolic network and that of non-oleaginous organisms. Deletion of either ACL gene results in reduced ability to accumulate lipids. Both subunits localise to the cytosol where they presumably interact to drive lipid accumulation from citrate. The overexpression of these subunits separately or concomitantly will be tested and effect on lipid production determined. Carbon can alternatively be stored by yeast as glycogen, as the major alternative to triacylglycerols. A deletion of the gene coding for glycogen synthase (GSY1) will be made and the effect on lipid production determined. In order to maximise lipid accumulation it is desirable to prevent lipid degradation. Degradation of triacylglycerols is catalysed by triacylglycerol lipase (TGL) enzymes, which release fatty acid molecules for energy production via beta-oxidation. Deletions of TGL4 will be made and the effect on lipid production determined. We will test lipid accumulation with DGA1, GapC, MCE2, GPD1 and CpDGA2 in different combinations and expressed under three different promoters: 1. pTEF, the standard strong constitutive promoter 2. pTEF-Kozak, with 3 copies of CACA in tandem immediately before the initiation codon AUG. This is considered a Kozak consensus ribosome binding site sequence and is believed to be important for eukaryotic translation. (Jiang et al 2024 Bioresource Technology 399: 130614). 3. pTEF-4UAS, with 8x additional upstream activation sequences. Upstream activation sequences enhance transcriptional activity, and have been shown to significantly improve protein expression for the pTEF promoter. Example 8 – Methods for Example 7 1. Design a. Backbones / Vectors Two different vectors have been used. At the first stage, we prepared our own homemade vector to use the golden gate assembly technique based on the PaqCI restriction enzyme. The backbone of this vector, containing the origin of replication and the ampicillin marker for bacterial selection, was derived from the plasmids obtained from AddGene’s EasyCloneYALI Collection (Addgene Kit #1000000140 and #1000000141). To this backbone we added zeta regions for random integration into Y. lipolytica’s genome, a marker for yeast selection, a strong, endogenous promoter (pTEF) and an endogenous terminator (tLIP2), as well as the restriction sites for PaqCI. After this we acquired the Yarrowia lipolytica Golden Gate tool kit (Addgene kit #1000000167), which offered their own golden gate assembly system based on the BsaI restriction enzyme. We ordered the backbones to be synthesised again to allow for marker recycling, by adding a missing loxP region. For this, the standard backbone used by ThermoFisher for cloned genes was used (pMA). This backbone thereafter was used instead of the other ones for all purposes. b. Markers used Three different markers were used. Two markers, to grant resistance to hygromycin (HygR marker) and nourseothricin (NtcR marker) antibiotics, were ordered as synthetic constructs from ThermoFisher. The URA3 marker, granting the ability to grow without uracil, was amplified from Y. lipolytica genomic DNA. c. Knockout Gene knockout or deletions were performed through homologous recombination. Two regions of 1 kb in length were used to target the exchange of the coding sequence of the gene to be deleted with the URA3 marker. d. Overexpression Endogenous genes were overexpressed by introducing them under the control of a strong constitutive promoter. Genomic integration was performed using zeta sequences, which can integrate randomly into the genome., increasing the efficiency of integration. e. Heterologous expression Expression of genes from other organisms was performed in a similar manner to the overexpression of endogenous genes. The gene sequence for the heterologous genes to be expressed was modified to tailor the codon composition to that of Y. lipolytica by using the GeneArt tool from ThermoFisher. 2. Genetic engineering a. Transformation Transformation of DNA fragments or plasmids was performed through the lithium acetate (LiAc) method. The day before, the yeast strains to be transformed were spread on solid growth media (YPD agar) to obtain fresh biomass. Also, DNA fragments to be transformed were obtained by digesting the plasmid containing them with the restriction enzyme NotI at room temperature overnight. The day of the transformation, a transformation mix was prepared containing PEG 40%, of LiAc 0.1M at pH 6, and Dithiothreitol (DTT) 0.1M. A spoonful of fresh yeast biomass was resuspended in 100 μl of transformation mix, after which 5 μl of single stranded DNA from salmon sperm (ssDNA) was added. After this, 2-3 μg of the DNA to be transformed was added, the whole solution was mixed with a vortex and incubated at 28°C while shaking for 30 minutes. After this, samples were mixed again and incubated at 39°C for another 30 minutes. Finally, samples were centrifuged to remove the transformation mix and the cells were resuspended in water before plating them into selective solid media. When performing knockouts or directed integration, a variation of this protocol involving hydroxyurea was used to increase the efficiency of homologous recombination. The fresh biomass was added to 10 mL of liquid growth media and incubated at 28°C and 200 rpm for 3 hours. Next,95 mg of hydroxyurea was added and the cells were incubated for another 2 hours. After this, cells were washed, centrifuged and resuspended in a volume of water equal to the volume of the cell pellet obtained. The protocol continued as above using 50 μl of the processed cells. b. Marker recycling To be able to perform serial rounds of genetic modifications, markers used to modify the Y. lipolytica genome contained loxP regions in their flanks. These regions were used to remove the marker from the genome by expressing a Cre recombinase from a replicative plasmid. After transforming the plasmid containing the recombinase, colonies were tested to check the loss of the marker, after which they were tested to check the loss of the replicative plasmid. 3. Validation and screening a. Colony PCR To validate the correct integration of genes, or the correct deletion of genes, colony PCR was used to check the presence of desired DNA sequences in the modified Y. lipolytica genome. Colonies obtained from the transformation were restreaked on fresh, solid media. After one day of growth, a small amount of the colony biomass was resuspended in 2 μl of water and lysed with the following thermocycler program: ● 30 seconds at 65°C ● 30 seconds at 8°C ● 1 minute 30 seconds at 65°C ● 3 minutes at 97°C ● 1 minute at 8°C ● 3 minutes at 65°C ● 1 minute at 97°C ● 1 minute at 65°C ● 10 minutes at 80°C ● Hold at 10°C The lysed cells were used as template for a touchdown PCR using OneTaq® Quick- Load® DNA Polymerase from NEB. The PCR product was checked through a 1% agarose gel electrophoresis. b. qPCR Quantitative PCR (qPCR) was performed to validate the expression changes at RNA level of engineered genes. RNA was extracted from yeast cultures with the RiboPure™ Yeast RNA Purification Kit from ThermoFisher. SuperScript™ IV Reverse Transcriptase from ThermoFisher was used to generate cDNA. The cDNA was used as a DNA template for a PCR reaction using OneTaq® Quick-Load® DNA Polymerase from NEB. This PCR was performed to check for the correct functioning of qPCR primers, as well as to check the correct deletion or heterologous expression of genes, for which no quantification is required. After validation of the cDNA samples and primers, qPCR reactions were set up in triplicates using the iTaq™ Universal SYBR® Green Supermix from BioRad and they were run on the CFX Opus 96 Real-Time PCR System from BioRad, using the protocol recommended by the manufacturer. The raw data obtained was used to calculate ΔΔCq values to compare the expression between the mutants and the controls. 4. Fermentation a. Shake-flasks Strains to be tested were grown in 5 mL of YPD (rich growth media) overnight. The optical density was measured to be able to inoculate the right volume of preculture into 250 mL flasks containing 50 mL of lipid production media (either 0.1 or 0.2 initial OD, for lipid production media see Table 1). Unless otherwise stated, shake flasks were incubated for 3 days at 28°C and 200 rpm. At the end of the run, 40 mL of each culture was transferred to falcon tubes and centrifuged at 4500 rpm for 10 min. The supernatant was separated and stored for glucose quantification, while the pellet was freeze dried for cell dry weight (CDW) measurement and lipid quantification. b. Benchtop bioreactor Benchtop bioreactors (Minifors by Infors-HT) with a working volume of 3 L were used to study the behaviour in larger scale conditions, as well as the effect of pH and aeration on lipid production by the mutant strains. It also allowed us to study the effect of changing process conditions during the growth and lipogenic phase in order to optimise them. To keep the pH at the desired setpoint, 4M NaOH and 6M H2SO4 were used. Struktol SB648 was used to prevent foaming. The tip speed of the impellers was manually set to 1.13-1.98 m / s, while the aeration was manually set at 0.5-1.5 vvm depending on the desired dissolved oxygen setpoint. For fermentation media see Table 1. c. 70L and 350L Bioreactors Stainless steel bioreactors (Applikon) were used to test strains, media and process conditions for large-scale production scenarios. In general, conditions from smaller scales (see Benchtop bioreactors) were tested on first 70L, then 350L scale. The working volumes were 50L and 250L, respectively. 3M NaOH and 3 M H3PO4 were used to control pH and Struktol SB2121 was used to prevent foaming events. To control the dissolved oxygen levels, a tip speed of 0.60-1.98 m / s and an air volume of 0.5-1.5 vvm was applied using an automated controller (Solaris). Table 1: Lipid production media used and their composition Media Content Addition in lipogenic phase YNB02 Glucose 50 g / L, YNB 1.7 g / L, (NH4)2SO4 1.1 g / L - BL14 Glucose 100 g / L, YNB 1.7 g / L, Urea 1 g / L - BL25 Glucose 40 g / L, YNB 1.7 g / L, Urea 0.5 g / L - FM04 Glucose 40 g / L, YNB 3.4 g / L, urea 1 g / L Glucose 90 g / L AI02 Glucose 90 g / L, YNB 3.4 g / L, urea 5 g / L Glucose 90 g / L AI05 Glucose 90 g / L, YNB 3.4 g / L, urea 2.5 g / L Glucose 90 g / L 5. Lipid quantification a. Gravimetry Screw-cap tubes were filled with ~400μL of 500-750 μm glass beads and with 100 mg of freeze dried yeast cells. The biomass was disrupted twice in a bead beater for 10x30s at 4500 rpm, after which 1 mL of a 2:1 (v:v) mixture of chloroform and methanol was added and the bead beater disruption was repeated. Samples were centrifuged and the supernatant was transferred to a 15 mL falcon tube. Adding the mixture, disrupting with the bead beater and centrifuging was repeated three times more, pooling the supernatants in the same tube. After four rounds of extraction, 2 mL of KCl 1M : methanol (4:1 v / v with 0.034% MgCl2) were added to the falcon tube containing the chloroform:methanol mixture. Samples were mixed and centrifuged, after which the bottom layer was transferred to a pre-weighed glass vial. The solvent was left to evaporate for two days before measuring the total weight of the extracted lipids. b. GC-FID Freeze dried cells were lysed using a stainless steel ball with a tissue lyser ball mill (e.g. Qiagen Tissuelyser II), in the presence of 0.5ml hexane for 5 minutes. 0.5 mL of 7% sodium methoxide solution and 50 μl of 10 mg / mL C17:0 methyl-ester analytical standard was then added to the sample tube and ground with a ball mill (e.g. Qiagen Tissuelyser II) for another 5 min, after which it was left at room temperature for 15 min. Soluble residues were removed by adding 0.5 mL of water, mixing and recovering the upper hexane-rich layer, which was added to a glass chromatography vial. Samples were analysed through GC-FID (Agilent 8890), using a Zebron ZB-FAME column (60m x 0.25mm ID x 0.20um), hydrogen as carrier gas with a constant flow of 2 mL / min. The initial temperature was 50°C, which was increased to 240°C with an increase of 10°C per minute. 6. Glucose quantification a. Colorimetric assay The D-Glucose Assay Kit (GOPOD Format) from Neogen / Megazyme was used. The reagent was prepared as indicated by the manufacturer. A glucose standard curve was prepared using the standard provided. Samples were diluted if necessary and 50 μl were mixed with 1.5 mL of the GOPOD reagent. They were mixed and incubated at 48°C for 20 min, after which the absorbance was measured at 510 nm and the glucose concentration calculated based on the standard curve. b. HPLC-RID Samples were centrifuged and the supernatant removed before being filtered and diluted in preparation for analysis. Each sample was then transferred to a liquid chromatography vial with micro insert and crimped. A calibration curve was prepared and run alongside the samples to allow accurate identification and quantification of glucose. Samples were run on an organic acid column at 35°C in a Vanquish HPLC stack equipped with a refractive index detector. The mobile phase used for the duration of the 20 minute run time was 0.005N sulphuric acid.

Claims

Claims 1. An oleaginous yeast cell that expresses a heterologous diacylglycerol acyltransferase 2 (DGA2).

2. The oleaginous yeast cell of claim 1 wherein the oleaginous yeast cell comprises a DGA2 expression cassette comprising a gene encoding a heterologous diacylglycerol acyltransferase 2 (DGA2) operably linked to a promoter.

3. The oleaginous yeast cell of claim 2 wherein the DGA2 expression cassette is integrated into the genome of the oleaginous yeast cell.

4. The oleaginous yeast cell of claim 2 or 3 wherein the oleaginous yeast cell comprises only one copy of the DGA2 expression cassette integrated into the genome.

5. The oleaginous yeast cell of claim 2 wherein the DGA2 expression cassette is maintained episomally within the oleaginous yeast cell, optionally wherein the heterologous DGA2 is expressed from a plasmid, optionally a multicopy plasmid, optionally a high copy plasmid.

6. The oleaginous yeast cell of any of claims 2-5 wherein the promoter is selected from the group comprising the pTEF-4UAS promoter (SEQ ID NO: 16); the 2UAS1- pTEF promoter; the 3UAS1-pTEF promoter; the 4UAS1-pTEF promoter; the 8UAS1- pTEF promoter; and the hp8d promoter.

7. The oleaginous yeast cell of any of claims 2-6 wherein the promoter is the pTEF- 4UAS promoter (SEQ ID NO: 16).

8. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell is a Yarrowia lipolytica cell.

9. The oleaginous yeast cell of any of the preceding claims wherein the DGA2 is from Claviceps purpurea.

10. The oleaginous yeast cell of any of the preceding claims wherein the DGA2 has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

11. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell has not been engineered to overexpress DGA1, optionally has not been engineered to overexpress the native DGA1 or a heterologous DGA1.

12. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell has not been engineered to add an additional copy of the native DGA1.

13. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell comprises the native DGA1 gene at the wild type locus under the control of the native DGA1 promoter and does not comprise an additional DGA1 gene elsewhere in the cell.

14. The oleaginous yeast cell of any of the preceding claims wherein where the oleaginous yeast cell is in a haploid state it comprises only one copy of the native DGA1 gene; and where the oleaginous yeast cell is in a diploid state the yeast cell comprises only two copies the native DGA1 gene.

15. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell does not comprise a heterologous DGA1 gene.

16. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell does not comprise a native or heterologous DGA1 gene operably linked to: a TEF promoter; or to a GPD1 promoter; a EXP1 promoter; pTEF-4UAS promoter (SEQ ID NO: 16); the 2UAS1-pTEF promoter; the 3UAS1-pTEF promoter; the 4UAS1- pTEF promoter; the 8UAS1-pTEF promoter; or the hp8d promoter.

17. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell does not comprise: a) an expression cassette comprising the DGA1 gene from Rhodotorula toruloides (rtDGA1) operably linked to a promoter; and / or b) an expression cassette comprising the DGA1 gene from L. starkeyi operably linked to a promoter.

18. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell does not comprise an overexpression of:a) native GPD1, SLC1 and / or LRO1; or b) heterologous GPD1, SLC1 and / or LRO1.

19. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and does not have a deletion or disruption of the TGL3 gene.

20. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell does not comprise a DGA2 expression cassette that comprises a DGA2 gene operably linked to a promoter that either i) consists of a pTEF promoter; or ii) is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2 optionally wherein the DGA2: a) is from Claviceps purpurea; and / or b) has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

21. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter is that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2 optionally wherein the DGA2 gene from Claviceps purpurea has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

22. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter optionally wherein the DGA2 gene from Claviceps purpurea has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

23. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell comprises: a) one copy and not more than one copy of a DGA2 expression cassette that comprises the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter; and b) no copies of a DGA2 gene operably linked to a promoter that consists of a pTEF promoter or that is a pTEF promoter that is not associated with one or more UAS sequences, optionally one or more UAS sequences from XRP2; and c) no copies of a DGA1 gene operably linked to a pTEF promoter or to a pTEF- 4UAS promoter; and d) the native DGA1 gene in its native locus under the control of the native DGA1 promoter optionally wherein the DGA2 gene from Claviceps purpurea has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

24. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell comprises the native Δ9 desaturase, optionally wherein the oleaginous yeastcell is a Yarrowia lipolytica cell and comprises the native Yarrowia lipolytica Δ9 desaturase.

25. The oleaginous yeast cell of any of claims wherein the oleaginous yeast cell: a) comprises the native Δ12 desaturase, optionally wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and comprises the native Yarrowia lipolytica Δ12 desaturase; and / or b) does not comprise an A. adeninivorans Δ9 desaturase, optionally wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and does not comprise an A. adeninivorans Δ9 desaturase.

26. The oleaginous yeast cell of any of claims wherein the oleaginous yeast cell does not comprise E. coli phosphotransferase, optionally wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and does not comprise E. coli phosphotransferase.

27. The oleaginous yeast cell of any of claims wherein the oleaginous yeast cell does not comprise Herpes simplex thymidine kinase.

28. The oleaginous yeast cell of any of the preceding claims wherein the oleaginous yeast cell natively has a hyphal growth phenotype and wherein the oleaginous yeast cell comprises a gene deletion or disruption which prevents or reduces hyphal growth.

29. The oleaginous yeast cell according to any of the preceding claims wherein the oleaginous yeast cell is a Yarrowia lipolytica cell and comprises a knockout or disruption of the MHY1 gene, wherein the disruption affects the function of the Mhy1p protein and prevents or reduces hyphal growth.

30. The oleaginous yeast cell according to any of the preceding claims wherein the oleaginous yeast cell comprises just one modification relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome.

31. The oleaginous yeast cell according to any of the preceding claims wherein the oleaginous yeast cell comprises just one modification that results in increased lipid production relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome.

32. The oleaginous yeast cell according to any of the preceding claims wherein the oleaginous yeast cell comprises just one modification that results in increased lipid production relative to a corresponding wildtype strain, wherein the modification is the insertion of the DGA2 expression cassette into the genome, and wherein the yeast cell also comprises a deletion or disruption of the MHY1 gene.

33. The oleaginous yeast cell according to any of the preceding claims wherein the oleaginous yeast cell comprises one or more insertions, deletions or substitutions for purposes other than increasing lipid production.

34. The oleaginous yeast cell according to any of the preceding claims wherein a population of the oleaginous yeast cells has a lipid content (% cell dry weight) of at least 50%, or at least 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 80% or more after culture in BL05M lipogenic medium for 96 hours.

35. The oleaginous yeast cell according to any of the preceding claims wherein a population of the oleaginous yeast cells has a lipid titre of at least 12g / L, or at least 12.2, 12.4, 12.6, 12.8, 13.0, 13.2, 13.4, 13.6, 13.8, 14.0, 14.2, 14.4, 14.6, 14.8, 15.0, 15.2, 15.4, 15.6, 15.8, 16.0, 16.2, 16.4, 16.6, 16.8, 17.0, 17.2, 17.4, 17.6, 17.8, 18.0, 18.2, 18.4, 18.6, 18.8, 19.0, 19.2, 19.4, 19.6, 19.8, or at least 20.0 g / L when cultured in BL05M lipogenic medium for 96 hours.

36. The oleaginous yeast cell according to any of the preceding claims wherein a population of the oleaginous yeast cells has a lipid content (% cell dry weight) of at least 74%, 76%, 80% or more after culture in BL05M lipogenic medium for 168 hours.

37. The oleaginous yeast cell according to any of the preceding claims wherein a population of the oleaginous yeast cells has a lipid titre of at least 25.0 g / L or at least 25.2, 25.4, 25.6, 25.8, 26.0, 26.2, 26.4, 26.6, 26.8, 27.0, 27.2, 27.4, 27.6, 27.8, 28.0, 28.2, 28.4, 28.6, 28.8, 29.0, 29.2, 29.4, 29.6, 29.8, 30.0, 30.2, 30.4, 30.6, 30.8, 31.0, 31.2, 31.4, 31.6, 31.8, or at least 32.0 g / L when cultured in BL05M lipogenic medium for 168 hours.

38. The oleaginous yeast cell according to any of the preceding claims wherein the yeast cell further comprises: i) Overexpression of NADP-dependant glyceraldehyde-3-phosphate dehydrogenase (GapC), for example GapC from Clostridium acetobutylicum, forexample a GapC from Clostridium acetobutylicum having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 3 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3; ii) Overexpression of NADP-dependant malic enzyme (MCE2), for example MCE2 from Mucor circinelloides, for example a MCE2 from Mucor circinelloides having a sequence that is codon optimised for expression in Yarrowia lipolytica, for example having the sequence of SEQ ID NO: 4 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 4; iii) Overexpression of acetyl-CoA-carboxylase (ACC1), for example ACC1 from Yarrowia lipolytica, for example ACC1 having the sequence of SEQ ID NO: 2 or having a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2; iv) express or overexpress ACC1 that has been engineered to be insensitive to deactivation via phosphorylation, for example ACC1 that comprises a S1178A substitution in the Yarrowia lipolytica ACC1 that has the sequence of SEQ ID NO: 2 or that has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2, provided that residue 1178 is an alanine; v) Overexpression of the ATP:citrate lyase (ACL), for example overexpression of ACL1 and / or ACL2, for example the ACL1 and / or ACL2 from Yarrowia lipolytica, for example: a) an ACL1 of sequence SEQ ID NO: 6 or having the sequence of SEQ ID NO: 6 or a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6; and / or b) an ACL2 of sequence SEQ ID NO: 7, or having the sequence of SEQ ID NO: 7 with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7; vi) Overexpression of NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), for example GPD1 from Yarrowia lipolytica, for example a GPD1 from Yarrowia with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 13;vii) a reduced ability to store carbon for example to store glycogen for example engineered so as to have an underexpression or knockout of glycogen synthase (GSY1); viii) a reduced ability to degrade lipids, for example engineered to have an underexpression or knockout of triacyglyceroal lipase (TGL); ix) an inability to produce hyphae, for example where the yeast is an oleaginous yeast for example where the yeast is Yarrowia lipolytica the knockout is of the Mhy1p gene; x) overexpression of fatty acid synthase (FAS), for example FAS from Yarrowia lipolytica, and / or overexpression of FAS from Rhodosporidium toruloides; xi) express or overexpress pyruvate dehydrogenase from a bacterial species, for example from E.coli; xii) overexpression of acyl-CoA-binding protein ACB1; xiii) reduced expression or a knockout of URE2; xiv) overexpression of 6-phosphogluconolactonase ; xv) overexpression of SLC1 (1-acylglycerol-3-phosphate O-acyltransferase); xvi) a reduced expression, or a gene knockout, of PEX10 (Peroxisomal membrane E3 ubiquitin ligase); and / or xvii) a reduced expression or a gene knockout of MFE1 (a Member of the Peroxisomal Hydroxyacyl Coenzyme A Dehydrogenase Family).

39. The yeast cell according to any of the preceding embodiments wherein: said overexpression of GapC is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of GapC under the control of the GPDp promoter; and / or said overexpression of MCE2 is an overexpression with respect to a Yarrowia lipolytica cell that comprises one copy of MCE2 under the control of the TEFin promoter. for example wherein the yeast cell comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more genomically integrated cassettes, for example wherein at least two of the genomically integrated cassettes encode the same gene.

40. The oleaginous yeast cell according to any of claims 38 or 39 wherein said overexpression is driven by any of the following promoters:pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) 41. The oleaginous yeast cell according to any of claims 38-40 wherein: a) the acetyl-coA-carboxylase (ACC1) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) b) the glyceraldehyde-3-phosphate dehydrogenase (GapC) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18) c) the NADP-dependant malic enzyme (MCE2) is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18); and / or d) the NAD-dependent cytosolic glycerol-3-phosphate dehydrogenase (GPD1), is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16)pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18).

42. The oleaginous yeast cell of any of the preceding embodiments wherein the yeast cell comprises an overexpression of all of ACC1, GapC and MCE2, for example where each of ACC1, GapC and MCE2 is operably linked to any of the following promoters: pTEF (SEQ ID NO: 14) pTEF Kozak (SEQ ID NO: 15)) pTEF-4UAS (SEQ ID NO: 16) pGPD1 (SEQ ID NO: 17) or pMnDH2 (SEQ ID NO: 18).

43. The oleaginous yeast cell according to any of the preceding claims wherein the expression cassette(s) comprise a transcription termination, for example a transcription terminator selected from the group comprising or consisting of the following terminators: i) tLip2 [SEQ ID NO: 20]; or ii) tXPR2.

44. The oleaginous yeast cell of any of the preceding embodiments wherein the cell does not comprise: i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and / or iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:

17.

45. The oleaginous yeast cell of any of the preceding claims where: the cell does not comprise an overexpression of any one or more of ACC1, GapC and / or MCE2.

46. The oleaginous yeast cell of any of the preceding embodiments wherein the cell does not comprise:i) an overexpression of ACC1 wherein ACC1 is operably linked to the h4pd promoter (SEQ ID NO: 19); ii) an overexpression of DGA1 wherein DGA1 is operably linked to the TEFin promoter; iii) an overexpression of MCE for example MCE from wherein the MCE is operably linked to the TEFin promoter; and iv) an overexpression of GapC, for example GapC from wherein the GapC is operably linked to the GPDp promoter, for example GPD promoter of SEQ ID NO:

17.

47. The oleaginous yeast cell according to any of the preceding claims wherein a culture of the yeast cells produces a lipid titre of at least 20 g / L, for example at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 g / L or more.

48. The oleaginous yeast cell according to any of the preceding claims wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more, optionally wherein the wherein a culture of the yeast cells produces a lipid productivity of at least 0.25g / L / h, optionally at least 0.33, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5g / L / h or more when the culture conditions comprise active agitation in an aerated fermenter.

49. The oleaginous yeast cell according to any of the preceding embodiments wherein a culture of the yeast cells produces a lipid content of at least 0.2 g / g CDW, for example at least 0.225, 0.25, 0.275, 0.30, 0.325, 0.35, 0.375, 0.40, 0.425, 0.45, 0.475, 0.50, 0.60, 0.70, 0.80, 0.90, 0.95 or 1.

00.

50. A method of producing lipids wherein the method comprises culturing a population of oleaginous yeast cells according to any of the preceding claims.

51. The method of claim 50 wherein the culture media comprises urea, for example where the yeast is a Yarrowia species, for example a Yarrowia lipolytica yeast, the culture media comprises urea.

52. The method of any of claims 50 or 51 wherein the method comprises culturing said population of yeast cells under conditions of nitrogen starvation.

53. The method of any of claims 50-52 wherein the method comprises extracting and / or purifying lipids from the culture media following culture of the population of oleaginous yeast cells.

54. A composition comprising lipids prepared by the method according to any of claims 50-53.

55. A nucleic acid that is a DGA2 expression cassette, wherein the expression cassette comprises or consists of the DGA2 gene from Claviceps purpurea operably linked to a pTEF-4UAS promoter, optionally wherein the DGA2 has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

56. A nucleic acid that is a DGA2 expression cassette, wherein the expression cassette comprises or consists of the DGA2 gene from Claviceps purpurea operably linked to a promoter selected from the group consisting of: pTEF-4UAS promoter (SEQ ID NO: 16); the 2UAS1-pTEF promoter; the 3UAS1-pTEF promoter; the 4UAS1-pTEF promoter; the 8UAS1-pTEF promoter; and the hp8d promoter, optionally wherein the DGA2 has the sequence of SEQ ID NO: 5 or has a sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 5 and retains the activity of diacylglycerol acyltransferase 2.

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