# Patent

## Title
Compositions and methods for lipid production

## Abstract

Described herein, inter alia, are compositions, oleagnious organisms, and methods useful for producing lipids, lipid precursors, and/or oleochemicals.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 61/819,476, filed May 3, 2013, which is incorporated herein by reference in its entirety and for all purposes.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under N000141110669 awarded by Office of Naval Research. The government has certain rights in the invention.


REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK
The Sequence Listing written in file 93331-003510US-907029_ST25.TXT, created on Apr. 29, 2014, 210,560 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated herein by reference in its entirety and for all purposes.
BACKGROUND
Increasing oil consumption makes continued dependence on petroleum reserves untenable. Microbial production of renewable alternatives can reduce petroleum footprints through the in vivo synthesis of ethanol, biodiesel, and industrial precursors (Curran et al. 2013; Elshahed 2010; Li et al. 2008; Xu et al. 2013; Yim et al. 2011). Economic viability is highly dependent upon microbial choice, and an ideal host efficiently generates high titers independent of fermentation condition, through native or imported biosynthetic metabolism (Alper and Stephanopoulos 2009). In this regard, Yarrowia lipolytica's genetic tractability, efficient utilization of many energy sources, and native capacity to accumulate lipids make it an ideal platform for oleo-chemical synthesis (Barth and Gaillardin 1996; Beopoulos et al. 2009a; Papanikolaou and Aggelis 2002).
Here we have employed a large-scale combinatorial approach to maximize lipid production in Y. lipolytica through both genomic engineering and combinatorial and inverse metabolic engineering multiplexed with phenotypic induction.
Y. lipolytica has a fully defined metabolic engineering toolbox that enables intracellular flux control through genomic manipulation (Blazeck et al. 2013b; Dujon et al. 2004; Fickers et al. 2003; Juretzek et al. 2001; Matsuoka et al. 1993). Y. lipolytica is commonly utilized for heterologous protein excretion and to examine and manipulate lipid and fatty acid metabolism (Beopoulos et al. 2009b; Beopoulos et al. 2008; Dulermo and Nicaud 2011; Madzak et al. 2004; Thevenieau et al. 2009), and has proven amenable to downstream manipulation of its fatty acid content to alter desaturation levels (Chuang et al. 2010) or to synthesize novel oleo-chemicals (Blazeck et al. 2013a). Thus, Y. lipolytica lipid reserves are ideal for in vivo catalysis to alkanes (Schirmer et al. 2010), fatty acid esters (Shi et al. 2012) or for standard transesterification-based conversion and use as biodiesel. In particular, biodiesel production grants a high net energy gain compared to other alternative fuels with minimal environmental impact, and harvesting lipid reserves from a microbial source such as Y. lipolytica enables easily scaled-up production without compromising food supply (Christophe et al. 2012; Hill et al. 2006; Kirstine and Galbally 2012; Subramaniam et al. 2010). Y. lipolytica's natural lipid content consists of predominantly C16:0, C16:1, C18:0, C18:1, and C18:2 fatty acids (Beopoulos et al. 2008; Blazeck et al. 2013a; Tai and Stephanopoulos 2013), very similar to the fatty acid content of biodiesel derived from soybeans and rapeseed (Gruzdiene and Anelauskaite 2011; Hammond et al. 2005). Economic viability can be greatly improved by fully utilizing all sugars from lignocellulosic biomass or by using carbon from industrial waste streams. In this regard, Y. lipolytica can efficient utilize hydrophobic and waste carbon sources, such as crude glycerol (Andre et al. 2009; Fickers et al. 2005; Makri et al. 2010; Rywinska et al. 2013), and has shown excellent heterologous gene expression when utilizing glucose, sucrose, glycerol, or oleic acid as a carbon source (Blazeck et al. 2013b). Finally, Y. lipolytica is regarded as a “safe-to-use” organism (Groenewald et al. 2013).
Lipid accumulation in Y. lipolytica can be induced by nitrogen starvation and has been associated with the activity of four enzymes: AMP Deaminase (AMPDp), ATP-Citrate Lyase (ACLp), Malic Enzyme (MAEp) and Acetyl-CoA Carboxylase (ACCp) (Beopoulos et al. 2009a; Dulermo and Nicaud 2011). AMPDp cleaves AMP into NH4+ and inosine 5′-monophosphate to replenish intracellular nitrogen levels; AMP deficiency inhibits the citric acid cycle resulting in citric acid accumulation. ACLp cleaves citric acid into oxaloacetate and acetyl-CoA, and ACCp carboxylates acetyl-CoA into malonyl-CoA fatty acid building blocks. Fatty acid synthesis is further encouraged by a MEAp-mediated increase in NADPH levels (Beopoulos et al. 2009a). Fatty acids can be directly stored in intracellular lipid bodies or further incorporated in triacylglycerides before storage (Beopoulos et al. 2008). Triacylglyceride synthesis follows the Kennedy Pathway to fuse three fatty acids to a glycerol-3-phosphate (G3P) backbone (Kennedy 1961). The ultimate step is catalyzed by the DGA1 or DGA2 acyl-CoA:diacylglycerol acyltransferases (Beopoulos et al. 2009a; Beopoulos et al. 2012). G3P backbone is synthesized from dihydroxyacetone phosphate (DHAP) by the cytosolic, NAD+-dependent glycerol-3-phosphate dehydrogenase (GPD1) and recycled into glycolysis by the mitochondrial, FAD+-dependent glycerol-3-phosphate dehydrogenase isoform (GUT2) (Dulermo and Nicaud 2011). TAG hydrolysis mobilizes free fatty acids for peroxisomal degradation through the four step β-oxidation cycle (Beopoulos et al. 2011)—oxidation by one of six acyl-CoA oxidases (POX1-6), hydration and dehydrogenation by the multifunctional enzyme (MFE1), and thiolysis by a 3-ketoacyl-CoA-thiolase (POT1 or PAT1) (Beopoulos et al. 2009a). The PEX10p transcription factor has been implicated in peroxisomal biogenesis and Δpex10 mutants display increased triacylglyceride content (Blazeck et al. 2013a; Hong et al. 2012; Zhu et al. 2012).
Genomic modifications to Y. lipolytica 's fatty acid, lipid, and central carbon metabolism have shown promise towards increasing lipid accumulation capacity. Deletion of the six POX genes increased ex novo incorporation of oleic acid in Y. lipolytica, while deletion of the single MFE1 gene had a similar effect (Beopoulos et al. 2008; Dulermo and Nicaud 2011). Increasing G3P backbone levels by combining GUT2p deletion and GPD1p overexpression in these β-oxidation deficient backgrounds further increased ex novo lipid accumulation to 65-75% triacylglyceride content (Dulermo and Nicaud 2011). Overexpression of DGA1p increased de novo triacylglyceride accumulation fourfold over control levels to 33.8% triacylglyceride content, and co-overexpression of ACC1p further increased triacylglyceride accumulation to a final yield of 41% triacylglyceride content (Tai and Stephanopoulos 2013). To date, no study has attempted to combine the beneficial effects of engineering Y. lipolytica's fatty acid, lipid and central metabolism in a single strain. Additionally, Y. lipolytica's dependence on media formulation for lipid accumulation has not been adequately explored, nor has its ability to randomly accumulate mutations that enhance lipid accumulation. Furthermore, no attempt has been made to utilize mutation-based evolutionary selection to identify novel lipogenic genotypes. Thus, the ultimate capacity of Y. lipolytica to accumulate lipids and other oleochemicals has not been unlocked. To this end, we have employed a large scale combinatorial approach to maximize lipid production while accounting for unexpected interactions between genotype and environmentally-induced phenotype. The present invention provides solutions to these and other problems in the art.
BRIEF SUMMARY
In a first aspect is provided a genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) wherein the dry weight of said yeast cell includes greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals.
In a second aspect is provided a method of producing a lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) including: 1) culturing a yeast cell as described herein (including embodiments or as described in the examples, tables, figures, and/or claims) in a growth medium; and 2) isolating the lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) (e.g. from the medium or yeast cell).
In a third aspect is provided a method of isolating a genetically modified yeast cell from a plurality of yeast cells, including greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals in dry weight, including allowing a genetically modified yeast cell to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium thereby isolating the genetically modified yeast cell, wherein the population of yeast cells includes a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals than said genetically modified yeast cell.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Nile Red assay quantifying lipid content of PO1f WT strain in C160N0.2 media supplemented with individual micronutrients after 2, 4, and 8 days of cultivation.
FIG. 2. Nile Red assay quantifying lipid content of PO1f WT strain in C160N0.2 media supplemented with multiple micronutrients after 2, 4, and 8 days of cultivation.
FIG. 3. Nile Red assay quantify lipid content of 46 rationally constructed genetically modified PO1f derivatives.
FIG. 4. Fold improvement of lipid accumulation (from Nile Red assay signal (RFU)) by enabling the capacity to synthesis leucine through incorporation of the LEU2 marker to different genotypic background. LEU2 expression can be from an episomal or an integrated sequence.
FIG. 5. Heat map of lipid content based on Nile Red signal of PO1f WT cultured in media formulations with different carbon to nitrogen ratios after 4 days.
FIG. 6. Heat map of lipid content based on Nile Red signal of PO1f-S1-S2-φ cultured in media formulations with different carbon to nitrogen ratios after 4 days.
FIG. 7. Heat map of lipid content based on Nile Red signal of ΔPEX10ΔMFE1 cultured in media formulations with different carbon to nitrogen ratios after 4 days.
FIG. 8. Heat map of lipid content based on Nile Red signal of ΔPEX10ΔMFE1-pMCS-DGA1 cultured in media formulations with different carbon to nitrogen ratios after 4 days.
FIG. 9. Nile Red assay quantify lipid content on Day 4 with different strains growing on different saccharides as carbon sources. Saccharide initial concentration was set at 80 g/L with 5 g/L ammonium sulfate.
FIG. 10. Nile Red assay quantify lipid content of isolated L36 strain cultured in C160N0.2 media supplemented with multiple micronutrients after 2, 4, and 8 days of cultivation.
FIG. 11. Nile Red assay quantify lipid content with EMS mutagenesis in evolved L36 strains and L36.
FIG. 12. Fluorescence light microscopy pictures of lipid accumulation in selected strains. Lipids were stained with Nile Red as usual. Strain ΔPEX10ΔMFE1-pMCS-DGA1 shows almost total lipid content while PO1f WT has very little.
FIG. 13. General lipid metabolism in yeast and a portion of selected targets to engineering lipid metabolism.
FIG. 14. The isolation and characterization of superior lipid production strain L36.
FIG. 15. Fatty acid profiles for different strains.
FIG. 16. Lipid accumulation in strain PO1f and PO1fΔaco1 DGA1 leu+ ura+ characterized with flow cytometry using cells stained with Nile Red on 48 hour and 96 hour time point. The starting OD of the culture is 2.5 and the cells were cultivated in yeast synthetic medium with 80 g/L glucose.
FIG. 17. Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 192 h time point. The starting OD of the culture is 5 and the cells were cultivated in yeast synthetic medium with 160 g/L glucose and 0.2 g/L ammonium sulfate. Illustrated in the bar graph, L36Δmga2 presented a significantly reduced lipid level comparing to L36 and L36Δmga2 MGA2-36 presented an elevated level of lipid accumulation comparing to L36Δmga2, indicating that mga2-36 is the reason of the high lipid accumulation phenotype in L36 strain. Combining the data with Δmga2 and Δmga2 MGA2-36 in PO1f, this set of data proves that Δmga2 can lead to improved lipid accumulation and further introduce the mutant transcriptional factor MGA2-36 can further elevate the level of lipid accumulation. (All strains in the set contain an episomal plasmid with LEU2). Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 192 h time point with yeast synthetic medium containing 160 g/L glucose and 0.2 g/L ammonium sulfate and 96 h time point with yeast synthetic medium containing 80 g/L glucose and 5 g/L ammonium sulfate. Introducing MGA2-36 to the engineered strain leads to elevated level of lipid accumulation, suggesting MGA2-36 can be used a lipid enhancer in the rationally engineered lipid production strain. Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 192 h time point with yeast synthetic medium containing 160 g/L glucose and 0.2 g/L ammonium sulfate. PO1fΔmga2 leu+ showed improved level of lipid accumulation comparing to PO1f leu+ indicating mga2 knockout could improve lipid accumulation. Introducing a transmembrane domain truncated MGA2-36 in PO1f could elevate the lipid level inside the cell.
FIG. 18. Gas chromatography characterization of major fatty acid species profile in PO1f, Engineered strain and L36. L36 overproduced C16:1n9 fatty acid which could be linked with the mutant of MGA2 gene, which plays an important function on activating/regulating delta9 desaturase expression.
FIG. 19. Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 96 h time point with yeast synthetic medium containing 80 g/L glucose and 5 g/L ammonium sulfate. 1st round EMS mutagenesis and floating cell transfer method selected strain E26 and E13 using final engineered strain PO1f Δpex10,mfe DGA1 leu+ ura+ presented a higher lipid accumulation level comparing to the engineered strain.
FIG. 20. Lipid production (g/L) in bioreaction with 160 g/L glucose and 13.4 g/L YNB with ammonium sulfate without amino acid (set control DO at 50% and pH=3.5) with engineered strain and evolved strain E26.
FIG. 21. List of consensus mutations in strain E26 and E13 identified in open reading frame through next generation sequencing analysis. Among them, YLOSH6; YLIRC20; YLRME1; YLYOX1; YLUGA2 contains missense mutations in annotated protein.
FIG. 22. Summary illustration of 1st and 2nd round of EMS mutagenesis and floating cells transfer selection with final engineered strain PO1f Δpex10,mfe DGA1 leu+ ura+ as starting strain for evolving and selecting high lipid production strain. Green indicating the final engineering strain, blue indicating the non-EMS treated control stains and red indicating the selected high lipid production strains. Strains were rank ordered based on the value cultured OD600*Nile Red mean fluorescence intensity*10−4.
FIG. 23. Fermentation profiles of pex10 mfe1 leucine+ uracil+ DGA1 and PO1f leucine+ uracil+. Time courses of the 1.5 L scale batch fermentation of the pex10 mfe1 leucine+ uracil+ DGA1 (a,b) and PO1f leucine+ uracil+ (c,d) strains in 80 g/L glucose, 6.7 g/L YNB (no amino acids, 1.365 g/L ammonium) are shown, including production of biomass, lipids, and citric acid (left axis a,c), consumption of glucose (right axis a,c), and ammonium level (b,d). (a) During the pex10 mfe1 leucine+ uracil+ DGA1 fermentation, negligible citric acid was produced, and lipid product accumulated during and after biomass production phases. This fermentation was run three times in identical conditions, reaching final yields of 15.25 g/L lipids and 20.3 g/L biomass (75% lipid content), 14.96 g/L lipids and 20.6 g/L biomass (73% lipid content), and 16.9 g/L lipids and 19.21 g/L biomass (88% lipid content). Most time points show average values from the former two fermentations (75% and 73% final lipid content), while endpoints represent averages from all three final values. Glucose and ammonium substrate were fully consumed after 72 hours, but surprisingly, (b) ammonium level was replenished to a steady state level of  ̃0.5 g/L, almost 40% of the original starting level. (c) During the PO1f leucine+ uracil+ fermentation, citric acid accumulated to more than 14 g/L after 72 hours before quickly reducing to 4 g/L. Lipid production did not trend with biomass production, reaching a final yield of only 3 g/L lipids, compared to 30 g/L biomass, and glucose was again consumed within 72 hours. (d) Ammonium was fully consumed after 72 hours with no replenishment as observed in the mutant strain.



DETAILED DESCRIPTION
Our work described herein represents the largest scale engineering effort in an oleaginous organism to date. We analyzed the effect of nitrogen starvation and carbon level on a wildtype Y. lipolytica strain and a strain with two genomic modifications to increase lipid (e.g. triacylglyceride) accumulation. By testing twenty media formulations containing between 10 g/L and 320 g/L glucose and 0.04 g/L and 10 g/L ammonium sulfate, we demonstrated that increasing carbon to nitrogen ratio (C:N ratio) generally induces lipid (e.g. triacylglyceride) accumulation, that carbon level is more important than nitrogen level towards this induction, and that this optimum carbon level is dependent upon genomic background. We further determined that lipid (e.g. triacylglyceride) accumulation could be increased through the addition of certain metallic cofactors in the wildtype background as well as for some Y. lipolytica strains already engineered for increased lipid (e.g. triacylglyceride) content. In an effort to rationally engineer Y. lipolytica for increased lipid (e.g. triacylglyceride) accumulation while accounting for unpredictable cumulative effects arising from simultaneously altering fatty acid, lipid, and central carbon metabolism, we overexpressed multiple (e.g. five) enzymes implicated in lipid (e.g. triacylglyceride) accumulation in multiple (e.g. four) background strains differentially deficient in fatty acid degradation. These native enzymatic overexpressions were driven by high-strength constitutive promoters, occurred singly or in tandem with a second enzyme overexpression, and alleviated one of two auxotrophies (leucine and uracil). This combinatorial approach generated over 50 distinct genotypes that produced a large range in lipid (e.g. triacylglyceride) accumulation ability, culminating in upwards of 40-fold above control when using Nile-red based fluorescence and nearly 5-fold when using concentration (g/L) or percent lipid by cell mass (% dcw). In the process, we discovered a correlation between the auxotrophic marker used to select for protein overexpression and a strain's capacity to accumulate oleo-content. Specifically, the ability to endogenously produce the amino acid leucine, conferred by a selectable leucine auxotrophic marker, is beneficial (e.g. essential) to enable high lipid titer. We further examined a few (e.g. thirteen) of these strains to determine how C:N ratio and genotype interacted towards producing lipid (e.g. triacylglyceride) content on a larger scale. We observed a strong tendency towards high lipid (e.g. triacylglyceride) levels in most high producers at a single media formulation—cultivated in 80 g/L glucose and 5 g/L ammonium sulfate. We selected a MFE1, PEX10 double knockout strain with no auxotrophies overexpressing the DGA1p lipid synthesis as our final rationally engineered strain, and demonstrated its triacylglyceride accumulation ability on a variety of carbon sources, demonstrating its robust capacity to accumulate triacylglycerides regardless of media composition.
Through our time working with Y. lipolytica, we became aware of its surprising capacity to randomly (or forcibly through the use of an exogenous mutagen such as EMS) generate isolatable sub-strains that reproducibly displayed higher than wildtype triacylglyceride levels. In fact, one such strain, dubbed L36, displayed remarkable accumulation ability. Whole-genome sequencing of this strain pinpointed a mutation in the MGA2 transcriptional regulator as the most likely genomic explanation. Complementation assays of an MGA2p truncation mutant into wildtype background reached 50% of L36 lipid levels. We sought to harness this general capacity for beneficial mutation by subjecting wildtype, L36, and two of our highest producing rationally engineered strains to ethylmethanesulfonate (EMS) mutagenesis and positive selection. By combining large-scale investigations of phenotypic induction, genomic engineering, and positive random mutations, this work establishes a framework for engineering oleaginous organisms for increased lipid production. In this regard, we have pinpointed specific media formulations, genomic modifications, and genomic mutations that positively effect lipid (e.g. triacylglyceride) biosynthesis. The resultant strains are ideal for direct biodiesel precursor synthesis, lipid synthesis, oleochemical synthesis, lipid precursor synthesis, or for in vivo catalysis of fatty acid reserves to value added chemicals. Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 192 h time point with yeast synthetic medium containing 160 g/L glucose and 0.2 g/L ammonium sulfate and 96 h time point with yeast synthetic medium containing 80 g/L glucose and 5 g/L ammonium sulfate. Introducing MGA2-36 to the engineered strain leads to elevated level of lipid accumulation, suggesting MGA2-36 can be used a lipid enhancer in the rationally engineered lipid production strain. Lipid accumulation characterized with flow cytometry using cells stained with Nile Red on 192 h time point with yeast synthetic medium containing 160 g/L glucose and 0.2 g/L ammonium sulfate. PO1fΔmga2 leu+ showed improved level of lipid accumulation comparing to PO1f leu+ indicating mga2 knockout could improve lipid accumulation. Introducing a transmembrane domain truncated MGA2-36 in PO1f could elevate the lipid level inside the cell.
I. DEFINITIONS
The term “oleaginous organism” means an organism (e.g. a cell such as a yeast cell) that is capable of producing a lipid, lipid precursor, oleochemical, or oil (or combinations thereof) at a level exceeding the amount required for normal cellular survival and propagation of the organism (e.g. cell, yeast cell), such as for example necessary for structural integrity (e.g. membrane formation and maintenance) and cellular maintenance. Examples of amounts exceeding the amount required for normal cellular survival and propagation include an amount of lipids, oils, lipid precursors, and oleochemicals greater than 20% wt/wt total dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%). In embodiments, the oleaginous organism is an oleaginous yeast. In some embodiments, the oleaginous yeast is from a genus selected from the group consisting of Apiotrichum, Candida, Cryptococcus, Debaromyces, Endomycopsis, Geotrichum, Hyphopichia, Lipomyces, Lypomyces, Pichia, Rodosporidium, Rhodotorula, Sporobolomyces, Starmerella, Torulaspora, Trichosporon, Wickerhamomyces, Yarrowia, and Zygoascus. In embodiments, the oleaginous yeast is selected from the group consisting of Apiotrichum curvatum, Candida apicola, Candida curvata, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Candida utilis, Cryptococcus curvatus, Cryptococcus terricolus, Debaromyces hansenii, Endomycopsis vernalis, Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichum histeridarum, Geotrichum silvicola, Geotrichum vulgare, Hyphopichia burtonii, Lipomyces lipoferus, Lipomyces lipofer, Lypomyces orentalis, Lipomyces starkeyi, Lipomyces tetrasporous, Pichia mexicana, Rodosporidium sphaerocarpum, Rhodosporidium toruloides, Rhodotorula aurantiaca, Rhodotorula dairenensis, Rhodotorula diffluens, Rhodotorula glutinus, Rhodotorula glutinis var. glutinis, Rhodotorula gracilis, Rhodotorula graminis, Rhodotorula minuta, Rhodotorula mucilaginosa, Rhodotorula mucilaginosa Rhodotorula mucilaginosa, Rhodotorula terpenoidalis, Rhodotorula toruloides, Sporobolomyces alborubescens, Starmerella bombicola, Torulaspora delbruekii, Torulaspora pretoriensis, Trichosporon behrend, Trichosporon brassicae, Trichosporon cutaneum, Trichosporon domesticum, Trichosporon fermentans, Trichosporon laibachii, Trichosporon loubieri, Trichosporon loubieri var. loubieri, Trichosporon montevideense, Trichosporon pullulans, Wickerhamomyces canadensis, Yarrowia lipolytica, and Zygoascus meyerae. 
The term “buoyancy” is used according to its plain ordinary meaning and refers to the upward force exerted by a fluid, which opposes the weight of an immersed object (e.g. oleaginous organism or oleaginous yeast cell). Pressure increases with depth, resulting in a net force tending to accelerate object upward, wherein the magnitude of the force is proportional to the difference between the top and bottom of the fluid and is equivalent to the weight of the fluid that would otherwise occupy the space occupied by the object (i.e. the displace fluid). In embodiments, an oleaginous organism or yeast cell is considered “buoyant” when it does not settle (e.g. due to gravitation force alone, due to centrifugal force, due to an applied force, or due to a combination of forces such as centrifugation) to the bottom of a vessel holding a liquid (e.g. media) in which the oleaginous organism or yeast cell resides. For example, a cell may be buoyant if it floats above the bottom of the vessel, at an intermediate position between the bottom level and top level of the liquid, or on top of the upper surface of the liquid. An example of a measurement of the buoyancy of an object (e.g. cell) is the weight of the fluid the object would displace if the object were placed in the fluid. Another example of a measurement of the buoyancy of an object (e.g. cell) is a comparison of the average density of the object and the average density of the liquid to be displaced, taking into account the depth of the liquid in a column of the liquid. The term “buoyant density” is used according to its plain ordinary meaning and refers to a measure of the tendency of a substance to float in some other substance.
The term “carbon substrate” means a carbon source that a microorganism (e.g. oleaginous organism or oleaginous yeast) will metabolize to derive energy (e.g. monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fatty acids, monoglycerides, carbon dioxide, methanol, formaldehyde, formate or carbon-containing amines). The term “carbon source” refers to a carbon containing composition (e.g. compound, mixture of compounds) that an organism (e.g. oleaginous organism, yeast cell) may metabolize for use by the organism or that may be used for organism viability. A “majority carbon source” refers to a carbon containing composition that accounts for greater than 50% of the available carbon sources for an organism (e.g. in a media, in a growth media, in a defined media for growing yeast cells, or in a defined media for producing lipids by yeast cells) at a specified time (e.g. media when starting a yeast culture, media in a bioreactor when growing yeast, or media when producing lipids from yeast). In embodiments, an oleaginous yeast may be cultured using a medium comprising one or more carbon sources selected from the group consisting of glucose, fructose, sucrose, lactose, galactose, xylose, mannose, rhamnose, arabinose, glycerol, acetate, depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, wheat, and mixtures thereof (e.g. mixtures of glycerol and glucose, mixtures of glucose and xylose, mixtures of fructose and glucose, mixtures of sucrose and depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, and/or wheat). In embodiments, an oleaginous yeast is cultured using a medium comprising one or more carbon sources selected from the group consisting of depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, thick cane juice, sugar beet juice, and wheat. In embodiments, an oleaginous yeast is cultured using a medium comprising lignocellulosic biomass. In embodiments carbon sources may be monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) or mixtures from renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor, sugar beet molasses, or barley malt). Additionally, carbon sources may include alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, various commercial sources of fatty acids including vegetable oils (e.g., soybean oil) or animal fats.
Nitrogen may be supplied from an inorganic (e.g., (NH4)2SO4) or organic source (e.g., urea, glutamate). The term “nitrogen source” refers to a nitrogen containing composition (e.g. compound, mixture of compounds, salt) that an organism (e.g. oleaginous organism, yeast cell) may metabolize for use by the organism or that may be used for organism viability. A “majority nitrogen source” refers to a nitrogen containing composition that accounts for greater than 50% of the available nitrogen sources for an organism (e.g. in a media, in a growth media, in a defined media for growing yeast cells, or in a defined media for producing lipids by yeast cells) at a specified time (e.g. media when starting a yeast culture, media in a bioreactor when growing yeast, or media when producing lipids from yeast).
The term “Biomass” refers to material produced by growth and/or propagation of cells. “Lignocellulosic biomass” is used according to it plain ordinary meaning and refers to plant dry matter comprising carbohydrate (e.g. cellulose or hemicellulose) and polymer (e.g. lignin). Lignocellulosic biomass may include agricultural residues (e.g. corn stover or sugarcane bagasse), energy crops (e.g. poplar trees, willow, Miscanthus purpureum, Pennisetum purpureum, elephant grass, maize, Sudan grass, millet, white sweet clover, rapeseed, giant miscanthus, switchgrass, jatropha, Miscanthus giganteus, or sugarcane), wood residues (e.g. sawmill or papermill discard), or municipal paper waste.
The term “Culture”, “cultivate”, and “ferment” are used interchangeably and refer to the intentional growth, propagation, proliferation, and/or enablement of metabolism, catabolism, and/or anabolism of one or more cells (e.g. oleaginous organism or oleaginous yeast). The combination of both growth and propagation may be termed proliferation. Examples include production by an organism of lipids, lipid precursors, and/or oleochemicals or production of a lipid, lipid precursor, and/or oleochemical of interest. Culture does not refer to the growth or propagation of microorganisms in nature or otherwise without human intervention.
The terms “dry weight” and “dry cell weight” are used interchangeably and refer to a weight determined in the relative absence of water. In embodiments, oleaginous yeast biomass comprising a fraction or percentage of a particular component by dry weight means that the fraction or percentage is calculated based on the weight of the biomass after substantially all water has been removed.
The term “growth” means an increase in cell size, total cellular contents, and/or cell mass or weight of a cell (e.g. oleaginous organism or oleaginous yeast).
The term “lipid” refers to a class of molecules that are soluble in nonpolar solvents (e.g. ether or chloroform), are relatively or completely insoluble in water, and include one or more hydrocarbon chains which are hydrophobic. In embodiments, a lipid may be a triacylglyeride (i.e. fat), fatty acid (e.g. saturated or unsaturated); glyceride or glycerolipid (e.g. monoglyceride, diglyceride, triglyceride, neutral fat, phosphoglyceride, or glycerophospholipid); sphingolipid; sterol lipid (e.g. cholesterol or a steroid hormone); prenol lipid (e.g. terpenoid); fatty alcohol; wax; polyketide; sugar-linked lipid, glycolipid, or protein-linked lipid.
The term “oil” means a triacylglyceride (or triglyceride oil), produced by an organism (e.g. oleaginous organism, oleaginous yeast, plant, and/or animal). An oil is generally liquid at normal ambient temperatures and pressures. In embodiments, oil may be vegetable or seed oils derived from plants (e.g. soy, rapeseed, canola, palm, palm kernel, coconut, corn, olive, sunflower, cotton seed, cuphea, peanut, camelina sativa, mustard seed, cashew nut, oats, lupine, kenaf, calendula, hemp, coffee, linseed, hazelnut, euphorbia, pumpkin seed, coriander, camellia, sesame, safflower, rice, tung oil tree, cocoa, copra, pium poppy, castor beans, pecan, jojoba, jatropha, macadamia, Brazil nuts, avocado, or combinations thereof). An oil may include a plurality of different triacylglycerides. For example, a vegetable or seed oil may include more than one triacylglyceride and use of the name of that vegetable or seed oil (e.g. soy, rapeseed, canola, palm, etc.) when referring to an oil generated by an oleaginous organism will be understood to mean an oil including most (e.g. all) of the triacylglycerides normally in the vegetable or seed oil (e.g. at different ratios relative to each other or the same or similar ratios relative to each other). In other embodiments, an oil may be a plurality of triacylglyceride and other lipid molecules produced by an oleaginous organism.
The term “propagation” refers to an increase in cell number via cell division.
The terms “V/V”, “vol/vol”, or “v/v”, referring to proportions by volume, means the ratio of the volume of one substance in a composition to the volume of the total composition including the substance.
The term “W/W”, “wt/wt”, or “w/w”, referring to proportions by weight, means the ratio of the weight of one substance in a composition to the weight of the total composition including the substance. For example, 5% w/w substance X means that 5% of the composition's weight is composed of substance X and the remainder of the weight of the composition (i.e. 95%) is composed of other substances.
The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of yeast origin, for example, promoters derived from viruses or from other organisms can be used in the compositions or methods described herein.
A polynucleotide sequence is “heterologous to” a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from naturally occurring allelic variants.
The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). In embodiments, a recombinant nucleic acid is a nucleic acid in an oleaginous organism (e.g. oleaginous yeast) that has been manipulated by a human, for example a recombinant nucleic acid comprising a coding region for a protein that is overexpressed in an oleaginous organism relative to the absence of the recombinant nucleic acid or a recombinant nucleic acid that results in disruption of a coding region or promoter region of an oleaginous organism and reduces or eliminates expression of a protein relative the absence of the recombinant nucleic acid. One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.
“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. The term “nucleic acid” includes single-, double-, or multiple-stranded DNA, RNA and analogs (derivatives) thereof. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. In certain embodiments, the nucleic acids herein contain phosphodiester bonds. In other embodiments, nucleic acid analogs are included that may have alternate backbones. The term encompasses nucleic acids containing known analogues of natural nucleotides which have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. A particular nucleic acid sequence also encompasses “splice variants.” Similarly, a particular protein encoded by a nucleic acid encompasses any protein encoded by a splice variant of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. An example of potassium channel splice variants is discussed in Leicher, et al., J. Biol. Chem. 273(52):35095-35101 (1998).
The term “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 10 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
One example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.
The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence with a higher affinity, e.g., under more stringent conditions, than to other nucleotide sequences (e.g., total cellular or library DNA or RNA).
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Polypeptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the level of activity or function of a target molecule or the physical state of the target of the molecule. In embodiments a modulator is a recombinant nucleic acid that is capable of increasing or decreasing the amount of a protein in a cell or the level of activity of a protein in a cell or transcription of a second nucleic acid in a cell. In embodiments, a modulator increases or decreases the level of activity of a protein or the amount of the protein in a cell. The term “modulate” is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule. In embodiments, a recombinant nucleic acid that modulates the level of activity of a protein may increase the activity or amount of the protein relative the absence of the recombinant nucleic acid. In embodiments, an increase in the activity or amount of a protein may include overexpression of the protein. “Overexpression” is used in accordance with its plain meaning and refers to an increased level of expression of a protein relative to a control (e.g. cell or expression system not including a recombinant nucleic acid that contributes to the overexpression of a protein). In embodiments, a decrease in the activity or amount of a protein may include a mutation (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid; all/any of which may be in the coding region for a protein or in an operably linked region (e.g. promoter)) of the protein. The term “increased” refers to a detectable increase compared to a control. In some embodiments, the increase is by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000%, or more compared to the control. In embodiments, the increase is by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000%, or more compared to the control. In embodiments, the increase is by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000%, compared to the control. Similarly, the term “decreased” refers to a measurable decrease compared to a control. In some embodiments, the decrease is by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more compared to the control. In embodiments, the decrease is by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more compared to the control. In embodiments, the decrease is by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, compared to the control. One of ordinary skill will be able to identify a relevant control.
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
“Transformation” refers to the transfer of a nucleic acid molecule into a host organism (e.g. oleaginous organism or oleaginous yeast). In embodiments, the nucleic acid molecule may be a plasmid that replicates autonomously or it may integrate into the genome of the host organism (e.g. oleaginous organism or oleaginous yeast). Host organisms containing the transformed nucleic acid molecule may be referred to as “transgenic” or “recombinant” or “transformed” organisms (e.g. oleaginous organism or oleaginous yeast). A “genetically modified” organism (e.g. genetically modified yeast cell) is an organism (e.g. yeast cell) that includes a nucleic acid that has been modified by human intervention. Examples of a nucleic acid that has been modified by human intervention include, but are not limited to, insertions, deletions, mutations, expression nucleic acid constructs (e.g. over-expression or expression from a non-natural promoter or control sequence or an operably linked promoter and gene nucleic acid distinct from a naturally occurring promoter and gene nucleic acid in an organism), extra-chromosomal nucleic acids, and genomically contained modified nucleic acids. Genetically modified organisms may be made by rational modification of a nucleic acid or may be made by use of a mutagen or mutagenesis protocol that results in a mutation that was not identified (e.g. intended or targeted) prior to the use of the mutagen or mutagenesis protocol (e.g. UV exposure, EMS exposure, mutagen exposure, random genomic mutagenesis, transformation of a library of different nucleic acid constructs). Genetically modified organisms that include a modification (e.g. modification, insertion, deletion, mutation) not previously known or intended prior to making of the genetically modified organism may be identified through screening a plurality of organism including one or more genetically modified organisms by using a selection criteria that identifies the genetically modified organism of interest (e.g. an increased level of lipids, lipid precursors, and/or oleochemicals; floats above an organism not including the same genetic modification). In embodiments, a genetically modified organism includes a recombinant nucleic acid.
Methods for synthesizing sequences and bringing sequences together are well established and known to those of skill in the art. For example, in vitro mutagenesis and selection, site-directed mutagenesis, error prone PCR (Melnikov et al., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), “gene shuffling” or other means can be employed to obtain mutations of naturally occurring genes.
Mutagenesis (e.g. chemical mutagenesis or site directed mutagenesis) may be used to modulate lipid production or storage in an oleaginous organism (e.g. oleaginous yeast). For example, a mutant construct or mutagen is transformed into an oleaginous yeast cell and the ability of the resulting transformed oleaginous yeast cell to produce or store one or more lipids is assayed and compared to the control cell. In some embodiments, it may be useful to disrupt or inactivate a host organism's native gene to modulate lipid production or storage. For example, a recombinant DNA fragment (e.g. a selectable marker gene) may be inserted into the gene to be disrupted in order to interrupt its coding sequence and the resulting recombinant nucleic acid then transformed into a host cell. Another example of a method of gene disruption is the use of transposable elements or transposons, which is well known to those of skill in the art.
In general, means for the purification of lipids, may include extraction with organic solvents, sonication, supercritical fluid extraction, saponification physical means such as presses, extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high-speed centrifugation or distillation, or combinations of these techniques.
In embodiments, the protein AMP Deaminase (AMPD) is a protein able to be translated from the nucleic acid corresponding to YALI0E11495 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene AMP Deaminase (AMPD) is the nucleic acid or gene corresponding to YALI0E11495 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, AMP Deaminase (AMPD) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E11495 of Yarrowia lipolytica as described above. In embodiments, AMP Deaminase (AMPD) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E11495 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Leucine Biosynthesis Gene (LEU2), also known as 3-isopropylmalate dehydrogenase, is a protein able to be translated from the nucleic acid corresponding to GenBank AF260230 or YALI0C00407 g of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Leucine Biosynthesis Gene (LEU2) is the nucleic acid or gene corresponding to GenBank AF260230 or YALI0C00407 g of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Leucine Biosynthesis Gene (LEU2) is a protein or nucleic acid/gene of a yeast strain corresponding to AF260230 of Yarrowia lipolytica as described above. In embodiments, Leucine Biosynthesis Gene (LEU2) is a protein or nucleic acid/gene of an oleaginous organism corresponding to AF260230 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Uracil Biosynthesis gene (URA3), also known as Orotidine 5′-phosphate decarboxylase, is a protein able to be translated from the nucleic acid corresponding to GenBank YLU40564 or YALI0E26741 g of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Uracil Biosynthesis gene (URA3) is the nucleic acid or gene corresponding to GenBank YLU40564 or YALI0E26741 g of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Uracil Biosynthesis gene (URA3) is a protein or nucleic acid/gene of a yeast strain corresponding to YLU40564 of Yarrowia lipolytica as described above. In embodiments, Uracil Biosynthesis gene (URA3) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YLU40564 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein ATP-Citrate Lyase (ACL) is a protein including the protein ACL1, also called ATP-Citrate Lyase 1, able to be translated from the nucleic acid corresponding to YALI0E34793 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene ATP-Citrate Lyase (ACL) includes the nucleic acid or gene ACL1 corresponding to YALI0E34793 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, the protein ATP-Citrate Lyase (ACL) is a protein including the protein ACL2, also called ATP-Citrate Lyase 2, able to be translated from the nucleic acid corresponding to YALI0D24431 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, the nucleic acid or gene ATP-Citrate Lyase (ACL) includes the nucleic acid or gene ACL2 corresponding to YALI0D24431 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, ATP-Citrate Lyase (ACL) includes a protein or nucleic acid/gene of a yeast strain corresponding to YALI0D24431 of Yarrowia lipolytica as described above. In embodiments, ATP-Citrate Lyase (ACL) includes a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0D24431 of Yarrowia lipolytica as described above. In embodiments, the protein ATP-Citrate Lyase (ACL) is a protein including the protein ACL1 able to be translated from the nucleic acid corresponding to YALI0E34793 of the Genolevures database and the protein ACL2 able to be translated from the nucleic acid corresponding to YALI0D24431 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, the nucleic acid or gene ATP-Citrate Lyase (ACL) includes the nucleic acid or gene ACL1 corresponding to YALI0E34793 and the nucleic acid or gene ACL2 corresponding to YALI0D24431 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, ATP-Citrate Lyase (ACL) includes proteins or nucleic acids/genes of a yeast strain corresponding to YALI0E34793 and YALI0D24431 of Yarrowia lipolytica as described above. In embodiments, ATP-Citrate Lyase (ACL) includes proteins or nucleic acids/genes of an oleaginous organism corresponding to YALI0E34793 and YALI0D24431 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Malic Enzyme (MAE, MEA, MEA1) is a protein able to be translated from the nucleic acid corresponding to YALI0E18634 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Malic Enzyme (MAE, MEA, MEA1) is the nucleic acid or gene corresponding to YALI0E18634 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Malic Enzyme (MAE, MEA, MEA1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E18634 of Yarrowia lipolytica as described above. In embodiments, Malic Enzyme (MAE, MEA, MEA1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E18634 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein acyl-CoA: diacylglycerol acyltransferase (DGA1), also called acyl-CoA:diacylglycerol acyltransfer 1 is a protein able to be translated from the nucleic acid corresponding to YALI0E32769 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene acyl-CoA:diacylglycerol acyltransferase (DGA1) is the nucleic acid or gene corresponding to YALI0E32769 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, acyl-CoA: diacylglycerol acyltransferase (DGA1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E32769 of Yarrowia lipolytica as described above. In embodiments, acyl-CoA:diacylglycerol acyltransferase (DGA1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E32769 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein acyl-CoA: diacylglycerol acyltransferase (DGA2), also called acyl-CoA:diacylglycerol acyltransfer 2, is a protein able to be translated from the nucleic acid corresponding to YALI0D07986 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene acyl-CoA:diacylglycerol acyltransferases (DGA2) is the nucleic acid or gene corresponding to YALI0D07986 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, acyl-CoA:diacylglycerol acyltransferases (DGA2) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0D07986 of Yarrowia lipolytica as described above. In embodiments, acyl-CoA:diacylglycerol acyltransferases (DGA2) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0D07986 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Lipid synthesis regulator (MGA2) is a protein able to be translated from the nucleic acid corresponding to YALI0B12342 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Lipid synthesis regulator (MGA2) is the nucleic acid or gene corresponding to YALI0B12342 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Lipid synthesis regulator (MGA2) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0B12342 of Yarrowia lipolytica as described above. In embodiments, Lipid synthesis regulator (MGA2) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0B12342 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Chromatin assembly gene (RLF2 subunit p90) is a protein able to be translated from the nucleic acid corresponding to YALI0F21637 g of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Chromatin assembly gene (RLF2 subunit p90) is the nucleic acid or gene corresponding to YALI0F21637 g of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Chromatin assembly gene (RLF2 subunit p90) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0F21637 g of Yarrowia lipolytica as described above. In embodiments, Chromatin assembly gene (RLF2 subunit p90) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0F21637 g of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Mitochondrial 2′ O-ribose methyltransferase (MRM2) is a protein able to be translated from the nucleic acid corresponding to YALI0E31933 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Mitochondrial 2′ O-ribose methyltransferase (MRM2) is the nucleic acid or gene corresponding to YALI0E31933 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Mitochondrial 2′ O-ribose methyltransferase (MRM2) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E31933 of Yarrowia lipolytica as described above. In embodiments, Mitochondrial 2′ O-ribose methyltransferase (MRM2) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E31933 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Transcription Factor (PEX10) is a protein able to be translated from the nucleic acid corresponding to YALI0C01023 g of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Transcription Factor (PEX10) is the nucleic acid or gene corresponding to YALI0C01023 g of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Transcription Factor (PEX10) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0C01023 g of Yarrowia lipolytica as described above. In embodiments, Transcription Factor (PEX10) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0C01023 g of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein multifunctional enzyme (MFE1) is a protein able to be translated from the nucleic acid corresponding to YALI0E15378 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene multifunctional enzyme (MFE1) is the nucleic acid or gene corresponding to YALI0E15378 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, multifunctional enzyme (MFE1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E15378 of Yarrowia lipolytica as described above. In embodiments, multifunctional enzyme (MFE1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E15378 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein O-6-methylguanine-DNA methyltransferase (MGMT, O6M) is a protein able to be translated from the nucleic acid corresponding to YALI0C10010p of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene O-6-methylguanine-DNA methyltransferase (MGMT, O6M) is the nucleic acid or gene corresponding to YALI0C10010p of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, O-6-methylguanine-DNA methyltransferase (MGMT, O6M) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0C10010p of Yarrowia lipolytica as described above. In embodiments, O-6-methylguanine-DNA methyltransferase (MGMT, O6M) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0C10010p of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Aconitase (ACO1) is a protein able to be translated from the nucleic acid corresponding to YALI0D09361 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Aconitase (ACO1) is the nucleic acid or gene corresponding to YALI0D09361 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Aconitase (ACO1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0D09361 of Yarrowia lipolytica as described above. In embodiments, O Aconitase (ACO1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0D09361 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Citrate Synthase (CIT1) is a protein able to be translated from the nucleic acid corresponding to YALI0E02684 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Citrate Synthase (CIT1) is the nucleic acid or gene corresponding to YALI0E02684 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Citrate Synthase (CIT1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E02684 of Yarrowia lipolytica as described above. In embodiments, Citrate Synthase (CIT1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E02684 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein Acetyl-CoA Carboxylase (ACC) is a protein able to be translated from the nucleic acid corresponding to YALI0C11407 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene Acetyl-CoA Carboxylase (ACC) is the nucleic acid or gene corresponding to YALI0C11407 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, Acetyl-CoA Carboxylase (ACC) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0C11407 of Yarrowia lipolytica as described above. In embodiments, Acetyl-CoA Carboxylase (ACC) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0C11407 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein RME1 zinc-finger transcription factor (RME1) is a protein able to be translated from the nucleic acid corresponding to YALI0E17215 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene RME1 zinc-finger transcription factor (RME1) is the nucleic acid or gene corresponding to YALI0E17215 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, RME1 zinc-finger transcription factor (RME1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E17215 of Yarrowia lipolytica as described above. In embodiments, RME1 zinc-finger transcription factor (RME1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E17215 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein YOX1 homeodomain protein (YOX1) is a protein able to be translated from the nucleic acid corresponding to YALI0E20449 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene YOX1 homeodomain protein (YOX1) is the nucleic acid or gene corresponding to YALI0E20449 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, YOX1 homeodomain protein (YOX1) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0E20449 of Yarrowia lipolytica as described above. In embodiments, YOX1 homeodomain protein (YOX1) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0E20449 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein UGA2 succinate semialdehyde dehydrogenase (UGA2) is a protein able to be translated from the nucleic acid corresponding to YALI0F26191 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene UGA2 succinate semialdehyde dehydrogenase (UGA2) is the nucleic acid or gene corresponding to YALI0F26191 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, UGA2 succinate semialdehyde dehydrogenase (UGA2) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0F26191 of Yarrowia lipolytica as described above. In embodiments, UGA2 succinate semialdehyde dehydrogenase (UGA2) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0F26191 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein OSH6 oxysterol-binding protein homolog 6 (OSH6) is a protein able to be translated from the nucleic acid corresponding to YALI0A02354 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene OSH6 oxysterol-binding protein homolog 6 (OSH6) is the nucleic acid or gene corresponding to YALI0A02354 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, OSH6 oxysterol-binding protein homolog 6 (OSH6) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0A02354 of Yarrowia lipolytica as described above. In embodiments, OSH6 oxysterol-binding protein homolog 6 (OSH6) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0A02354 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
In embodiments, the protein IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) is a protein able to be translated from the nucleic acid corresponding to YALI0C07150 of the Genolevures database (i.e. found at http://www.genolevures.org/), wherein YALI0 stands for Yarrowia lipolytica and A,B,C,D,E,F specifies chromosome. In embodiments, the nucleic acid or gene IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) is the nucleic acid or gene corresponding to YALI0C07150 of the Genolevures database (i.e. found at http://www.genolevures.org/). In embodiments, IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) is a protein or nucleic acid/gene of a yeast strain corresponding to YALI0C07150 of Yarrowia lipolytica as described above. In embodiments, IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) is a protein or nucleic acid/gene of an oleaginous organism corresponding to YALI0C07150 of Yarrowia lipolytica as described above. In embodiments, the protein and/or nucleic acid is a wildtype version of the protein or nucleic acid. In embodiments, the protein and/or nucleic acid is a mutant form of the protein and/or nucleic acid (e.g. point mutant, loss of function mutation, missense mutation, deletion, or insertion of heterologous nucleic acid).
As used to describe a protein or nucleic/acid of another organism in comparison to a protein or nucleic/acid of Yarrowia lipolytica, the term “corresponds” or “corresponding” is used according to its ordinary meaning and refers to a protein or nucleic acid/gene that includes similar or identical sequence of amino acid or nucleotides respectively and/or performs a similar or identical function and/or has a similar of identical activity as the protein or nucleic acid/gene in Yarrowia lipolytica as described above. In some embodiments, a protein or nucleic acid corresponding to a protein or nucleic acid from Yarrowia lipolytica is a homolog. In embodiments, the protein and/or nucleic acid of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) includes an amino acid and/or nucleotide sequence included in the protein and/or nucleic acid sequence for Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) respectively, described herein (e.g. Examples section and/or sequence listing). In embodiments, the protein and/or nucleic acid of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) is the amino acid and/or nucleotide sequence of the protein and/or nucleic acid sequence for Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) respectively, described herein (e.g. Examples section and/or sequence listing).
The term “wildtype” as used herein when referring to an oleaginous organism (e.g. yeast strain or Yarrowia lipolytica strain) means an organism that has not been genetically modified to improve production of a lipid (e.g. increase yield of a lipid, alter the structure of a lipid produced by the organism, reduce production of one lipid to improve production of a second lipid, or modulate the production of a lipid). In embodiments, a wildtype yeast strain may be auxotrophic for one or more compounds (e.g. leucine and/or uracil). In embodiments, a wildtype Yarrowia lipolytica strain is PO1f (ATCC #MYA-2613), a leucine and uracil auxotroph devoid of any secreted protease activity (Madzak et al., 2000).
The term “oleochemical” is used herein in accordance with its well known meaning and refers to chemicals or compounds derived from lipids or fats. In embodiments, an oleochemical is a lipid or fat derived from a different lipid or fat. In embodiments an oleochemical is a chemical or compound produced by an oleaginous organism. In embodiments, an oleochemical is a chemical or compound derived from a lipid or lipid precursor produced by an oleaginous organism (e.g., fatty acid esters such as methyl esters, ethyl esters, propyl esters, or butyl esters that are derived from a fatty acid produced by an oleaginous organism by transesterification). In embodiments, an oleochemical may include further in vivo or in vitro modification of a lipid or lipid precursor enabled by endogenous or heterologous modifying enzymes or chemical reactions.
The term “lipid precursor” is used in accordance with its well known meaning and refers to a pathway intermediate (e.g., acetyl-CoA or malonyl-CoA) in the biosynthesis of a lipid. In embodiments, a lipid precursor may be any molecule along the biosynthetic pathway making triglycerides including free citrate, acetyl-CoA, free fatty acids, pyruvate, citric acid cycle intermediates, diacylglycerides, and/or triacylglycerides.
The term “micronutrient” is used in accordance with its well known meaning and refers to nutrients used by an organism (e.g. oleaginous organisms, yeast, oleaginous yeast) for growth, proliferation, propagation, survival, one or more essential biological functions, production of a lipid, lipid precursor, or oleochemical, which are required for such functions in small quantities. Examples of micronutrients include, but are not limited to, minerals, vitamins, and elements (e.g. cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, and/or boron).
II. OLEAGINOUS ORGANISMS
In a first aspect is provided a genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) wherein the dry weight of the oleaginous organism includes greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 30% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 40% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 50% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 70% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 80% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 90% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes about an average of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 g/L culture (e.g. in a bioreactor) of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 g/L culture (e.g. in a bioreactor) of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 g/L culture (e.g. in a bioreactor) of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
In embodiments, the oleaginous organism is a yeast cell. In embodiments, the oleaginous organism is an oleaginous yeast cell. In embodiments, the yeast cell is selected from the group consisting of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In embodiments, the yeast cell is selected from the group consisting of Rhodosporidium toruloides, Lipomyces starkeyii, Lipomyces lipoferus, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Candida utilis, Trichosporon pullans, Trichosporon cutaneum, Rhodotorula glutinus, Rhodotorula graminis and Yarrowia lipolytica. In embodiments, the yeast cell is selected from the group consisting of Lipomyces starkeyii, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Rhodotorula glutinis, and Yarrowia lipolytica. In embodiments, the yeast cell is Yarrowia lipolytica. 
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is buoyant in an aqueous medium. In embodiments, the yeast cell includes a greater buoyancy (i.e. greater tendency to float, lower density) than a yeast cell that includes less than about 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) by dry weight (e.g. less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight). In embodiments, the yeast cell includes a greater buoyancy (i.e. greater tendency to float, lower density) than a yeast cell that includes less than 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) by dry weight (e.g. less than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) does not sediment to the bottom of a column of liquid (e.g. water, buffer, growth media, minimal media) that is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 mm tall due to gravitation force alone. The term “about” when used in connection with a defined amount refers to an amount up to and including greater than and/or less than 10% of the associated value and includes the associated value. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) does not sediment to the bottom of a column of liquid (e.g. water, buffer, growth media, minimal media) that is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 mm tall due to gravitation force alone. In embodiments, the yeast cell includes a greater buoyancy (i.e. greater tendency to float, lower density) than a yeast cell that does not include the same recombinant nucleic acid or combination of recombinant nucleic acids as the buoyant yeast cell. In embodiments, the yeast cell is buoyant following centrifugation (e.g. at about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950. 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10000×g).
In embodiments of the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) including more than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. more than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight), included are lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) selected from the group consisting of a fatty acid, wax, sterol, vitamin, monoglyceride, diglyceride, triglyceride, phospholipid, glycerolipid, glycerophospholipid, sphingolipid, saccharolipid, polyketide, sterol lipid, triacylglyceride, wax ester, fatty acid ethyl ester, fatty acid methyl ester, component of biodiesel, saturated hydrocarbon, unsaturated hydrocarbon, branched hydrocarbon, and a prenol lipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a fatty acid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a wax. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a sterol. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a vitamin. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a monoglyceride. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a diglyceride. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a triglyceride. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a phospholipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a glycerolipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a glycerophospholipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a sphingolipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a saccharolipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a polyketide. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a sterol lipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a triacylglyceride. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a prenol lipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a wax ester. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a fatty acid ethyl ester. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a fatty acid methyl ester. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a component of biodiesel. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a saturated hydrocarbon. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is an unsaturated hydrocarbon. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a branched hydrocarbon. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a lipid. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a lipid precursor. In embodiments, the majority lipid, lipid precursor, or oleochemical in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is an oleochemical.
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C5:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C5:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C5:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C5:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C6:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C6:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C6:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C6:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C7:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C7:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C7:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C7:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C8:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C8:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C8:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C8:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C9:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C9:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C9:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C9:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C10:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C10:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C10:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C10:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C11:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C11:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C11:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C11:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C12:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C12:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C12:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C12:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C13:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C13:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C13:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C13:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C14:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C14:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C14:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C14:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C15:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C15:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C15:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C15:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C16:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C16:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C16:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C16:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C17:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C17:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C17:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C17:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C18:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C18:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C18:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C18:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C19:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C19:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C19:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C19:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C20:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C20:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C20:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C20:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C21:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C21:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C21:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C21:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C22:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C22:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C22:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C22:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C23:0 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C23:1 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C23:2 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces C23:3 fatty acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces a fatty acid described herein above at a greater level (e.g. 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 1000000 fold) compared to the same oleaginous organism lacking the genetic modification. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces a lipid including a fatty acid selected from the group consisting of C5:0, C5:1, C5:2, C5:3, C6:0, C6:1, C6:2, C6:3, C7:0, C7:1, C7:2, C7:3, C8:0, C8:1, C8:2, C8:3, C9:0, C9:1, C9:2, C9:3, C10:0, C10:1, C10:2, C10:3, C11:0, C11:1, C11:2, C11:3, C12:0, C12:1, C12:2, C12:3, C13:0, C13:1, C13:2, C13:3, C14:0, C14:1, C14:2, C14:3, C15:0, C15:1, C15:2, C15:3, C16:0, C16:1, C16:2, C16:3, C17:0, C17:1, C17:2, C17:3, C18:0, C18:1, C18:2, C18:3, C19:0, C19:1, C19:2, C19:3, C20:0, C20:1, C20:2, C20:3, C21:0, C21:1, C21:2, C21:3, C22:0, C22:1, C22:2, C22:3, C23:0, C23:1, C23:2, and C23:3. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces a lipid derived from an endogenously produced fatty acid selected from the group consisting of C5:0, C5:1, C5:2, C5:3, C6:0, C6:1, C6:2, C6:3, C7:0, C7:1, C7:2, C7:3, C8:0, C8:1, C8:2, C8:3, C9:0, C9:1, C9:2, C9:3, C10:0, C10:1, C10:2, C10:3, C11:0, C11:1, C11:2, C11:3, C12:0, C12:1, C12:2, C12:3, C13:0, C13:1, C13:2, C13:3, C14:0, C14:1, C14:2, C14:3, C15:0, C15:1, C15:2, C15:3, C16:0, C16:1, C16:2, C16:3, C17:0, C17:1, C17:2, C17:3, C18:0, C18:1, C18:2, C18:3, C19:0, C19:1, C19:2, C19:3, C20:0, C20:1, C20:2, C20:3, C21:0, C21:1, C21:2, C21:3, C22:0, C22:1, C22:2, C22:3, C23:0, C23:1, C23:2, and C23:3. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces a lipid, lipid precursor, or oleochemical (e.g. fatty acid) described herein at a greater level (e.g. 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 1000000 fold) compared to the same oleaginous organism lacking the genetic modification.
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a recombinant nucleic acid, wherein the recombinant nucleic acid modulates the level of activity of a protein in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to the absence of the recombinant nucleic acid. In embodiments, the protein is selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA:diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), and IRC20 E3 ubiquitin-protein ligase and helicase (IRC20). In embodiments, the protein is Leucine Biosynthesis Gene (LEU2). In embodiments, the protein is Uracil Biosynthesis gene (URA3). In embodiments, the protein is multifunctional enzyme (MFE1). In embodiments, the protein is Transcription Factor (PEX10). In embodiments, the protein is AMP Deaminase (AMPD). In embodiments, the protein is ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2). In embodiments, the protein is Malic Enzyme (MAE). In embodiments, the protein is Acetyl-CoA Carboxylase (ACC). In embodiments, the protein is acyl-CoA: diacylglycerol acyltransferase (DGA1). In embodiments, the protein is acyl-CoA:diacylglycerol acyltransferases (DGA2). In embodiments, the protein is Mitochondrial 2′ O-ribose methyltransferase (MRM2). In embodiments, the protein is Lipid synthesis regulator (MGA2). In embodiments, the protein is Chromatin assembly gene (RLF2 subunit p90). In embodiments, the protein is O-6-methylguanine-DNA methyltransferase (MGMT). In embodiments, the protein is Aconitase (ACO1). In embodiments, the protein is Citrate Synthase (CIT1). In embodiments, the protein is RME1 zinc-finger transcription factor (RME1). In embodiments, the protein is YOX1 homeodomain protein (YOX1). In embodiments, the protein is UGA2 succinate semialdehyde dehydrogenase (UGA2). In embodiments, the protein is OSH6 oxysterol-binding protein homolog 6 (OSH6). In embodiments, the protein is IRC20 E3 ubiquitin-protein ligase and helicase (IRC20). In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the function of the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the amount of the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the transcription of the mRNA encoding the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the translation of the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the coding sequence of the gene encoding the protein (e.g. mutating (e.g. point mutant or missense mutant), truncating, inserting into, or deleting). In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the regulatory elements (e.g. promoter) of the gene encoding the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the stability of the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is modulating the stability of the transcript encoding the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is reducing the level of activity of the protein. In embodiments, modulating the level of activity of a protein in an oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is increasing the level of activity of the protein.
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein in the citric acid cycle in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in the Kennedy Pathway in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in fatty acid synthesis in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in fatty acid storage (e.g. accumulation) in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that that modulates (e.g. reduces or increases) the level of activity of a protein involved in lipid synthesis in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in lipid storage (e.g. accumulation) in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in triacylglyceride storage (e.g. accumulation) in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in triacylglyceride synthesis in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in peroxisomal biogenesis in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in the beta-oxidation cycle in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in fatty acid degradation in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in lipid degradation in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in triacylglyceride degradation in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid). In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a genetic modification (e.g. recombinant nucleic acid) that modulates (e.g. reduces or increases) the level of activity of a protein involved in central carbon metabolism in the oleaginous organism relative to the absence of the genetic modification (e.g. recombinant nucleic acid).
In embodiments, the recombinant nucleic acid increases the level of activity of a protein in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the protein is selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), and IRC20 E3 ubiquitin-protein ligase and helicase (IRC20). In embodiments, the protein is selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), 0-6-methylguanine-DNA methyltransferase (MGMT), and Citrate Synthase (CIT1). In embodiments, the protein is Leucine Biosynthesis Gene (LEU2). In embodiments, the protein is Uracil Biosynthesis gene (URA3). In embodiments, the protein is AMP Deaminase (AMPD). In embodiments, the protein is ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2). In embodiments, the protein is Malic Enzyme (MAE). In embodiments, the protein is Acetyl-CoA Carboxylase (ACC). In embodiments, the protein is acyl-CoA:diacylglycerol acyltransferase (DGA1). In embodiments, the protein is acyl-CoA:diacylglycerol acyltransferases (DGA2). In embodiments, the protein is Mitochondrial 2′ O-ribose methyltransferase (MRM2). In embodiments, the protein is Lipid synthesis regulator (MGA2). In embodiments, the protein is Chromatin assembly gene (RLF2 subunit p90). In embodiments, the protein is O-6-methylguanine-DNA methyltransferase (MGMT). In embodiments, the protein is Citrate Synthase (CIT1). In embodiments, the protein is RME1 zinc-finger transcription factor (RME1). In embodiments, the protein is YOX1 homeodomain protein (YOX1). In embodiments, the protein is UGA2 succinate semialdehyde dehydrogenase (UGA2). In embodiments, the protein is OSH6 oxysterol-binding protein homolog 6 (OSH6). In embodiments, the protein is IRC20 E3 ubiquitin-protein ligase and helicase (IRC20). In embodiments, the protein is selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), Malic Enzyme (MAE), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), and O-6-methylguanine-DNA methyltransferase (MGMT) or said nucleic acid decreases the level of activity of Lipid synthesis regulator (MGA2).
In embodiments, the genetic modification (e.g. recombinant nucleic acid) decreases the level of activity of a protein in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the protein is selected from the group consisting of multifunctional enzyme (MFE1), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), Transcription Factor (PEX10), and Aconitase (ACO1). In embodiments, the protein is multifunctional enzyme (MFE1). In embodiments, the protein is Lipid synthesis regulator (MGA2). In embodiments, the protein is Chromatin assembly gene (RLF2 subunit p90). In embodiments, the protein is Transcription Factor (PEX10). In embodiments, the protein is Aconitase (ACO1). In embodiments, the protein is RME1 zinc-finger transcription factor (RME1). In embodiments, the protein is YOX1 homeodomain protein (YOX1). In embodiments, the protein is UGA2 succinate semialdehyde dehydrogenase (UGA2). In embodiments, the protein is OSH6 oxysterol-binding protein homolog 6 (OSH6). In embodiments, the protein is IRC20 E3 ubiquitin-protein ligase and helicase (IRC20).
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a plurality of genetic modifications (e.g. recombinant nucleic acids) that collectively modulate one, two, three, four, five, six, seven, eight, nine, ten, or more of the group of proteins consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), and IRC20 E3 ubiquitin-protein ligase and helicase (IRC20).
In embodiments, the recombinant nucleic acid encodes a protein comprising a mutation relative to the wildtype protein. In embodiments, the mutation is a point mutation. In embodiments, the mutation is a deletion. In embodiments, the mutation is an insertion. In embodiments, the mutation is a fusion with a second protein. In embodiments, the recombinant nucleic acid encodes a mutant of a protein selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20).
In embodiments, the recombinant nucleic acid encodes a mutant of a protein selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), or IRC20 E3 ubiquitin-protein ligase and helicase (IRC20).
In embodiments, the recombinant nucleic acid is an AMP Deaminase (AMPD) having the nucleotide sequence of SEQ ID NO.:33. In embodiments, the recombinant nucleic acid is an AMP Deaminase (AMPD) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or the entire sequence) with SEQ ID NO.:33, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Leucine Biosynthesis Gene (LEU2) having the nucleotide sequence of SEQ ID NO.:35. In embodiments, the recombinant nucleic acid is a Leucine Biosynthesis Gene (LEU2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or the entire sequence) with SEQ ID NO.:35, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Uracil Biosynthesis gene (URA3) having the nucleotide sequence of SEQ ID NO.:37. In embodiments, the recombinant nucleic acid is a Uracil Biosynthesis gene (URA3) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, or the entire sequence) with SEQ ID NO.:37, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is an ATP-Citrate Lyase (ACL) (subunit 1) having the nucleotide sequence of SEQ ID NO.:39. In embodiments, the recombinant nucleic acid is an ATP-Citrate Lyase (ACL) (subunit 1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or the entire sequence) with SEQ ID NO.:39, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is an ATP-Citrate Lyase (ACL) (subunit 2) having the nucleotide sequence of SEQ ID NO.:41. In embodiments, the recombinant nucleic acid is an ATP-Citrate Lyase (ACL) (subunit 2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or the entire sequence) with SEQ ID NO.:41, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Malic Enzyme (MEA, MAE, MEA1) having the nucleotide sequence of SEQ ID NO.:43. In embodiments, the recombinant nucleic acid is a Malic Enzyme (MEA, MAE, MEA1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, or the entire sequence) with SEQ ID NO.:43, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a acyl-CoA:diacylglycerol acyltransferase (DGA1) having the nucleotide sequence of SEQ ID NO.:45. In embodiments, the recombinant nucleic acid is a acyl-CoA: diacylglycerol acyltransferase (DGA1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or the entire sequence) with SEQ ID NO.:45, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a acyl-CoA:diacylglycerol acyltransferase (DGA2) having the nucleotide sequence of SEQ ID NO.:47. In embodiments, the recombinant nucleic acid is a acyl-CoA:diacylglycerol acyltransferase (DGA2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or the entire sequence) with SEQ ID NO.:47, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Lipid synthesis regulator (MGA2) having the nucleotide sequence of SEQ ID NO.:49. In embodiments, the recombinant nucleic acid is a Lipid synthesis regulator (MGA2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, or the entire sequence) with SEQ ID NO.:49, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a mutant Lipid synthesis regulator (MGA2-L36 mutant) having the nucleotide sequence of SEQ ID NO.:51. In embodiments, the recombinant nucleic acid is a mutant Lipid synthesis regulator (MGA2-L36 mutant) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, or the entire sequence) with SEQ ID NO.:51, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a truncated Lipid synthesis regulator (MGA2-truncated) having the nucleotide sequence of SEQ ID NO.:53. In embodiments, the recombinant nucleic acid is a truncated Lipid synthesis regulator (MGA2-truncated) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, or the entire sequence) with SEQ ID NO.:53, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Chromatin assembly gene (RLF2 subunit p90) having the nucleotide sequence of SEQ ID NO.:58. In embodiments, the recombinant nucleic acid is a Chromatin assembly gene (RLF2 subunit p90) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or the entire sequence) with SEQ ID NO.:58, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Mitochondrial 2′ O-ribose methyltransferase (MRM2) having the nucleotide sequence of SEQ ID NO.:63. In embodiments, the recombinant nucleic acid is a Mitochondrial 2′ O-ribose methyltransferase (MRM2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, or the entire sequence) with SEQ ID NO.:63, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Citrate Synthase (CIT1) having the nucleotide sequence of SEQ ID NO.:67. In embodiments, the recombinant nucleic acid is a Citrate Synthase (CIT1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or the entire sequence) with SEQ ID NO.:67, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Acetyl-CoA Carboxylase (ACC) having the nucleotide sequence of SEQ ID NO.:69. In embodiments, the recombinant nucleic acid is a Acetyl-CoA Carboxylase (ACC) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, or the entire sequence) with SEQ ID NO.:69, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Transcription Factor (PEX10) having the nucleotide sequence of SEQ ID NO.:71. In embodiments, the recombinant nucleic acid is a Transcription Factor (PEX10) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or the entire sequence) with SEQ ID NO.:71, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a multifunctional enzyme (MFE1) having the nucleotide sequence of SEQ ID NO.:73. In embodiments, the recombinant nucleic acid is a multifunctional enzyme (MFE1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or the entire sequence) with SEQ ID NO.:73, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a Aconitase (ACO1) having the nucleotide sequence of SEQ ID NO.:75. In embodiments, the recombinant nucleic acid is a Aconitase (ACO1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, or the entire sequence) with SEQ ID NO.:75, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a YOX1 homeodomain protein (YOX1) having the nucleotide sequence of SEQ ID NO.:77. In embodiments, the recombinant nucleic acid is a YOX1 homeodomain protein (YOX1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or the entire sequence) with SEQ ID NO.:77, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a UGA2 succinate semialdehyde dehydrogenase (UGA2) having the nucleotide sequence of SEQ ID NO.:78. In embodiments, the recombinant nucleic acid is a UGA2 succinate semialdehyde dehydrogenase (UGA2) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or the entire sequence) with SEQ ID NO.:78, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a RME1 zinc-finger transcription factor (RME1) having the nucleotide sequence of SEQ ID NO.:79. In embodiments, the recombinant nucleic acid is a RME1 zinc-finger transcription factor (RME1) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, or the entire sequence) with SEQ ID NO.:79, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a OSH6 oxysterol-binding protein homolog 6 (OSH6) having the nucleotide sequence of SEQ ID NO.:80. In embodiments, the recombinant nucleic acid is a OSH6 oxysterol-binding protein homolog 6 (OSH6) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or the entire sequence) with SEQ ID NO.:80, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) having the nucleotide sequence of SEQ ID NO.:81. In embodiments, the recombinant nucleic acid is a IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, or the entire sequence) with SEQ ID NO.:81, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the recombinant nucleic acid is a O-6-methylguanine-DNA methyltransferase (MGMT) having the nucleotide sequence of SEQ ID NO.:65. In embodiments, the recombinant nucleic acid is a O-6-methylguanine-DNA methyltransferase (MGMT) having at least 60% identity (e.g. at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a region of at least 100 nucleotides (e.g. at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or the entire sequence) with SEQ ID NO.:65, (e.g. using the same length of nucleotides for comparison or the entirety of both nucleic acids).
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a recombinant nucleic acid that decreases the level of activity of multifunctional enzyme (MFE1) protein and Transcription Factor (PEX10) protein, increases the level of activity of acyl-CoA: diacylglycerol acyltransferase (DGA1) protein, or increases the level of activity of Leucine Biosynthesis Gene (LEU2) protein relative to a oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that does not include the recombinant nucleic acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes recombinant nucleic acids that decrease the level of activity of multifunctional enzyme (MFE1) protein and Transcription Factor (PEX10) protein, increase the level of activity of acyl-CoA:diacylglycerol acyltransferase (DGA1) protein, and increase the level of activity of Leucine Biosynthesis Gene (LEU2) protein relative to a oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that does not include the recombinant nucleic acids. In embodiments, the level of activity is the level of expression of the protein.
In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes an extra-chromosomal recombinant nucleic acid. In embodiments, the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes a recombinant nucleic acid integrated into the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) genome. In embodiments, the extra-chromosomal recombinant nucleic acid includes a gene that is also included in the genome of the yeast cell oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) (e.g. Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA:diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Aconitase (ACO1), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6 oxysterol-binding protein homolog 6 (OSH6), IRC20 E3 ubiquitin-protein ligase and helicase (IRC20), a wildtype version thereof, or a mutant version thereof). In embodiments, the extra-chromosomal recombinant nucleic acid includes a gene that is also included in the genome of the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) (e.g. Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL) (e.g. ACL subunit 1, ACL subunit 2, or subunit 1 and 2), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), O-6-methylguanine-DNA methyltransferase (MGMT), Citrate Synthase (CIT1), RME1 zinc-finger transcription factor (RME1), YOX1 homeodomain protein (YOX1), UGA2 succinate semialdehyde dehydrogenase (UGA2), OSH6-oxysterol-binding protein homolog 6 (OSH6), IRC20 E3 ubiquitin-protein ligase and helicase (IRC20), a wildtype version thereof, or a mutant version thereof). In embodiments, a recombinant nucleic acid integrated into the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) genome replaces (e.g. partially or completely) a promoter included in the oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) genome pror to integration of the recombinant nucleic acid.
In embodiments, the yeast cell is a yeast cell including one or more genetic modifications (e.g. recombinant nucleic acids), as described herein (including in the Examples section below, the tables, the figures, and the claims herein). In embodiments, the yeast cell is a yeast cell described herein, including in an example, table, figure, or claim. In embodiments, the oleaginous yeast cell is L36 as described herein (e.g. examples, tables, and figures). In embodiments, the oleaginous yeast cell is derived from L36 as described herein (e.g. examples, tables, and figures). In embodiments, the oleaginous yeast cell is E26 as described herein (e.g. examples, tables, and figures). In embodiments, the oleaginous yeast cell is E13 as described herein (e.g. examples, tables, and figures). In embodiments, the oleaginous yeast cell is derived from E26 or E13.
In embodiments, the dry weight of the genetically modified yeast cell described herein includes greater than about 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., greater than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%; greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%; of lipid; lipids; lipid precursors; lipid precursor, oleochemical, and/or oleochemicals).
In embodiments, the genetically modified yeast cell described herein includes a recombinant Leucine Biosynthesis Gene (LEU2). In embodiments, the genetic modification increases the level of activity of the Leucine Biosynthesis Gene (LEU2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein is capable of de novo synthesis of leucine (e.g. at sufficient levels to meet the leucine requirements of the yeast cell). In embodiments, the genetically modified yeast cell described herein is capable of de novo synthesis of leucine independent of the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Uracil Biosynthesis gene (URA3). In embodiments, the genetic modification increases the level of activity of the Uracil Biosynthesis gene (URA3) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein is capable of de novo synthesis of uracil (e.g. at sufficient levels to meet the uracil requirements of the yeast cell). In embodiments, the genetically modified yeast cell described herein is capable of de novo synthesis of uracil independent of the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified multifunctional enzyme (MFE1) gene. In embodiments, the genetic modification decreases the level of activity of the multifunctional enzyme (MFE1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified PEX10 Transcription Factor (PEX10) gene. In embodiments, the genetic modification decreases the level of activity of the PEX10 Transcription Factor (PEX10) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant AMP Deaminase (AMPD) protein. In embodiments, the genetic modification increases the level of activity of the AMP Deaminase (AMPD) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant ATP-Citrate Lyase 1 (ACL1) protein. In embodiments, the genetic modification increases the level of activity of the ATP-Citrate Lyase 1 (ACL1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant ATP-Citrate Lyase 2 (ACL2) protein. In embodiments, the genetic modification increases the level of activity of the ATP-Citrate Lyase 2 (ACL2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant ATP-Citrate Lyase 1 (ACL1) protein and ATP-Citrate Lyase 2 (ACL2) protein. In embodiments, the genetic modification increases the level of activity of the ATP-Citrate Lyase 1 (ACL1) protein and ATP-Citrate Lyase 2 (ACL2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Malic Enzyme (MAE) protein. In embodiments, the genetic modification increases the level of activity of the Malic Enzyme (MAE) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Acetyl-CoA Carboxylase (ACC) protein. In embodiments, the genetic modification increases the level of activity of the Acetyl-CoA Carboxylase (ACC) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant acyl-CoA:diacylglycerol acyltransferase 1 (DGA1) protein. In embodiments, the genetic modification increases the level of activity of the acyl-CoA:diacylglycerol acyltransferase 1 (DGA1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant acyl-CoA:diacylglycerol acyltransferase 2 (DGA2) protein. In embodiments, the genetic modification increases the level of activity of the acyl-CoA:diacylglycerol acyltransferase 2 (DGA2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Mitochondrial 2′ O-ribose methyltransferase (MRM2) protein. In embodiments, the genetic modification increases the level of activity of the Mitochondrial 2′ O-ribose methyltransferase (MRM2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Lipid synthesis regulator (MGA2) protein. In embodiments, the genetically modified yeast cell described herein includes a genetically modified Lipid synthesis regulator (MGA2) gene. In embodiments, the genetically modified yeast cell described herein includes at least one nucleotide deletion in the genomic Lipid synthesis regulator (MGA2) gene and expression of a Lipid synthesis regulator (MGA2) protein including a mutation corresponding to G643R in Yarrowia lipolytica Lipid synthesis regulator (MGA2) In embodiments, the genetic modification decreases the level of activity of the Lipid synthesis regulator (MGA2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified Chromatin assembly gene (RLF2 subunit p90) gene. In embodiments, the genetic modification decreases the level of activity of the Chromatin assembly gene (RLF2 subunit p90) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant O-6-methylguanine-DNA methyltransferase (MGMT) protein. In embodiments, the genetic modification increases the level of activity of the O-6-methylguanine-DNA methyltransferase (MGMT) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified Aconitase (ACO1) gene. In embodiments, the genetic modification decreases the level of activity of the Aconitase (ACO1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a recombinant Citrate Synthase (CIT1) gene. In embodiments, the genetic modification increases the level of activity of the Citrate Synthase (CIT1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified RME1 zinc-finger transcription factor (RME1) gene. In embodiments, the genetic modification decreases the level of activity of the RME1 zinc-finger transcription factor (RME1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified YOX1 homeodomain protein (YOX1) gene. In embodiments, the genetic modification decreases the level of activity of the YOX1 homeodomain protein (YOX1) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified UGA2 succinate semialdehyde dehydrogenase (UGA2) gene. In embodiments, the genetic modification decreases the level of activity of the UGA2 succinate semialdehyde dehydrogenase (UGA2) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified OSH6 oxysterol-binding protein homolog 6 (OSH6) gene. In embodiments, the genetic modification decreases the level of activity of the OSH6 oxysterol-binding protein homolog 6 (OSH6) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the genetically modified yeast cell described herein includes a genetically modified IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) gene. In embodiments, the genetic modification decreases the level of activity of the IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) protein relative to an otherwise identical yeast cell lacking the genetic modification. In embodiments, the gene or protein described herein is a Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is a yeast gene or protein corresponding to the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is a gene or protein from an oleaginous organism corresponding to the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is the Yarrowia lipolytica gene or protein identified by sequence herein. In embodiments, the gene or protein is a mutant gene or protein of a wildtype gene or protein corresponding to the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is a mutant gene or protein of a wildtype yeast gene or protein corresponding to the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is a homolog of the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein is a homolog of the Yarrowia lipolytica gene or protein identified by sequence herein. In embodiments, the gene or protein is a mutant of the Yarrowia lipolytica gene or protein. In embodiments, the gene or protein described in this paragraph is LEU2, URA3, MFE1, PEX10, AMPD, ACL, ACL1, ACL2, MAE, ACC, DGA, DGA1, DGA2, MRM2, MGA2, RLF2 subunit p90, MGMT, ACO1, CIT1, RME1, YOX1, UGA2, OSH6, or IRC20). In embodiments, the gene or protein described in this paragraph is LEU2, URA3, MFE1, PEX10, AMPD, ACL, ACL1, ACL2, MAE, ACC, DGA, DGA1, DGA2, MRM2, MGA2, RLF2 subunit p90, MGMT, ACO1, CIT1, RME1, YOX1, UGA2, OSH6, or IRC20), having the sequence identified herein.
In embodiments, the genetic modification modulates the level of activity of a component of a lipid biosynthetic pathway. In embodiments, the genetic modification modulates the level of activity of a component of a lipid precursor biosynthetic pathway. In embodiments, the genetic modification modulates the level of activity of a component of an oleochemical biosynthetic pathway. In embodiments, the genetic modification modulates the level of activity of a component of a pathway incorporating Acetyl-CoA into a lipid, lipid precursor, or oleochemical. In embodiments, the genetic modification modulates the level of activity of a component of a pathway incorporating malonyl-CoA into a lipid, lipid precursor, or oleochemical. In embodiments, the genetic modification increases the level of activity of a component of a lipid biosynthetic pathway. In embodiments, the genetic modification increases the level of activity of a component of a lipid precursor biosynthetic pathway. In embodiments, the genetic modification increases the level of activity of a component of an oleochemical biosynthetic pathway. In embodiments, the genetic modification increases the level of activity of a component of a pathway incorporating acetyl-CoA into a lipid, lipid precursor, or oleochemical. In embodiments, the genetic modification increases the level of activity of a component of a pathway incorporating malonyl-CoA into a lipid, lipid precursor, or oleochemical. In embodiments, the genetic modification modulates the level of activity of a component of a lipid, or lipid precursor, metabolic pathway. In embodiments, the genetic modification decreases the level of activity of a component of a lipid, or lipid precursor, metabolic pathway. In embodiments, the genetic modification decreases the level of activity of a component of a lipid, or lipid precursor, metabolic pathway. In embodiments, the genetic modification increases the level of acetyl-CoA in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification increases the level of malonyl-CoA in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification increases the level of triglyceride production in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification decreases the level of beta-oxidation activity in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification decreases the level of fatty acid catabolism in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification decreases the level of peroxisome biogenesis activity in the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification produces a lipid, lipid precursor, or oleochemical at a higher level than by a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) produces a lipid, lipid precursor, or oleochemical at a higher level than by a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the genetic modification modulates the level of activity of a component of the citric acid cycle. In embodiments, the genetic modification modulates the level of activity of a component of the TCA cycle. In embodiments, the genetic modification modulates the level of activity of a component of the Kennedy pathway. In embodiments, the genetic modification reduces the level of activity of the TCA cycle. In embodiments, the genetic modification increases the level of activity of the Kennedy pathway.
In embodiments, the lipid, lipid precursor, or oleochemical produced at a higher level by the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) is a fatty acid, wax, sterol, vitamin, monoglyceride, diglyceride, triglyceride, phospholipid, glycerolipid, glycerophospholipid, sphingolipid, saccharolipid, polyketide, sterol lipid, triacylglyceride, prenol lipid, fatty acid ester, fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty alcohol, fatty amine, glycerol, alcohol ethoxylate, alcohol sulfate, or alcohol ether sulfate. In embodiments, the genetic modification includes a mutation relative to the wild type gene. In embodiments, the genetic modification includes a deletion of a portion of a gene. In embodiments, the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) includes an increased level of a fatty acid selected from the group consisting of C5:0, C5:1, C5:2, C5:3, C6:0, C6:1, C6:2, C6:3, C7:0, C7:1, C7:2, C7:3, C8:0, C8:1, C8:2, C8:3, C9:0, C9:1, C9:2, C9:3, C10:0, C10:1, C10:2, C10:3, C11:0, C11:1, C11:2, C11:3, C12:0, C12:1, C12:2, C12:3, C13:0, C13:1, C13:2, C13:3, C14:0, C14:1, C14:2, C14:3, C15:0, C15:1, C15:2, C15:3, C16:0, C16:1, C16:2, C16:3, C17:0, C17:1, C17:2, C17:3, C18:0, C18:1, C18:2, C18:3, C19:0, C19:1, C19:2, C19:3, C20:0, C20:1, C20:2, C20:3, C21:0, C21:1, C21:2, C21:3, C22:0, C22:1, C22:2, C22:3, C23:0, C23:1, C23:2, and C23:3, relative to a genetically unmodified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell) that is otherwise identical (e.g. genetically) to the genetically modified oleaginous organism (e.g. yeast cell, oleaginous yeast cell, Yarrowia lipolytica, algae, or plant cell). In embodiments, the fatty acid is C17:0 C17:1. In embodiments, the fatty acid is C16:1n9.
In embodiments, the genetic modification is an engineered genetic modification. In embodiments, the engineered genetic modification includes modulated expression of a protein. In embodiments, the engineered genetic modification includes increased expression of a protein. In embodiments, the engineered genetic modification includes decreased expression of a protein. In embodiments, the genetic modification is associated with exposure to a mutagen. In embodiments, the genetic modification includes modulated expression of a protein in a lipid, or lipid precursor, or oleochemical biosynthetic pathway.
III. METHODS OF MAKING AND PURIFYING LIPIDS, LIPID PRECURSORS, AND/OR OLEOCHEMICALS
Lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) produced by cells of the invention can be harvested, or otherwise collected, by any convenient method (e.g. centrifugation of extracellular secreted lipids, exposure to solvent, whole cell extraction (e.g. cell disruption and collection), hydrophobic solvent extraction (e.g. hexane), liquefaction, supercritical carbon dioxide extraction, freeze drying, mechanical pulverization, secretion (e.g. by addition of effective exporter proteins), or combinations thereof).
In embodiments, reduced nitrogen conditions promote accumulation of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical). In embodiments, cells (e.g. oleaginous organisms or oleaginous yeast) are first cultured in standard conditions and then cultured in low nitrogen conditions where harvesting is desired. In embodiments, oleaginous yeast species are grown in a medium including a carbon substrate and/or nitrogen source, optionally in the absence of light, optionally in an aerobic environment. In embodiments, media for culturing oleaginous yeast may include a carbon substrate, a fixed nitrogen source, trace elements, a buffer for pH maintenance, phosphate, or a combination thereof.
In embodiments, the carbon substrate may be selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fatty acids, monoglycerides, carbon dioxide, methanol, formaldehyde, formate, carbon-containing amines, glucose, fructose, sucrose, lactose, galactose, xylose, mannose, rhamnose, arabinose, glycerol, acetate, depolymerized sugar beet pulp, black liquor, corn starch, depolymerized cellulosic material, corn stover, sugar beet pulp, switchgrass, milk whey, molasses, potato, rice, sorghum, sugar cane, wheat, thick cane juice, sugar beet juice, wheat, lignocellulosic biomass, and combinations thereof.
Examples of cellulosic material that may be depolymerized and used as a carbon substrate (e.g. fixed carbon source) include sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves, husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp, citrus peels; hardwood and softwood thinnings; hardwood and softwood residues; saw mill wastes (wood chips, sawdust) and pulp mill waste; paper fractions of municipal solid waste; municipal grass clippings; wood construction waste; and cellulosic crops such as switchgrass, hybrid poplar wood, and miscanthus, fiber cane, and fiber sorghum.
Oleaginous yeast cultures may yield oleaginous yeast biomass in fermentation media. To extract lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) from the biomass, the biomass may be harvested, concentrated, dewatered (i.e. separation of the biomass from the liquid medium) (e.g. through centrifugation, filtration, use of mechanical pressure, simple sedimentation, or sedimentation), or combinations thereof. Centrifugation does not always remove significant amounts of intracellular water from the oleaginous yeast and so is often considered a dewatering, not a drying, step. The biomass can optionally be dried (oven dried, lyophilized, and the like) and conditioned prior to cell disruption (lysis).
In a second aspect is provided a method of producing a lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) including: 1) culturing a yeast cell as described herein (including embodiments or as described in the examples, tables, figures, and/or claims) in a growth medium; and 2) isolating the lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) (e.g. from the medium or yeast cell).
In embodiments, the lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) is isolated from the yeast cell. In embodiments, the lipid, lipid precursor, or oleochemical (e.g., lipid, lipid precursor, oleochemical) is isolated from the medium. In embodiments, the growth medium includes a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellulosic biomass. In embodiments, the majority carbon source is glucose. In embodiments, the majority carbon source is glycerol. In embodiments, the majority carbon source is xylose. In embodiments, the majority carbon source is fructose. In embodiments, the majority carbon source is mannose. In embodiments, the majority carbon source is ribose. In embodiments, the majority carbon source is sucrose. In embodiments, the majority carbon source is lignocellulosic biomass. In embodiments, the carbon source is glucose. In embodiments, the carbon source is glycerol. In embodiments, the carbon source is xylose. In embodiments, the carbon source is fructose. In embodiments, the carbon source is mannose. In embodiments, the carbon source is ribose. In embodiments, the carbon source is sucrose. In embodiments, the carbon source is lignocellulosic biomass. In embodiments, the majority carbon source is not glucose. In embodiments, the majority nitrogen source is ammonium sulfate ((NH4)2SO4).
In embodiments, the growth medium includes a carbon source and a nitrogen source wherein the carbon source is at a concentration at least 2-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 3-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 4-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 5-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 6-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 7-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 8-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 9-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 10-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 11-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 12-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 13-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 14-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 15-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 16-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 17-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 18-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 19-fold greater than the concentration of the nitrogen source. In embodiments, the carbon source is at a concentration at least 20-fold greater than the concentration of the nitrogen source. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is about 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512, 1024, 1600, 2048, 4096, 8192, or 16284. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000. In embodiments, the ratio of the carbon source to the nitrogen source (wt/wt) is at least 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, 200, 250, 300, 350, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000. In embodiments, the carbon source to nitrogen source ratio corresponds to a ratio calculated from one or more of the ratios described above when the ratios described above are for a carbon source of glucose (g/L) and a nitrogen source of ammonium sulfate (g/L) for a carbon source that may not be glucose and a nitrogen source that may not be ammonium sulfate. In embodiments, the ratio of the concentration of the carbon source to the concentration of the nitrogen source is as described herein, including in embodiments, examples, tables, figures, and claims. In embodiments, the amount and ratio of the carbon source to the nitrogen source (wt/wt) is equivalent to 160:0.2 glucose:ammonium sulfate. In embodiments, the amount and ratio of the carbon source to the nitrogen source (wt/wt) is equivalent to 80:5 glucose:ammonium sulfate.
In embodiments, the carbon source is at a concentration (g/L) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 100, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500. In embodiments, the carbon source is at a concentration (g/L) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 100, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500. In embodiments, the carbon source, which is optionally not glucose, is at a concentration for the carbon source that would provide an equal amount of carbon as one of the amounts described above where the amount described above is for glucose.
In embodiments, the nitrogen source is at a concentration (g/L) of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100. In embodiments, the nitrogen source is at a concentration (g/L) of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100. In embodiments, the nitrogen source, which is optionally not ammonium sulfate, is at a concentration for the nitrogen source that would provide an equal amount of nitrogen as one of the amounts described above where the amount described above is for ammonium sulfate.
In embodiments, the growth medium includes a micronutrient. In embodiments, the growth medium includes a plurality of micronutrients. In embodiments, the growth medium includes cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, and/or boron. In embodiments, the growth medium includes iron and copper or molybdenum. In embodiments, the growth medium includes copper and nickel. In embodiments, the growth medium includes copper, iron, and either molybdenum or nickel. In embodiments, the growth medium includes copper, iron, molybdenum, and nickel. In embodiments, the growth medium includes cobalt. In embodiments, the growth medium includes iron. In embodiments, the growth medium includes magnesium. In embodiments, the growth medium includes potassium. In embodiments, the growth medium includes zinc. In embodiments, the growth medium includes nickel. In embodiments, the growth medium includes molybdenum. In embodiments, the growth medium includes manganese. In embodiments, the growth medium includes copper. In embodiments, the growth medium includes boron. In embodiments, the growth medium is supplemented with cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, and/or boron. In embodiments, the growth medium is supplemented with iron and copper or molybdenum. In embodiments, the growth medium is supplemented with copper and nickel. In embodiments, the growth medium is supplemented with copper, iron, and either molybdenum or nickel. In embodiments, the growth medium is supplemented with copper, iron, molybdenum, and nickel. In embodiments, the growth medium is supplemented with cobalt. In embodiments, the growth medium is supplemented with iron. In embodiments, the growth medium is supplemented with magnesium. In embodiments, the growth medium is supplemented with potassium. In embodiments, the growth medium is supplemented with zinc. In embodiments, the growth medium is supplemented with nickel. In embodiments, the growth medium is supplemented with molybdenum. In embodiments, the growth medium is supplemented with manganese. In embodiments, the growth medium is supplemented with copper. In embodiments, the growth medium is supplemented with boron. In embodiments, the growth medium includes CoCl2 at a concentration of about 15 mg/L. In embodiments, the growth medium includes MgSO4 at a concentration of about 250 mg/L. In embodiments, the growth medium includes KI at a concentration of about 15 mg/L. In embodiments, the growth medium includes ZnSO4.7H2O at a concentration of about 20 mg/L. In embodiments, the growth medium includes MnSO4.H2O at a concentration of about 12.5 mg/L. In embodiments, the growth medium includes Boric acid at a concentration of about 12.5 mg/L. In embodiments, the growth medium includes (NH4)2Mo.4H2O at a concentration of about 15 mg/L. In embodiments, the growth medium includes NiSO4.6H2O at a concentration of about 12.5 mg/L. In embodiments, the growth medium includes FeSO4.7H2O at a concentration of about 20 mg/L. In embodiments, the growth medium includes CuSO4 at a concentration of about 15 mg/L. In embodiments, the growth medium includes CoCl2 at a concentration of 15 mg/L. In embodiments, the growth medium includes MgSO4 at a concentration of 250 mg/L. In embodiments, the growth medium includes KI at a concentration of 15 mg/L. In embodiments, the growth medium includes ZnSO4.7H2O at a concentration of 20 mg/L. In embodiments, the growth medium includes MnSO4.H2O at a concentration of 12.5 mg/L. In embodiments, the growth medium includes Boric acid at a concentration of 12.5 mg/L. In embodiments, the growth medium includes (NH4)2Mo.4H2O at a concentration of 15 mg/L. In embodiments, the growth medium includes NiSO4.6H2O at a concentration of 12.5 mg/L. In embodiments, the growth medium includes FeSO4.7H2O at a concentration of 20 mg/L. In embodiments, the growth medium includes CuSO4 at a concentration of 15 mg/L. In embodiments, the growth medium is supplemented with CoCl2 at a concentration of about 15 mg/L. In embodiments, the growth medium is supplemented with MgSO4 at a concentration of about 250 mg/L. In embodiments, the growth medium is supplemented with KI at a concentration of about 15 mg/L. In embodiments, the growth medium is supplemented with ZnSO4.7H2O at a concentration of about 20 mg/L. In embodiments, the growth medium is supplemented with MnSO4.H2O at a concentration of about 12.5 mg/L. In embodiments, the growth medium is supplemented with Boric acid at a concentration of about 12.5 mg/L. In embodiments, the growth medium is supplemented with (NH4)2Mo.4H2O at a concentration of about 15 mg/L. In embodiments, the growth medium is supplemented with NiSO4.6H2O at a concentration of about 12.5 mg/L. In embodiments, the growth medium is supplemented with FeSO4.7H2O at a concentration of about 20 mg/L. In embodiments, the growth medium is supplemented with CuSO4 at a concentration of about 15 mg/L. In embodiments, the growth medium is supplemented with CoCl2 at a concentration of 15 mg/L. In embodiments, the growth medium is supplemented with MgSO4 at a concentration of 250 mg/L. In embodiments, the growth medium is supplemented with KI at a concentration of 15 mg/L. In embodiments, the growth medium is supplemented with ZnSO4.7H2O at a concentration of 20 mg/L. In embodiments, the growth medium is supplemented with MnSO4.H2O at a concentration of 12.5 mg/L. In embodiments, the growth medium is supplemented with Boric acid at a concentration of 12.5 mg/L. In embodiments, the growth medium is supplemented with (NH4)2Mo.4H2O at a concentration of 15 mg/L. In embodiments, the growth medium is supplemented with NiSO4.6H2O at a concentration of 12.5 mg/L. In embodiments, the growth medium is supplemented with FeSO4.7H2O at a concentration of 20 mg/L. In embodiments, the growth medium is supplemented with CuSO4 at a concentration of 15 mg/L. In embodiments, the growth medium includes CoCl2 at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium includes MgSO4 at a concentration of about 125 to 375 mg/L. In embodiments, the growth medium includes KI at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium includes ZnSO4.7H2O at a concentration of about 10 to 30 mg/L. In embodiments, the growth medium includes MnSO4.H2O at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium includes Boric acid at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium includes (NH4)2Mo.4H2O at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium includes NiSO4.6H2O at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium includes FeSO4.7H2O at a concentration of about 10 to 30 mg/L. In embodiments, the growth medium includes CuSO4 at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium includes CoCl2 at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium includes MgSO4 at a concentration of 125 to 375 mg/L. In embodiments, the growth medium includes KI at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium includes ZnSO4.7H2O at a concentration of 10 to 30 mg/L. In embodiments, the growth medium includes MnSO4.H2O at a concentration of 6 to 18 mg/L. In embodiments, the growth medium includes Boric acid at a concentration of 6 to 18 mg/L. In embodiments, the growth medium includes (NH4)2Mo.4H2O at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium includes NiSO4.6H2O at a concentration of 6 to 18 mg/L. In embodiments, the growth medium includes FeSO4.7H2O at a concentration of 10 to 30 mg/L. In embodiments, the growth medium includes CuSO4 at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with CoCl2 at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with MgSO4 at a concentration of about 125 to 375 mg/L. In embodiments, the growth medium is supplemented with KI at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with ZnSO4.7H2O at a concentration of about 10 to 30 mg/L. In embodiments, the growth medium is supplemented with MnSO4.H2O at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium is supplemented with Boric acid at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium is supplemented with (NH4)2Mo.4H2O at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with NiSO4.6H2O at a concentration of about 6 to 18 mg/L. In embodiments, the growth medium is supplemented with FeSO4.7H2O at a concentration of about 10 to 30 mg/L. In embodiments, the growth medium is supplemented with CuSO4 at a concentration of about 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with CoCl2 at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with MgSO4 at a concentration of 125 to 375 mg/L. In embodiments, the growth medium is supplemented with KI at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with ZnSO4.7H2O at a concentration of 10 to 30 mg/L. In embodiments, the growth medium is supplemented with MnSO4.H2O at a concentration of 6 to 18 mg/L. In embodiments, the growth medium is supplemented with Boric acid at a concentration of 6 to 18 mg/L. In embodiments, the growth medium is supplemented with (NH4)2Mo.4H2O at a concentration of 7.5 to 22.5 mg/L. In embodiments, the growth medium is supplemented with NiSO4.6H2O at a concentration of 6 to 18 mg/L. In embodiments, the growth medium is supplemented with FeSO4.7H2O at a concentration of 10 to 30 mg/L. In embodiments, the growth medium is supplemented with CuSO4 at a concentration of 7.5 to 22.5 mg/L.
In embodiments, the method does not include nitrogen starvation of the oleaginous organism (e.g. oleaginous yeast cell).
In embodiments, the oleaginous yeast is cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 hours. In embodiments, the oleaginous yeast is cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 hours. In embodiments, the oleaginous yeast is cultured for about 48, 96, 144, or 192 hours. In embodiments, the oleaginous yeast is cultured for 48, 96, 144, or 192 hours. In embodiments, the oleaginous yeast is cultured for about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days. In embodiments, the oleaginous yeast is cultured for 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days.
In an aspect is provided a method of producing a lipid, lipid precursor, or oleochemical including culturing a yeast cell described herein in a growth medium; and isolating the lipid, lipid precursor, or oleochemical.
In embodiments, the lipid, lipid precursor, or oleochemical is isolated from the yeast cell. In embodiments, the lipid, lipid precursor, or oleochemical is isolated from the growth medium. In embodiments, the growth medium includes a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellulosic biomass. In embodiments, the growth medium includes lignocellulosic biomass as the majority carbon source. In embodiments, the growth medium includes a carbon source and a nitrogen source wherein the carbon source is at a concentration at least 10-fold greater than the concentration of the nitrogen source (wt/wt). In embodiments, the growth medium includes a carbon source and a nitrogen source wherein the carbon source is at a concentration at least 16-fold greater than the concentration of the nitrogen source (wt/wt). In embodiments, the growth medium includes a carbon source and a nitrogen source wherein the carbon source is at a concentration at least 320-fold greater than the concentration of the nitrogen source (wt/wt).
In embodiments, the growth medium includes cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, or boron. In embodiments, the growth medium includes any combination of two or more of cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, or boron. In embodiments, the growth medium includes cobalt in an amount equivalent to 7.5 to 22.5 mg/L CoCl2, magnesium in an amount equivalent to 125 to 375 mg/L MgSO4, potassium in an amount equivalent to 7.5 to 22.5 mg/L KI, zinc in an amount equivalent to 10 to 30 mg/L ZnSO4.7H2O, manganese in an amount equivalent to 6 to 18 mg/L MnSO4.H2O, boron in an amount equivalent to 6 to 18 mg/L Boric acid, molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4. In embodiments, the growth medium includes about 5.77×10−5 M to 1.73×10−4M cobalt, about 0.001 M to 0.003 M magnesium, about 4.52×10−5 M to 1.35×10−4 M potassium, about 4.05×10−5 M to 1.22×10−4 M zinc, about 3.55×10−5 to 1.06×10−4M manganese, about 9.07×10−5 M to 2.91×10−4 M boron, about 3.76×10−5 M to 1.10×10−4M molybdenum, about 2.28×10−5 M to 6.84×10−5 M nickel, about 3.60×10−5 M to 1.08×10−4M iron, or about 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes 5.77×10−5 M to 1.73×10−4 M cobalt, 0.001 M to 0.003 M magnesium, 4.52×10−5 M to 1.35×10−4 M potassium, 4.05×10−5 M to 1.22×10−4 M zinc, 3.55×10−5 to 1.06×10−4 M manganese, 9.07×10−5 M to 2.91×10−4 M boron, 3.76×10−5 M to 1.10×10−4 M molybdenum, 2.28×10−5 M to 6.84×10−5 M nickel, 3.60×10−5 M to 1.08×10−4 M iron, or 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes about 5.77×10−5 M to 1.73×10−4M cobalt. In embodiments, the growth medium includes about 0.001 M to 0.003 M magnesium. In embodiments, the growth medium includes about 4.52×10−5 M to 1.35×10−4 M potassium. In embodiments, the growth medium includes about 4.05×10−5 M to 1.22×10−4 M zinc. In embodiments, the growth medium includes about 3.55×10−5 to 1.06×10−4 M manganese. In embodiments, the growth medium includes about 9.07×10−5 M to 2.91×10−4 M boron. In embodiments, the growth medium includes about 3.76×10−5 M to 1.10×10−4M molybdenum. In embodiments, the growth medium includes about 2.28×10−5 M to 6.84×10−5 M nickel. In embodiments, the growth medium includes about 3.60×10−5 M to 1.08×10−4M iron. In embodiments, the growth medium includes about 4.70×10−5 M to 1.41×10−4M copper. In embodiments, the growth medium includes 5.77×10−5 M to 1.73×10−4 M cobalt. In embodiments, the growth medium includes 0.001 M to 0.003 M magnesium. In embodiments, the growth medium includes 4.52×10−5 M to 1.35×10−4 M potassium. In embodiments, the growth medium includes 4.05×10−5 M to 1.22×10−4 M zinc. In embodiments, the growth medium includes 3.55×10−5 to 1.06×10−4 M manganese. In embodiments, the growth medium includes 9.07×10−5 M to 2.91×10−4 M boron. In embodiments, the growth medium includes 3.76×10−5 M to 1.10×10−4 M molybdenum. In embodiments, the growth medium includes 2.28×10−5 M to 6.84×10−5 M nickel. In embodiments, the growth medium includes 3.60×10−5 M to 1.08×10−4 M iron. In embodiments, the growth medium includes 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes iron, copper, and molybdenum. In embodiments, the growth medium includes molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4. In embodiments, the growth medium includes 3.76×10−5 M to 1.10×10−4 M molybdenum, 3.60×10−5 M to 1.08×10−4 M iron, or 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes copper and nickel. In embodiments, the growth medium includes nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4. In embodiments, the growth medium includes 2.28×10−5 M to 6.84×10−5 M nickel or 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes copper, iron, and either molybdenum or nickel. In embodiments, the growth medium includes molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4. In embodiments, the growth medium includes 3.76×10−5 M to 1.10×10−4 M molybdenum, 2.28×10−5 M to 6.84×10−5 M nickel, 3.60×10−5 M to 1.08×10−4 M iron, or 4.70×10−5 M to 1.41×10−4 M copper. In embodiments, the growth medium includes copper, iron, molybdenum, and nickel.
In another aspect is provided a method of isolating a yeast cell including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight from a plurality of yeast cells, including allowing a yeast cell including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium thereby isolating the yeast cell including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical), wherein the population of yeast cells includes a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical). In another aspect is provided a method of isolating a genetically modified yeast cell from a plurality of yeast cells including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a genetically modified yeast cell to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium thereby isolating the genetically modified yeast cell, wherein the population of yeast cells includes a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the genetically modified yeast cell.
In embodiments is a method of isolating a yeast cell (e.g. genetically modified yeast cell), including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, from a plurality of yeast cells, including allowing a yeast cell (e.g. genetically modified yeast cell) to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium thereby isolating the yeast cell (e.g. genetically modified yeast cell), wherein the population of yeast cells includes a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the genetically modified yeast cell.
In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 30% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 40% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 50% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 70% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 80% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) includes greater than 90% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) is floating on the top surface of the aqueous medium. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) is above the bottom of a vessel containing the aqueous medium. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) is floating above the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 mm in the aqueous medium. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) is floating above the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 mm in the aqueous medium. In embodiments, the genetically modified yeast cell including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) floating above the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight has a buoyant density greater than the buoyant density of the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight by about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, g/mL. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight (e.g. greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight) floating above the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight has a buoyant density greater than the buoyant density of the population of yeast cells including a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight by 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, g/mL. In embodiments, the yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical), includes a mutation created by natural genetic drift.
In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 0.5 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 1.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 2.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 2.5 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 3.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 4.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 0.5 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 1.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 2.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 2.5 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 3.0 vvm (volume per volume per minute). In embodiments of the method, the plurality of yeast cells are in a bioreactor with agitation and aeration rates of 4.0 vvm (volume per volume per minute).
In embodiments of the method, the aqueous medium includes a yeast growth medium, minimal media, complete supplement media, or greater than 50 g/L carbon source (e.g. glucose) and less than 5 g/L of a nitrogen source (e.g. ammonium sulfate). In embodiments of the method, the aqueous medium includes a yeast growth medium. In embodiments of the method, the aqueous medium includes a minimal media. In embodiments of the method, the aqueous medium includes a complete supplement media. In embodiments of the method, the aqueous medium includes greater than 50 g/L carbon source (e.g. glucose) and less than 5 g/L of a nitrogen source (e.g. ammonium sulfate). In embodiments of the method, the aqueous medium is a yeast growth medium. In embodiments of the method, the aqueous medium is a minimal media. In embodiments of the method, the aqueous medium is a complete supplement media.
In embodiments of the method of isolating a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium, the allowing is performed by centrifugation or simple sedimentation. In embodiments of the method of isolating a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium, the allowing is performed by centrifugation. In embodiments of the method of isolating a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium, the allowing is performed by simple sedimentation. In embodiments of the method of isolating a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium, the allowing is performed by sedimentation. In embodiments of the method of isolating a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, including allowing a yeast cell (e.g. genetically modified yeast cell) including greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight to separate from a population of yeast cells within the plurality of yeast cells by floating above the population of yeast cells within an aqueous medium, the allowing is performed by sedimentation due to gravity.
In embodiments of the method, the genetically modified yeast cell is formed by transforming a yeast cell with a recombinant nucleic acid (e.g. as described herein, including in embodiments, examples, tables, figures, and/or claims). In embodiments, the genetically modified yeast cell is formed by mutagenizing a yeast cell. In embodiments, the yeast cell (e.g. genetically modified yeast cell includes a mutation created by natural genetic drift.
In embodiments, the method is a method described herein, including in embodiments, examples, tables, figures, and claims.
IV. ADDITIONAL EMBODIMENTS
1p. A genetically modified yeast cell wherein the dry weight of said yeast cell comprises greater than 20% wt/wt lipid.
2p. The yeast cell of embodiment 1p comprising greater than 30% wt/wt lipid.
3p. The yeast cell of embodiment 1p comprising greater than 40% wt/wt lipid.
4p. The yeast cell of embodiment 1p comprising greater than 50% wt/wt lipid.
5p. The yeast cell of embodiment 1p comprising greater than 60% wt/wt lipid.
6p. The yeast cell of embodiment 1p comprising greater than 70% wt/wt lipid.
7p. The yeast cell of embodiment 1p comprising greater than 80% wt/wt lipid.
8p. The yeast cell of embodiment 1p comprising greater than 90% wt/wt lipid.
9p. The yeast cell of any one of embodiments 1p to 8p, selected from the group consisting of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. 
10p. The yeast cell of any one of embodiments 1p to 8p, selected from the group consisting of Rhodosporidium toruloides, Lipomyces starkeyii, Lipomyces lipoferus, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Candida utilis, Trichosporon pullans, Trichosporon cutaneum, Rhodotorula glutinus, Rhodotorula graminis and Yarrowia lipolytica. 

11p. The yeast cell of any one of embodiments 1p to 8p, selected from the group consisting of Lipomyces starkeyii, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Rhodotorula glutinis, and Yarrowia lipolytica. 

12p. The yeast cell of any one of embodiments 1p to 8p, wherein said yeast cell is Yarrowia lipolytica. 

13p. The yeast cell of any one of embodiments 1p to 12p, wherein said yeast cell is buoyant in an aqueous medium.

14p. The yeast cell of any one of embodiments 1p to 13p, wherein said lipid is selected from the group consisting of a fatty acid, wax, sterol, vitamin, monoglyceride, diglyceride, triglyceride, phospholipid, glycerolipid, glycerophospholipid, sphingolipid, saccharolipid, polyketide, sterol lipid, triacylglyceride, and a prenol lipid.

15p. A yeast cell comprising a recombinant nucleic acid, wherein said recombinant nucleic acid modulates the level of activity of a protein in said yeast cell relative to the absence of the recombinant nucleic acid, and wherein said protein is selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), multifunctional enzyme (MFE1), Transcription Factor (PEX10), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA:diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), and O-6-methylguanine-DNA methyltransferase (MGMT).

16p. The yeast cell of embodiment 15p, wherein said recombinant nucleic acid increases the level of activity of a protein in said yeast cell selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), AMP Deaminase (AMPD), ATP-Citrate Lyase (ACL), Malic Enzyme (MAE), Acetyl-CoA Carboxylase (ACC), acyl-CoA: diacylglycerol acyltransferase (DGA1), acyl-CoA: diacylglycerol acyltransferases (DGA2), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), and O-6-methylguanine-DNA methyltransferase (MGMT).

17p. The yeast cell of any one of embodiments 15p to 16p, wherein said recombinant nucleic acid decreases the level of activity of a protein in said yeast cell selected from the group consisting of multifunctional enzyme (MFE1), Lipid synthesis regulator (MGA2), Chromatin assembly gene (RLF2 subunit p90), and Transcription Factor (PEX10).

18p. The yeast cell of any one of embodiments 15p to 17p, wherein said recombinant nucleic acid increases the level of activity of a protein in said yeast cell selected from the group consisting of Leucine Biosynthesis Gene (LEU2), Uracil Biosynthesis gene (URA3), Malic Enzyme (MAE), Mitochondrial 2′ O-ribose methyltransferase (MRM2), Lipid synthesis regulator (MGA2), and O-6-methylguanine-DNA methyltransferase (MGMT) or said nucleic acid decrease the level of activity of Lipid synthesis regulator (MGA2).

19p. The yeast cell of any one of embodiments 15p to 18p, wherein said recombinant nucleic acid encodes a protein comprising a mutation relative to the wildtype protein.

20p. The yeast cell of any one of embodiments 15p to 18p, wherein said nucleic acid modulates the level of expression of a protein.

21p. The yeast cell of embodiment 15p, wherein said yeast cell comprises a recombinant nucleic acid that decreases the level of activity of multifunctional enzyme (MFE1) protein and Transcription Factor (PEX10) protein, increases the level of activity of acyl-CoA:diacylglycerol acyltransferase (DGA1) protein, or increases the level of activity of Leucine Biosynthesis Gene (LEU2) protein relative to a yeast cell that does not comprise said recombinant nucleic acids.

22p. The yeast cell of any one of embodiments 1p to 21p, wherein said yeast cell comprises a fatty acid selected from the group consisting of C5:0, C5:1, C5:2, C5:3, C6:0, C6:1, C6:2, C6:3, C7:0, C7:1, C7:2, C7:3, C8:0, C8:1, C8:2, C8:3, C9:0, C9:1, C9:2, C9:3, C10:0, C10:1, C10:2, C10:3, C11:0, C11:1, C11:2, C11:3, C12:0, C12:1, C12:2, C12:3, C13:0, C13:1, C13:2, C13:3, C14:0, C14:1, C14:2, C14:3, C15:0, C15:1, C15:2, C15:3, C16:0, C16:1, C16:2, C16:3, C17:0, C17:1, C17:2, C17:3, C18:0, C18:1, C18:2, C18:3, C19:0, C19:1, C19:2, C19:3, C20:0, C20:1, C20:2, C20:3, C21:0, C21:1, C21:2, C21:3, C22:0, C22:1, C22:2, C22:3, C23:0, C23:1, C23:2, and C23:3.

23p. The yeast cell of any one of embodiments 1p to 21p, wherein said yeast cell comprises a fatty acid selected from the group consisting of C17:0 and C17:1.

24p. A method of producing a lipid comprising:

1) culturing a yeast cell of any one of embodiments 1p to 23p in a growth medium;

2) isolating said lipid.

25p. The method of embodiment 24p, wherein said lipid is isolated from said yeast cell. 26p. The method of embodiment 24p, wherein said lipid is isolated from the medium.

27p. The method of any one of embodiments 24p to 26p, wherein said growth medium comprises a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellulosic biomass.

28p. The method of any one of embodiments 24p to 26p, wherein said growth medium comprises lignocellulosic biomass as the majority carbon source.

29p. The method of any one of embodiments 24p to 28p, wherein said growth medium comprises a carbon source and a nitrogen source wherein said carbon source is at a concentration at least 10-fold greater than the concentration of the nitrogen source.

30p. The method of any one of embodiments 24p to 29p, wherein said growth medium comprises cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, or boron.

31p. The method of embodiment 30p, wherein the growth medium comprises iron, copper, and molybdenum.

32p. The method of embodiment 30p, wherein the growth medium comprises copper and nickel.

33p. The method of embodiment 30p, wherein the growth medium comprises copper, iron, and either molybdenum or nickel.

34p. The method of embodiment 30p, wherein the growth medium comprises copper, iron, molybdenum, and nickel.

35p. A method of isolating a genetically modified yeast cell from a plurality of yeast cells comprising greater than 20% wt/wt lipids in dry weight, comprising allowing a genetically modified yeast cell to separate from a population of yeast cells within said plurality of yeast cells by floating above said population of yeast cells within an aqueous medium thereby isolating said genetically modified yeast cell, wherein said population of yeast cells comprises a lower percentage wt/wt of lipids than said genetically modified yeast cell.

36p. The method of any embodiment 35p, wherein said genetically modified yeast cell comprises greater than 30% wt/wt lipids in dry weight.

37p. The method of embodiment 35p, wherein said genetically modified yeast cell comprises greater than 40% wt/wt lipids in dry weight.

38p. The method of embodiment 35p, wherein said genetically modified yeast cell comprises greater than 50% wt/wt lipids in dry weight.

39p. The method of embodiment 35p, wherein said genetically modified yeast cell comprises greater than 60% wt/wt lipids in dry weight.

40p. The method of any one of embodiments 35p to 39p, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 0.5 vvm (volume per volume per minute).

41p. The method of any one of embodiments 35p to 39p, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 1.0 vvm (volume per volume per minute).

42p. The method of any one of embodiments 35p to 39p, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 2.0 vvm (volume per volume per minute).

43p. The method of any one of embodiments 35p to 39p, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 3.0 vvm (volume per volume per minute).

44p. The method of any one of embodiments 35p to 39p, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 4.0 vvm (volume per volume per minute).

45p. The method of any one of embodiments 35p to 44p, wherein said aqueous medium comprises a yeast growth medium, minimal media, complete supplement media, or greater than 50 g/L glucose and less than 5 g/L of a nitrogen source.

46p. The method of any one of embodiments 35p to 45p, wherein said allowing is performed by centrifugation or simple sedimentation.

47p. The method of any one of embodiments 35p to 46p, wherein said genetically modified yeast cell was formed by transforming a yeast cell with a recombinant nucleic acid.
48p. The method of any one of embodiments 35p to 4′7p, wherein said genetically modified yeast cell was formed by mutagenizing a yeast cell.
1. A genetically modified yeast cell wherein the dry weight of said yeast cell comprises greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
2. The genetically modified yeast cell of embodiment 1 comprising greater than 30% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
3. The genetically modified yeast cell of embodiment 1 comprising greater than 40% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
4. The genetically modified yeast cell of embodiment 1 comprising greater than 50% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
5. The genetically modified yeast cell of embodiment 1 comprising greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
6. The genetically modified yeast cell of embodiment 1 comprising greater than 70% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
7. The genetically modified yeast cell of embodiment 1 comprising greater than 80% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
8. The genetically modified yeast cell of embodiment 1 comprising greater than 90% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).
9. The genetically modified yeast cell of any one of embodiments 1 to 8, selected from the group consisting of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. 
10. The genetically modified yeast cell of any one of embodiments 1 to 8, selected from the group consisting of Rhodosporidium toruloides, Lipomyces starkeyii, Lipomyces lipoferus, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Candida utilis, Trichosporon pullans, Trichosporon cutaneum, Rhodotorula glutinus, Rhodotorula graminis and Yarrowia lipolytica. 

11. The genetically modified yeast cell of any one of embodiments 1 to 8, selected from the group consisting of Lipomyces starkeyii, Rhodosporidium toruloides, Apiotrichum curvatum, Candida curvata, Cryptococcus curvatus, Trichosporon fermentans, Rhodotorula glutinis, and Yarrowia lipolytica. 

12. The genetically modified yeast cell of any one of embodiments 1 to 8, wherein said yeast cell is Yarrowia lipolytica. 

13. The genetically modified yeast cell of any one of embodiments 1 to 12, wherein said yeast cell is buoyant in an aqueous medium.

14. The genetically modified yeast cell of one of embodiments 1 to 13, comprising a recombinant Leucine Biosynthesis Gene (LEU2).

15. The genetically modified yeast cell of one of embodiments 1 to 13, wherein said genetic modification increases the level of activity of the Leucine Biosynthesis Gene (LEU2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

16. The genetically modified yeast cell of one of embodiments 1 to 15, comprising a recombinant Uracil Biosynthesis gene (URA3).

17. The genetically modified yeast cell of one of embodiments 1 to 15, wherein said genetic modification increases the level of activity of the Uracil Biosynthesis gene (URA3) protein relative to an otherwise identical yeast cell lacking said genetic modification.

18. The genetically modified yeast cell of one of embodiments 1 to 17, comprising a genetically modified multifunctional enzyme (MFE1) gene.

19. The genetically modified yeast cell of one of embodiments 1 to 17, wherein said genetic modification decreases the level of activity of the multifunctional enzyme (MFE1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

20. The genetically modified yeast cell of one of embodiments 1 to 19, comprising a genetically modified PEX10 Transcription Factor (PEX10) gene.

21. The genetically modified yeast cell of one of embodiments 1 to 19, wherein said genetic modification decreases the level of activity of the PEX10 Transcription Factor (PEX10) protein relative to an otherwise identical yeast cell lacking said genetic modification.

22. The genetically modified yeast cell of one of embodiments 1 to 21, comprising a recombinant AMP Deaminase (AMPD) protein.

23. The genetically modified yeast cell of one of embodiments 1 to 21, wherein said genetic modification increases the level of activity of the AMP Deaminase (AMPD) protein relative to an otherwise identical yeast cell lacking said genetic modification.

24. The genetically modified yeast cell of one of embodiments 1 to 23, comprising a recombinant ATP-Citrate Lyase (ACL1) protein.

25. The genetically modified yeast cell of one of embodiments 1 to 23, wherein said genetic modification increases the level of activity of the ATP-Citrate Lyase (ACL1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

26. The genetically modified yeast cell of one of embodiments 1 to 25, comprising a recombinant ATP-Citrate Lyase (ACL2) protein.

27. The genetically modified yeast cell of one of embodiments 1 to 25, wherein said genetic modification increases the level of activity of the ATP-Citrate Lyase (ACL2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

28. The genetically modified yeast cell of one of embodiments 1 to 27, comprising a recombinant Malic Enzyme (MAE) protein.

29. The genetically modified yeast cell of one of embodiments 1 to 27, wherein said genetic modification increases the level of activity of the Malic Enzyme (MAE) protein relative to an otherwise identical yeast cell lacking said genetic modification.

30. The genetically modified yeast cell of one of embodiments 1 to 29, comprising a recombinant Acetyl-CoA Carboxylase (ACC) protein.

31. The genetically modified yeast cell of one of embodiments 1 to 29, wherein said genetic modification increases the level of activity of the Acetyl-CoA Carboxylase (ACC) protein relative to an otherwise identical yeast cell lacking said genetic modification.

32. The genetically modified yeast cell of one of embodiments 1 to 31, comprising a recombinant acyl-CoA: diacylglycerol acyltransferase 1 (DGA1) protein.

33. The genetically modified yeast cell of one of embodiments 1 to 31, wherein said genetic modification increases the level of activity of the acyl-CoA:diacylglycerol acyltransferase 1 (DGA1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

34. The genetically modified yeast cell of one of embodiments 1 to 33, comprising a recombinant acyl-CoA: diacylglycerol acyltransferase 2 (DGA2) protein.

35. The genetically modified yeast cell of one of embodiments 1 to 33, wherein said genetic modification increases the level of activity of the acyl-CoA:diacylglycerol acyltransferase 2 (DGA2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

36. The genetically modified yeast cell of one of embodiments 1 to 35, comprising a recombinant Mitochondrial 2′ O-ribose methyltransferase (MRM2) protein.

37. The genetically modified yeast cell of one of embodiments 1 to 35, wherein said genetic modification increases the level of activity of the Mitochondrial 2′ O-ribose methyltransferase (MRM2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

38. The genetically modified yeast cell of one of embodiments 1 to 37, comprising a recombinant Lipid synthesis regulator (MGA2) protein.

39. The genetically modified yeast cell of one of embodiments 1 to 37, comprising a genetically modified Lipid synthesis regulator (MGA2) gene.

40. The genetically modified yeast cell of one of embodiments 1 to 37, comprising at least one nucleotide deletion in the genomic Lipid synthesis regulator (MGA2) gene and expression of a Lipid synthesis regulator (MGA2) protein comprising a mutation corresponding to G643R in Yarrowia lipolytica. Lipid synthesis regulator (MGA2)

41. The genetically modified yeast cell of one of embodiments 1 to 37, wherein said genetic modification decreases the level of activity of the Lipid synthesis regulator (MGA2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

42. The genetically modified yeast cell of one of embodiments 1 to 41, comprising a genetically modified Chromatin assembly gene (RLF2 subunit p90) gene.

43. The genetically modified yeast cell of one of embodiments 1 to 41, wherein said genetic modification decreases the level of activity of the Chromatin assembly gene (RLF2 subunit p90) protein relative to an otherwise identical yeast cell lacking said genetic modification.

44. The genetically modified yeast cell of one of embodiments 1 to 43, comprising a recombinant O-6-methylguanine-DNA methyltransferase (MGMT) protein.

45. The genetically modified yeast cell of one of embodiments 1 to 43, wherein said genetic modification increases the level of activity of the O-6-methylguanine-DNA methyltransferase (MGMT) protein relative to an otherwise identical yeast cell lacking said genetic modification.

46. The genetically modified yeast cell of one of embodiments 1 to 45, comprising a genetically modified Aconitase (ACO1) gene.

47. The genetically modified yeast cell of one of embodiments 1 to 45, wherein said genetic modification decreases the level of activity of the Aconitase (ACO1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

48. The genetically modified yeast cell of one of embodiments 1 to 47, comprising a recombinant Citrate Synthase (CIT1) gene.

49. The genetically modified yeast cell of one of embodiments 1 to 47, wherein said genetic modification increases the level of activity of the Citrate Synthase (CIT1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

50. The genetically modified yeast cell of one of embodiments 1 to 49, comprising a genetically modified RME1 zinc-finger transcription factor (RME1) gene.

51. The genetically modified yeast cell of one of embodiments 1 to 49, wherein said genetic modification decreases the level of activity of the RME1 zinc-finger transcription factor (RME1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

52. The genetically modified yeast cell of one of embodiments 1 to 51, comprising a genetically modified YOX1 homeodomain protein (YOX1) gene.

53. The genetically modified yeast cell of one of embodiments 1 to 51, wherein said genetic modification decreases the level of activity of the YOX1 homeodomain protein (YOX1) protein relative to an otherwise identical yeast cell lacking said genetic modification.

54. The genetically modified yeast cell of one of embodiments 1 to 53, comprising a genetically modified UGA2 succinate semialdehyde dehydrogenase (UGA2) gene.

55. The genetically modified yeast cell of one of embodiments 1 to 53, wherein said genetic modification decreases the level of activity of the UGA2 succinate semialdehyde dehydrogenase (UGA2) protein relative to an otherwise identical yeast cell lacking said genetic modification.

56. The genetically modified yeast cell of one of embodiments 1 to 55, comprising a genetically modified OSH6 oxysterol-binding protein homolog 6 (OSH6) gene.

57. The genetically modified yeast cell of one of embodiments 1 to 55, wherein said genetic modification decreases the level of activity of the OSH6 oxysterol-binding protein homolog 6 (OSH6) protein relative to an otherwise identical yeast cell lacking said genetic modification.

58. The genetically modified yeast cell of one of embodiments 1 to 57, comprising a genetically modified IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) gene.

59. The genetically modified yeast cell of one of embodiments 1 to 57, wherein said genetic modification decreases the level of activity of the IRC20 E3 ubiquitin-protein ligase and helicase (IRC20) protein relative to an otherwise identical yeast cell lacking said genetic modification.

60. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification modulates the level of activity of a component of a lipid biosynthetic pathway.

61. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification modulates the level of activity of a component of a pathway incorporating Acetyl-CoA into a lipid, lipid precursor, or oleochemical.

62. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification modulates the level of activity of a component of a pathway incorporating malonyl-CoA into a lipid, lipid precursor, or oleochemical.

63. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of activity of a component of a lipid biosynthetic pathway.

64. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of activity of a component of a pathway incorporating acetyl-CoA into a lipid, lipid precursor, or oleochemical.

65. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of activity of a component of a pathway incorporating malonyl-CoA into a lipid, lipid precursor, or oleochemical.

66. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification modulates the level of activity of a component of a lipid, lipid precursor, or oleochemical, metabolic pathway.

67. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification decreases the level of activity of a component of a lipid, lipid precursor, or oleochemical, metabolic pathway.

68. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification decreases the level of activity of a component of a lipid, lipid precursor, or oleochemical, metabolic pathway.

69. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of acetyl-CoA in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

70. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of malonyl-CoA in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

71. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification increases the level of triglyceride production in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

72. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification decreases the level of beta-oxidation activity in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

73. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification decreases the level of fatty acid catabolism in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

74. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification decreases the level of peroxisome biogenesis activity in the genetically modified yeast cell relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

75. The genetically modified yeast cell of any one of embodiments 1 to 59, wherein said genetic modification produces a lipid, lipid precursor, or oleochemical at a higher level than by a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

76. The genetically modified yeast cell of embodiment 75, wherein said lipid, lipid precursor, or oleochemical produced at a higher level by said genetically modified yeast cell is a fatty acid, wax, sterol, vitamin, monoglyceride, diglyceride, triglyceride, phospholipid, glycerolipid, glycerophospholipid, sphingolipid, saccharolipid, polyketide, sterol lipid, triacylglyceride, prenol lipid, fatty acid ester, fatty acid methyl ester, fatty acid ethyl ester, fatty acid propyl ester, fatty acid butyl ester, fatty alcohol, fatty amine, glycerol, alcohol ethoxylate, alcohol sulfate, or alcohol ether sulfate.

77. The genetically modified yeast cell of any one of embodiments 1 to 76, wherein said genetic modification comprises a mutation relative to the wild type gene.

78. The genetically modified yeast cell of any one of embodiments 1 to 76, wherein said genetic modification comprises a deletion of a portion of a gene.

79. The genetically modified yeast cell of one of embodiments 1 to 78, wherein said yeast cell comprises an increased level of a fatty acid selected from the group consisting of C5:0, C5:1, C5:2, C5:3, C6:0, C6:1, C6:2, C6:3, C7:0, C7:1, C7:2, C7:3, C8:0, C8:1, C8:2, C8:3, C9:0, C9:1, C9:2, C9:3, C10:0, C10:1, C10:2, C10:3, C11:0, C11:1, C11:2, C11:3, C12:0, C12:1, C12:2, C12:3, C13:0, C13:1, C13:2, C13:3, C14:0, C14:1, C14:2, C14:3, C15:0, C15:1, C15:2, C15:3, C16:0, C16:1, C16:2, C16:3, C17:0, C17:1, C17:2, C17:3, C18:0, C18:1, C18:2, C18:3, C19:0, C19:1, C19:2, C19:3, C20:0, C20:1, C20:2, C20:3, C21:0, C21:1, C21:2, C21:3, C22:0, C22:1, C22:2, C22:3, C23:0, C23:1, C23:2, and C23:3, relative to a genetically unmodified yeast cell that is otherwise identical to said genetically modified yeast cell.

80. The genetically modified yeast cell of embodiment 79, wherein said fatty acid is C17:0 C17:1.

81. The genetically modified yeast cell of embodiment 79, wherein said fatty acid is C16:1n9.

82. The genetically modified yeast cell of one of embodiments 1 to 81, wherein said genetic modification is an engineered genetic modification.

83. The genetically modified yeast cell of embodiment 82, wherein said engineered genetic modification comprises modulated expression of a protein.

84. The genetically modified yeast cell of embodiment 82, wherein said engineered genetic modification comprises increased expression of a protein.

85. The genetically modified yeast cell of embodiment 82, wherein said engineered genetic modification comprises decreased expression of a protein.

86. The genetically modified yeast cell of one of embodiments 1 to 81, wherein said genetic modification is associated with exposure to a mutagen.

87. The genetically modified yeast cell of one of embodiments 1 to 86, wherein said genetic modification comprises modulated expression of a protein in a lipid, or lipid precursor, biosynthetic pathway.

88. A method of producing a lipid, lipid precursor, or oleochemical comprising:

1) culturing a yeast cell of any one of embodiments 1 to 87 in a growth medium; and

2) isolating said lipid, lipid precursor, or oleochemical.

89. The method of embodiment 88, wherein said lipid, lipid precursor, or oleochemicalis isolated from said yeast cell.

90. The method of embodiment 88, wherein said lipid, lipid precursor, or oleochemical is isolated from the growth medium.

91. The method of any one of embodiments 88 to 90, wherein said growth medium comprises a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellulosic biomass.

92. The method of any one of embodiments 88 to 90, wherein said growth medium comprises lignocellulosic biomass as the majority carbon source.

93. The method of any one of embodiments 88 to 92, wherein said growth medium comprises a carbon source and a nitrogen source wherein said carbon source is at a concentration at least 10-fold greater than the concentration of the nitrogen source (wt/wt).

94. The method of any one of embodiments 88 to 92, wherein said growth medium comprises a carbon source and a nitrogen source wherein said carbon source is at a concentration at least 16-fold greater than the concentration of the nitrogen source (wt/wt).

95. The method of any one of embodiments 88 to 92, wherein said growth medium comprises a carbon source and a nitrogen source wherein said carbon source is at a concentration at least 320-fold greater than the concentration of the nitrogen source (wt/wt).

96. The method of any one of embodiments 88 to 95, wherein said growth medium comprises micronutrients (e.g. cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, or boron).

97. The method of any one of embodiments 88 to 95, wherein said growth medium comprises cobalt in an amount equivalent to 7.5 to 22.5 mg/L CoCl2, magnesium in an amount equivalent to 125 to 375 mg/L MgSO4, potassium in an amount equivalent to 7.5 to 22.5 mg/L KI, zinc in an amount equivalent to 10 to 30 mg/L ZnSO4.7H2O, manganese in an amount equivalent to 6 to 18 mg/L MnSO4.H2O, boron in an amount equivalent to 6 to 18 mg/L Boric acid, molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4.

98. The method of any one of embodiments 88 to 95, wherein said growth medium comprises 5.77×10−5 M to 1.73×10−4 M cobalt, 0.001 M to 0.003 M magnesium, 4.52×10−5 M to 1.35×10−4 M potassium, 4.05×10−5 M to 1.22×10−4 M zinc, 3.55×10−5 M to 1.06×10−4 M manganese, 9.07×10−5 M to 2.91×10−4 M boron, 3.76×10−5 M to 1.10×10−4 M molybdenum, 2.28×10−5 M to 6.84×10−5 M nickel, 3.60×10−5 M to 1.08×10−4 M iron, or 4.70×10−5 M to 1.41×10−4 M copper.

99. The method of any one of embodiments 88 to 95, wherein the growth medium comprises iron, copper, and molybdenum.

100. The method of any one of embodiments 88 to 95, wherein said growth medium comprises molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4.

101. The method of any one of embodiments 88 to 95, wherein said growth medium comprises 3.76×10−5 M to 1.10×10−4 M molybdenum, 3.60×10−5 M to 1.08×10−4 M iron, or 4.70×10−5 M to 1.41×10−4 M copper.

102. The method of any one of embodiments 88 to 95, wherein the growth medium comprises copper and nickel.

103. The method of any one of embodiments 88 to 95, wherein said growth medium comprises nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4.

104. The method of any one of embodiments 88 to 95, wherein said growth medium comprises 2.28×10−5 M to 6.84×10−5 M nickel or 4.70×10−5 M to 1.41×10−4 M copper.

105. The method of any one of embodiments 88 to 95, wherein the growth medium comprises copper, iron, and either molybdenum or nickel.

106. The method of any one of embodiments 88 to 95, wherein said growth medium comprises molybdenum in an amount equivalent to 7.5 to 22.5 mg/L (NH4)2Mo.4H2O, nickel in an amount equivalent to 6 to 18 mg/L NiSO4.6H2O, iron in an amount equivalent to 10 to 30 mg/L FeSO4.7H2O, or copper in an amount equivalent to 7.5 to 22.5 mg/L CuSO4.

107. The method of any one of embodiments 88 to 95, wherein said growth medium comprises 3.76×10−5 M to 1.10×10−4 M molybdenum, 2.28×10−5 M to 6.84×10−5 M nickel, 3.60×10−5 M to 1.08×10−4M iron, or 4.70×10−5 M to 1.41×10−4M copper.

108. The method of any one of embodiments 88 to 95, wherein the growth medium comprises copper, iron, molybdenum, and nickel.

109. A method of isolating a genetically modified yeast cell from a plurality of yeast cells, comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight, comprising allowing a genetically modified yeast cell to separate from a population of yeast cells within said plurality of yeast cells by floating above said population of yeast cells within an aqueous medium thereby isolating said genetically modified yeast cell, wherein said population of yeast cells comprises a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than said genetically modified yeast cell.

110. The method of embodiment 109, wherein said genetically modified yeast cell comprises greater than 30% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

111. The method of embodiment 109, wherein said genetically modified yeast cell comprises greater than 40% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

112. The method of embodiment 109, wherein said genetically modified yeast cell comprises greater than 50% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

113. The method of embodiment 109, wherein said genetically modified yeast cell comprises greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

114. The method of any one of embodiments 109 to 113, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 0.5 vvm (volume per volume per minute).

115. The method of any one of embodiments 109 to 113, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 1.0 vvm (volume per volume per minute).

116. The method of any one of embodiments 109 to 113, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 2.0 vvm (volume per volume per minute).

117. The method of any one of embodiments 109 to 113, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 3.0 vvm (volume per volume per minute).

118. The method of any one of embodiments 109 to 113, wherein said plurality of yeast cells are in a bioreactor with agitation and aeration rates of about 4.0 vvm (volume per volume per minute).

119. The method of any one of embodiments 109 to 118, wherein said aqueous medium comprises a yeast growth medium, minimal media, complete supplement media, or greater than 50 g/L glucose and less than 5 g/L of a nitrogen source.

120. The method of any one of embodiments 109 to 119, wherein said allowing is performed by centrifugation or simple sedimentation.

121. The method of any one of embodiments 109 to 120, wherein said genetically modified yeast cell was formed by transforming a yeast cell with a recombinant nucleic acid.

122. The method of any one of embodiments 109 to 120, wherein said genetically modified yeast cell was formed by mutagenizing a yeast cell.

123. The method of any one of embodiments 109 to 120, wherein said genetically modified yeast cell is created by first exposing a yeast cell to a mutagen (e.g. a chemical mutagen, radiation, UV, or a biological mutagen).

124. The method of any one of embodiments 109 to 120, wherein said genetically modified yeast cell was formed by mutagenizing a yeast cell.

125. A method of isolating a yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight from a plurality of yeast cells, comprising allowing a yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) to separate from a population of yeast cells within said plurality of yeast cells by floating above said population of yeast cells within an aqueous medium thereby isolating said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical), wherein said population of yeast cells comprises a lower percentage wt/wt of lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) than said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical).

126. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 30% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

127. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 40% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

128. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 50% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

129. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 60% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

130. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 70% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

131. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 80% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

132. The method of embodiment 125, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises greater than 90% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) in dry weight.

133. The method of one of embodiments 125 to 132, wherein said yeast cell comprising greater than 20% wt/wt lipids, lipid precursors, and/or oleochemicals (e.g., lipid, lipids, lipid precursors, lipid precursor, oleochemicals, or oleochemical) comprises a mutation created by natural genetic drift.

134. The method of any one of embodiments 88 to 95, wherein said growth medium comprises cobalt.

135. The method of any one of embodiments 88 to 95 and 134, wherein said growth medium comprises iron.

136. The method of any one of embodiments 88 to 95 and 134 to 135, wherein said growth medium comprises magnesium.

137. The method of any one of embodiments 88 to 95 and 134 to 136, wherein said growth medium comprises potassium.

138. The method of any one of embodiments 88 to 95 and 134 to 137, wherein said growth medium comprises zinc.

139. The method of any one of embodiments 88 to 95 and 134 to 138, wherein said growth medium comprises nickel.

140. The method of any one of embodiments 88 to 95 and 134 to 139, wherein said growth medium comprises molybdenum.

141. The method of any one of embodiments 88 to 95 and 134 to 140, wherein said growth medium comprises manganese.

142. The method of any one of embodiments 88 to 95 and 134 to 141, wherein said growth medium comprises copper.

143. The method of any one of embodiments 88 to 95 and 134 to 142, wherein said growth medium comprises boron.

V. EXAMPLES
The following examples are meant to illustrate certain embodiments of the invention and not to limit the scope of the invention described herein.
A. MATERIALS AND METHODS
Base Strains and Media.
E. coli strain DH10B was used for cloning and plasmid propagation. DH10B was grown at 37° C. with constant shaking in Luria-Bertani Broth (Teknova) supplemented with 50 μg/ml of ampicillin for plasmid propagation. Yarrowia lipolytica strain PO1f (ATCC #MYA-2613), a leucine and uracil auxotroph devoid of any secreted protease activity (Madzak et al., 2000), was used as the base strain for all studies. Table 1 contains a list of PO1f derivatives produced in this study. Y. lipolytica was cultivated at 30° C. with constant agitation. 2 mL cultures of Y. lipolytica used in large-scale screens were grown in a rotary drum (CT-7, New Brunswick Scientific) at speed seven, and larger culture volumes were shaken in flasks at 225 rpm.
YSC media consisted of 20 g/L glucose (Fisher Scientific), 0.79 g/L CSM supplement (MP Biomedicals), and 6.7 g/L Yeast Nitrogen Base w/o amino acids (Becton, Dickinson, and Company). YSC-URA, YSC-LEU, and YSC-LEU-URA media contained 0.77 g/L CSM-Uracil, 0.69 g/L CSM-Leucine, or 0.67 g/L CSM-Leucine-Uracil in place of CSM, respectively. YPD media contained 10 g/L yeast extract (Fisher Scientific), 20 g/L peptone (Fisher Scientific) and 20 g/L glucose, and was often supplemented with 300 μg/ml Hygromycin B (Invitrogen) for knockout selection. Lipid accumulation response towards media formulation was investigated by cultivation in varying concentrations of glucose and nitrogen. These media formulations contained 0.79 g/L CSM, 1.7 g/L Yeast Nitrogen Base w/o amino acid and w/o (NH4)2SO4 (Becton, Dickinson, and Company), between 10 g/L and 320 g/L glucose, and between 0.04 g/L and 10 g/L ammonium sulfate—(NH4)2SO4 (Fisher Scientific). These media are routinely referred to by their ratio of carbon content (glucose) to nitrogen content (ammonium sulfate). For instance, media containing 80 g/L glucose and 5 g/L ammonium sulfate is called C80:N5 media. When utilizing alternative carbon sources, glucose was replaced by 80 g/L arabinose, 80 g/L fructose, 80 g/L galactose, 80 g/L glycerol (Fisher Scientific), 80 g/L mannose, 80 g/L maltose 80 g/L ribose, 80 g/L sucrose (Acros Organics), 80 g/L Xylose, or 80 g/L of a saccharide mix resembling the composition of lignocellulosic biomass (57% Glucose, 32% Xylose, 5% Arabinose, 3% Mannose, and 3% Galactose by weight). Solid media for E. coli and Yarrowia lipolytica was prepared by adding 20 g/L agar (Teknova) to liquid media formulations.
When analyzing the effect of micronutrient supplementation, CoCl2 (15 mg/L), MgSO4 (250 mg/L), KI (15 mg/L), ZnSO4.7H2O (20 mg/L), MnSO4.H2O (12.5 mg/L), Boric acid (12.5 mg/L), (NH4)2Mo.4H2O (15 mg/L), NiSO4.6H2O (12.5 mg/L), FeSO4.7H2O (20 mg/L), or CuSO4 (15 mg/L) were added to the stated media formulation. Concentrations given are the final concentrations of the metal ion.
Initial Optimization of Media Formulation for Wildtype and Engineered Strains.
Nitrogen starvation is the accepted impetus for effecting a state of lipid accumulation in oleaginous organisms (Ratledge 2002). As a preliminary analysis of this induction potential, we selected seven media variations wildly variant in their ratios' of carbon content (glucose) to nitrogen content (ammonium sulfate) to assay for their ability to induce lipid accumulation. These media formulations are routinely referred to by this carbon to nitrogen ratio (C:N ratio), i.e., media containing 160 g/L glucose and 0.2 g/L ammonium sulfate is called C160:N0.2 media. We cultivated wildtype Y. lipolytica strain PO1f in these seven media formulations and assayed for relative lipid (e.g. triacylglyceride) accumulation using nile red fluorescence flow cytometry after 2, 4, 6, and 8 days. We observed a strong correlation between increasing carbon to nitrogen ratio and increased lipid (e.g. triacylglyceride) accumulation that spanned a 10-fold range, and we were able to increase nile red fluorescence levels by three-fold compared to levels induced in standard minimal (YSC) media. Thus, we confirmed the beneficial effect of increasing C:N ratio towards lipid (e.g. triacylglyceride) accumulation in non-engineered Y. lipolytica, so we sought to further improve oleo-content with additional media supplementation. In particular, FeSO4 supplementation has been implicated in enabling increased citric acid accumulation in Y. lipolytica (Kamzolova et al. 2003), specifically under oxygen limiting conditions. Citric acid and fatty acid accumulation are closely linked in Y. lipolytica, so we hypothesized that this iron-responsive citric acid accumulation could also increase downstream lipid (e.g. triacylglyceride) accumulation. To fully analyze the potential benefits of micronutrient addition towards lipid (e.g. triacylglyceride) accumulation (Song et al. 2012; Zhao et al. 2008), we cultivated PO1f in minimal media supplemented with cobalt, magnesium, potassium, zinc, manganese, boric acid, molybdenum, nickel, iron, and copper (FIG. 1), and saw increased lipid (e.g. triacylglyceride) accumulation with iron, nickel, copper, molybdenum, and zinc. We performed a combinatorial screening of iron, nickel, copper, and molybdenum supplementation to detect cumulative beneficial effects towards increasing cellular lipid content. Triple supplementation with copper, nickel, and iron increased lipid accumulation levels to the highest observed at that time (FIG. 2).
Thus, manipulating media formulation effectively increased lipid formulation in a wildtype strain, however, the relationship between strain genotype and this effect has yet to be explored. We sought to determine if a strain rationally engineered for increased lipid accumulation would benefit in the same manner from increasing C:N ratio. In our initial attempts to engineer a Y. lipolytica strain for increased lipid accumulation, we overexpressed the AMPDp in a ΔPEX10 background to create a strain with a 17-fold increase in nile red fluorescence levels. To determine if genomic modifications could affect differential responses towards media-induced lipid accumulation, we cultivated unmodified PO1f and our engineered high lipid producer in twenty media formulations that varied in carbon and nitrogen levels (Table 3) and analyzed for lipid content with nile red fluorescence flow cytometry after two days, four days, and eight days. Two days was insufficient time to induce lipid accumulation, while lipid accumulation is evident a majority of media formulation for the PO1f ΔPEX10 AMPDp overexpression strain after eight days. Heat graphs of relative fluorescent values illustrate that the PO1f ΔPEX10 AMPDp overexpression strain accumulates lipids efficiently at an optimum value of 80 g/L glucose after 4 days, while PO1f is only slight induced in any condition, most noticeably after six to eight days in C160N0.2 media. In general, the 320 g/L glucose condition is too high to induce lipid accumulation effectively, most likely because the high sugar content prevents cell growth. Likewise, formulations 0.04 and 0.2 g/L ammonium sulfate tend to poorly induce lipid accumulation, especially within four days or less. Finally, an optimum C:N ratio of  ̃10 to 40 can be observed when discounting these highest glucose and lowest ammonium sulfate.
B. CLONING AND TRANSFORMATION PROCEDURES
All restriction enzymes were purchased from New England Biolabs and all digestions were performed according to standard protocols. PCR reactions were set up with recommended conditions using Phusion high fidelity DNA polymerase (Finnzymes), or LongAmp Taq DNA polymerase (New England Biolabs). Ligation reactions were performed overnight at room temperature using T4 DNA Ligase (Fermentas). Gel extractions were performed using the Fermentas GeneJET extraction kit purchased from Fisher ThermoScientific. E. coli minipreps were performed using the Zyppy Plasmid Miniprep Kit (Zymo Research Corporation). E. coli maxipreps were performed using the Qiagen HiSpeed Plasmid Maxi Kit. Transformation of E. coli strains was performed using standard electroporator protocols (Sambrook and Russell, 2001). Large amounts of linearized DNA (>20n), necessary for Y. lipolytica PO1f transformation were cleaned and precipitated using a standard phenol:chloroform extraction followed by an ethanol precipitation (Kirby, 1956).
Genomic DNA (gDNA) was extracted from Y. lipolytica using the Wizard Genomic DNA Purification kit (Promega). Transformation of Y. lipolytica with replicative plasmids was performed using the Zymogen Frozen EZ Yeast Transformation Kit II (Zymo Research Corporation), with plating on YSC-LEU plates. Transformation of Y. lipolytica PO1f with linearized cassettes was performed as described previously (Blazeck et al. 2013a), with selection on appropriate plates. All auxotrophic or antibiotic selection markers were flanked with LoxP sites to allow for retrieval of integrated markers the pMCS-UAS1B16-TEF-Cre replicative vector (Blazeck et al. 2013a).
Plasmid Construction.
Primer sequences can be found in the Table 2. All Y. lipolytica episomal plasmids were centromeric, replicative vectors derived from plasmid pS116-Cen1-1(227) (Yamane et al. 2008) after it had been modified to include a multi-cloning site, a hrGFP green fluorescent reporter gene (pIRES-hrGFP, Agilent) driven by the strong UAS1B16-TEF promoter (Blazeck et al. 2011), and a cycl terminator (Mumberg et al. 1995) to create plasmid pMCS-UAS1B16-TEF-hrGFP. Integrative plasmids were derived from plasmids pUC-S1-UAS1B16-Leum or pUC-S1-UAS1B16-TEF (Blazeck et al. 2013a) that contained 5′ and 3′ rDNA integrative sequences surrounding the following elements—(from 5′ to 3′) a uracil section marker surrounded by LoxP sites for marker retrieval, the strong UAS1B16-Leum or UAS1B16-TEF promoter, AscI and Pad restriction enzyme sites for gene insertion, and a XPR2 minimal terminator. These integrative plasmids were also designed to contain two identical NotI restriction enzyme sites directly outside of the rDNA regions so that plasmid linearization would simultaneously remove E. coli pUC19-based DNA. All plasmids containing expression cassettes were sequenced confirmed before transformation into Y. lipolytica. 
Construction of Episomal Expression Cassettes:
The following genes were PCR amplified from Y. lipolytica PO1f gDNA and inserted into vector pMCS-UAS1B16-TEF-hrGFP in place of hrGFP with an AscI/PacI digest: AMPD, ACL subunit 1 (ACL1), ACL subunit 2 (ACL2), MEA1, DGA1, DGA2, the TupI general transcriptional repressor (Morin et al. 2011), and the HAC1 basic leucine zipper transcription factor involved in unfolded protein response (Morin et al. 2011) with primers, respectively. This formed plasmids pMCS-UAS1B16-TEF-AMPD, pMCS-UAS1B16-TEF-ACL1, pMCS-UAS1B16-TEF-ACL2, pMCS-UAS1B16-TEF-MEA, pMCS-UAS1B16-TEF-DGA1, pMCS-UAS1B16-TEF-DGA2, pMCS-UAS1B16-TEF-TUP1, and pMCS-UAS1B16-TEF-HAC1.
Construction of Integrative Expression Cassettes:
The following genes were gel extracted from the previously constructed episomal expression vectors and inserted into vector pUC-S1-UAS1B16-TEF with an AscI/PacI digest: AMPD, ACL subunit 1 (ACL1), ACL subunit 2 (ACL2), MEA1, DGA1, and DGA2. This formed plasmids pUC-S1-UAS1B16-TEF-AMPD, pUC-S1-UAS1B16-TEF-ACL1, pUC-S1-UAS1B16-TEF-ACL2, pUC-S1-UAS1B16-TEF-MEA1, and pUC-S1-UAS1B16-TEF-DGA1, and pUC-S1-UAS1B16-TEF-DGA2. The loxP-surrounded uracil marker of these integrative plasmids was replaced with a loxP-surrounded leucine marker to enable integrative selection with leucine auxotrophy and co-expression of two enzymes without marker retrieval. These leucine marker integrative plasmids were dubbed plasmids pUC-S2-UAS1B16-TEF-AMPD, pUC-S2-UAS1B16-TEF-ACL1, pUC-S2-UAS1B16-TEF-ACL2, pUC-S2-UAS1B16-TEF-MEA1, and pUC-S2-UAS1B16-TEF-DGA1, and pUC-S2-UAS1B16-TEF-DGA2.
ACL1 and ACL2 were similarly inserted into pUC-S1-UAS1B16-Leum with primers, respectively, to form plasmids pUC-S1-UAS1B16-Leum-ACL1 and pUC-S1-UAS1B16-Leum-ACL2.
Strain Construction.
All strains were confirmed through gDNA extraction and PCR confirmation and are listed in Table 1. We previously constructed two markerless single-gene deletion strains in the Y. lipolytica PO1f background, PO1f-Δmfe1 and PO1f-Δpex10, deficient in their β-oxidation and peroxisomal biogenesis capacity, respectively (Blazeck et al. 2013a). Following our previous protocol, the PEX10 gene was deleted from strain PO1f-Δmfe1 to form the markerless double mutant PO1f-Δmfe1-Δpex10. These four strains, PO1f, PO1f-Δmfe1, PO1f-Δpex10, and PO1f-Δmfe1-Δpex10 were utilized as backgrounds for single and double overexpression of the AMPD, ACL1, ACL2, MEA, DGA1, and DGA2 genes, including variation in selective marker utilized, i.e., leucine (S2 integrative cassette or pMCS episomal cassette) vs. uracil (S1 integrative cassette). S2 and S1 integrative cassettes were linearized, transformed into our four background strains, and selected for on appropriate dropout plates. Table 1 contains a list of rationally engineered strains derived in this manner. ORF-less plasmids pUC-S1-UAS1B16-TEF and pUC-S1-UAS1B16-TEF were utilized to create strains lacking leucine, uracil, or both leucine and uracil auxotrophies, dubbed S1-Ø, S2-Ø, and S1-S2-Ø (Table 1).
Combinatorial Genome Engineering.
Prior engineering efforts have successfully increased lipid accumulation in Y. lipolytica by manipulating fatty acid, lipid, or central carbon metabolism, but no attempt has been made to simultaneously alter these metabolic functionalities (Beopoulos et al. 2008; Dulermo and Nicaud 2011; Tai and Stephanopoulos 2013). We sought to concurrently control these aspects of lipid synthesis by overexpressing three enzymes that control metabolic flux from central carbon metabolism into fatty acid synthesis (AMPDp, ACLp, and MEA1p) or two isozymes that control lipid synthesis (DGA1p and DGA2p) in four genomic backgrounds with altered fatty acid catabolic ability. These four genomic backgrounds included the PO1f (WT) strain, a PO1f MFE1 deletion strain (ΔMFE1), a PO1f PEX10 deletion strain (ΔPEX10), and a MFE1 PEX10 double knockout strain (ΔPEX10ΔMFE1). The majority of enzymatic overexpressions were driven by the high strength UAS1B16-TEF constitutive promoter (Blazeck et al. 2011), were integrated into Y. lipolytica 's genomic rDNA repeats (Blazeck et al. 2013a; Ledall et al. 1994), and alleviated either PO1f s uracil or leucine auxotrophy. In our previous work, we noticed that alleviation of the leucine auxotrophy tended to increase lipid (e.g. triacylglyceride) accumulation far more than alleviation of the uracil auxotrophy. Therefore, nearly identical strains were routinely created differing only in the marker utilized to integrate an enzymatic overexpression cassette, enabling either uracil synthesis (S1) or leucine synthesis (S2). Initial overexpressions of the DGA1p and DGA2p enzymes occurred episomally with an identical UAS1B16-TEF promoter on a leucine-marker containing plasmid, though final strain construction entailed integrating these cassettes. Strain names included background (WT, ΔMFE1, ΔPEX10, or ΔPEX10ΔMFE1), markers used (S1, S2, S1-S2, or pMCS), and enzymes overexpressed (AMPD, MEA, ACL1, ACL2, DGA1, DGA2) so a strain overexpressing the AMPDp enzyme with a leucine marker in the ΔPEX10ΔMFE1 background is called ΔPEX10ΔMFE1 S2-AMPD. S1-Ø, S2-Ø, and S1, 2-Ø refer to strains without protein overexpressions but with uracil, leucine, or uracil+ leucine auxotrophies alleviated. ACL1p and ACL2p form a heterodimer in vivo so were tested as concurrent overexpressions.
Our combinatorial approach generated over 46 distinct genotypes that were analyzed for lipid (e.g. triacylglyceride) accumulation with nile red fluorescence flow cytometry after four days growth in C80N5 media and produced a large range in lipid (e.g. triacylglyceride) accumulation ability, culminating in a 60-fold improvement over PO1f WT control (FIG. 3). We saw that the deletion of the pex10 peroxisomal biogenesis transcription factor combined with overexpression of a acyl-CoA:diacylglycerol acyltransferase (DGA1 or DGA2) are essential for the highest lipid (e.g. triacylglyceride) production (FIG. 3). When comparing ammonia depletion in PO1f WT and our highest lipid producer, ΔPEX10ΔMFE1 pMCSDGA1, we observed a pronounced reduction in steady state nitrogen concentration in the ΔPEX10ΔMFE1 pMCSDGA1 strain. We saw a very noticeable correlation between the ability to synthesize leucine and lipid (e.g. triacylglyceride) accumulation ability, with an average increase of five fold in lipid content between comparable strains with and without a leucine marker present (FIG. 4). Deletion of mfe1 drastically reduced this increase in lipid (e.g. triacylglyceride) content. ΔMFE1 and ΔPEX10ΔMFE1 saw only a 1.42 fold and 2.58 fold increases in lipid (e.g. triacylglyceride) content granted from the capacity to synthesize leucine compared to 8.16 and 7.45 fold increases in WT and ΔPEX10 backgrounds (FIG. 4). In three of our four backgrounds, DGA1p outperformed DGA2p (FIG. 3); WT pMCSDGA2 was not included, but subsequent testing showed WT pMCSDGA1 to give higher lipid (e.g. triacylglyceride) levels than WT pMCSDGA2. Overall, fluorescence levels were highest in the ΔPEX10 and ΔPEX10ΔMFE1 backgrounds ( ̃3-fold WT), and lowest in the ΔMFE1 background ( ̃65% of WT), although mfe1 deletion has been shown to increase lipid (e.g. triacylglyceride) accumulation in media containing higher C:N ratio in eight day cultivation periods (Blazeck et al. 2013a). Because mfe1 deletion should further inhibit fatty acid degradation in the ΔPEX10ΔMFE1 background in long-scale fermentations, the DGA1p was integrated into the ΔPEX10ΔMFE1 background with S2 cassette and a S1-Ø to form our final fully heterotrophic rationally engineered strain. This ΔPEX10ΔMFE1 S1-S2-DGA1 strain displayed similar lipid (e.g. triacylglyceride) content to strains containing episomally expressed DGA1p and could accumulate lipids (e.g. triacylglyceride) effectively without any amino acid supplementation (Table 4) and yielded are highest % lipid (e.g. triacylglyceride) content of 32% dry cell weight for a total of 1.32 g/L. Furthermore, we saw no significant difference in LEU3 or DGA1 mRNA levels between these two strains. During bioreactor runs, these strains are able to produce significant amounts of lipids and cells exhibit 88% by dry cell weight lipids. Improved lipid production with one of the highest producing strains, ΔPEX10ΔMFE1-S1-S2-DGA1 in a bioreactor. Lipid levels have reached 22 g/L in media containing only 80 g/L glucose, 5 g/L ammonium sulfate, and 1.7 g/L Yeast Nitrogen Base (without amino acids or ammonium sulfate). Increasing dissolved oxygen content and maintaining pH at or above 5.0 enabled this yield. This represents  ̃86% of the theoretical yield. Furthermore, in these strains, we identify the presence of unique C17 fatty acids (FIG. 15).
Complex control of cellular processes, like lipid accumulation, is coordinated by transcription factors that regulate gene networks. In particular, the TupI general transcriptional repressor and the Had leucine zipper transcription factor involved in unfolded protein response have been shown to be upregulated in lipid (e.g. triacylglyceride) accumulation cell states (Morin et al. 2011). However, overexpression of these two proteins decreased lipid (e.g. triacylglyceride) accumulation in the PO1f WT background.
Dissection of Genotype-Dependence Towards Media Induction.
We more fully examined how C:N ratio and genotype interacted towards enabling lipid (e.g. triacylglyceride) accumulate on a larger scale by examining the response of twelve strains grown in thirteen different C:N ratios (Table 5). We were pleased to observe a strong tendency towards high lipid (e.g. triacylglyceride) levels in most high producers at a single media formulation—C80N5 (FIG. 5-8), allowing us to pinpoint a formulation for later use. Two trends stand out—(1) The 0.2 g/L ammonium sulfate formulations rarely enable lipid (e.g. triacylglyceride) accumulation, so that (2) the difference in induction from media containing 1 g/L and 5 g/L is slight, making glucose concentration seem more important towards increasing content than nitrogen content (after a certain threshold is reached).
Lipid Accumulation on Multiple Carbon Sources.
Viability of lipid (e.g. triacylglyceride) production depends on the capacity to fully convert all sugars from lignocellulosic biomass to lipids or to use carbon from industrial waste streams for lipid production. We analyzed the ability PO1f WT, ΔPEX10 S1-MEA, ΔPEX10 S2-AMPD, ΔPEX10ΔMFE1 S2-DGA1, and ΔPEX10ΔMFE pMCSDGA1 to generate lipids (e.g. triacylglyceride) when utilizing glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, or a lignocellulosic sugar blend as their carbon source (FIG. 9). ΔPEX10ΔMFE1 S2-DGA1 and ΔPEX10ΔMFE1 pMCSDGA1 generated the highest lipid (e.g. triacylglyceride) content across the board under conditions tested, and all engineered strains demonstrated the capacity to utilize each carbon source for lipid (e.g. triacylglyceride) production. Glucose, mannose, and the lignocellulosic saccharide blend were utilized easiest while ribose utilizations generated the least lipid (e.g. triacylglyceride) content of the conditions tested. The PO1f WT and ΔPEX10ΔMFE1 S2-DGA1 strain were tested to determine if decreasing carbon content or increasing initial inoculum amount could increase xylose-generate lipid (e.g. triacylglyceride) accumulation. Increasing xylose concentration and decreasing inoculum amount increased lipid (e.g. triacylglyceride) content in the ΔPEX10ΔMFE1 S2-DGA1 strain, while little difference was noticeable in the PO1f WT strain. However, PO1f WT demonstrated a surprising capacity to utilize pure glycerol for lipid (e.g. triacylglyceride) generation.
Isolation of a Novel MGA2 Mutation with Whole Genome Sequencing.
During the screening of a gDNA overexpression library intended to increase Y. lipolytica's lipid (e.g. triacylglyceride) production, we isolated a strain, dubbed L36, with incredible lipid (e.g. triacylglyceride) accumulation ability (FIG. 10). L36's lipid (e.g. triacylglyceride) production could be enhanced with micronutrient supplementation (FIG. 10). Complete sequencing of the L36 genome revealed a missense mutation in the MGA2 lipid synthesis regulator (MGA2G643R) as the most likely potential cause for L36's lipid (e.g. triacylglyceride) production capacity. Overexpression of a truncated MGA2p in a PO1f WT background reconstituted 58% of the observed L36 phenotype.
Directed Evolution with EMS Mutagenesis to Increase Lipid Accumulation
Direct evolution is commonly utilized to increase growth rate or to decrease sensitivity to a toxic metabolite. However, directed evolution has never been evaluated as a tool to increase lipid (e.g. triacylglyceride) production in oleaginous organisms. As evidenced by the isolation of strain L36, it is likely that Y. lipolytica is amenable to this approach. We subjected both L36 (FIG. 11) and ΔPEX10ΔMFE1 S2-DGA1 to EMS mutagenesis followed by serial selection via subculturing and then nile red staining. Both backgrounds proved highly responsive towards the directed evolution approach, and an increase in fluorescence with a large increase in final cell concentration (Table 6).
Besides the minerals, during the experiments, we also observed a critical phenotype for lipid (e.g. triacylglyceride) production in Yarrowia lipolytica: the lipid (e.g. triacylglyceride) de novo lipid (e.g. triacylglyceride) accumulation is close related to leucine biosynthesis pathway. A 5 fold lipid (e.g. triacylglyceride) level increase was achieved with strain harboring complete LEU biosynthesis pathway comparing to the one without complete pathway. Although this phenotype has been reported with engineered Saccharomyces cerevisiae (Kamisaka et al. 2007), this is the first observation in oleaginous yeast to our best knowledge. Understanding of this phenotype could be essential to understand the basic differences between oleaginous microbes and normal ones. However, to the date, the fundamental reason is still missing. Two possible routes may contribute to this, one is through TOR pathway (Kim and Guan 2011; Laplante and Sabatini 2009) and the other one is through leucine degradation and ketone body generation (Endemann et al. 1982). Either pathway heavily interacts with the whole cell metabolism which requires deep analysis to reveal the true mechanism behind.
Engineering with Known: Biosynthesis pathways and basic regulations. Rational systematic engineering Yarrowia lipolytica for high lipid production. Engineering with Unknown: Pathway interactions and complex regulation networks. Engineering lipid production in Yarrowia lipolytica through Inverse combinatorial metabolic Engineering. Confirmed lipid enhancers include DGA1 (Diacylglycerol acyltransferase) 300% improvement, MRM2 (Mitochondrial 2′ O-ribose methyltransferase) 25% improvement, MGMT (O-6-methylguanine-DNA methyltransferase) 15% improvement.
C. FATTY ACID CHARACTERIZATION BY NILE RED STAINING COUPLE WITH FLOW CYTOMETRY OR FLUORESCENCE MICROSCOPY
Nile Red is commonly utilized to stain oleaginous cellular material, and can be coupled with fluorescence flow cytometry to gauge relative lipid content (Greenspan et al. 1985). Y. lipolytica strains were routinely inoculated from glycerol stock in biological triplicate in appropriate media for 72 hours at 30° C. with shaking Cell concentrations were normalized to a specific OD600 for reinoculation in fresh media and further incubation. In general, 2 mL cultures were inoculated to an OD600=2.5, and larger volume cultures were inoculated to an OD600=0.1. Cultures were incubated for two to eight days at 30° C. with constant agitation. 2 mL cultures were incubated in a rotary drum (CT-7, New Brunswick Scientific) at speed seven and flasks were shaken at 225 rpm in a standing incubator. To harvest, one OD600 unit of each cultures was spun down at 1000 g for three minutes and resuspended in 500 μL Phosphate Buffered Saline solution (PBS) (Sigma Aldrich). 6 μL of 1 mM Nile Red (dissolved in DMSO) was added, and then cells were incubated in the dark at room temperature for 15 minutes. Cells were spun down at 1000 g for three minutes, resuspended in 800 μL ice cold water, spun down again, and resuspended again in 800 μL ice cold water. 300 μL of stained cells were added to 1 ml ice cold water and tested with a FACS Fortessa (BD Biosciences), a voltage of 350, a 10,000 cell count, a forward scatter of 125, a side scatter of 125, and the 535LP and 585/42BP filters for fluorescence detection using the GFP fluorochrome. Samples were kept on ice and in the dark during the test and the data was analyzed using FlowJo software (Tree Star Inc., Ashland, Oreg.) to compute mean fluorescence values. Day-to-day variability was mitigated by analyzing all comparable strains on the same day. An average fluorescence and standard deviation were calculated from the mean values of biological replicates. Stained cells were routinely examined with fluorescence microscopy under a 100× oil immersion objective using the FITC channel on an Axiovert 200M microscope (Zeiss).
D. LIPID QUANTIFICATION AND FATTY ACID PROFILE ANALYSIS
Lipids from  ̃20-30 OD600 equivalents were extracted following the procedure described by (Folch et al. 1957) and modified for yeast (Schneiter and Daum 2006). Dried lipids were transesterified with N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (Sigma-Aldrich) following the procedure of (Paik et al., 2009), and 2 μL samples were injected into a GC-FID (Agilent Technologies 6890 Network GC System) equipped with an Agilent HP-5 column (5% phenyl-95% methylsiloxane—product number 19091J-413) to analyze fatty acid fractions. Briefly, the following settings were used: Detector Temp=300° C., He Flow=1.0 mL/min, Oven Temp=80° C. for 2 min, increased at 30° C./min to 200° C., increased at 2° C./min to 229° C., increased at 1° C./min to 232° C., increased at 50° C./min to 325° C. Fatty acid standards for C16:0 palmitic acid, C16:1(n-7) palmitoleic acid, C18:0 stearic acid, C18:1 (n-9) oleic acid, and C18:2 (n-6) linoleic acid were purchased from Sigma-Aldrich, transesterified, and analyzed by GC to identify fatty acid peaks.
E. CITRIC ACID QUANTIFICATION
A 2 mL culture sample was pelleted down for 5 minutes at 3000×g, and the supernatant was filtered using a 0.2 mm syringe filter (Corning Incorporated). Filtered supernatant was analyzed with a HPLC Ultimate 3000 (Dionex) and a Zorbax SB-Aq column (Agilent Technologies). A 2.0 μL injection volume was used in a mobile phase composed of a 99.5:0.5 ratio of 25 mM potassium phosphate buffer (pH=2.0) to acetonitrile with a flow rate of 1.25 mL/min. The column temperature was maintained at 30° C. and UV-Vis absorption was measured at 210 nm. A citric acid standard (Sigma-Aldrich) was used to detect and quantify citric acid production.
F. EMS MUTAGENESIS AND ISOLATION OF HIGH LIPID PRODUCING STRAINS
10 OD units from cultures grown overnight were spun down in sterile microcentrifuge tubes at 5000 g for 10 seconds. Cell pellets were resuspended in 1 mL H2O, repelleted, and resuspended in 1 mL PBS. Two samples were spun down from each culture, one for EMS mutagenesis (30 μl of EMS added) and one as a control to determine the prevalence of spontaneous beneficial mutation (no EMS added). Cells were incubated for 1 hr at 30° C., with agitation, pelleted and resuspended in 200 μl of 5% sodium thiosulfate, transferred to fresh microcentrifuge tubes, washed twice in 200 μl of 5% sodium thiosulfate, and resuspended in 1 mL H2O. Cells were then grown to stationary phase in YSC media, and then reinoculated at an OD600=2.5 in 1 mL C80N5 media and grown for four days. Three to six serial transfers of the cell cultures followed in which the 1 mL cultures were spun down at 1000 g for two minutes, and the top 200 μL of the supernatant was transferred to 1 mL of fresh YSC media and allowed to grow to stationary phase before again spinning down and transferring. Final cultures (top 200 μL after spin down) were plated on YSC plates containing 0.01 mM Nile Red. After four days, high lipid producers were selected by viewing plates under a blue fluorescent light and picking colonies with brighter pink fluorescent color. Lipid amount was determined by coupling Nile Red staining with flow cytometry as described above.
The EMS mutagenesis procedures were performed following the protocol described by Winston (Winston 2001). Briefly, an overnight culture was cultivated to OD about 10. Cells were then harvested, washed and resuspended with 0.1 M sodium phosphate buffer (pH 7). 30 μl of EMS were added and incubated with unmutagenized control for 1 hr at 30° C., with agitation. The cells were then washed with 5% sodium thiosulfate and ready for serial transfer experiments to enrich the high lipid population. The EMS treated cells and unmutagenized cells were first cultured YSC media for 72 hours and then cultured in high glucose media with starting OD at 2.5 for 96 hours. The cells were centrifuged down with 100 g, the unclear supernatant, which contains high lipid accumulation strains, was used as seed for another round of cultivation. After five rounds of transfer, the cells were plated on Nile Red YSC plate to facilitate the isolation of high lipid production strains. Individual colonies were picked from the EMS treated cells as well as unmutagenized cells for characterization.
Characterization of EMS mutagenesis and floating cell transfer selection procedure selected strain E13 and E26. Second generation sequencing platform illumina paired ended sequencing PE 2×100 were performed with genomic DNA extracted from strain E26, E13 as well as PO1f by Genomic Sequencing and Analysis Facility in The University of Texas at Austin. 6424381 reads for strain E26 and 6565093 reads for strain E13 were collected from illumina HiSeq, which lead to a coverage approximately 65×. The Illumina reads were mapped to the CLIB122 genome using BWA (Li and Durbin 2009) and analyzed with Samtools (Beopoulos, Cescut et al. 2009) and BEDTools (Quinlan and Hall 2010). The SNPs identified were then filtered with SnpSift with QUAL>=30 (Pablo, Viral et al. 2012) The SNPs identified from PO1f, EMS26 and EMS13 were compared to extract the authentic SNPs in EMS26 and EMS13. The identified SNPs were then visualized in the IGV genome visualization software to validate as well as study the location of the SNPs in the genome due to the high false error rate in SNP calling process (Liu, Guo et al. 2012).
Information on identified targets in E26 and E13 strains following mutagenesis. Succinate semialdehyde dehydrogenase (SSADH), which coverts succinate semialdehyde to succinate after UGA1,4-aminobutyrate aminotransferase, deaminates GABA to succinate (Ramos, El Guezzar et al. 1985). Higher levels of accumulation of α-ketoglutarate were found in uga2 mutants in Saccharomyces cerevisiae (Cao, Barbosa et al. 2013) (3VZ1; 3VZ3). In the same time, lower levels of succinic acid (more than 5 fold decrease) were also identified in the yeast (Kamei, Tamura et al. 2011). The identified mutation in UGA2 in sequenced strains of Proline 209 is a highly conserved residual and close to a hydrogen bond forming Serine (Yuan, Yin et al. 2013). GABA metabolism is closely related to nitrogen assimilation in yeast and nitrogen limitation has been studied as a key function for triggering lipogenesis in Yarrowia lipolytica (Beopoulos, Cescut et al. 2009). Nitrogen sources have also been proven as an important factor for lipid accumulation inside cells (Evans and Ratledge 1984). A relationship between GABA metabolism and the TOR pathway, an important signaling pathway for lipid accumulation (Blazeck, Hill et al. 2014), has also been suggested (Cardenas, Cutler et al. 1999; Staschke, Dey et al. 2010). YALI0E17215 g codes for a protein with similarity to Saccharomyces cerevisiae RME1, which is a zinc finger protein involved in the control of meiosis (Covitz, Herskowitz et al. 1991). A similar protein has shown significant levels of increase in mRNA levels in a lipid accumulation-improved snf1 mutant in Yarrowia lipolytica (Xue, Sharpe et al. 2013). YALI0E20449p shows limited similarity to known protein sequences except the homeodomain, a DNA binding domain involved in the transcriptional regulation of key eukaryotic developmental processes, which shows similarities. Mutation V289G in YALI0E20449p exists outside of the homeodomain. S. cerevisiae homeodomain protein yox1 is able to bind leucine-tRNA (Kaufmann 1993) and leucine-tRNA synthase plays an important role (Han, Jeong et al. 2012) in the TOR pathway. Leucine has been suggested to be a critical lipid production enhancer (Blazeck, Hill et al. 2014). Recently, IRC20 containing a Snf2/Swi2 family ATPase/helicase and a RING finger domain, has been shown to be an E3 ubiquitin ligase (Richardson, Gardner et al. 2013) as well as a putative helicase. OSH6 overexpression has shown lifespan extension effect on yeast by increasing vacuole fusion and may relate to TORC (Gebre, Connor et al. 2012).
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TABLE 1







List of select strains used in this study











Reference or


Host Strain Name
Genotype
Source










Escherichia coli strains









DH10B
F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80d/acZΔM15
Open Biosystems



ΔlacX74 endA1 recA1 deoR Δ(ara, leu)7697



araD139 galU galK nupG rpsL λ







Yarrowia lipolytica base strains









WT (PO1f)
MatA, leu2-270, ura3-302, xpr2-322, axp1-2
Madzak et al.




2000


ΔMFE1 (PO1f-Δmfe1)
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
Blazeck et al. 2013



Δmfe1


ΔPEX10 (PO1f-Δpex10)
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
Blazeck et al. 2013



Δpex10


ΔPEX10ΔMFE1 (PO1f-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


Δpex10-Δmfe1)
Δpex10, Δmfe1


ΔACO1 (PO1f-Δaco1)
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δaco1







Selected Yarrowia lipolytica overexpression strains









WT-S1-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



URA3 (S1)


WT-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (S2)


WT-S1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



URA3, LEU2 (S1, S2)


WT-pMCS
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS)


WT-pMCS-TUP1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-TUP1


WT-pMCS-HAC1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-HAC1


WT-S1-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



URA3 (S1), UAS1B16-TEF-AMPD


WT-S2-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (S2), UAS1B16-TEF-AMPD


WT-S1-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



URA3 (S1), UAS1B16-TEF-MEA1


WT-S2-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (S2), UAS1B16-TEF-MEA1


WT-S1-S2-AMPD-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



URA3, LEU2 (S1, S2), UAS1B16-TEF-AMPD,



UAS1B16-TEF-MEA1


WT-pMCS-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-DGA1


ΔMFE1-S1-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3 (S1)


ΔMFE1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (S2)


ΔMFE1-S1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3, LEU2 (S1, S2)


ΔMFE1-S1-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3 (S1), UAS1B16-TEF-AMPD


ΔMFE1-S2-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (S2), UAS1B16-TEF-AMPD


ΔMFE1-S1-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3 (S1), UAS1B16-TEFMEA1


ΔMFE1-S2-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (S2), UAS1B16-TEF-MEA1


ΔMFE1-S1-S2-AMPD-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3, LEU2 (S1, S2), UAS1B16-TEF-



AMPD, UAS1B16-TEF-MEA1


ΔMFE1-S1-S2-ACL1-ACL2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, URA3, LEU2 (S1, S2), UAS1B16-TEF-



ACL1, UAS1B16-TEF-ACL2


ΔMFE1-pMCS-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (pMCS), UAS1B16-TEF-DGA1


ΔMFE1-pMCS-DGA2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (pMCS), UAS1B16-TEF-DGA2


ΔPEX10-S1-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, URA3 (S1)


ΔPEX10-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (S2)


ΔPEX10-S1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, URA3, LEU2 (S1, S2)


ΔPEX10-S1-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, URA3 (S1), UAS1B16-TEF-AMPD


ΔPEX10-S2-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (S2), UAS1B16-TEF-AMPD


ΔPEX10-S1-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, URA3 (S1), UAS1B16-TEFMEA1


ΔPEX10-S2-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (S2), UAS1B16-TEF-MEA1


ΔPEX10-S1-S2-AMPD-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, URA3, LEU2 (S1, S2), UAS1B16-TEF-



AMPD, UAS1B16-TEF-MEA1


ΔPEX10-pMCS-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (pMCS), UAS1B16-TEF-DGA1


ΔPEX10-pMCS-DGA2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (pMCS), UAS1B16-TEF-DGA2


ΔPEX10ΔMFE1-S1-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1)


ΔPEX10ΔMFE1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (S2)


ΔPEX10ΔMFE1-S1-S2-Ø
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3, LEU2 (S1, S2)


ΔPEX10ΔMFE1-S1-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), UAS1B16-TEF-



AMPD


ΔPEX10ΔMFE1-S2-AMPD
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (S2), UAS1B16-TEF-



AMPD


ΔPEX10ΔMFE1-S1-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), UAS1B16-TEFMEA1


ΔPEX10ΔMFE1-S2-MEA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (S2), UAS1B16-TEF-



MEA1


ΔPEX10ΔMFE1-S1-S2-AMPD-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


MEA1
Δpex10, Δmfe1, URA3, LEU2 (S1, S2), UAS1B16-



TEF-AMPD, UAS1B16-TEF-MEA1


ΔPEX10ΔMFE1-S1-S2-ACL1-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


ACL2
Δpex10, Δmfe1, URA3, LEU2 (S1, S2), UAS1B16-



TEF-ACL1, UAS1B16-TEF-ACL2


ΔPEX10ΔMFE1-pMCS-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (pMCS), UAS1B16-TEF-



DGA1


ΔPEX10ΔMFE1-pMCS-DGA2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (pMCS), UAS1B16-TEF-



DGA2


ΔPEX10ΔMFE1-S2-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, LEU2 (S2), UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-Ø-S2-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


DGA1
Δpex10, Δmfe1, URA3 (S1), LEU2 (S2),



UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-AMPD-S2-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


DGA1
Δpex10, Δmfe1, URA3 (S1), LEU2 (S2),



UAS1B16-TEF-AMPD, UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-MEA1-S2-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


DGA1
Δpex10, Δmfe1, URA3 (S1), LEU2 (S2),



UAS1B16-TEF-MEA1, UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), UAS1B16-TEF-DGA1


WT-S2-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Leu2 (S2), UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), UAS1B16-TEF-DGA1


ΔPEX10-S2-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, LEU2 (S2), UAS1B16-TEF-DGA1


WT-pMCS-DGA2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-DGA1


ΔPEX10ΔMFE1-S1-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3, UAS1B16-TEF-DGA1


ΔMFE1-S2-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2(S2), UAS1B16-TEF-DGA1


ΔMFE1-S2-DGA2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δmfe1, LEU2 (S2), UAS1B16-TEF-DGA2


ΔPEX10ΔMFE1-S1-Ø-pMCS-
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work


DGA1
Δpex10, Δmfe1, URA3 (S1), LEU2 (pMCS),



UAS1B16-TEF-DGA1


Po1f pMCSMga2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2
This work



LEU2 (pMCS), UAS1B16-TEF-Mga2


Po1f pMCSMga2dTM
MatA, leu2-270, ura3-302, xpr2-322, axp1-2
This work



LEU2 (pMCS), UAS1B16-TEF-Mga2dTM



(truncated of transmembrane span)


Po1f pMCSMga2L36
MatA, leu2-270, ura3-302, xpr2-322, axp1-2
This work



LEU2 (pMCS), UAS1B16-TEF-Mga2L36 (has



SNP found in L36 strain)


Po1f pMCSMRM2
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-MRM2


Po1f pMCSO6M
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS), UAS1B16-TEF-O6M


ΔACO1-DGA1
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δaco1, URA3 (S1), LEU2 (S2), UAS1B16-TEF-



DGA1







L36 and EMS derived strains









L36
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



LEU2 (pMCS) - isolated and fully sequenced to



determine source of high lipid accumulation -



most likely from mutation in MGA2 ORF.


L36 E1S6-4
L36 strain mutagenized further with EMS
This work


L36 E1S6-5
L36 strain mutagenized further with EMS
This work


L36 E1S6-6
L36 strain mutagenized further with EMS
This work


ΔPEX10ΔMFE1-S2-DGA1 E1
ΔPEX10ΔMFE1-S2-DGA1 strain mutagenized
This work



with EMS


ΔPEX10ΔMFE1-S2-DGA1 E6
ΔPEX10ΔMFE1-S2-DGA1 strain mutagenized
This work



with EMS


ΔPEX10ΔMFE1-S2-DGA1 E12
ΔPEX10ΔMFE1-S2-DGA1 strain mutagenized
This work



with EMS


E13
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), LEU2 (S2),



UAS1B16-TEF-DGA1 strain mutagenized with



EMS and selected


E26
MatA, leu2-270, ura3-302, xpr2-322, axp1-2,
This work



Δpex10, Δmfe1, URA3 (S1), LEU2 (S2),



UAS1B16-TEF-DGA1 strain mutagenized with



EMS and selected
















TABLE 2





List of primers used in this study
















JB387 YL AMPD 5′
TTGGCGCGCCatgccgcagcaagcaatgg


AscI
(SEQ ID NO.: 1)


 


JB388 YL AMPD 3′
CCTTAATTAAttaaccatgcagccgctcaaac


PacI
(SEQ ID NO.: 2)


 


JB402 YL ACL1 5′
TTGGCGCGCCatgtctgccaacgagaacat


AscI
(SEQ ID NO.: 3)


 


JB403 YL ACL1 + 
TTGGCGCGCCtctgccaacgagaacatctc


4 5′ AscI
(SEQ ID NO.: 4)


 


JB404 YL ACL1 3′
CCTTAATTAActatgatcgagtcttggccttg


PacI
(SEQ ID NO.: 5)


 


JB405 YL ACL2 5′
TTGGCGCGCCATGTCAGCGAAATCCATTCACG


AscI
(SEQ ID NO.: 6)


 


JB406 YL ACL2 + 
TTGGCGCGCCTCAGCGAAATCCATTCACGAG


4 5′ AscI
(SEQ ID NO.: 7)


 


JB407 YL ACL2 3′
CCTTAATTAATTAAACTCCGAGAGGAGTGGAA


PacI
(SEQ ID NO.: 8)


 


JB862 Loxleu 5′
CCAccgcggataacttcgtataatgtatgctatacgaagttatgagtctttattggtgatgggaaga


SacII
(SEQ ID NO.: 9)


 


JB863 Loxleu 3′
CGGTTCGAAataacttcgtatagcatacattatacgaagttatcagtcgccagcttaaagatatcta


Bstb1
(SEQ ID NO.: 10)


 


JB865 hygR 3′ 
GgaacggtAGATCtCGAGCGTCCCAAAACCTTCTC


bglII
(SEQ ID NO.: 11)


 


JB883 hygR 5′ 
GtggacGGgccggcgtttggcgcccgttttttcg


Nae
(SEQ ID NO.: 12)


 


JB911 DGA1 5′ 
CattcaaaGGCGCGCCatgactatcgactcacaatactaca


AscI
(SEQ ID NO.: 13)


 


JB912 DGA1 3′ 
GcGGATCCTTAATTAAttactcaatcattcggaactctgg


PacI
(SEQ ID NO.: 14)


 


JB913 DGA2 5′ 
CattcaaaGGCGCGCCATGGAAGTCCGACGACGAAA


AscI
(SEQ ID NO.: 15)


 


JB914 DGA2 3′ 
GcGGATCCTTAATTAACTACTGGTTCTGCTTGTAGTTGT


PacI
(SEQ ID NO.: 16)


 


AH011 Tup1 5′ 
GACTGGCGCGCATGAGCTTCCCCCAACAAGTA


Asc
(SEQ ID NO.: 17)


 


AH012 Tup1 3′ 
GTCCTTAATTAATTATCTGTTGACAGGAAAGTATCGC


PacI
(SEQ ID NO.: 18)


 


AH007 HacI 5′ 
GACTGGCGCGCATGTCTATCAAGCGAGAAGAGT


AscI
(SEQ ID NO.: 19)


 


AH008 HacI 3′ 
GTCCTTAATTAACTAGATCAGCAATAAAGTCGTGCT


PacI
(SEQ ID NO.: 20)


 


AH020 MAE 5′ 
GACTGGCGCGCCATGTTACGACTACGAACCATGC


AscI
(SEQ ID NO.: 21)


 


AH021 MAE 3′ 
GTCCTTAATTAACTAGTCGTAATCCCGCACATG


PacI
(SEQ ID NO.: 22)


 


LQ310 Mga2 5′ 
ACTGGGCGCGCC atggctaaagacaaggaaatcgactttgac


AscI
(SEQ ID NO.: 23)


 


LQ303 Mga2TM 3′
ACTGTTAATTAA tcagtaaatgtaagccagaacatcgt


PacI
(SEQ ID NO.: 24)


 


LQ309 Mga2 3′ 
ACTGTTAATTAA tcatgcagcctgggcctgg


PacI
(SEQ ID NO.: 25)


 


LQ294 O6M 5′ 
ACTGGGCGCGCC atgttttacaccaagcccgacccg


AscI
(SEQ ID NO.: 26)


 


LQ295 O6M 3′ 
ACTGTTAATTAA ttagagagtcccccacatgtcaccc


PacI
(SEQ ID NO.: 27)


 


LQ259 MRM2 5′ 
ACTGGGCGCGCC Atgcgccaaaagctgccgttcaac


AscI
(SEQ ID NO.: 28)


 


LQ260 MRM2 3′ 
ACTGTTAATTAA ttatggcttcccttctgccacatc


PacI
(SEQ ID NO.: 29)


 


LQ261 DGA1 5′ 
ACTGGGCGCGCC Atgactatcgactcacaatactac


AscI
(SEQ ID NO.: 30)


 


LQ262 DGA1 3′ 
ACTGTTAATTAA ttactcaatcattcggaactctgg


PacI
(SEQ ID NO.: 31)
















TABLE 3







Media formulations used for two strain testing












Carbon Source
Nitrogen Source




Glucose
Ammonium Sulfate



Media Name
(g/L)
(g/L)















C10N5
10
5



C20N0.04
20
0.04



C20N0.2
20
0.2



C20N1
20
1



C20N5 (YSC)
20
5



C20N10
20
10



C40N0.2
40
0.2



C40N1
40
1



C40N5
40
5



C80N0.04
80
0.04



C80N0.2
80
0.2



C80N1
80
1



C80N5
80
5



C80N10
80
10



C160N0.2
160
0.2



C160N1
160
1



C160N5
160
5



C320N0.2
320
0.2



C320N1
320
1



C320N5
320
5

















TABLE 4







ΔPex10, Mfe S1Ø, S2-DGA1 CSM vs Minimal Media (-CSM)


Comparison


ΔPex10, Mfe S1Ø, S2-DGA1 CSM vs Minimal Media (-CSM)


Comparison


Strain: ΔPex10, Mfe S1-φ, S2-DGA1










Media
Sample
Day 4 OD
Day 4 GFP Fluorescence













CSM - C80N5
A
16.83
36696


CSM - C80N5
B
16.76
34397


CSM - C80N5
C
16.31
39166


Minimal Media - C80N5
A
11.7
29365


Minimal Media - C80N5
B
11.46
52520


Minimal Media - C80N5
C
11.87
32427
















TABLE 5







Media Formulations used for 12 strain testing












Carbon Source
Nitrogen Source



Media
Glucose
Ammonium Sulfate



Name
(g/L)
(g/L)















C20N0.2
20
0.2



C20N1
20
1



C20N5
20
5



(YSC)



C40N0.2
40
0.2



C40N1
40
1



C40N5
40
5



C80N0.2
80
0.2



C80N1
80
1



C80N5
80
5



C80N10
80
10



C160N0.2
160
0.2



C160N1
160
1



C160N5
160
5

















TABLE 6







RFU and OD for EMS data












RFU
OD















ΔPEX10ΔMFE1 S2-
23750
8.81



DGA1 Control



E1
31800
21.91



E6
35400
18.86



E12
37100
22.5



L36 Control
23133.33
11.83



E1S6 4
34350
20.61



E1S6 6
34250
20.58



E1S6 8
28750
18.31

















TABLE 7







List of genes and genetic changes








Gene
Type of Modification





Leucine Biosynthesis Gene (LEU2) - Note
Over-expression


may also be able to include rest of genes of


leucine biosynthetic pathway, have yet to test


these additional ones


Uracil Biosynthesis gene (URA3)
Over-expression


multifunctional enzyme (MFE1) in b-oxidation


Deletion pathway


Transcription Factor (PEX10)
Deletion


AMP Deaminase (AMPD)
Over-expression


ATP-Citrate Lyase (ACL1 and/or ACL2)
Over-expression


Malic Enzyme (MAE/MEA)
Over-expression


Acetyl-CoA Carboxylase (ACC)
Over-expression


acyl-CoA:diacylglycerol acyltransferases
Over-expression


(DGA1 and/or DGA2)


Mitochondrial 2′ O-ribose
Over-expression


methyltransferase(MRM2)


O-6-methylguanine-DNA methyltransferase
Over-expression


(MGMT)


Aconitase (ACO1)
Deletion


Citrate Synthase (CIT1)
Over-expression
















TABLE 8







Strain L36 important SNP list














Mutation


Accession


Chromosome
Position
type
sequence
Gene
numbers





B
1644655
SNP
C > T
mga2
12342g


 


D
2401168
Insertion
A > AG
sorbitol utilization
18964g






protein SOU2



 


E
1837892
SNP
C > A
CEN0E
15444s


 



1837894
SNP
T > A
CEN0E
15444s


 



4025540
SNP
C > A
DEHA0A1298g IPF
33891g






95.1



 



4025542
SNP
G > C
DEHA0A1298g IPF
33891g






95.1



 


F
2861334
Insertion
A > AGAGGG
RLF2 chromatin
21637g





CTAGAGAG
asembly complex






AGGGAGA
subunit p90






A







(SEQ ID







NO.: 32)









Gene Targets: The reference number given for each name corresponds to the Genolevures database: http://www.genolevures.org/. YALI0 stands for Yarrowia lipolytica. A,B,C,D,E,F specifies chromosome, and the following number specifies location. Note: Leu2 and Ura3 given as GenBank Accession numbers








AMPD - YALI0E11495



(SEQ ID NO.: 33)



Nucleotide =



atgccgcagcaagcaatggatatcaagggcaaggccaagtctgtgcccatgcccgaagaagacgacctgg


 


actcgcattttgtgggtcccatctctccccgacctcacggagcagacgagattgctggctacgtgggctg


 


cgaagacgacgaagacgagcttgaagaactgggaatgctgggccgatctgcgtccacccacttctcttac


 


gcggaagaacgccacctcatcgaggttgatgccaagtacagagctcttcatggccatctgcctcatcagc


 


actctcagagtcccgtgtccagatcttcgtcatttgtgcgggccgaaatgaaccacccccctcccccacc


 


ctccagccacacccaccaacagccagaggacgatgacgcatcttccactcgatctcgatcgtcgtctcga


 


gcttctggacgcaagttcaacagaaacagaaccaagtctggatcttcgctgagcaagggtctccagcagc


 


tcaacatgaccggatcgctcgaagaagagccctacgagagcgatgacgatgcccgactatctgcggaaga


 


cgacattgtctatgatgctacccagaaagacacctgcaagcccatatctcctactctcaaacgcacccgc


 


accaaggacgacatgaagaacatgtccatcaacgacgtcaaaatcaccaccaccacagaagatcctcttg


 


tggcccaggagctgtccatgatgttcgaaaaggtgcagtactgccgagacctccgagacaagtaccaaac


 


cgtgtcgctacagaaggacggagacaaccccaaggatgacaagacacactggaaaatttaccccgagcct


 


ccaccaccctcctggcacgagaccgaaaagcgattccgaggctcgtccaaaaaggagcaccaaaagaaag


 


acccgacaatggatgaattcaaattcgaggactgcgaaatccccggacccaacgacatggtcttcaagcg


 


agatcctacctgtgtctatcaggtctatgaggatgaaagctctctcaacgaaaataagccgtttgttgcc


 


atcccctcaatccgagattactacatggatctggaggatctcattgtggcttcgtctgacggacctgcca


 


agtcttttgctttccgacgactgcaatatctagaagccaagtggaacctctactacctgctcaacgagta


 


cacggagacaaccgagtccaagaccaacccccatcgagacttttacaacgtacgaaaggtcgacacccac


 


gttcaccactctgcctgcatgaaccagaagcatctgctgcgattcatcaaatacaagatgaagaactgcc


 


ctgatgaagttgtcatccaccgagacggtcgggagctgacactctcccaggtgtttgagtcacttaactt


 


gactgcctacgacctgtctatcgatacccttgatatgcatgctcacaaggactcgttccatcgatttgac


 


aagttcaacctcaagtacaaccctgtcggtgagtctcgactgcgagaaatcttcctaaagaccgacaact


 


acatccagggtcgatacctagctgagatcacaaaggaggtgttccaggatctcgagaactcgaagtacca


 


gatggcggagtaccgtatttccatctacggtcggtccaaggacgagtgggacaagctggctgcctgggtg


 


ctggacaacaaactgttttcgcccaatgttcggtggttgatccaggtgcctcgactgtacgacatttaca


 


agaaggctggtctggttaacacctttgccgacattgtgcagaacgtctttgagcctcttttcgaggtcac


 


caaggatcccagtacccatcccaagctgcacgtgttcctgcagcgagttgtgggctttgactctgtcgat


 


gacgagtcgaagctggaccgacgtttccaccgaaagttcccaactgcagcatactgggacagcgcacaga


 


accctccctactcgtactggcagtactatctatacgccaacatggcctccatcaacacctggagacagcg


 


tttgggctataatacttttgagttgcgaccccatgctggagaggctggtgacccagagcatcttctgtgc


 


acttatctggttgctcagggtatcaaccacggtattctgttgcgaaaggtgcccttcattcagtaccttt


 


actacctggaccagatccccattgccatgtctcctgtgtccaacaatgcgctgttcctcacgttcgacaa


 


gaaccccttctactcatacttcaagcggggtctcaacgtgtccttgtcatcggatgatcctctgcagttt


 


gcttacactaaggaggctctgattgaggagtactctgtggctgcgctcatttacaagctttccaacgtgg


 


atatgtgtgagcttgctcgaaactcggtactgcaatctggctttgagcgaatcatcaaggagcattggat


 


cggcgaaaactacgagatccatggccccgagggcaacaccatccagaagacaaacgtgcccaatgtgcgt


 


ctggccttccgagacgagactttgacccacgagcttgctctggtggacaagtacaccaatcttgaggagt


 


ttgagcggctgcatggtta


 


(SEQ ID NO.: 34)



Amino Acid =



MPQQAMDIKGKAKSVPMPEEDDLDSHFVGPISPRPHGADEIAGYVGCEDDEDELEELGMLGRSASTHFSY


 


AEERHLIEVDAKYRALHGHLPHQHSQSPVSRSSSFVRAEMNHPPPPPSSHTHQQPEDDDASSTRSRSSSR


 


ASGRKFNRNRTKSGSSLSKGLQQLNMTGSLEEEPYESDDDARLSAEDDIVYDATQKDTCKPISPTLKRTR


 


TKDDMKNMSINDVKITTTTEDPLVAQELSMMFEKVQYCRDLRDKYQTVSLQKDGDNPKDDKTHWKIYPEP


 


PPPSWHETEKRFRGSSKKEHQKKDPTMDEFKFEDCEIPGPNDMVFKRDPTCVYQVYEDESSLNENKPFVA


 


IPSIRDYYMDLEDLIVASSDGPAKSFAFRRLQYLEAKWNLYYLLNEYTETTESKTNPHRDFYNVRKVDTH


 


VHHSACMNQKHLLRFIKYKMKNCPDEVVIHRDGRELTLSQVFESLNLTAYDLSIDTLDMHAHKDSFHRFD


 


KFNLKYNPVGESRLREIFLKTDNYIQGRYLAEITKEVFQDLENSKYQMAEYRISIYGRSKDEWDKLAAWV


 


LDNKLFSPNVRWLIQVPRLYDIYKKAGLVNTFADIVQNVFEPLFEVTKDPSTHPKLHVFLQRVVGFDSVD


 


DESKLDRRFHRKFPTAAYWDSAQNPPYSYWQYYLYANMASINTWRQRLGYNTFELRPHAGEAGDPEHLLC


 


TYLVAQGINHGILLRKVPFIQYLYYLDQIPIAMSPVSNNALFLTFDKNPFYSYFKRGLNVSLSSDDPLQF


 


AYTKEALIEEYSVAALIYKLSNVDMCELARNSVLQSGFERIIKEHWIGENYEIHGPEGNTIQKTNVPNVR


 


LAFRDETLTHELALVDKYTNLEEFERLHG*


 


Leu2 - AF260230


(SEQ ID NO.: 35)



Nucleotide =



atggaacccgaaactaagaagaccaagactgactccaagaagattgttcttctcggcggcgacttctgtg


 


gccccgaggtgattgccgaggccgtcaaggtgctcaagtctgttgctgaggcctccggcaccgagtttgt


 


gtttgaggaccgactcattggaggagctgccattgagaaggagggcgagcccatcaccgacgctactctc


 


gacatctgccgaaaggctgactctattatgctcggtgctgtcggaggcgctgccaacaccgtatggacca


 


ctcccgacggacgaaccgacgtgcgacccgagcagggtctcctcaagctgcgaaaggacctgaacctgta


 


cgccaacctgcgaccctgccagctgctgtcgcccaagctcgccgatctctcccccatccgaaacgttgag


 


ggcaccgacttcatcattgtccgagagctcgtcggaggtatctactttggagagcgaaaggaggatgacg


 


gatctggcgtcgcttccgacaccgagacctactccgttcctgaggttgagcgaattgcccgaatggccgc


 


cttcctggcccttcagcacaacccccctcttcccgtgtggtctcttgacaaggccaacgtgctggcctcc


 


tctcgactttggcgaaagactgtcactcgagtcctcaaggacgaattcccccagctcgagctcaaccacc


 


agctgatcgactcggccgccatgatcctcatcaagcagccctccaagatgaatggtatcatcatcaccac


 


caacatgtttggcgatatcatctccgacgaggcctccgtcatccccggttctctgggtctgctgccctcc


 


gcctctctggcttctctgcccgacaccaacgaggcgttcggtctgtacgagccctgtcacggatctgccc


 


ccgatctcggcaagcagaaggtcaaccccattgccaccattctgtctgccgccatgatgctcaagttctc


 


tcttaacatgaagcccgccggtgacgctgttgaggctgccgtcaaggagtccgtcgaggctggtatcact


 


accgccgatatcggaggctcttcctccacctccgaggtcggagacttgttgccaacaaggtcaaggagct


 


gctcaagaaggagtaagtcgtttctacgacgcattgatggaaggagcaaactgacgcgcctgcgggttgg


 


tctaccggcagggtccgctagtgtataa


 


(SEQ ID NO.: 36)



Amino Acid =



MEPETKKTKTDSKKIVLLGGDFCGPEVIAEAVKVLKSVAEASGTEFVFEDRLIGGAAIEKEGEPITDATL


 


DICRKADSIMLGAVGGAANTVWTTPDGRTDVRPEQGLLKLRKDLNLYANLRPCQLLSPKLADLSPIRNVE


 


GTDFIIVRELVGGIYFGERKEDDGSGVASDTETYSVPEVERIARMAAFLALQHNPPLPVWSLDKANVLAS


 


SRLWRKTVTRVLKDEFPQLELNHQLIDSAAMILIKQPSKMNGIIITTNMFGDIISDEASVIPGSLGLLPS


 


ASLASLPDTNEAFGLYEPCHGSAPDLGKQKVNPIATILSAAMMLKFSLNMKPAGDAVEAAVKESVEAGIT


 


TADIGGSSSTSEVGDLLPTRSRSCSRRSKSFLRRIDGRSKLTRLRVGLPAGSASV*


 


Ura3 - YLU40564


(SEQ ID NO.: 37)



Nucleotide =



atgccctcctacgaagctcgagctaacgtccacaagtccgcctttgccgctcgagtgctcaagctcgtgg


 


cagccaagaaaaccaacctgtgtgcttctctggatgttaccaccaccaaggagctcattgagcttgccga


 


taaggtcggaccttatgtgtgcatgatcaagacccatatcgacatcattgacgacttcacctacgccggc


 


actgtgctccccctcaaggaacttgctcttaagcacggtttcttcctgttcgaggacagaaagttcgcag


 


atattggcaacactgtcaagcaccagtacaagaacggtgtctaccgaatcgccgagtggtccgatatcac


 


caacgcccacggtgtacccggaaccggaatcattgctggcctgcgagctggtgccgaggaaactgtctct


 


gaacagaagaaggaggacgtctctgactacgagaactcccagtacaaggagttcctggtcccctctccca


 


acgagaagctggccagaggtctgctcatgctggccgagctgtcttgcaagggctctctggccactggcga


 


gtactccaagcagaccattgagcttgcccgatccgaccccgagtttgtggttggcttcattgcccagaac


 


cgacctaagggcgactctgaggactggcttattctgacccccggggtgggtcttgacgacaagggagacg


 


ctctcggacagcagtaccgaactgttgaggatgtcatgtctaccggaacggatatcataattgtcggccg


 


aggtctgtacggccagaaccgagatcctattgaggaggccaagcgataccagaaggctggctgggaggct


 


taccagaagattaactgttag


 


(SEQ ID NO.: 38)



Amino Acid =



MPSYEARANVHKSAFAARVLKLVAAKKTNLCASLDVTTTKELIELADKVGPYVCMIKTHIDIIDDFTYAG


 


TVLPLKELALKHGFFLFEDRKFADIGNTVKHQYKNGVYRIAEWSDITNAHGVPGTGIIAGLRAGAEETVS


 


EQKKEDVSDYENSQYKEFLVPSPNEKLARGLLMLAELSCKGSLATGEYSKQTIELARSDPEFVVGFIAQN


 


RPKGDSEDWLILTPGVGLDDKGDALGQQYRTVEDVMSTGTDIIIVGRGLYGQNRDPIEEAKRYQKAGWEA


 


YQKINC*


 


ACLsubunit1 - YALI0E34793


(SEQ ID NO.: 39)



Nucleotide =



atgtctgccaacgagaacatctcccgattcgacgcccctgtgggcaaggagcaccccgcctacgagctct


 


tccataaccacacacgatctttcgtctatggtctccagcctcgagcctgccagggtatgctggacttcga


 


cttcatctgtaagcgagagaacccctccgtggccggtgtcatctatcccttcggcggccagttcgtcacc


 


aagatgtactggggcaccaaggagactcttctccctgtctaccagcaggtcgagaaggccgctgccaagc


 


accccgaggtcgatgtcgtggtcaactttgcctcctctcgatccgtctactcctctaccatggagctgct


 


cgagtacccccagttccgaaccatcgccattattgccgagggtgtccccgagcgacgagcccgagagatc


 


ctccacaaggcccagaagaagggtgtgaccatcattggtcccgctaccgtcggaggtatcaagcccggtt


 


gcttcaaggttggaaacaccggaggtatgatggacaacattgtcgcctccaagctctaccgacccggctc


 


cgttgcctacgtctccaagtccggaggaatgtccaacgagctgaacaacattatctctcacaccaccgac


 


ggtgtctacgagggtattgctattggtggtgaccgataccctggtactaccttcattgaccatatcctgc


 


gatacgaggccgaccccaagtgtaagatcatcgtcctccttggtgaggttggtggtgttgaggagtaccg


 


agtcatcgaggctgttaagaacggccagatcaagaagcccatcgtcgcttgggccattggtacttgtgcc


 


tccatgttcaagactgaggttcagttcggccacgccggctccatggccaactccgacctggagactgcca


 


aggctaagaacgccgccatgaagtctgctggcttctacgtccccgataccttcgaggacatgcccgaggt


 


ccttgccgagctctacgagaagatggtcgccaagggcgagctgtctcgaatctctgagcctgaggtcccc


 


aagatccccattgactactcttgggcccaggagcttggtcttatccgaaagcccgctgctttcatctcca


 


ctatttccgatgaccgaggccaggagcttctgtacgctggcatgcccatttccgaggttttcaaggagga


 


cattggtatcggcggtgtcatgtctctgctgtggttccgacgacgactccccgactacgcctccaagttt


 


cttgagatggttctcatgcttactgctgaccacggtcccgccgtatccggtgccatgaacaccattatca


 


ccacccgagctggtaaggatctcatttcttccctggttgctggtctcctgaccattggtacccgattcgg


 


aggtgctcttgacggtgctgccaccgagttcaccactgcctacgacaagggtctgtccccccgacagttc


 


gttgataccatgcgaaagcagaacaagctgattcctggtattggccatcgagtcaagtctcgaaacaacc


 


ccgatttccgagtcgagcttgtcaaggactttgttaagaagaacttcccctccacccagctgctcgacta


 


cgcccttgctgtcgaggaggtcaccacctccaagaaggacaacctgattctgaacgttgacggtgctatt


 


gctgtttcttttgtcgatctcatgcgatcttgcggtgcctttactgtggaggagactgaggactacctca


 


agaacggtgttctcaacggtctgttcgttctcggtcgatccattggtctcattgcccaccatctcgatca


 


gaagcgactcaagaccggtctgtaccgacatccttgggacgatatcacctacctggttggccaggaggct


 


atccagaagaagcgagtcgagatcagcgccggcgacgtttccaaggccaagactcgatcatag


 


(SEQ ID NO.: 40)



Amino Acid =



MSANENISRFDAPVGKEHPAYELFHNHTRSFVYGLQPRACQGMLDFDFICKRENPSVAGVIYPFGGQFVT


 


KMYWGTKETLLPVYQQVEKAAAKHPEVDVVVNFASSRSVYSSTMELLEYPQFRTIAIIAEGVPERRAREI


 


LHKAQKKGVTIIGPATVGGIKPGCFKVGNTGGMMDNIVASKLYRPGSVAYVSKSGGMSNELNNIISHTTD


 


GVYEGIAIGGDRYPGTTFIDHILRYEADPKCKIIVLLGEVGGVEEYRVIEAVKNGQIKKPIVAWAIGTCA


 


SMFKTEVQFGHAGSMANSDLETAKAKNAAMKSAGFYVPDTFEDMPEVLAELYEKMVAKGELSRISEPEVP


 


KIPIDYSWAQELGLIRKPAAFISTISDDRGQELLYAGMPISEVFKEDIGIGGVMSLLWFRRRLPDYASKF


 


LEMVLMLTADHGPAVSGAMNTIITTRAGKDLISSLVAGLLTIGTRFGGALDGAATEFTTAYDKGLSPRQF


 


VDTMRKQNKLIPGIGHRVKSRNNPDFRVELVKDFVKKNFPSTQLLDYALAVEEVTTSKKDNLILNVDGAI


 


AVSFVDLMRSCGAFTVEETEDYLKNGVLNGLFVLGRSIGLIAHHLDQKRLKTGLYRHPWDDITYLVGQEA


 


IQKKRVEISAGDVSKAKTRS*


 


ACLsubunit2 - YALI0D24431


(SEQ ID NO.: 41)



Nucleotide =



atgtcagcgaaatccattcacgaggccgacggcaaggccctgctcgcacactttctgtccaaggcgcccg


 


tgtgggccgagcagcagcccatcaacacgtttgaaatgggcacacccaagctggcgtctctgacgttcga


 


ggacggcgtggcccccgagcagatcttcgccgccgctgaaaagacctacccctggctgctggagtccggc


 


gccaagtttgtggccaagcccgaccagctcatcaagcgacgaggcaaggccggcctgctggtactcaaca


 


agtcgtgggaggagtgcaagccctggatcgccgagcgggccgccaagcccatcaacgtggagggcattga


 


cggagtgctgcgaacgttcctggtcgagccctttgtgccccacgaccagaagcacgagtactacatcaac


 


atccactccgtgcgagagggcgactggatcctcttctaccacgagggaggagtcgacgtcggcgacgtgg


 


acgccaaggccgccaagatcctcatccccgttgacattgagaacgagtacccctccaacgccacgctcac


 


caaggagctgctggcacacgtgcccgaggaccagcaccagaccctgctcgacttcatcaaccggctctac


 


gccgtctacgtcgatctgcagtttacgtatctggagatcaaccccctggtcgtgatccccaccgcccagg


 


gcgtcgaggtccactacctggatcttgccggcaagctcgaccagaccgcagagtttgagtgcggccccaa


 


gtgggctgctgcgcggtcccccgccgctctgggccaggtegtcaecattgacgccggctccaccaaggtg


 


tccatcgacgccggccccgccatggtcttccccgctcctttcggtcgagagctgtccaaggaggaggcgt


 


acattgcggagctcgattccaagaccggagcttctctgaagctgactgttctcaatgccaagggccgaat


 


ctggacccttgtggctggtggaggagcctccgtcgtctacgccgacgccattgcgtctgccggctttgct


 


gacgagctcgccaactacggcgagtactctggcgctcccaacgagacccagacctacgagtacgccaaaa


 


ccgtactggatctcatgacccggggcgacgctcaccccgagggcaaggtactgttcattggcggaggaat


 


cgccaacttcacccaggttggatccaccttcaagggcatcatccgggccttccgggactaccagtcttct


 


ctgcacaaccacaaggtgaagatttacgtgcgacgaggcggtcccaactggcaggagggtctgcggttga


 


tcaagtcggctggcgacgagctgaatctgcccatggagatttacggccccgacatgcacgtgtcgggtat


 


tgttcctttggctctgcttggaaagcggcccaagaatgtcaagccttttggcaccggaccttctactgag


 


gcttccactcctctcggagtttaa


 


(SEQ ID NO.: 42)



Amino Acid =



MSAKSIHEADGKALLAHFLSKAPVWAEQQPINTFEMGTPKLASLTFEDGVAPEQIFAAAEKTYPWLLESG


 


AKFVAKPDQLIKRBGKAGLLVLNKSWEECKPWIAERAAKPINVEGIDGVLRTFLVEPFVPHDQKHEYYIN


 


IHSVREGDWILFYHEGGVDVGDVDAKAAKILIPVDIENEYPSNATLTKELLAHVPEDQHQTLLDFINRLY


 


AVYVDLQFTYLEINPLVVIPTAQGVEVHYLDLAGKLDQTAEFECGPKWAAARSPAALGQVVTIDAGSTKV


 


SIDAGPAMVFPAPFGRELSKEEAYIAELDSKTGASLKLTVLNAKGRIWTLVAGGGASVVYADAIASAGFA


 


DELANYGEYSGAPNETQTYEYAKTVLDLMTRGDAHPEGKVLFIGGGIANFTQVGSTFKGIIRAFRDYQSS


 


LHNHKVKIYVRRGGPNWQEGLRLIKSAGDELNLPMEIYGPDMHVSGIVPLALLGKRPKNVKPFGTGPSTE


 


ASTPLGV*


 


MEA1 - YALI0E18634


(note: 4 nucleotide difference compared to the reference sequence. 


In embodiments, MEA1 is the reference sequence associated with


YALI0E18634. In embodiments, MEA1 is the reference sequence with 


the four nucleotide differences from the reference sequence 


shown below.)


(SEQ ID NO.: 43)



Nucleotide =



atgttacgactacgaaccatgcgacccacacagaccagcgtcagggcggcgcttgggcccaccgctgcgg


 


cccgaaacatgtcctcctccagcccctccagcttcgaatactcgtcctacgtcaagggcacgcgggaaat


 


cggccaccgaaaggcgcccacaacccgtctgtcggttgagggccccatctacgtgggcttcgacggcatt


 


cgtcttctcaacctgccgcatctcaacaagggctcgggattccccctcaacgagcgacgggaattcggac


 


tcagtggtcttctgccctctgccgaagccaccctggaggaacaggtcgaccgagcataccaacaattcaa


 


aaagtgtggcactcccttagccaaaaacgggttctgcacctcgctcaagttccaaaacgaggtgctctac


 


tacgccctgctgctcaagcacgttaaggaggtcttccccatcatctatacaccgactcagggagaagcca


 


ttgaacagtactcgcggctgttccggcggcccgaaggctgcttcctcgacatcaccagtccctacgacgt


 


ggaggagcgtctgggagcgtttggagaccatgacgacattgactacattgtcgtgactgactccgagggt


 


attctcggaattggagaccaaggagtgggcggtattggtatttccatcgccaagctggctctcatgactc


 


tatgtgctggagtcaacccctcacgagtcattcctgtggttctggatacgggaaccaacaaccaggagct


 


gctgcacgaccccctgtatctcggccgacgaatgccccgagtgcgaggaaagcagtacgacgacttcatc


 


gacaactttgtgcagtctgcccgaaggctgtatcccaaggcggtgatccatttcgaggactttgggctcg


 


ctaacgcacacaagatcctcgacaagtatcgaccggagatcccctgcttcaacgacgacatccagggcac


 


tggagccgtcactctggcctccatcacggccgctctcaaggtgctgggcaaaaatatcacagatactcga


 


attctcgtgtacggagctggttcggccggcatgggtattgctgaacaggtctatgataacctggttgccc


 


agggtctcgacgacaagactgcgcgacaaaacatctttctcatggaccgaccgggtctactgaccaccgc


 


acttaccgacgagcagatgagcgacgtgcagaagccgtttgccaaggacaaggccaattacgagggagtg


 


gacaccaagactctggagcacgtggttgctgccgtcaagccccatattctcattggatgttccactcagc


 


ccggcgcctttaacgagaaggttgtcaaggagatgcttaaacacacccctcgacccatcattctccctct


 


ttccaaccccacacgtcttcatgaggctgtccctgcagatctgtacaagtggaccgacggcaaggctctg


 


gttgccaccggctcgccctttgacccagtcaacggcaaggagacgtctgagaacaataactgctttgttt


 


tccccggaatcgggctgggagccattctgtctcgatcaaagctcatcaccaacaccatgattgctgctgc


 


catcgagtgcctcgccgaacaggcccccattctcaagaaccacgacgagggagtacttcccgacgtagct


 


ctcatccagatcatttcggcccgggtggccactgccgtggttcttcaggccaaggctgagggcctagcca


 


ctgtcgaggaagagctcaagcccggcaccaaggaacatgtgcagattcccgacaactttgacgagtgtct


 


cgcctgggtcgagactcagatgtggcggcccgtctaccggcctctcatccatgtgcgggattacgactag


 


(SEQ ID NO.: 44)



Amino Acid =



MLRLRTMRPTQTSVRAALGPTAAARNMSSSSPSSFEYSSYVKGTREIGHRKAPTTRLSVEGPIYVGFDGI


 


RLLNLPHLNKGSGFPLNERREFGLSGLLPSAEATLEEQVDRAYQQFKKCGTPLAKNGFCTSLKFQNEVLY


 


YALLLKHVKEVFPIIYTPTQGEAIEQYSRLFRRPEGCFLDITSPYDVEERLGAFGDHDDIDYIVVTDSEG


 


ILGIGDQGVGGIGISIAKLALMTLCAGVNPSRVIPVVLDTGTNNQELLHDPLYLGRRMPRVRGKQYDDFI


 


DNFVQSARRLYPKAVIHFEDFGLANAHKILDKYRPEIPCFNDDIQGTGAVTLASITAALKVLGKNITDTR


 


ILVYGAGSAGMGIAEQVYDNLVAQGLDDKTARQNIFLMDRPGLLTTALTDEQMSDVQKPFAKDKANYEGV


 


DTKTLEHVVAAVKPHILIGCSTQPGAFNEKVVKEMLKHTPRPIILPLSNPTRLHEAVPADLYKWTDGKAL


 


VATGSPFDPVNGKETSENNNCFVFPGIGLGAILSRSKLITNTMIAAAIECLAEQAPILKNHDEGVLPDVA


 


LIQIISARVATAVVLQAKAEGLATVEEELKPGTKEHVQIPDNFDECLAWVETQMWRPVYRPLIHVRDYD*


 


DGA1 - YALI0E32769


(SEQ ID NO.: 45)



Nucleotide =



atgactatcgactcacaatactacaagtcgcgagacaaaaacgacacggcacccaaaatcgcgggaatcc


 


gatatgccccgctatcgacaccattactcaaccgatgtgagaccttctctctggtctggcacattttcag


 


cattcccactttcctcacaattttcatgctatgctgcgcaattccactgctctggccatttgtgattgcg


 


tatgtagtgtacgctgttaaagacgactccccgtccaacggaggagtggtcaagcgatactcgcctattt


 


caagaaacttcttcatctggaagctctttggccgctacttccccataactctgcacaagacggtggatct


 


ggagcccacgcacacatactaccctctggacgtccaggagtatcacctgattgctgagagatactggccg


 


cagaacaagtacctccgagcaatcatctccaccatcgagtactttctgcccgccttcatgaaacggtctc


 


tttctatcaacgagcaggagcagcctgccgagcgagatcctctcctgtctcccgtttctcccagctctcc


 


gggttctcaacctgacaagtggattaaccacgacagcagatatagccgtggagaatcatctggctccaac


 


ggccacgcctcgggctccgaacttaacggcaacggcaacaatggcaccactaaccgacgacctttgtcgt


 


ccgcctctgctggctccactgcatctgattccacgcttcttaacgggtccctcaactcctacgccaacca


 


gatcattggcgaaaacgacccacagctgtcgcccacaaaactcaagcccactggcagaaaatacatcttc


 


ggctaccacccccacggcattatcggcatgggagcctttggtggaattgccaccgagggagctggatggt


 


ccaagctctttccgggcatccctgtttctcttatgactctcaccaacaacttccgagtgcctctctacag


 


agagtacctcatgagtctgggagtcgcttctgtctccaagaagtcctgcaaggccctcctcaagcgaaac


 


cagtctatctgcattgtcgttggtggagcacaggaaagtcttctggccagacccggtgtcatggacctgg


 


tgctactcaagcgaaagggttttgttcgacttggtatggaggtcggaaatgtcgcccttgttcccatcat


 


ggcctttggtgagaacgacctctatgaccaggttagcaacgacaagtcgtccaagctgtaccgattccag


 


cagtttgtcaagaacttccttggattcacccttcctttgatgcatgcccgaggcgtcttcaactacgatg


 


tcggtcttgtcccctacaggcgacccgtcaacattgtggttggttcccccattgacttgccttatctccc


 


acaccccaccgacgaagaagtgtccgaataccacgaccgatacatcgccgagctgcagcgaatctacaac


 


gagcacaaggatgaatatttcatcgattggaccgaggagggcaaaggagccccagagttccgaatgattg


 


agtaa


 


(SEQ ID NO.: 46)



Amino Acid =



MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFSIPTFLTIFMLCCAIPLLWPFVIA


 


YVVYAVKDDSPSNGGVVKRYSPISRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWP


 


QNKYLRAIISTIEYFLPAFMKRSLSINEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSRYSRGESSGSN


 


GHASGSELNGNGNNGTTNRRPLSSASAGSTASDSTLLNGSLNSYANQIIGENDPQLSPTKLKPTGRKYIF


 


GYHPHGIIGMGAFGGIATEGAGWSKLFPGIPVSLMTLTNNFRVPLYREYLMSLGVASVSKKSCKALLKRN


 


QSICIVVGGAQESLLARPGVMDLVLLKRKGFVRLGMEVGNVALVPIMAFGENDLYDQVSNDKSSKLYRFQ


 


QFVKNFLGFTLPLMHARGVFNYDVGLVPYRRPVNIVVGSPIDLPYLPHPTDEEVSEYHDRYIAELQRIYN


 


EHKDEYFIDWTEEGKGAPEFRMIE*


 


DGA2 - YALI0D07986


(SEQ ID NO.: 47)



Nucleotide =



atggaagtccgacgacgaaaaatcgacgtgctcaaggcccagaaaaacggctacgaatcgggcccaccat


 


ctcgacaatcgtcgcagccctcctcaagagcatcgtccagaacccgcaacaaacactcctcgtccaccct


 


gtcgctcagcggactgaccatgaaagtccagaagaaacctgcgggacccccggcgaactccaaaacgcca


 


ttcctacacatcaagcccgtgcacacgtgctgctccacatcaatgctttcgcgcgattatgacggctcca


 


accccagcttcaagggcttcaaaaacatcggcatgatcattctcattgtgggaaatctacggctcgcatt


 


cgaaaactacctcaaatacggcatttccaacccgttcttcgaccccaaaattactccttccgagtggcag


 


ctctcaggcttgctcatagtcgtggcctacgcacatatcctcatggcctacgctattgagagcgctgcca


 


agctgctgttcctctctagcaaacaccactacatggccgtggggcttctgcataccatgaacactttgtc


 


gtccatctcgttgctgtcctacgtcgtctactactacctgcccaaccccgtggcaggcacaatagtcgag


 


tttgtggccgttattctgtctctcaaactcgcctcatacgccctcactaactcggatctccgaaaagccg


 


caattcatgcccagaagctcgacaagacgcaagacgataacgaaaaggaatccacctcgtcttcctcttc


 


ttcagatgacgcagagactttggcagacattgacgtcattcctgcatactacgcacagctgccctacccc


 


cagaatgtgacgctgtcgaacctgctgtacttctggtttgctcccacactggtctaccagcccgtgtacc


 


ccaagacggagcgtattcgacccaagcacgtgatccgaaacctgtttgagctcgtctctctgtgcatgct


 


tattcagtttctcatcttccagtacgcctaccccatcatgcagtcgtgtctggctctgttcttccagccc


 


aagctcgattatgccaacatctccgagcgcctcatgaagttggcctccgtgtctatgatggtctggctca


 


ttggattctacgctttcttccagaacggtctcaatcttattgccgagctcacctgttttggaaacagaac


 


cttctaccagcagtggtggaattcccgctccattggccagtactggactctatggaacaagccagtcaac


 


cagtactttagacaccacgtctacgtgcctcttctcgctcggggcatgtcgcggttcaatgcgtcggtgg


 


tggttttctttttctccgccgtcatccatgaactgcttgtcggcatccccactcacaacatcatcggagc


 


cgccttcttcggcatgatgtcgcaggtgcctctgatcatggctactgagaaccttcagcatattaactcc


 


tctctgggccccttccttggcaactgtgcattctggttcacctttttcctgggacaacccacttgtgcat


 


tcctttattatctggcttacaactacaagcagaaccagtag


 


(SEQ ID NO.: 48)



Amino Acid =



MEVRRRKIDVLKAQKNGYESGPPSRQSSQPSSRASSRTRNKHSSSTLSLSGLTMKVQKKPAGPPANSKTP


 


FLHIKPVHTCCSTSMLSRDYDGSNPSFKGFKNIGMIILIVGNLRLAFENYLKYGISNPFFDPKITPSEWQ


 


LSGLLIVVAYAHILMAYAIESAAKLLFLSSKHHYMAVGLLHTMNTLSSISLLSYVVYYYLPNPVAGTIVE


 


FVAVILSLKLASYALTNSDLRKAAIHAQKLDKTQDDNEKESTSSSSSSDDAETLADIDVIPAYYAQLPYP


 


QNVTLSNLLYFWFAPTLVYQPVYPKTERIRPKHVIRNLFELVSLCMLIQFLIFQYAYPIMQSCLALFFQP


 


KLDYANISERLMKLASVSMMVWLIGFYAFFQNGLNLIAELTCFGNRTFYQQWWNSRSIGQYWTLWNKPVN


 


QYFRHHVYVPLLARGMSRFNASVVVFFFSAVIHELLVGIPTHNIIGAAFFGMMSQVPLIMATENLQHINS


 


SLGPFLGNCAFWFTFFLGQPTCAFLYYLAYNYKQNQ*


 


MGA2- YALI0B12342


(SEQ ID NO.: 49)



Nucleotide =



atggctaaagacaaggaaatcgactttgactacacgggagaactggtgatggacgatttcgagttcccca


 


tcgacgacatgctccacaacgacggagatgactttgtcaagaaggaaacgtgggacgagggttttggttt


 


cggaacaaatggcgccgtgggtgcgcagatggacgtccagaccagcccatttagcgaccctgtttttggc


 


ggcgtgggagcaggccctgacatgatgggtctcatggatacaaacatgaaccacatcaacggtagtcaca


 


acatgaacagcgtcgtcaagcaggaggactactacacaccgtccatgggcactcccatgaacccccaaca


 


gcaacagtccatgacccctcaacagcagcatcacatgaaccacaaccagccctctcagctccaatctttg


 


catcaacagtcccagaaggctcaaccacagcagcaacaacaacagccacatcagtcgacaggagtcgata


 


gcataatcacaaaggcatacaccagggcagcaggagacctaccgtacggacgaaagtactcacgacaact


 


caacaagtaccccgaggacgtggagtattcatctttcgacccatcgctatggagcaatttgctgaccaac


 


tcggaaactccgtaccaataccagatacatgtccattccatgcccggaaaatcacgtgtggagacccaaa


 


tcaaatgtgcattatcaatctaccctccgcctccacagcagtccgttcgacttccgacagacaccatttc


 


gcgtcccaagttccagctcaagcagggccacattccagactcgtgtctctccttggaagtatacattgtg


 


ggcgagcagaaccccagcaagcccgtcaatttgtgttctagatgcatcaaacgagaacagaagcgagcct


 


gtcgaaagaaactctttgacgagtcggaggagctgtcgtgggtcgagactcgtcaacgacgtctggctgt


 


cttcaactgctccgaggtgcttgagttcaaggatgtggaacggcgagtatacatccccgagtccggcact


 


acagttaccgccaagcagctggttctgcccctgcgtctggcttgctactgtagacaccacggggagaaaa


 


agggatttcgaatcctcttttgtcttagagacgagggaggccagattgtgggtgtgggccagagtggaac


 


gaccgtcatgatcactgacgaccacaaggttgtgggagacgcggttgccatgccgactacagccactgct


 


cctgccaccgctggctcttcacaaccccccacccaggttcctacccccgctgcatcttcgtcgacgagct


 


atcgtcctcgaaactcgcttcctctatcgcctacttccatggaagactcttcgtcggagttcacctcgga


 


ccattctcattactccaactatggttctaaacgacgacgagacggctcttccatcagcgattggagcggc


 


atgatgaacgtgcgaggcatggatagacaggcttccattaccagcattcccgaaatggttggtggcatgt


 


cgaacatgactgtggccagtgcttcgggtagcgccactaatctggctgctcacaacatgaacaaccccgc


 


agacgaaaacctgcccgtcatcaagcgaatcatcccctcgcagggttccattcgaggcggcattgaagta


 


accctgcttggatctggcttcaagtccaatctggtggctgttttcggtgacaacaaggccgtgggcaccc


 


actgctggtctgattcgaccatcgtgacccatctgccgccttcgaccatcgtgggtcccgttgtggtgtc


 


tttcgaaggttttgtgctcgacaagcctcagatttttacctattttgacgacacagacggccagttgatt


 


gagttggcgctccaggttgtgggtctcaagatgaacggacggctggaagacgcccgaaacattgccatgc


 


gaatcgtgggcaacaatggaggcgttgcgggcgcacaaggcgccatggcaggcgggaacatgtctaacgg


 


agacgttggaatggaaagtgctgctgcagacagttcggttcaacccgtatcgcctcccacagaccacgaa


 


gatgtggttctgcgatgtctggctctcacagacattcctggaggccgaattgccaactggcaactcacca


 


acgccgagggacagaccatggttcatctggccagtattctgggttactcgcgtgttctggtggctcttgt


 


ggctcgaggagctcgtgtggatgtttccgacaatggtggattcactcctcttcatttcgctgctctcttt


 


ggccgtcgaaagattgccaagaaactacttcggtgcaacgctgacccctacaaacgtaaccgaattggcg


 


aaaccgtgtttgatgttgcttgtcctcacattctcgatcttctggtcggtcctcagggcatgcctatggc


 


cgttcagacgtcgtatactcccgattaccatcgtcagcgtcgatcttcatcttcttccactctggcttcc


 


attgcatccatccaggattcgcgtgagtacggtttctatgaccatggaatgatttccaacctgtcgcata


 


ttccgtccacgtgctccattcgatcatcgacttctcagtttgacgctgaagacgagtgggacgagcgaga


 


tgaggaggatggagactttgacgacgattcagatgaggactcagacgatgactcagacgcgctcttcatg


 


tctgttagaaagcacgccaaggccaagtctgtggaatctcctctctctgaggaggaagagcgacttgtgc


 


gacacattgaggccgaagaccaggctgtggaggcccgtgtggctgccggaatcgtcagtagcaatgtacc


 


cgacgtggtgtcttccaatgactcggatcacgtgagatctgacacttccactgagaacaagtccttttca


 


cggtactttgaccgtactctcagcatggcatcttgggacgatgttctggcttacatttacagacccaagc


 


gagctactgtgcccaacaagcggtcttctggagctcctccttcagtcagatccacaagatcgcctctttc


 


ggaccatcccatcacgtcttcgggagacgagtccgaccgaaccatttctgcacatgccccttccggcggt


 


gccggtcgaggccggtctcattcgtccatctcgcgaatgtggcgatacctgaagaactcgtctgccgatg


 


aggccacccggtctcgatctcgagatgcaaacggagccggtgctccccctgcctacgaagaaatcttccc


 


tggccatggggttgtccacgacaagaaggttgtgcagatggccgctgcttctgctgccgagaactcgtct


 


gggcctgttggagcctcatcttcagcagttgcgtccacttctgcggctgccgctgtggtgccctccccac


 


tagcccccattgtggaggacgaggagcagctggtagaggcctggagacgacagcgacgatccatggctaa


 


cgatcgcatgttatttgccttctggctgcctgtgctgctcatggctattggttatatggtcatcaaggcg


 


tttggtctgttccccgaccaggtctctgccgttgagtctgtggctgagactgtgggtgtccactgccgtg


 


gagcagttgccaagctatggttcaagcagtaccctgttcaccgaggccagccactcaaggacacctgttc


 


atttgagcccaacagtctggtagagtcagctcttcgtcagatgaatgggtggtccgaccgggaggttccc


 


attcatcaagcccaggcccaggctgcatga


 


(SEQ ID NO.: 50)



Amino Acid =



MAKDKEIDFDYTGELVMDDFEFPIDDMLHNDGDDFVKKETWDEGFGFGTNGAVGAQMDVQTSPFSDPVFG


 


GVGAGPDMMGLMDTNMNHINGSHNMNSVVKQEDYYTPSMGTPMNPQQQQSMTPQQQHHMNHNQPSQLQSL


 


HQQSQKAQPQQQQQQPHQSTGVDSIITKAYTRAAGDLPYGRKYSRQLNKYPEDVEYSSFDPSLWSNLLTN


 


SETPYQYQIHVHSMPGKSRVETQIKCALSIYPPPPQQSVRLPTDTISRPKFQLKQGHIPDSCLSLEVYIV


 


GEQNPSKPVNLCSRCIKREQKRACRKKLFDESEELSWVETRQRRLAVFNCSEVLEFKDVERRVYIPESGT


 


TVTAKQLVLPLRLACYCRHHGEKKGFRILFCLRDEGGQIVGVGQSGTTVMITDDHKVVGDAVAMPTTATA


 


PATAGSSQPPTQVPTPAASSSTSYRPRNSLPLSPTSMEDSSSEFTSDHSHYSNYGSKRRRDGSSISDWSG


 


MMNVRGMDRQASITSIPEMVGGMSNMTVASASGSATNLAAHNMNNPADENLPVIKRIIPSQGSIRGGIEV


 


TLLGSGFKSNLVAVFGDNKAVGTHCWSDSTIVTHLPPSTIVGPVVVSFEGFVLDKPQIFTYFDDTDGQLI


 


ELALQVVGLKMNGRLEDARNIAMRIVGNNGGVAGAQGAMAGGNMSNGDVGMESAAADSSVQPVSPPTDHE


 


DVVLRCLALTDIPGGRIANWQLTNAEGQTMVHLASILGYSRVLVALVARGARVDVSDNGGFTPLHFAALF


 


GRRKIAKKLLRCNADPYKRNRIGETVFDVACPHILDLLVGPQGMPMAVQTSYTPDYHRQRRSSSSSTLAS


 


IASIQDSREYGFYDHGMISNLSHIPSTCSIRSSTSQFDAEDEWDERDEEDGDFDDDSDEDSDDDSDALFM


 


SVRKHAKAKSVESPLSEEEERLVRHIEAEDQAVEARVAAGIVSSNVPDVVSSNDSDHVRSDTSTENKSFS


 


RYFDRTLSMASWDDVLAYIYRPKRATVPNKRSSGAPPSVRSTRSPLSDHPITSSGDESDRTISAHAPSGG


 


AGRGRSHSSISRMWRYLKNSSADEATRSRSRDANGAGAPPAYEEIFPGHGVVHDKKVVQMAAASAAENSS


 


GPVGASSSAVASTSAAAAVVPSPLAPIVEDEEQLVEAWRRQRRSMANDRMLFAFWLPVLLMAIGYMVIKA


 


FGLFPDQVSAVESVAETVGVHCRGAVAKLWFKQYPVHRGQPLKDTCSFEPNSLVESALRQMNGWSDREVP


 


IHQAQAQAA*


 


Mga2-L36-mutant version


(SEQ ID NO.: 51)



Nucleotide =



atggctaaagacaaggaaatcgactttgactacacgggagaactggtgatggacgatttcgagttcccca


 


tcgacgacatgctccacaacgacggagatgactttgtcaagaaggaaacgtgggacgagggttttggttt


 


cggaacaaatggcgccgtgggtgcgcagatggacgtccagaccagcccatttagcgaccctgtttttggc


 


ggcgtgggagcaggccctgacatgatgggtctcatggatacaaacatgaaccacatcaacggtagtcaca


 


acatgaacagcgtcgtcaagcaggaggactactacacaccgtccatgggcactcccatgaacccccaaca


 


gcaacagtccatgacccctcaacagcagcatcacatgaaccacaaccagccctctcagctccaatctttg


 


catcaacagtcccagaaggctcaaccacagcagcaacaacaacagccacatcagtcgacaggagtcgata


 


gcataatcacaaaggcatacaccagggcagcaggagacctaccgtacggacgaaagtactcacgacaact


 


caacaagtaccccgaggacgtggagtattcatctttcgacccatcgctatggagcaatttgctgaccaac


 


tcggaaactccgtaccaataccagatacatgtccattccatgcccggaaaatcacgtgtggagacccaaa


 


tcaaatgtgcattatcaatctaccctccgcctccacagcagtccgttcgacttccgacagacaccatttc


 


gcgtcccaagttccagctcaagcagggccacattccagactcgtgtctctccttggaagtatacattgtg


 


ggcgagcagaaccccagcaagcccgtcaatttgtgttctagatgcatcaaacgagaacagaagcgagcct


 


gtcgaaagaaactctttgacgagtcggaggagctgtcgtgggtcgagactcgtcaacgacgtctggctgt


 


cttcaactgctccgaggtgcttgagttcaaggatgtggaacggcgagtatacatccccgagtccggcact


 


acagttaccgccaagcagctggttctgcccctgcgtctggcttgctactgtagacaccacggggagaaaa


 


agggatttcgaatcctcttttgtcttagagacgagggaggccagattgtgggtgtgggccagagtggaac


 


gaccgtcatgatcactgacgaccacaaggttgtgggagacgcggttgccatgccgactacagccactgct


 


cctgccaccgctggctcttcacaaccccccacccaggttcctacccccgctgcatcttcgtcgacgagct


 


atcgtcctcgaaactcgcttcctctatcgcctacttccatggaagactcttcgtcggagttcacctcgga


 


ccattctcattactccaactatggttctaaacgacgacgagacggctcttccatcagcgattggagcggc


 


atgatgaacgtgcgaggcatggatagacaggcttccattaccagcattcccgaaatggttggtggcatgt


 


cgaacatgactgtggccagtgcttcgggtagcgccactaatctggctgctcacaacatgaacaaccccgc


 


agacgaaaacctgcccgtcatcaagcgaatcatcccctcgcagggttccattcgaggcggcattgaagta


 


accctgcttggatctggcttcaagtccaatctggtggctgttttcggtgacaacaaggccgtgggcaccc


 


actgctggtctgattcgaccatcgtgacccatctgccgccttcgaccatcgtgggtcccgttgtggtgtc


 


tttcgaaggttttgtgctcgacaagcctcagatttttacctattttgacgacacagacggccagttgatt


 


gagttggcgctccaggttgtgggtctcaagatgaacagacggctggaagacgcccgaaacattgccatgc


 


gaatcgtgggcaacaatggaggcgttgcgggcgcacaaggcgccatggcaggcgggaacatgtctaacgg


 


agacgttggaatggaaagtgctgctgcagacagttcggttcaacccgtatcgcctcccacagaccacgaa


 


gatgtggttctgcgatgtctggctctcacagacattcctggaggccgaattgccaactggcaactcacca


 


acgccgagggacagaccatggttcatctggccagtattctgggttactcgcgtgttctggtggctcttgt


 


ggctcgaggagctcgtgtggatgtttccgacaatggtggattcactcctcttcatttcgctgctctcttt


 


ggccgtcgaaagattgccaagaaactacttcggtgcaacgctgacccctacaaacgtaaccgaattggcg


 


aaaccgtgtttgatgttgcttgtcctcacattctcgatcttctggtcggtcctcagggcatgcctatggc


 


cgttcagacgtcgtatactcccgattaccatcgtcagcgtcgatcttcatcttcttccactctggcttcc


 


attgcatccatccaggattcgcgtgagtacggtttctatgaccatggaatgatttccaacctgtcgcata


 


ttccgtccacgtgctccattcgatcatcgacttctcagtttgacgctgaagacgagtgggacgagcgaga


 


tgaggaggatggagactttgacgacgattcagatgaggactcagacgatgactcagacgcgctcttcatg


 


tctgttagaaagcacgccaaggccaagtctgtggaatctcctctctctgaggaggaagagcgacttgtgc


 


gacacattgaggccgaagaccaggctgtggaggcccgtgtggctgccggaatcgtcagtagcaatgtacc


 


cgacgtggtgtcttccaatgactcggatcacgtgagatctgacacttccactgagaacaagtccttttca


 


cggtactttgaccgtactctcagcatggcatcttgggacgatgttctggcttacatttacagacccaagc


 


gagctactgtgcccaacaagcggtcttctggagctcctccttcagtcagatccacaagatcgcctctttc


 


ggaccatcccatcacgtcttcgggagacgagtccgaccgaaccatttctgcacatgccccttccggcggt


 


gccggtcgaggccggtctcattcgtccatctcgcgaatgtggcgatacctgaagaactcgtctgccgatg


 


aggccacccggtctcgatctcgagatgcaaacggagccggtgctccccctgcctacgaagaaatcttccc


 


tggccatggggttgtccacgacaagaaggttgtgcagatggccgctgcttctgctgccgagaactcgtct


 


gggcctgttggagcctcatcttcagcagttgcgtccacttctgcggctgccgctgtggtgccctccccac


 


tagcccccattgtggaggacgaggagcagctggtagaggcctggagacgacagcgacgatccatggctaa


 


cgatcgcatgttatttgccttctggctgcctgtgctgctcatggctattggttatatggtcatcaaggcg


 


tttggtctgttccccgaccaggtctctgccgttgagtctgtggctgagactgtgggtgtccactgccgtg


 


gagcagttgccaagctatggttcaagcagtaccctgttcaccgaggccagccactcaaggacacctgttc


 


atttgagcccaacagtctggtagagtcagctcttcgtcagatgaatgggtggtccgaccgggaggttccc


 


attcatcaagcccaggcccaggctgcatga


 


(SEQ ID NO.: 52)



Amino Acid =



MAKDKEIDFDYTGELVMDDFEFPIDDMLHNDGDDFVKKETWDEGFGFGTNGAVGAQMDVQTSPFSDPVFG


 


GVGAGPDMMGLMDTNMNHINGSHNMNSVVKQEDYYTPSMGTPMNPQQQQSMTPQQQHHMNHNQPSQLQSL


 


HQQSQKAQPQQQQQQPHQSTGVDSIITKAYTRAAGDLPYGRKYSRQLNKYPEDVEYSSFDPSLWSNLLTN


 


SETPYQYQIHVHSMPGKSRVETQIKCALSIYPPPPQQSVRLPTDTISRPKFQLKQGHIPDSCLSLEVYIV


 


GEQNPSKPVNLCSRCIKREQKRACRKKLFDESEELSWVETRQRRLAVFNCSEVLEFKDVERRVYIPESGT


 


TVTAKQLVLPLRLACYCRHHGEKKGFRILFCLRDEGGQIVGVGQSGTTVMITDDHKVVGDAVAMPTTATA


 


PATAGSSQPPTQVPTPAASSSTSYRPRNSLPLSPTSMEDSSSEFTSDHSHYSNYGSKRRRDGSSISDWSG


 


MMNVRGMDRQASITSIPEMVGGMSNMTVASASGSATNLAAHNMNNPADENLPVIKRIIPSQGSIRGGIEV


 


TLLGSGFKSNLVAVFGDNKAVGTHCWSDSTIVTHLPPSTIVGPVVVSFEGFVLDKPQIFTYFDDTDGQLI


 


ELALQVVGLKMNRRLEDARNIAMRIVGNNGGVAGAQGAMAGGNMSNGDVGMESAAADSSVQPVSPPTDHE


 


DVVLRCLALTDIPGGRIANWQLTNAEGQTMVHLASILGYSRVLVALVARGARVDVSDNGGFTPLHFAALF


 


GRRKIAKKLLRCNADPYKRNRIGETVFDVACPHILDLLVGPQGMPMAVQTSYTPDYHRQRRSSSSSTLAS


 


IASIQDSREYGFYDHGMISNLSHIPSTCSIRSSTSQFDAEDEWDERDEEDGDFDDDSDEDSDDDSDALFM


 


SVRKHAKAKSVESPLSEEEERLVRHIEAEDQAVEARVAAGIVSSNVPDVVSSNDSDHVRSDTSTENKSFS


 


RYFDRTLSMASWDDVLAYIYRPKRATVPNKRSSGAPPSVRSTRSPLSDHPITSSGDESDRTISAHAPSGG


 


AGRGRSHSSISRMWRYLKNSSADEATRSRSRDANGAGAPPAYEEIFPGHGVVHDKKVVQMAAASAAENSS


 


GPVGASSSAVASTSAAAAVVPSPLAPIVEDEEQLVEAWRRQRRSMANDRMLFAFWLPVLLMAIGYMVIKA


 


FGLFPDQVSAVESVAETVGVHCRGAVAKLWFKQYPVHRGQPLKDTCSFEPNSLVESALRQMNGWSDREVP


 


IHQAQAQAA*


 


Mga2-truncated version removing of transmembrane span.


(SEQ ID NO.: 53)



Nucleotide =



atggctaaagacaaggaaatcgactttgactacacgggagaactggtgatggacgatttcgagttcccca


 


tcgacgacatgctccacaacgacggagatgactttgtcaagaaggaaacgtgggacgagggttttggttt


 


cggaacaaatggcgccgtgggtgcgcagatggacgtccagaccagcccatttagcgaccctgtttttggc


 


ggcgtgggagcaggccctgacatgatgggtctcatggatacaaacatgaaccacatcaacggtagtcaca


 


acatgaacagcgtcgtcaagcaggaggactactacacaccgtccatgggcactcccatgaacccccaaca


 


gcaacagtccatgacccctcaacagcagcatcacatgaaccacaaccagccctctcagctccaatctttg


 


catcaacagtcccagaaggctcaaccacagcagcaacaacaacagccacatcagtcgacaggagtcgata


 


gcataatcacaaaggcatacaccagggcagcaggagacctaccgtacggacgaaagtactcacgacaact


 


caacaagtaccccgaggacgtggagtattcatctttcgacccatcgctatggagcaatttgctgaccaac


 


tcggaaactccgtaccaataccagatacatgtccattccatgcccggaaaatcacgtgtggagacccaaa


 


tcaaatgtgcattatcaatctaccctccgcctccacagcagtccgttcgacttccgacagacaccatttc


 


gcgtcccaagttccagctcaagcagggccacattccagactcgtgtctctccttggaagtatacattgtg


 


ggcgagcagaaccccagcaagcccgtcaatttgtgttctagatgcatcaaacgagaacagaagcgagcct


 


gtcgaaagaaactctttgacgagtcggaggagctgtcgtgggtcgagactcgtcaacgacgtctggctgt


 


cttcaactgctccgaggtgcttgagttcaaggatgtggaacggcgagtatacatccccgagtccggcact


 


acagttaccgccaagcagctggttctgcccctgcgtctggcttgctactgtagacaccacggggagaaaa


 


agggatttcgaatcctcttttgtcttagagacgagggaggccagattgtgggtgtgggccagagtggaac


 


gaccgtcatgatcactgacgaccacaaggttgtgggagacgcggttgccatgccgactacagccactgct


 


cctgccaccgctggctcttcacaaccccccacccaggttcctacccccgctgcatcttcgtcgacgagct


 


atcgtcctcgaaactcgcttcctctatcgcctacttccatggaagactcttcgtcggagttcacctcgga


 


ccattctcattactccaactatggttctaaacgacgacgagacggctcttccatcagcgattggagcggc


 


atgatgaacgtgcgaggcatggatagacaggcttccattaccagcattcccgaaatggttggtggcatgt


 


cgaacatgactgtggccagtgcttcgggtagcgccactaatctggctgctcacaacatgaacaaccccgc


 


agacgaaaacctgcccgtcatcaagcgaatcatcccctcgcagggttccattcgaggcggcattgaagta


 


accctgcttggatctggcttcaagtccaatctggtggctgttttcggtgacaacaaggccgtgggcaccc


 


actgctggtctgattcgaccatcgtgacccatctgccgccttcgaccatcgtgggtcccgttgtggtgtc


 


tttcgaaggttttgtgctcgacaagcctcagatttttacctattttgacgacacagacggccagttgatt


 


gagttggcgctccaggttgtgggtctcaagatgaacggacggctggaagacgcccgaaacattgccatgc


 


gaatcgtgggcaacaatggaggcgttgcgggcgcacaaggcgccatggcaggcgggaacatgtctaacgg


 


agacgttggaatggaaagtgctgctgcagacagttcggttcaacccgtatcgcctcccacagaccacgaa


 


gatgtggttctgcgatgtctggctctcacagacattcctggaggccgaattgccaactggcaactcacca


 


acgccgagggacagaccatggttcatctggccagtattctgggttactcgcgtgttctggtggctcttgt


 


ggctcgaggagctcgtgtggatgtttccgacaatggtggattcactcctcttcatttcgctgctctcttt


 


ggccgtcgaaagattgccaagaaactacttcggtgcaacgctgacccctacaaacgtaaccgaattggcg


 


aaaccgtgtttgatgttgcttgtcctcacattctcgatcttctggtcggtcctcagggcatgcctatggc


 


cgttcagacgtcgtatactcccgattaccatcgtcagcgtcgatcttcatcttcttccactctggcttcc


 


attgcatccatccaggattcgcgtgagtacggtttctatgaccatggaatgatttccaacctgtcgcata


 


ttccgtccacgtgctccattcgatcatcgacttctcagtttgacgctgaagacgagtgggacgagcgaga


 


tgaggaggatggagactttgacgacgattcagatgaggactcagacgatgactcagacgcgctcttcatg


 


tctgttagaaagcacgccaaggccaagtctgtggaatctcctctctctgaggaggaagagcgacttgtgc


 


gacacattgaggccgaagaccaggctgtggaggcccgtgtggctgccggaatcgtcagtagcaatgtacc


 


cgacgtggtgtcttccaatgactcggatcacgtgagatctgacacttccactgagaacaagtccttttca


 


cggtactttgaccgtactctcagcatggcatcttgggacgatgttctggcttacatttactga


 


(SEQ ID NO.: 54)



Amino Acid =



MAKDKEIDFDYTGELVMDDFEFPIDDMLHNDGDDFVKKETWDEGFGFGTNGAVGAQMDVQTSPFSDPVFG


 


GVGAGPDMMGLMDTNMNHINGSHNMNSVVKQEDYYTPSMGTPMNPQQQQSMTPQQQHHMNHNQPSQLQSL


 


HQQSQKAQPQQQQQQPHQSTGVDSIITKAYTRAAGDLPYGRKYSRQLNKYPEDVEYSSFDPSLWSNLLTN


 


SETPYQYQIHVHSMPGKSRVETQIKCALSIYPPPPQQSVRLPTDTISRPKFQLKQGHIPDSCLSLEVYIV


 


GEQNPSKPVNLCSRCIKREQKRACRKKLFDESEELSWVETRQRRLAVFNCSEVLEFKDVERRVYIPESGT


 


TVTAKQLVLPLRLACYCRHHGEKKGFRILFCLRDEGGQIVGVGQSGTTVMITDDHKVVGDAVAMPTTATA


 


PATAGSSQPPTQVPTPAASSSTSYRPRNSLPLSPTSMEDSSSEFTSDHSHYSNYGSKRRRDGSSISDWSG


 


MMNVRGMDRQASITSIPEMVGGMSNMTVASASGSATNLAAHNMNNPADENLPVIKRIIPSQGSIRGGIEV


 


TLLGSGFKSNLVAVFGDNKAVGTHCWSDSTIVTHLPPSTIVGPVVVSFEGFVLDKPQIFTYFDDTDGQLI


 


ELALQVVGLKMNGRLEDARNIAMRIVGNNGGVAGAQGAMAGGNMSNGDVGMESAAADSSVQPVSPPTDHE


 


DVVLRCLALTDIPGGRIANWQLTNAEGQTMVHLASILGYSRVLVALVARGARVDVSDNGGFTPLHFAALF


 


GRRKIAKKLLRCNADPYKRNRIGETVFDVACPHILDLLVGPQGMPMAVQTSYTPDYHRQRRSSSSSTLAS


 


IASIQDSREYGFYDHGMISNLSHIPSTCSIRSSTSQFDAEDEWDERDEEDGDFDDDSDEDSDDDSDALFM


 


SVRKHAKAKSVESPLSEEEERLVRHIEAEDQAVEARVAAGIVSSNVPDVVSSNDSDHVRSDTSTENKSFS


 


RYFDRTLSMASWDDVLAYIY*


 


Sou2L36 YALI0D18964g


(SEQ ID NO.: 55)



Nucleotide =



Atgtctggaccttccaccctcgccacgggactgcaccctctccccacagagaccccaaagttccccacca


 


acatcatggaccgattctccctcaagggtaaggttgcctccgtcaccggctcctcgtcaggtatcggcta


 


ctgcgtggccgaggcctacgcccaggccggtgccgacgtggccatctggtacaactcccaccccgccgac


 


gcaaaggctgagcacctcgctaagacctacggcgtcaaggccaaggcctacaagtgccctgtcaccgacg


 


ccgccgccgtggagtccaccatccagcagatcgagaaggactttggcaccattgacatcttcgtcgccaa


 


cgctggtgtcccctggaccgccggccccatgatcgacgtgcccgacaacaaggagtgggacaaggtcatc


 


aacctggatctcaacggtgcctactactgcgccaagtacgccggccagatcttcaagaagaagggcaagg


 


gatccttcatcttcaccgcctccatgtccggccacattgtcaacatcccccagatgcaggcctgctacaa


 


cgccgccaaggccgctctgctgcacctgtctcgatcgctggccgtcgagtgggccggctttgcccgatgc


 


aacacagtctcccctggctacatggccaccgagatctccgactttgtccccaaggagaccaaggagaagt


 


ggtggcagctcattcccatgggccgagagggagacccctccgagctctagcctacctctaccttgcctct


 


ga


 


CEN0EL36 YALI0D15444s


(SEQ ID NO.: 56)



Nucleotide =



Cacaaatattcttgatttactttggttttgccctattcggaaattttattgatatctaatagaagtatta


 


aagtaaaaatgtactaatacttaattgtaatgtcatcagaaataacatttgaggaaaatatttcaaacct


 


aattgatatatatattagagatgtcccgcttctctgtcattaatatattcaagcaatcga


 


DEHA0Al298g IPF 95.1 YALI0E33891g


(SEQ ID NO.: 57)



Nucleotide =



Atgaagtccacctccgctactctcctcgcccttgccgcccttgtcgttgccgacaacgccgtcgtctctc


 


agatcaacgatggccagatccaggctcctcccgctggtggtgagggtgccaagcccgcccctgctccttc


 


tggagctgcccccggtgcccccggtgctggtgctcccggcgctggtgctcccggcgctggtgcccctggc


 


gctggcgagggtgctaagccctctggagctgcccccggtgcccccggcgctggtgctcccggtgctggtg


 


agggtgctaagccttctggcggtgcccccggtgctggcgctcctggtgctggcgagggtgctaagccctc


 


tggtggtgcccctggtgcccccggcgctggtgctcccggtgctggtgagggtgctaagccctctggtggt


 


gcccccggtgcccccggcgctggtgagggtgccaagccctccggctctgctcccggtgctcctggcgctg


 


gtgagggtgccaagccctccggctctgctcccggtgctcctggcgctggtgagggtgccaagccctctgg


 


ctctgctcccggtgctcctggtgctggtgagggtgccaagccctctggctctgctcccggtgctcctgga


 


gctggtgcaggtgctaagccctccgctggaggtgagcaccccgctgctgaggccactggtgtcgtcactc


 


agatccacgacggccagatccaggctcccgagcagacccagccccccgctgccggccctgcccaggctaa


 


cggtgctgccaccctcggtgcccagatcgttgccggtgttgtcgccgctgccggtgtcgctctcttctaa


 


RLF2 chromatin assembly complex subunit p90 YALI0F21637g


(SEQ ID NO.: 58)



Nucleotide =



atggccgacaacaagcctctgtgcacgattaccacgcccgaaccgtcacccaagcgtcgaaagatctctg


 


ccgaggagaaagaaaagatgcgacttgaaaaggaacagatcaagaagcagaaagaggaagagcgagagca


 


gcttcgaagacagaaggaagaagagaaagagctactgagaaagcagaaagaggaggagaaggaacaactg


 


aggaaacagaaggaggaggagaagagggctaaagaggaggagagagggctagagagagggagaaaacgac


 


gacgagaagaggaacgaaagaaggctgccgaagagaaggagcttgagcgagccaagattgcagaggagaa


 


ggctaagttggctgaagagaaggaggccaagagacttgaaaaagaagctgaactcaagaagaaggagcaa


 


gaacagactcgaatcatgtctttctttaacaagaagaccaaaaagaagaccaagaaggaagctgttaaca


 


gtgacaagtgtttggactttgataaagacttcctacccttccacatcaaagataccgtgtgtatggcaga


 


caagacggagtgtgaagtgatggatcaggatcctgttgactggctcaacagtctcaacctttctgatgac


 


agcaacaccgccgaagcagaagaaccacctgttcccgtcaaaaccatcattactcacatccagaccgctg


 


ccactctgggtctcaatcctgataattacaacggtactcctttagacacgctggtcaatgctcttcctag


 


acgatacttgcagttctatggtgacgagcgacccgcatacctgggcacgtactccaagagctgctcgcgt


 


gatctgttgcagaaccctctcttccaggtgcctggtttggactacgagtacgacagtgaggcagactggg


 


aagatgaaggagaagatattgaagatgatgaaattagtggagacgaggagatggaggacgacgaaatggc


 


cgactttgtgtgttctgatgatgccaagagtcccagcaccatgacttcaaaggtcacgacagcccaggaa


 


cctgttgttgtctggggctgctcagatatggttggtatgacttttggaggactgattgtccagggggcaa


 


ttgacccattcaaagactattggactgttgcaaaagttgagcagaagaccgatactaagagtgacgtgac


 


aatgactagtgcgacatcagcttctggtacagctattaaatctactacaaccaaaaccgaactcagcccg


 


tttgaagtcctctccaaaactctgtcaccttccccagcggttgcttcagccacgaaacagtttctggctg


 


ctgccaagcctcagaagctcattgctggagacgacctgactgctcttttgaagcgagtagatggatccga


 


cgataacaagacgctgttgaccgagctgctttgtaagcagtatccccagtacacacgcaagatggtcacg


 


gccaccattcagcactatgctgagcgacagggtcctaagagcgacaagcggtgggttctgaaggatatct


 


ag


 


TUP1 - YALI0A14542


(SEQ ID NO.: 59)



Nucleotide =



atgagcttcccccaacaagtaatagcgccgggccaacggctcaacgagcttctggaggccatcaaacagg


 


agttcgactccgtgaccaacgaggcgtccgtctaccggctgcacaaggacgagtttgacgtcaaggtgaa


 


ccagcagacgtcagatctgggccagattcgacagtcggtctacgagctagaaatggcgcaccgaaagatg


 


aaggagcgctacgaggaggaaatcatgcggctcaagagcgagctggaggcccgaggtggacccgctgcga


 


accccgcacactcccagcagcagcaacagcagcaacagcaacagcagcaacagcagcagcagaaccagca


 


ggcacaggaccaacaagcacgggccgcgcaacaacaggcagcccagcagcaggccctcgcccagcagcag


 


gccgcccagcagcaggctctggcccaacagcaggcccaggctcaacagcaggcccaggcccaggcccacc


 


acatgggtggtgtgcccccttcgcaaggacagcccccgtcgctgctgcgtccatcatccaacgtgttcag


 


cggcatcatgtccggtcagcccggcacctcttctctggctcccccgcagggacagcccggtcagccccag


 


cctggtcagccccaacctggtcaaccccagccctactccggctacgtgggtgctaacggctacacgtctt


 


cgccacataacggaccccccgtcatcagcgcaatggcctcgcccaacagcaagaagcgacaggtgtcgac


 


ccccgttcccggcaaggcgtctccccaggtggccccccaagagatgcaacagcagcagcaacagcagggc


 


cctccacagcagcagcaacctccccagcagcagcaacagagccccgaagagatgggcaactacctgggcg


 


acatggacattgagcgggtacctccggagctcaaaaaacaaaaggccgactggtttgtcgtttacaacca


 


gcgagcaccacggctgctggacgtggatattgtgcagtcgctggaccacaactctgtagtgtgctgtgtg


 


cggttctccgctgacggcaagtacattgccactggctgtaaccgatctgcccagattttcgacgtgcaga


 


ctggccagctcatctgccggctgcaggacgactcggtcgaccgagaaggcgacctgtacatccggtccgt


 


gtgtttctcgccggacggtaagtacctggccaccggcgccgaggacaagcagatccgagtgtgggacatt


 


aaatctcagagcatacggcacgtgttcactggccacgagcaggacatttactcgctggacttttcgcgaa


 


acggccgacacattgcctctggctctggcgaccgcacagtccgaatgtgggatattgagagcggccagtg


 


tactctaaccctgtcgatcgaggacggcgtcaccacggtggccatctcgcccgacggcaagtttgtggct


 


gcaggcagcttggacaagtctgtgcgaatctgggacacctctaccggtttcctggttgagcgtctggagg


 


cccctgatggacacaaggactccgtctatagtgtagctttcacccccaacggtatggatcttgtttccgg


 


ctcgctggacaagacgatcaagctgtgggagctgcaggctcctcgaggcattcaggccaaccagcgagga


 


ggcgtctgcgtcaagacgctgtgtggacacaaggactttgttctgagtgtggccagcacgctggatgggc


 


agtggattctttccggctccaaggaccggggtgtgcaattctgggaccctcgaacgggccaggtgcaact


 


catgctgcagggtcatcgaaattcggtcatcagtgtggctcctagtcccatgggcgggttgtttgctact


 


ggaagtggagattgcaaggctcgaatctggcgatactttcctgtcaacagataa


 


(SEQ ID NO.: 60)



Amino Acid =



MSFPQQVIAPGQRLNELLEAIKQEFDSVTNEASVYRLHKDEFDVKVNQQTSDLGQIRQSVYELEMAHRKM


 


KERYEEEIMRLKSELEARGGPAANPAHSQQQQQQQQQQQQQOQQNQQAQDQQARAAQQQAAQQQALAQQQ


 


AAQQQALAQQQAQAQQQAQAQAHHMGGVPPSQGQPPSLLRPSSNVFSGIMSGQPGTSSLAPPQGQPGQPQ


 


PGQPQPGQPQPYSGYVGANGYTSSPHNGPPVISAMASPNSKKRQVSTPVPGKASPQVAPQEMQQQQQQQG


 


PPQQQQPPQQQQQSPEEMGNYLGDMDIERVPPELKKQKADWFVVYNQRAPRLLDVDIVQSLDHNSVVCCV


 


RFSADGKYTATGCNRSAQIFDVQTGQLICRLQDDSVDREGDLYIRSVCFSPDGKYLATGAEDKQIRVWDI


 


KSQSIRHVFTGHEQDIYSLDFSRNGRHIASGSGDRTVRMWDIESGQCTLTLSIEDGVTTVAISPDGKFVA


 


AGSLDKSVRIWDTSTGFLVERLEAPDGHKDSVYSVAFTPNGMDLVSGSLDKTIKLWELQAPRGIQANQRG


 


GVCVKTLCGHKDFVLSVASTLDGQWILSGSKDRGVQFWDPRTGQVQLMLQGHRNSVISVAPSPMGGLFAT


 


GSGDCKARIWRYFPVNR*


 


HAC1 - YALI0B12716


(SEQ ID NO.: 61)



Nucleotide =



atgtctatcaagcgagaagagtcctttactcccacccccgaggacctgggatctcccctgacagctgatt


 


ctcctggctctcccgagtctggagacaagcgaaagaaggatctcactctgccccttcctgctggtgctct


 


tccccctcgaaagagagctaagacagagaacgaaaaggagcagagacgcatcgagcggatcatgcgaaac


 


cggcaggcggcacatgcgtctcgagagaagaagcgacgacatttggaggacctggagaagaagtgctcgg


 


agttgtcgtccgaaaacaacgatctacaccaccaggtgactgagtccaagaagaccaacatgcacctcat


 


ggaacaacactactcgctggtggccaagctgcagcagctctcgtcgctcgtcaacatggccaagtcttcc


 


ggagctttggccggcgttgatgtccccgacatgagcgatgtgtctatggcccccaagttggagatgccca


 


ccgcggctccttcccagcccatgggtctcgccagcgcgcccaccctcttcaaccacgataatgagaccgt


 


cgtccccgactctcctattgtgaagaccgaggaagtcgactctacaaactttctcctccacacggagtcc


 


tcctccccccccgaactagctgagagcactggctcaggctcgccatcgtcgactctgtcctgcgacgaaa


 


ctgattatcttgtggaccgggcgcgtcatccagcagtgatgactgtcgcaactactgaccagcagcgtcg


 


gcacaagatttcattttcatcaaggacgagcccgttgacgacgagcttggactgcatggactgtcggatg


 


acttcaccctgtttgaagacaacaagcagcctgcccagcacgactttattgctgatctag


 


(SEQ ID NO.: 62)



Amino Acid =



MSIKREESFTPTPEDLGSPLTADSPGSPESGDKRKKDLTLPLPAGALPPRKRAKTENEKEQRRIERIMRN


 


RQAAHASREKKRRHLEDLEKKCSELSSENNDLHHQVTESKKTNMHLMEQHYSLVAKLQQLSSLVNMAKSS


 


GALAGVDVPDMSDVSMAPKLEMPTAAPSQPMGLASAPTLFNHDNETVVPDSPIVKTEEVDSTNFLLHTES


 


SSPPELAESTGSGSPSSTLSCDETDYLVDRARHPAVMTVATTDQQRRHKISFSSRTSPLTTSLDCMDCRM


 


TSPCLKTTSSLPSTTLLLI*


 


MRM2- YALI0E31933


(SEQ ID NO.: 63)



Nucleotide =



Atgcgccaaaagctgccgttcaacccgctccagtcgcttctcccgcgaatctttgtgcggggcaaaaaac


 


acgatgcgcgcagccgctgggaaatgcgccagatgaaagacaagcatgtggccatggccaaggctgacgg


 


attccggtctcgagccgcgtacaagctacaggaactcgactccatgttccggctgttcaagcccggcatg


 


acggtggtggatttgggctttgcgcccggcgcatggagtcaagtggctgctcagcgagtgcggcctggag


 


gcagagttattggagtggatatccttccttgcattcctcctccaggagtgtccagcatccagggaaattt


 


cctgtccaaagaaacacaaaacgagctcaaacgtgtgctggccgtctcggcgatgggagttcccaaggac


 


aaggactctggtggcgccataggcactgctcctccgtcttatctggacactgaacgcgagcttggcagta


 


ttaacagcaacagcaacgaaccccaatttggcgacgactacccggtagatatagtgcttagtgacatgtg


 


cgaaacgttaccccaggaacacggattttttcaaagaactattaatgacccatactataggatggccaat


 


gtttccggcatagctgtgagggaccatgctgccagtattgtgagtgaaggaaggaagcgcattgggtgtg


 


gtgcagccagcttcgatgtggcagaagggaagccataa


 


(SEQ ID NO.: 64)



Amino Acid =



MRQKLPFNPLQSLLPRIFVRGKKHDARSRWEMRQMKDKHVAMAKADGFRSRAAYKLQELDSMFRLFKPGM


 


TVVDLGFAPGAWSQVAAQRVRPGGRVIGVDILPCIPPPGVSSIQGNFLSKETQNELKRVLAVSAMGVPKD


 


KDSGGAIGTAPPSYLDTERELGSINSNSNEPQFGDDYPVDIVLSDMCETLPQEHGFFQRTINDPYYRMAN


 


VSGIAVRDHAASIVSEGRKRIGCGAASFDVAEGKP*


 


O6M- YALI0C10010p


(SEQ ID NO.: 65)



Nucleotide =



atgttttacaccaagcccgacccggtggttgattattcccgcctcaaggacatggacatgtatcctgagt


 


acgacaatggccagaacatgggcttttccaacatgaacatgaccgatctttacgacggcggtcttaacat


 


gtcgtcgatggcgcaacccgtggcgttgaaccagatgggcagcatgggccccatgggctctttaagtaac


 


atgcccatgggttttgtgtcccagaaccagcctcaaactcaggctcaggcccaggcccagagccagaacc


 


agaatcagaaccagaaccagaaccagaaccagcctcagaatcacaacacccatgttatgagcgataacca


 


caaccatacccacaccaacaatactcacaacaccaacgtcacccacaacaccccctccatgggtggtcac


 


acaacctctgtcgggggccacgacaccaatgactcggcccatgttgggggtcacgccagcaatgtcacat


 


ccccgaccccggcaacccctgcctccacatcttccgtacccgcaacctcgcctcagattcccttcacggt


 


cgcgccacccgcaccgtcaggcaaatatgtgaccgatgacgagcgatggcaggcactggtcgaccgagac


 


cccgaggctgacggcgccttcatctactgcgtcaccagcaccaaggtgtactgccggcccacgtgctcgg


 


cccggctcgcgctgcggtccaacattgtgtattttgacaccatgaaggaggctgtggccgccggctaccg


 


cccctgccgacggtgcaaccccgacgtgagcgagatgaactcgcagcgacgcgccgtgggctccgtgtgt


 


aacctcatccactcgctggagcccgacaaggtgccacgtgtcaagaagctagccgagtccgtcggcctca


 


cgctctggcactttcaccgtctcttcaagcggtacacgggcctcacgcctcgacagtacatcactgagtt


 


ccacaagcgaaagcgccttgggctgccgcagttgcaagtcagcaaggtggtaaccaagaagagctatgag


 


cgacagcagcgtcgccagggcagcaacggttccacgccccagcagtctccccaagtcggcgcctcttcgc


 


cagccggcgaggtggaggccatcaagctcgagacccccgtcgaaaccgtccagccgctatactacgacag


 


caacggcgtgactcacaacgctgccaacgtcggggctcacagctccaatgtcactcacaacactagccat


 


gtcggaagcaacgcaacctccgccacgagctccattgccactcctctttccaacacaacgtcacccgaca


 


cctcgacgccggcccaggactcggcatacatcattgcccacggttccaacgccagcaacgccgctcctgt


 


ggttgctccggggcctgccaccggctctggcgacaactggatcaagacggagccctcgatggattttatg


 


cctcggtacgagccgcggtacgaccagtctatctccattgacgcccccatgtttattcctgatggtaacg


 


agtatcatcacaacggggagatgttgggtgacatgtgggggactctctaa


 


(SEQ ID NO.: 66)



Amino Acid =



MFYTKPDPVVDYSRLKDMDMYPEYDNGQNMGFSNMNMTDLYDGGLNMSSMAQPVALNQMGSMGPMGSLSN


 


MPMGFVSQNQPQTQAQAQAQSQNQNQNQNQNQNQPQNHNTHVMSDNHNHTHTNNTHNTNVTHNTPSMGGH


 


TTSVGGHDTNDSAHVGGHASNVTSPTPATPASTSSVPATSPQIPFTVAPPAPSGKYVTDDERWQALVDRD


 


PEADGAFIYCVTSTKVYCRPTCSARLALRSNIVYFDTMKEAVAAGYRPCRRCNPDVSEMNSQRRAVGSVC


 


NLIHSLEPDKVPRVKKLAESVGLTLWHFHRLFKRYTGLTPRQYITEFHKRKRLGLPQLQVSKVVTKKSYE


 


RQQRRQGSNGSTPQQSPQVGASSPAGEVEAIKLETPVETVQPLYYDSNGVTHNAANVGAHSSNVTHNTSH


 


VGSNATSATSSIATPLSNTTSPDTSTPAQDSAYIIAHGSNASNAAPVVAPGPATGSGDNWIKTEPSMDFM


 


PRYEPRYDQSISIDAPMFIPDGNEYHHNGEMLGDMWGTL*


 


CIT1 - YALI0E02684


(SEQ ID NO.: 67)



Nucleotide =



atgatttctgctattcgtcccgccgttcgatcttccgttcgtgttgcccctatggccaacaccgccttcc


 


gggcctactctacccaggatgtgagtatttcttttctttcatcaattggttgctgtgcgacggatttcgt


 


tgcgtcagcctgattgcaacagccttaggccccattttcgacctgttcttgcctcggcaaaagtttttcc


 


gaatgcatgtgacacgtcgaatgtggtgctttcaagcagcagcagcagcataaaatatggaatgtgttgt


 


gtgcagaagtcgacattacataaccccgcggcaaccatacgagatggcagtcataacaattgcaattgag


 


caatacaaaccacactgcaacccactaaaaagaaacacgactaacaaatagggtcttaaggagcgattcg


 


ccgagctcatccccgagaacgtcgagaagatcaagaagctccgaaaggagaagggtaacaccgtcatcgg


 


cgaggtcatcctcgaccaggcttacggtggtatgcgaggtattaagggtctcgtctgggagggatccgtc


 


ctcgaccccgaggagggtatccgattccgaggtctgactatccccgacctccagaagcagctcccccacg


 


cccctggcggaaaggagcctctccccgagggtcttttctggctcctgctcaccggcgagatccccactga


 


tgctcaggtcaagggtctgtccgctgactgggcctctcgagccgagatccccaagcatgttgaggagctc


 


atcgaccgatgcccccccaccctccaccccatggctcagctcggtattgccgtcaacgctctggagtccg


 


agtctcagttcaccaaggcttacgagaagggtgttaacaagaaggagtactggcagtacacctacgagga


 


ttccatgaacctcattgccaagctccccgtcattgcttctcgaatctaccgaaaccttttcaaggacgga


 


aagattgttggctccattgacaactctcttgactactctgctaacttcgcctctctgctcggctttggcg


 


acaacaaggagttcattgagcttctgcgactctacctcaccatccacgctgaccacgagggaggtaacgt


 


ctctgcccacaccaccaagcttgttggttctgctctctcctctcccttcctctctctgtccgctggtctc


 


aacggtcttgccggtcctctccacggccgagctaaccaggaggtccttgagtggattctcgagatgaagt


 


ccaagattggctctgatgtcaccaaggaggacattgagaagtacctctgggatacccttaaggccggtcg


 


agtcgtccccggttacggacacgccgttctccgaaagaccgatcctcgatacaccgcccagcgagagttc


 


gccctcgagcacatgcccgactacgacctcttccacctcgtttccaccatctacgaggttgcccccaagg


 


ttctcaccgagcacggcaagaccaagaacccctggcccaatgtggactcccactccggtgtcctcctcca


 


gtactacggtctcactgagcagtcttactacactgttctcttcggtgtttcccgagctatcggtgtcctg


 


ccccagctcatcatggaccgagcttacggtgctcccatcgagcgacccaagtccttctctaccgagaagt


 


acgctgagctcgttggcctcaagctctaa


 


(SEQ ID NO.: 68)



Amino acid =



MISAIRPAVRSSVRVAPMANTAFRAYSTQDGLKERFAELIPENVEKIKKLRKEKGNTVIGEVILDQAYGG


 


MRGIKGLVWEGSVLDPEEGIRFRGLTIPDLQKQLPHAPGGKEPLPEGLFWLLLTGEIPTDAQVKGLSADW


 


ASRAEIPKHVEELIDRCPPTLHPMAQLGIAVNALESESQFTKAYEKGVNKKEYWQYTYEDSMNLIAKLPV


 


IASRIYRNLFKDGKIVGSIDNSLDYSANFASLLGFGDNKEFIELLRLYLTIHADHEGGNVSAHTTKLVGS


 


ALSSPFLSLSAGLNGLAGPLHGRANQEVLEWILEMKSKIGSDVTKEDIEKYLWDTLKAGRVVPGYGHAVL


 


RKTDPRYTAQREFALEHMPDYDLFHLVSTIYEVAPKVLTEHGKTKNPWPNVDSHSGVLLQYYGLTEQSYY


 


TVLFGVSRAIGVLPQLIMDRAYGAPIERPKSFSTEKYAELVGLKL*


 


ACC - YALI0C11407


(SEQ ID NO.: 69)



Nucleotide =



atgcgactgcaattgaggacactaacacgtcggtttttcaggtgagtaaacgacggtggccgtggccacg


 


acagccgaggcgtcacgatgggccagacgagcacattctcgccgccacaacctcgccagcacaagaaact


 


aacccagtatggcttcaggatcttcaacgccagatgtggctcccttggtggaccccaacattcacaaagg


 


tctcgcctctcatttctttggactcaattctgtccacacagccaagccctcaaaagtcaaggagtttgtg


 


gcttctcacggaggtcatacagttatcaacaaggtgagtatttgacgtttagactgtataacaggcggcc


 


gcagtgcaacaacgaccaaaaagggtcgaaaaagggtcgaaaacggacacaaaagctggaaaacaagagt


 


gtaatacattcttacacgtccaattgttagacaaacacggctgttcggtcccaaaaccaccagtatcacc


 


tattttccacttgtgtctcggatctgatcataatctgatctcaagatgaaatttacgccaccgacatgat


 


attgtgattttcggattctccagaccgagcagattccagcaataccaccacttgcccaccttcagcggcc


 


tctcggcgcgattcgccactttccccaacgagtgttactaacccaggtcctcatcgctaacaacggtatt


 


gccgcagtaaaggagatccgttcagtacgaaaatgggcctacgagacctttggcgacgagcgagcaatct


 


cgttcaccgtcatggccacccccgaagatctcgctgccaacgccgactacattagaatggccgatcagta


 


cgtcgaggtgcccggaggaaccaacaacaacaactacgccaacgtcgagctgattgtcgacgtggctgag


 


cgattcggcgtcgatgccgtgtgggccggatggggccatgccagtgaaaatcccctgctccccgagtcgc


 


tagcggcctctccccgcaagattgtcttcatcggccctcccggagctgccatgagatctctgggagacaa


 


aatttcttctaccattgtggcccagcacgcaaaggtcccgtgtatcccgtggtctggaaccggagtggac


 


gaggttgtggttgacaagagcaccaacctcgtgtccgtgtccgaggaggtgtacaccaagggctgcacca


 


ccggtcccaagcagggtctggagaaggctaagcagattggattccccgtgatgatcaaggcttccgaggg


 


aggaggaggaaagggtattcgaaaggttgagcgagaggaggacttcgaggctgcttaccaccaggtcgag


 


ggagagatccccggctcgcccatcttcattatgcagcttgcaggcaatgcccggcatttggaggtgcagc


 


ttctggctgatcagtacggcaacaatatttcactgtttggtcgagattgttcggttcagcgacggcatca


 


aaagattattgaggaggctcctgtgactgtggctggccagcagaccttcactgccatggagaaggctgcc


 


gtgcgactcggtaagcttgtcggatatgtctctgcaggtaccgttgaatatctgtattcccatgaggacg


 


acaagttctacttcttggagctgaatcctcgtcttcaggtcgaacatcctaccaccgagatggtcaccgg


 


tgtcaacctgcccgctgcccagcttcagatcgccatgggtatccccctcgatcgaatcaaggacattcgt


 


ctcttttacggtgttaaccctcacaccaccactccaattgatttcgacttctcgggcgaggatgctgata


 


agacacagcgacgtcccgtcccccgaggtcacaccactgcttgccgaatcacatccgaggaccctggaga


 


gggtttcaagccctccggaggtactatgcacgagctcaacttccgatcctcgtccaacgtgtggggttac


 


ttctccgttggtaaccagggaggtatccattcgttctcggattcgcagtttggtcacatcttcgccttcg


 


gtgagaaccgaagtgcgtctcgaaagcacatggttgttgctttgaaggaactatctattcgaggtgactt


 


ccgaaccaccgtcgagtacctcatcaagctgctggagacaccggacttcgaggacaacaccatcaccacc


 


ggctggctggatgagcttatctccaacaagctgactgccgagcgacccgactcgttcctcgctgttgttt


 


gtggtgctgctaccaaggcccatcgagcttccgaggactctattgccacctacatggcttcgctagagaa


 


gggccaggtccctgctcgagacattctcaagacccttttccccgttgacttcatctacgagggccagcgg


 


tacaagttcaccgccacccggtcgtctgaggactcttacacgctgttcatcaacggttctcgatgcgaca


 


ttggagttagacctctttctgacggtggtattctgtgtcttgtaggtgggagatcccacaatgtctactg


 


gaaggaggaggttggagccacgcgactgtctgttgactccaagacctgccttctcgaggtggagaacgac


 


cccactcagcttcgatctccctctcccggtaagctggttaagttcctggtcgagaacggcgaccacgtgc


 


gagccaaccagccctatgccgagattgaggtcatgaagatgtacatgactctcactgctcaggaggacgg


 


tattgtccagctgatgaagcagcccggttccaccatcgaggctggcgacatcctcggtatcttggccctt


 


gatgatccttccaaggtcaagcatgccaagccctttgagggccagcttcccgagcttggaccccccactc


 


tcagcggtaacaagcctcatcagcgatacgagcactgccagaacgtgctccataacattctgcttggttt


 


cgataaccaggtggtgatgaagtccactcttcaggagatggttggtctgctccgaaaccctgagcttcct


 


tatctccagtgggctcatcaggtgtcttctctgcacacccgaatgagcgccaagctggatgctactcttg


 


ctggtctcattgacaaggccaagcagcgaggtggcgagtttcctgccaagcagcttctgcgagcccttga


 


gaaggaggcgagctctggcgaggtcgatgcgctcttccagcaaactcttgctcctctgtttgaccttgct


 


cgagagtaccaggacggtcttgctatccacgagcttcaggttgctgcaggccttctgcaggcctactacg


 


actctgaggcccggttctgcggacccaacgtacgtgacgaggatgtcattctcaagcttcgagaggagaa


 


ccgagattctcttcgaaaggttgtgatggcccagctgtctcattctcgagtcggagccaagaacaacctt


 


gtgctggcccttctcgatgaatacaaggtggccgaccaggctggcaccgactctcctgcctccaacgtgc


 


acgttgcaaagtacttgcgacctgtgctgcgaaagattgtggagctggaatctcgagcttctgccaaggt


 


atctctgaaagcccgagagattctcatccagtgcgctctgccctctctaaaggagcgaactgaccagctt


 


gagcacattctgcgatcttctgtcgtcgagtctcgatacggagaggttggtctggagcaccgaactcccc


 


gagccgatattctcaaggaggttgtcgactccaagtacattgtctttgatgtgcttgcccagttctttgc


 


ccacgatgatccctggatcgtccttgctgccctggagctgtacatccgacgagcttgcaaggcctactcc


 


atcctggacatcaactaccaccaggactcggacctgcctcccgtcatctcgtggcgatttagactgccta


 


ccatgtcgtctgctttgtacaactcagtagtgtcttctggctccaaaacccccacttccccctcggtgtc


 


tcgagctgattccgtctccgacttttcgtacaccgttgagcgagactctgctcccgctcgaaccggagcg


 


attgttgccgtgcctcatctggatgatctggaggatgctctgactcgtgttctggagaacctgcccaaac


 


ggggcgctggtcttgccatctctgttggtgctagcaacaagagtgccgctgcttctgctcgtgacgctgc


 


tgctgctgccgcttcatccgttgacactggcctgtccaacatttgcaacgttatgattggtcgggttgat


 


gagtctgatgacgacgacactctgattgcccgaatctcccaggtcattgaggactttaaggaggactttg


 


aggcctgttctctgcgacgaatcaccttctccttcggcaactcccgaggtacttatcccaagtatttcac


 


gttccgaggccccgcatacgaggaggaccccactatccgacacattgagcctgctctggccttccagctg


 


gagctcgcccgtctgtccaacttcgacatcaagcctgtccacaccgacaaccgaaacatccacgtgtacg


 


aggctactggcaagaacgctgcttccgacaagcggttcttcacccgaggtatcgtacgacctggtcgtct


 


tcgagagaacatccccacctcggagtatctcatttccgaggctgaccggctcatgagcgatattttggac


 


gctctagaggtgattggaaccaccaactcggatctcaaccacattttcatcaacttctcagccgtctttg


 


ctctgaagcccgaggaggttgaagctgcctttggcggtttcctggagcgatttggccgacgtctgtggcg


 


acttcgagtcaccggtgccgagatccgaatgatggtatccgaccccgaaactggctctgctttccctctg


 


cgagcaatgatcaacaacgtctctggttacgttgtgcagtctgagctgtacgctgaggccaagaacgaca


 


agggccagtggattttcaagtctctgggcaagcccggctccatgcacatgcggtctatcaacactcccta


 


ccccaccaaggagtggctgcagcccaagcggtacaaggcccatctgatgggtaccacctactgctatgac


 


ttccccgagctgttccgacagtccattgagtcggactggaagaagtatgacggcaaggctcccgacgatc


 


tcatgacttgcaacgagctgattctcgatgaggactctggcgagctgcaggaggtgaaccgagagcccgg


 


cgccaacaacgtcggtatggttgcgtggaagtttgaggccaagacccccgagtaccctcgaggccgatct


 


ttcatcgtggtggccaacgatatcaccttccagattggttcgtttggccctgctgaggaccagttcttct


 


tcaaggtgacggagctggctcgaaagctcggtattcctcgaatctatctgtctgccaactctggtgctcg


 


aatcggcattgctgacgagctcgttggcaagtacaaggttgcgtggaacgacgagactgacccctccaag


 


ggcttcaagtacctttacttcacccctgagtctcttgccaccctcaagcccgacactgttgtcaccactg


 


agattgaggaggagggtcccaacggcgtggagaagcgtcatgtgatcgactacattgtcggagagaagga


 


cggtctcggagtcgagtgtctgcggggctctggtctcattgcaggcgccacttctcgagcctacaaggat


 


atcttcactctcactcttgtcacctgtcgatccgttggtatcggtgcttaccttgttcgtcttggtcaac


 


gagccatccagattgagggccagcccatcattctcactggtgcccccgccatcaacaagctgcttggtcg


 


agaggtctactcttccaacttgcagcttggtggtactcagatcatgtacaacaacggtgtgtctcatctg


 


actgcccgagatgatctcaacggtgtccacaagatcatgcagtggctgtcatacatccctgcttctcgag


 


gtcttccagtgcctgttctccctcacaagaccgatgtgtgggatcgagacgtgacgttccagcctgtccg


 


aggcgagcagtacgatgttagatggcttatttctggccgaactctcgaggatggtgctttcgagtctggt


 


ctctttgacaaggactctttccaggagactctgtctggctgggccaagggtgttgttgttggtcgagctc


 


gtcttggcggcattcccttcggtgtcattggtgtcgagactgcgaccgtcgacaatactacccctgccga


 


tcccgccaacccggactctattgagatgagcacctctgaagccggccaggtttggtaccccaactcggcc


 


ttcaagacctctcaggccatcaacgacttcaaccatggtgaggcgcttcctctcatgattcttgctaact


 


ggcgaggcttttctggtggtcagcgagacatgtacaatgaggttctcaagtacggatctttcattgttga


 


tgctctggttgactacaagcagcccatcatggtgtacatccctcccaccggtgagctgcgaggtggttct


 


tgggttgtggttgaccccaccatcaactcggacatgatggagatgtacgctgacgtcgagtctcgaggtg


 


gtgtgctggagcccgagggaatggtcggtatcaagtaccgacgagacaagctactggacaccatggctcg


 


tctggatcccgagtactcctctctcaagaagcagcttgaggagtctcccgattctgaggagctcaaggtc


 


aagctcagcgtgcgagagaagtctctcatgcccatctaccagcagatctccgtgcagtttgccgacttgc


 


atgaccgagctggccgaatggaggccaagggtgtcattcgtgaggctcttgtgtggaaggatgctcgtcg


 


attcttcttctggcgaatccgacgacgattagtcgaggagtacctcattaccaagatcaatagcattctg


 


ccctcttgcactcggcttgagtgtctggctcgaatcaagtcgtggaagcctgccactcttgatcagggct


 


ctgaccggggtgttgccgagtggtttgacgagaactctgatgccgtctctgctcgactcagcgagctcaa


 


gaaggacgcttctgcccagtcgtttgcttctcaactgagaaaggaccgacagggtactctccagggcatg


 


aagcaggctctcgcttctctttctgaggctgagcgggctgagctgctcaaggggttgtga


 


(SEQ ID NO.: 70)



Amino Acid =



MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVASHGGHTVINKV


 


LIANNGIAAVKEIRSVRKWAYETFGDERAISFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVE


 


LIVDVAERFGVDAVWAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKISSTIVAQHAKVPCIP


 


WSGTGVDEVVVDKSTNLVSVSEEVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGIRKVEREEDFE


 


AAYHQVEGEIPGSPIFIMQLAGNARHLEVQLLADQYGNNISLFGRDCSVQRRHQKIIEEAPVTVAGQQTF


 


TAMEKAAVRLGKLVGYVSAGTVEYLYSHEDDKFYFLELNPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPL


 


DRIKDIRLFYGVNPHTTTPIDFDFSGEDADKTQRRPVPRGHTTACRITSEDPGEGFKPSGGTMHELNFRS


 


SSNVWGYFSVGNQGGIHSFSDSQFGHIFAFGENRSASRKHMVVALKELSIRGDFRTTVEYLIKLLETPDF


 


EDNTITTGWLDELISNKLTAERPDSFLAVVCGAATKAHRASEDSIATYMASLEKGQVPARDILKTLFPVD


 


FIYEGQRYKFTATRSSEDSYTLFINGSRCDIGVRPLSDGGILCLVGGRSHNVYWKEEVGATRLSVDSKTC


 


LLEVENDPTQLRSPSPGKLVKFLVENGDHVRANQPYAEIEVMKMYMTLTAQEDGIVQLMKQPGSTIEAGD


 


ILGILALDDPSKVKHAKPFEGQLPELGPPTLSGNKPHQRYEHCQNVLHNILLGFDNQVVMKSTLQEMVGL


 


LRNPELPYLQWAHQVSSLHTRMSAKLDATLAGLIDKAKQRGGEFPAKQLLRALEKEASSGEVDALFQQTL


 


APLFDLAREYQDGLAIHELQVAAGLLQAYYDSEARFCGPNVRDEDVILKLREENRDSLRKVVMAQLSHSR


 


VGAKNNLVLALLDEYKVADQAGTDSPASNVHVAKYLRPVLRKIVELESRASAKVSLKAREILIQCALPSL


 


KERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVFDVLAQFFAHDDPWIVLAALELYIR


 


RACKAYSILDINYHQDSDLPPVISWRFRLPTMSSALYNSVVSSGSKTPTSPSVSRADSVSDFSYTVERDS


 


APARTGAIVAVPHLDDLEDALTRVLENLPKRGAGLAISVGASNKSAAASARDAAAAAASSVDTGLSNICN


 


VMIGRVDESDDDDTLIARISQVIEDFKEDFEACSLRRITFSFGNSRGTYPKYFTFRGPAYEEDPTIRHIE


 


PALAFQLELARLSNFDIKPVHTDNRNIHVYEATGKNAASDKRFFTRGIVRPGRLRENIPTSEYLISEADR


 


LMSDILDALEVIGTTNSDLNHIFINFSAVFALKPEEVEAAFGGFLERFGRRLWRLRVTGAEIRMMVSDPE


 


TGSAFPLRAMINNVSGYVVQSELYAEAKNDKGQWIFKSLGKPGSMHMRSINTPYPTKEWLQPKRYKAHLM


 


GTTYCYDFPELFRQSIESDWKKYDGKAPDDLMTCNELILDEDSGELQEVNREPGANNVGMVAWKFEAKTP


 


EYPRGRSFIVVANDITFQIGSFGPAEDQFFFKVTELARKLGIPRIYLSANSGARIGIADELVGKYKVAWN


 


DETDPSKGFKYLYFTPESLATLKPDTVVTTEIEEEGPNGVEKRHVIDYIVGEKDGLGVECLRGSGLIAGA


 


TSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQIEGQPIILTGAPAINKLLGREVYSSNLQLGGTQIMY


 


NNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHKTDVWDRDVTFQPVRGEQYDVRWLISGRTLE


 


DGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPADPANPDSIEMSTSEAGQ


 


VWYPNSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRDMYNEVLKYGSFIVDALVDYKQPIMVYIPPT


 


GELRGGSWVVVDPTINSDMMEMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARLDPEYSSLKKQLEESP


 


DSEELKVKLSVREKSLMPIYQQISVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRIRRRLVEEYLI


 


TKINSILPSCTRLECLARIKSWKPATLDQGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQLRKDR


 


QGTLQGMKQALASLSEAERAELLKGL*


 


Knockouts:


PEX10 - YALI0C01023g


(SEQ ID NO.: 71)



Nucleotide =



atgtggggaagttcacatgcattcgctggtgaatctgatctgacactacaactacacaccaggtccaaca


 


tgagcgacaatacgacaatcaaaaagccgatccgacccaaaccgatccggacggaacgcctgccttacgc


 


tggggccgcagaaatcatccgagccaaccagaaagaccactactttgagtccgtgcttgaacagcatctc


 


gtcacgtttctgcagaaatggaagggagtacgatttatccaccagtacaaggaggagctggagacggcgt


 


ccaagtttgcatatctcggtttgtgtacgcttgtgggctccaagactctcggagaagagtacaccaatct


 


catgtacactatcagagaccgaacagctctaccgggggtggtgagacggtttggctacgtgctttccaac


 


actctgtttccatacctgtttgtgcgctacatgggcaagttgcgcgccaaactgatgcgcgagtatcccc


 


atctggtggagtacgacgaagatgagcctgtgcccagcccggaaacatggaaggagcgggtcatcaagac


 


gtttgtgaacaagtttgacaagttcacggcgctggaggggtttaccgcgatccacttggcgattttctac


 


gtctacggctcgtactaccagctcagtaagcggatctggggcatgcgttatgtatttggacaccgactgg


 


acaagaatgagcctcgaatcggttacgagatgctcggtctgctgattttcgcccggtttgccacgtcatt


 


tgtgcagacgggaagagagtacctcggagcgctgctggaaaagagcgtggagaaagaggcaggggagaag


 


gaagatgaaaaggaagcggttgtgccgaaaaagaagtcgtcaattccgttcattgaggatacagaagggg


 


agacggaagacaagatcgatctggaggaccctcgacagctcaagttcattcctgaggcgtccagagcgtg


 


cactctgtgtctgtcatacattagtgcgccggcatgtacgccatgtggacactttttctgttgggactgt


 


atttccgaatgggtgagagagaagcccgagtgtcccttgtgtcggcagggtgtgagagagcagaacttgt


 


tgcctatcagataa


 


(SEQ ID NO.: 72)



Amino acid =



MWGSSHAFAGESDLTLQLHTRSNMSDNTTIKKPIRPKPIRTERLPYAGAAEIIRANQKDHYFESVLEQHL


 


VTFLQKWKGVRFIHQYKEELETASKFAYLGLCTLVGSKTLGEEYTNLMYTIRDRTALPGVVRRFGYVLSN


 


TLFPYLFVRYMGKLRAKLMREYPHLVEYDEDEPVPSPETWKERVIKTFVNKFDKFTALEGFTAIHLAIFY


 


VYGSYYQLSKRIWGMRYVFGHRLDKNEPRIGYEMLGLLIFARFATSFVQTGREYLGALLEKSVEKEAGEK


 


EDEKEAVVPKKKSSIPFIEDTEGETEDKIDLEDPRQLKFIPEASRACTLCLSYISAPACTPCGHFFCWDC


 


ISEWVREKPECPLCRQGVREQNLLPIR*


 


MFE1 - YALI0E15378


(SEQ ID NO.: 73)



Nucleotide =



atgaccgacaaggactgggatcttgtctacaaggtccacgttttcggtgcctacaaggttacccgagctg


 


cctggccttacttccgaaagcagaagtacggtcgagttatctctacctcttccgctgctggtctttacgg


 


aaacttcggccagaccaactactccgctgccaagctcgccctggttggtttcggtgagactctcgccaag


 


gagggtgccaagtacaacattacttccaacgtcatcgctcctcttgctgcttcccgaatgaccgagacag


 


tcatgcccgaggatatcctcaagctcctcaagcctgagtacgttgttcctctggtcggctacctcaccca


 


cgactctgtcaccgagtcttatggtatttacgaggtcggtgctggttacatggctaaaatccgatgggag


 


cgaggcaacggtgctgttttcaagggcgacgacactttcaccccgtctgctattctgaagcgatgggatg


 


aggtcacctcttttgagagccccacctaccctaacggccctgctgacttcttcaaatacgctgaggagtc


 


tgttaagcgacccgagaacccccagggacccaccgtctccttcaaggaccaggttgtcattgtcactgga


 


gccggtgctggcattggccgagcttactctcacctccttgctaagcttggtgccaaggtcgttgttaacg


 


atttcggtaaccctcagaaggttgtcgatgaaattaaggccctcggtggtatcgccgtcgctgacaagaa


 


caacgtcatccacggtgagaaggttgttcagaccgctatcgacgccttcggtgctgtccacgccgttgtc


 


aacaacgctggtattctccgagacaagtctttcgccaacatggatgatgagatgtggcagctgatctttg


 


atgtccacctcaacggtacttactccgttaccaaggccgcgtggccccacttccttaagcagaagtacgg


 


ccgtgtcatcaacaccacctcaacttctggtatctacggtaacttcggccaggccaactactctgccgcc


 


aaggctggtatcctcggtttctcccgagctcttgctcgagagggtgagaagtacaacattcttgtcaaca


 


ccattgcccctaacgctggtactgccatgactgcttctgtcttcactgaggagatgctcgagctcttcaa


 


gcccgatttcatcgcacccatcaccgtcctgcttgcttccgatcaggctcccgtcaccggtgatctgttt


 


gagactggttctgcttggatcggacagactcgatggcagcgagctggtggtaaggccttcaacaccaaga


 


agggtgtcacccccgaaatggttcgagacagctgggctaagatcgtcgacttcgatgatggtaactccac


 


ccatcccaccactccctccgagtctactactcagattcttgagaacatcttcaacgtgcctgatgaggag


 


gttgaggagactgctctcgttgctggtcccggtggtcccggtatcctcaacaaggagggcgaacctttcg


 


actacacttacacttaccgagacctcattctttacaaccttggtctcggtgccaaggctaatgagctcaa


 


gtatgtcttcgagggtgatgatgacttccagaccgtgcccactttcggtgttatcccttacatgggtggc


 


ctcatcactaccaactatggcgacttcgttcctaacttcaaccctatgatgcttctccacggtgagcagt


 


accttgaaatccgacagtggcctattcctaccaatgctacattggagaacaaggctaaggtcatcgatgt


 


cgttgacaagggcaaggctgccctccttgtcactgctaccaccaccacgaacaaggagactggtgaggag


 


gttttctacaacgagtcttctctcttcatccgaggctctggtggtttcggtggtaagtctaccggtactg


 


accgtggcgctgccactgctgccaacaagccccctgctcgagctcctgacttcgttaaggagatcaagat


 


ccaggaggaccaggctgccatttaccgactttctggtgattacaaccctcttcacatcgaccctgctttt


 


gctgctgttggtaactttgaccgacctattctccacggtctctgctcttttggtgtctccggtaaggctc


 


tttacgatcagtttggtcctttcaagaacgctaaggtccgatttgctggtcacgtcttccctggtgagac


 


cctgaaggttgagggctggaaggagggcaacaaggtcattttccagaccaaggttgttgagcgaggtact


 


accgccatcagcaatgccgccattgagctcttccccaaggatgctaagctctaa


 


(SEQ ID NO.: 74)



Amino Acid =



MTDKDWDLVYKVHVFGAYKVTRAAWPYFRKQKYGRVISTSSAAGLYGNFGQTNYSAAKLALVGFGETLAK


 


EGAKYNITSNVIAPLAASRMTETVMPEDILKLLKPEYVVPLVGYLTHDSVTESYGIYEVGAGYMAKIRWE


 


RGNGAVFKGDDTFTPSAILKRWDEVTSFESPTYPNGPADFFKYAEESVKRPENPQGPTVSFKDQVVIVTG


 


AGAGIGRAYSHLLAKLGAKVVVNDFGNPQKVVDEIKALGGIAVADKNNVIHGEKVVQTAIDAFGAVHAVV


 


NNAGILRDKSFANMDDEMWQLIFDVHLNGTYSVTKAAWPHFLKQKYGRVINTTSTSGIYGNFGQANYSAA


 


KAGILGFSRALAREGEKYNILVNTIAPNAGTAMTASVFTEEMLELFKPDFIAPITVLLASDQAPVTGDLF


 


ETGSAWIGQTRWQRAGGKAFNTKKGVTPEMVRDSWAKIVDFDDGNSTHPTTPSESTTQILENIFNVPDEE


 


VEETALVAGPGGPGILNKEGEPFDYTYTYRDLILYNLGLGAKANELKYVFEGDDDFQTVPTFGVIPYMGG


 


LITTNYGDFVPNFNPMMLLHGEQYLEIRQWPIPTNATLENKAKVIDVVDKGKAALLVTATTTTNKETGEE


 


VFYNESSLFIRGSGGFGGKSTGTDRGAATAANKPPARAPDFVKEIKIQEDQAAIYRLSGDYNPLHIDPAF


 


AAVGNFDRPILHGLCSFGVSGKALYDQFGPFKNAKVRFAGHVFPGETLKVEGWKEGNKVIFQTKVVERGT


 


TAISNAAIELFPKDAKL*


 


ACO1- YALI0D09361


(SEQ ID NO.: 75)



Nucleotide =



atgctggcttctcgagtttccatcaaggctgtgagtatcgatggtgaagaaagacaccgacaatcgccac


 


gttgtgccacagacacagacgcgtttctacacacacacacacaagagtcgacgtgtggtttagccgaggt


 


atttcgacagggaggaaaaacgacaacgaaaggaccgacagataccaaagcaacccaatcaccacctcaa


 


tcaatgatccccgcccgcgggaatgcggaaaaggcttctgcgacattacaacaaagccaactctgttgat


 


ttgttgtttgcgacattggctttgtgccggtcccaaaattacctcgaccaaccacacggcggcaattgaa


 


gacaatgcaaattaaatagcacatactaacccagccccgccttgcacgatctctcgcgactaccactaat


 


gcctccctcaacttggactccaaggtccgaatgaacaactgggaggccaacaacttcctcaacttcaaga


 


agcacaccgagaacgtccagattgtcaaggagcgactcaaccgacccctgacctacgctgagaagattct


 


ctacggccatctcgacaagccccatgagcaggagattgtccgaggtcagtcctacctcaagctgcgaccc


 


gatcgagccgcctgccaggatgccaccgcccagatggccattctgcagttcatgtctgccggtatcccca


 


ccgtccagacccccaccaccgtccactgtgaccatcttatccaggcccaggttggtggtgagcaggatct


 


tgctcgagccatcgacatcaacaaggaggtctacaacttccttggcaccgcctccgccaagtacgacatt


 


ggtttctggaaggccggatccggtattatccaccagatcattctcgagaactacgccttccccggtgccc


 


ttctcattggttccgactctcatacccccaacgccggtggtctcggtatgctcgccatcggtgtcggtgg


 


tgccgatgtcgtcgacgtcatggccggtctcccctgggagcttaaggcccccaagattatcggtgtcaag


 


ctgaccggtaagctctctggctggacctcccccaaggatattatcctgaaggtcgctggtatcctcaccg


 


tcaagggtggaaccggtgctatcgtcgagtacttcggtgatggtgtcgataacctgtcctgcactggtat


 


gggaaccatctgtaacatgggtgccgagattggtgctaccacctccaccttccccttcaacgagcgaatg


 


gccgactaccttaacgccactggccgaaaggagattgccgactttgctcgactttacaaccacttcctct


 


ctgccgatgagggttgtgagtacgatcagctcatcgagattgacctgaacacccttgagccttacgtcaa


 


cggtcccttcactcccgatcttgccacccccatctccaagctcaaggatgtcgccgtcgagaacggatgg


 


ccccttgaggtcaaggtcggtcttatcggctcttgcaccaactcctcttacgaggatatggagcgatccg


 


cctccattgccaaggacgccatggcccacggtcttaagtccaagtccatctacaccgtcacccccggttc


 


cgagcagatccgagccaccattgagcgagatggtcagctccagaccttcctcgacttcggtggtatcgtc


 


cttgctaacgcttgtggcccctgcattggtcagtgggaccgacgagacatcaagaagggtgagaagaaca


 


ccattgtctcttcttacaaccgaaacttcactggccgaaacgattctaaccctgccacccacgctttcgt


 


cacctctcccgatctcgtcaccgctttcgccattgctggtgacctccgattcaaccctctcactgactcc


 


ctgaaggattctgagggtaaggagttcaagctcaaggagcccactggaaagggtctgcccgaccgaggtt


 


acgaccccggcatggacacctaccaggctccccccgccgaccgatctgccgtcgaggttgatgtttcccc


 


cacttccgaccgactccagatcctcaagcccttcaagccttgggacggcaaggacggtattgacatgccc


 


atcctcatcaagtctcttggtaagaccaccactgaccatatctctcaggccggtccctggcttaagtacc


 


gaggccatctccagaacatctccaacaactacatgattggagccatcaacgctgagaacgaggaggccaa


 


caacgtccgaaaccagatcactggcgagtggggaggagttcccgagactgccattgcttaccgagacaac


 


ggtatccgatgggttgttgtcggaggtgataacttcggtgagggttcttctcgagagcacgctgctcttg


 


agccccgattcctcggtggtttcgccatcatcaccaagtcttttgcccgaattcacgagactaacctgaa


 


gaagcagggtctcctgccccttaacttcgtcaacggtgctgactacgacaagatccagccctccgataag


 


atctccattcttggtcttaaggaccttgcccccggcaagaacgtcaccattgaggttacccccaaggacg


 


gtgccaagtggaccaccgaggtttctcacacctacaactctgagcagctcgagtggttcaagtacggctc


 


tgccctcaacaagatggctgcctccaagaaataa


 


(SEQ ID NO.: 76)



Amino Acid =



MLASRVSIKAPRLARSLATTTNASLNLDSKVRMNNWEANNFLNFKKHTENVQIVKERLNRPLTYAEKILY


 


GHLDKPHEQEIVRGQSYLKLRPDRAACQDATAQMAILQFMSAGIPTVQTPTTVHCDHLIQAQVGGEQDLA


 


RAIDINKEVYNFLGTASAKYDIGFWKAGSGIIHQIILENYAFPGALLIGSDSHTPNAGGLGMLAIGVGGA


 


DVVDVMAGLPWELKAPKIIGVKLTGKLSGWTSPKDIILKVAGILTVKGGTGAIVEYFGDGVDNLSCTGMG


 


TICNMGAEIGATTSTFPFNERMADYLNATGRKEIADFARLYNHFLSADEGCEYDQLIEIDLNTLEPYVNG


 


PFTPDLATPISKLKDVAVENGWPLEVKVGLIGSCTNSSYEDMERSASIAKDAMAHGLKSKSIYTVTPGSE


 


QIRATIERDGQLQTFLDFGGIVLANACGPCIGQWDRRDIKKGEKNTIVSSYNRNFTGRNDSNPATHAFVT


 


SPDLVTAFAIAGDLRFNPLTDSLKDSEGKEFKLKEPTGKGLPDRGYDPGMDTYQAPPADRSAVEVDVSPT


 


SDRLQILKPFKPWDGKDGIDMPILIKSLGKTTTDHISQAGPWLKYRGHLQNISNNYMIGAINAENEEANN


 


VRNQITGEWGGVPETAIAYRDNGIRWVVVGGDNFGEGSSREHAALEPRFLGGFAIITKSFARIHETNLKK


 


QGLLPLNFVNGADYDKIQPSDKISILGLKDLAPGKNVTIEVTPKDGAKWTTEVSHTYNSEQLEWFKYGSA


 


LNKMAASKK*


 


YLYOX1 YALI0E20449g


(SEQ ID NO.: 77)



Nucleotide =



atggatctggcgaaaatcaccgacggcttcgtcaagcacgagacctcgtcgtcgtcctcttcttgctcca


 


ccaccaacacagggcccaccccagacttgtctccagtgacgccctccaaggaatgtgagaagcggccacg


 


agaggacgaccctgaagagtcgcacgacacgagcgccggcgccaacagcaacaacaacgctagcgtgtct


 


ctcatgtccaccccagagcccaagtcgtcgtctccccccggactgtcgcatttcgcacacctgatgcaaa


 


agtcggacaccatgtaccgacagaacctcaactcggaccagtacatctactcggacgaggagaaggagaa


 


ccacaagacttcgggcaagccccacaccccccaggtgcctcatacgccctccagtgtgccgacacaacaa


 


ccccaatatgcatttatttcacattccatcacctcgtacccgtcgaacgagcctcagattgacaacgcac


 


ggctggcgcgccgaaaacgacgccgaacgtctcccacggaactcgcgctgctggagcaggagtttgcccg


 


caaccagaagcctcccaagcacattcgcgtcgacattgcccgccgagtcgacatgactgaaaaggctgtg


 


caggtgtggttccagaacaagcggcagagcgtgcgaaagagcatgaacaagagcatgaccgatgacacct


 


ctttcgccgactcttcgttcgctgaaactacctttgacgagacagacggtaactccacattcctgtccaa


 


ttccaacgtcagcaccagcgtaagcaacaagtcaatcacttcttccatcacagacaacaagtcgcccctg


 


gcacagtcaaccaccgccgactctggtgccaacgccaacgccaacgccaacgccaacgccaacaacaaca


 


ccgcatccacttcctccacaaacgactccgaaattgcatccgtcgcccccaaaacaaacggcagctcatt


 


ctctgttttcgaagatacccccgagactcccgcgaaaaagaaacccagtgctccgcgactgtccatgcgt


 


ggtgggaaggctactgttatctacgccggcaagcccaagggtgtcacgctgtcctcgggaagacgtcttg


 


gggtccctgccacaccctcctctcccgccaacaacaatcttggcctgggaggctcgcctctggccacatc


 


gtctcctatgacccagcggaccgcgtcgcaactgaaccaggcatctgcatcttctcccctatcggctgtt


 


aagtccaagtcttttggaactgccgaggaaagcctggctgcgacgctcaagaagcggcttccgtccatgc


 


actacgacctgcccgtgaccaacaagacgtcgtctgtgcgccatggcgtgagctctcccgtggtcgacgc


 


cggcagccgtgaggccgagtgtatttccaatctcctctctcttcgaaacggaggacgatggtaa


 


YLUGA2 YALI0F26191g


(SEQ ID NO.: 78)



Nucleotide =



atgttgcgagccctgaataccgtccagcgactttccagcacccgagccatgtccacctcttccatttcgt


 


ctctgcttaagaaccccaatcttctgcgaaaccagggctatgtcaatggtcagtgggtctcctccaagac


 


cggagacactttcagcgttgagaacccagccactggcgagactctgggccaggtgcccgagttctctgtc


 


gccgaggccgatgaggctgtccagcacgcacagactgccttcaagaccttcaaacataccactggacgag


 


agcgatccaagatgctgcgaaagtggtacgatctgatgcaggagaatgctggtgatctggccaccctggt


 


gactctggagaacggtaagtccctcgctgacgccaagggcgagattggctacggagcatctttcttcgag


 


tggttctccgaggaagctcctcgaatctacggagacatcattccatccgccaaccccgccaaccgaatct


 


acacaatcaagcagcccatcggagtctgcggaatcatcaccccctggaacttcccctcggccatgatcac


 


ccgaaaggctgctgctgctgttgctgctggctgtaccatggtgatcaagcctggttccgaaacctcctac


 


tctgcccttgctctggcttacctggctgaacaggccggcatccctaagggtgttgtcaacgtggtcacta


 


ctaagaagaacactcgagcttttggtaacgccctgtgcgagaacccgaccgtcaaaaaggtttctttcac


 


gggctccactggtgtcggaaagacccttatgggcgcatcggcctccactcttaagaagctgtcctttgag


 


ctcggtggcaacgctcccttcattgtgtttgaggacgccgatattgaccgggctgtcgacggagctattg


 


cgtccaagttccgaggcactggccagacctgtgtctgtgcaaaccgaatttatgtgcacgagagcatcgc


 


cgagaagtttgctgagcgaatggcagccgtggtcaaggacttcaaggttggaaacggtctcgaccctaac


 


accacccatggccctcttatccacgagggagccaagggcaagatccaggagcaggttgacgatgctgtca


 


agaagggaggaaaggtactcattggaggctccgacgcccctgagatcggaaaggcctttttccagcctac


 


cgtcatttccggggccaagtctgatatgctgattgcctccgaggagacgtttggtcccattgctgccatc


 


ttcccctttaagaccgacgctgaggtcattgagcttgccaacaaggcagaggtcggtctggccggctact


 


tctactccaaggacgtgtaccgaatccaacaggttgccgaggctctcgaggtcggaatggtcggtgttaa


 


caccggtctgatgacggagtgtgctctgccctttggcggtatcaaggagtctggctttggccgagagggc


 


tccaagtacggcctggatgactacatggtgctcaagactattgttgtgtctggcgtcgagccccacattc


 


agccttaa


 


YLRME1 YALI0E17215g


(SEQ ID NO.: 79)



Nucleotide =



Atgtattcattcgacttcaactttgacacggcatatccgccacagactgaatattccaaacaagacgact


 


gtctgggatacatgcccatcacgcctccttacctggactggagctcgctgacattcccgccggttgaata


 


cgcacccatcgtcgataacgtgctcccggaagaaccctcggagccctcggacgtgtcttcttcttccgga


 


gaagaaagcccctactttttcgacgaatactgcaccattccctctctggtcgaccagctcaaagaaaacc


 


ccaacatttgggccatggcaaacaccgtcaagaaaggagcctacgtgtgtagccactgcactaagcaggg


 


cacccccgtcaagttcaaaaccatggtcgactttgccacccacctcgactcgcattctcatgaccgaagc


 


tgcaaatgcgccgacacaaaatgtccctggtccattgtgggcttctctactcgatcggaaatgcgaagac


 


acacaaactcggtccatcgacaaacacccttcacatgcaaaatctgtgaccgcgggtttgtacgagaaga


 


ctctctcaaacggcatgtcaaactactccacatttctcccctcaaaaccagacgaaagagtacctga


 


YLOSH6 YALI0A02354g


(SEQ ID NO.: 80)



Nucleotide =



atgcaccaccacctcaaccccaaggcgctcttttctggtgagtatggcggacagaaatggacggaggaac


 


gtggcagagccgattgaccagccacgcaggccgaccaagccccattgagtgagccattggacgtccttgg


 


cccgaatagacgctctctcccaggtttgccggaaaaacgagctgttatatccgaacgagctgtttgtgcc


 


caaaaaagcccctactaacccccaggccgaaaggagagcacctctccccagacacaagccgcgtccggct


 


ccggagccgtgtctccaggccgacctctggattcgtccaccaacgtcgaagatgtggatgagcttgacgg


 


agacggccagaacatcatcatgggaattatcgcgcagctgcgacccggcgctgatctgtctcgaatcaca


 


cttcccaccttcattctcgagcgaaagtccatgctcgagcgaatcacaaactccctgcagcaccccacat


 


atgtcattgaggcccacgccaccaaggaccccatgcagcggttcatccaagtggtaaagtggtaccactc


 


cggctggcacatcacccccaaggccgtcaaaaagcccctgaaccccattctcggcgagttcttcacatgc


 


tactgggactacgacgacggttcccacggatactacatctccgagcagacctcccaccaccctcccaagt


 


catcctacttttacatgatccctgagcacaacatccgagtcgacggtacactggctcccaagtcccgttt


 


cctgggtaactcagctgcttctctcatggagggcgccaccattctcaagttcctggacattgtagatgcc


 


aagggcgctcccgaggagtacgaaatcacttcgcccaatgcctacgcccgaggtattctctttgaacggc


 


tcaagtacgagtactgcgaccactcgatcatcaagtgtcccgctctggacctgactctggacctggactt


 


caaggccaagggcttcatttccggtacatacaatgccttcgagggccagatcaagaagatctccaccggc


 


gaggccttttacgatgtttatggaaagtgggatgaaatcatcgagctcaagaacctcaagaccggcgaga


 


agtcggtgctgtttgacgtgactaaggccgccctgcaccctcccaaggtgcgacccatcgctgagcaggc


 


cgccaccgagtcccgacgactgtgggagcccgtcaccgacgctcttgctaagcgagaccacaccgttgct


 


accgacgaaaagttcaagattgaggacaaacagcgaacgctggccaaggagcgagaagagcacggcgtca


 


agttcctgcccaaactgttcaagcccgcccccgctcccctggacttcattctgtataaggatctgcacgg


 


cactcccgaagagatcaccaaggagattctcagcatagtccccattctgcccggccaacagttcaccaag


 


gactttgaaatgtccggcgagaagaaatacaagctggagaagagcggccaggccagcagcgagactcagc


 


ccaccgccacgaccactgcggctgccccccaagcaggcgctgtccccacaacccctgctaacggccagac


 


tcccctggccaagacttctgatcttcaggaggctcttcccaccgaagaggacgagttccacgacgcccag


 


tag


 


YLIRC20 YALI0C07150g


(SEQ ID NO.: 81)



Nucleotide =



atgacaagtgatgcgataaacgccatggaaaacgacagtacgacggtggtagaggtggaaacgacatttg


 


tgaacgataacgtggtccgtggcttcctcgatgttgcacgtgatacgctgccagacgtccaaggactcct


 


tccactggtccaagtgcagctggtggcggatatctcaagagagatgctggagggcgaagaagtgctggaa


 


atcaccgatccagagtcacatggcgtcaaaaataccgaagcaggtgacgaaacgaactcacgtgacccca


 


tcgtcgcttctgcgcctgctaccctggttcctaacgagagcacattagagattcatgtcacgcccaagta


 


caccaccaaggacaagaaacgaggccgcaaaaagaccaagaaggacgaagattggttggtaacatgcttg


 


ggtgttgttcaactaggaaacgtggaaaccagtgaccacgtgcttaccgctttgaaacaggctctttcgg


 


tagccaaatttaacccgcgaaatcgagtcagtgtcttttcagtgaatcctcacgtcactgttaccaaaaa


 


caatggtgtttacagcatttccatcacttttggagtctttgcgaagccttttgatggccacgtcaaccct


 


gagatccatatggcaggtcacctcaacattgtgaatgtcatccgacagttcctgggcgtaactaagataa


 


aacagctacataagaacgactatgtgactcctgaatacttctacgagtgcctggaactcaaggatgatac


 


cgaggttgagatcaacagagatcttcagccggaagggatgagatcaaaacttttggattaccagcttgaa


 


actgtggggtgggttctggatagagaaaagggagaatcgcgtgagaagacgatagagggaattccttcac


 


catggaaacggttcagggctcatggtatcaactggttggttgattttgtgggtctcaacattggtcctga


 


gaaggaggtgatggagattttgacacgagatacgaaaccaacaactgaggaccccgagattcaagcagta


 


tcacgtgacgcagattttaaggctggatatggacttattgctgatgaaatgggtcttggaaagacagttg


 


agctactagctgtagtcctgaataaccccagacctgaatttccaccgcaaacacactacgatctgtactc


 


tgacagagacgtgttacctaccaagacgactctcattttatgtcctgccagtatcagtcaacagtggatt


 


gctgaggttactaaacatgctcccagtctctctgtctttctgtacactggtcgagcagctttggatgctc


 


aaagagagaaggaaggtactcccgataccgatattgaggttggaattgactcagatactgattcagaagg


 


ccctcttgtttcaaaacatgcacaatttctctctcagttcgacattgtagtcacatcctatgaagttgca


 


tctcgcgaggttgccaacgctctttacaaccctctgagaggtcgtgtaactcgcaccaagacgaagctaa


 


agtcgaaagatacccgagatgtcgatctcgtgcaagaccggctttccctccaatctccactgagtcagct


 


tcagttctggcgtgtgattctggacgaggttcagatggtgggaaacacggtctccaacgcagctgttgta


 


gctcgtattattccccgagtgcatgcatggggagtcagtggtactcctataaagaagggcatgcctgact


 


tacttggcatgtgtgtgtttttgagatgtgaacccggcgagttttatggaagaagtgattgtgagtatac


 


taaaggaacagtcagagtggcatgtgacaaaaaaacaaaaaaccatatggctcaatgaagggctacgact


 


aacacagatgcgtataactactcttggcaaaacggatacctcagcacaactatgacagcatctggagtaa


 


gtcatcaaaaacactgggagatgctcatgcttgacaagcctcggtttcgagacgttattcgtcaaatgtc


 


tattcgacatactaagcgacaggtcagagatcaactagtattgcctcctcaggaaagacaccatgtgaga


 


ctcagattcaatctagtcgaggaagaaaactaccgacacctgcgtgaaggtgttgagagtgccgtcagtg


 


aggcagtggctagttctctcatgagagaagagagggaagctacacgtgaggcagctgtggtggataggta


 


tggcgttctgccttcaagtgtcactccccctgtgagcaacagacctagaggcactttcaacatcggaggc


 


tctaatccctatgctagtatcatggcgaatatcaacaacacagtcattgaacctgaaattgagattgatc


 


ccagtatcacttctagtggagagggtgacggccaacatgtctacactacctggtcgggtgctgtagacac


 


gtatggtggtgagtctagcggtacagctgctagtagcaccgatgctgacggcgatgataacgctcaatct


 


cccacatctgatacagctagcaacactgacatcaatgttagtgctattcccgatatagaggtatccccga


 


ctgccacccctacagcctccaccagatcccaaaatggaacttctgctcctccagcatcttccgctcctgc


 


ggatttaacaacagcaaccctctcttcctggctgttacggttacgacaaacctgctgccatcctcgagtc


 


ggttctggtaacaagaaggctctcggaaacggtattcttcaaactgtcagtcacgtgctggacgccatgt


 


gcgaccaggcgctcacccagctgctgaacgacgagcgaagtctgtttgtcgaagagctggagaaggcacg


 


agttcacgagttcaacaaacaaccagacattggactcacagtgcttcagtcacgtgtttctgaagtcgag


 


gttcgaactggtgagatccgagatatggctgttgctgcggctacgcggtatgctatgaagaagaaggagg


 


taatttccgagtggaagcgtattggtgaggttgataacaagcgcaagttggaggagagtgatgacggtgc


 


tgctaatgttaagaaagtcaaggtcgaaaaagaggagaaggaggaagaagtggcaaaggaggaggtttcc


 


gaagattttaaaatggagggaactgagaacaactccatttttggagctccaactgcttttctgggctctg


 


attcggagtctgagagcactggtaagatgtccaaaccattacaaaagtacctgaacaactccgaggaact


 


tcagacggagaaggagcgaaaacaggcttttctgcaccggtacaggagctggatggatcttatgcatcgg


 


tactattttttcattgctacttttcatttccaagttggagaagcgtgagtatgacaaagatttgtaatga


 


cgtggtggttctactggggtcatgagaggtcatgagacatactaacacagtaaaaaagtggctgaggaga


 


agaaagaaaaagaggatgggaaggacgatgaagagaaggaagatgaagagaaggaagagattgaggtcaa


 


gaaagaggaggatgaagggaccaagagtgacgagtgagtatagagatatcatgagtggcagaataacttg


 


tgccattcgctcctcttatgtatatgtgtactaacacagtctggaaacgcactattacacgctggcagaa


 


caaatccgaacccagctacttcaacgccctattgagagagtagaccaagacgtgggtcgacttgaacggg


 


ccaaggagctggagatggttcagatccctgttgataccttgactcgagatctagtacaggcttctccttt


 


ccttgaggcacgtgtttcgggtctactcgagatcatcaaccaacagtccgaatatcttgaagaatggatg


 


accagagttcgagagctgttggttgcacgtgacgagaaggacgtgaaagaaacagataagaagaagaata


 


aaggagatgtcgagaaagttgaaggcgaaaacactgatccttatgcttctggattagacaaccaacaata


 


tgcgtcggactaccttgatgctatatcgtacctgctgcaactcagagatgaagctctcaatgccaagact


 


acggcctcagcagccgacaagatccaagttaacttgtggtaccacaatgactacgaagaagagcttaccg


 


atcttcaggtggccctcaaggaagctctggacgcttgtcatgtgagtcccactcttggtgccctcaaacc


 


tatcgttgctgctctgaagacggactctggagctgtttcattgtcaatttacaacccgaaatggcctccc


 


aagttgctgtccaagctcaatccgatcgttaagacagttacctcgacaaccaaggcttgcagagacctgt


 


tgtcagtcgttagaagctgtttcaactcgaaggttgtctattacaagcagctgcagcaactgtctgacaa


 


tgtgagcagtctggaggaactcatcgagcctggttatgtcacactggaacgcctgaacgccaaaataaac


 


catctcgtacctttaatcaagcgtacaaagggccgaatcacatacttacagagtctcaaaggtgatgatg


 


acacaactggagtttccaacatgactggaattcataaaatgtgtgtcatctgtcaggatgattatattat


 


cgtgggatccatcactgtctgtggccattacttttgcagaaactgcctggaagagtggtggcagacacat


 


aatacgtgtccaatgtgcaagactgtattgtcccgcgacgatgtgttctctttcacccaacaggacaagg


 


aagacaagtcacgtgcaggttctttcgctgctcggatcaatcaagatgacgccattggagcaatgtatgc


 


gccagtgtcggaggacactcaacagttgatgagcaaacagagcatcaagagtgcgtatggcacaaagatt


 


gaccacgttatcaagtatatcaagatgctcactcatcgggctcctggcactcagattgtcatcttttctc


 


agtgggcagagattctcacattgttagcttcagccctcactgagaacaagattgcatacgcggagccgaa


 


aacactgatgtctttcttgcaatcggaagaagtcacgtgtttcctcttgaacgcaaagttccagtccact


 


ggcctgactcttgtaaatgccactcacgtcattctatgcgagcccattctcaacgctgctcttgaggctc


 


aggccatcagtcgaatccaccgaatgggccagactcagactacccacgtgactatcttcactatggccga


 


tactgttgaagaagaggttctgcgtcttgctattaacaagcggttgaaaagtatggacggtgatgagacg


 


tttgaggagaatgaatctcgacatgtgacatcaggagtgggtgcgctcgccaccgataaatccggagagg


 


tggtcaaccgtcaggatatgtgggacgctttgtttcccagtgacgggtaa






It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A method of producing a lipid, lipid precursor, or oleochemical comprising:
a) culturing a genetically modified yeast cell in a growth medium; and
b) isolating said lipid, lipid precursor, or oleochemical,

wherein the dry weight of said genetically modified yeast cell comprises greater than 60% wt/wt lipids, lipid precursors, and oleochemicals; and
wherein said genetically modified yeast cell comprises (i) a recombinant acyl-CoA:diacylglycerol acyltransferase 1 (DGA1) gene and a UGA2 succinate semialdehyde dehydrogenase (UGA2) gene comprising a mutation, wherein said mutation results in a loss of function of succinate semialdehyde dehydrogenase of 20% or more compared to succinate semialdehyde dehydrogenase encoded by UGA2 without the mutation; or (ii) a recombinant acyl-CoA:diacylglycerol acyltransferase 2 (DGA2) gene and a UGA2 succinate semialdehyde dehydrogenase (UGA2) gene comprising a mutation, wherein said mutation results in a loss of function of succinate semialdehyde dehydrogenase of 20% or more compared to succinate semialdehyde dehydrogenase encoded by UGA2 without the mutation.

2. The method of claim 1, wherein said growth medium comprises a majority carbon source selected from the group consisting of glucose, glycerol, xylose, fructose, mannose, ribose, sucrose, and lignocellulosic biomass.

3. The method of claim 1, wherein said growth medium comprises lignocellulosic biomass as the majority carbon source.

4. The method of claim 1, wherein said growth medium comprises cobalt, iron, magnesium, potassium, zinc, nickel, molybdenum, manganese, copper, or boron.

5. The method of claim 1, wherein said growth medium comprises 5.77×10−5 M to 1.73×10−4 M cobalt, 0.001 M to 0.003 M magnesium, 4.52×10−5 M to 1.35×10−4 M potassium, 4.05×10−5 M to 1.22×10−4 zinc, 3.55×10−5 M to 1.06×10−4 manganese, 9.07×10−5 M to 2.91×10−4 boron, 3.76×10−5 M to 1.10×10−5 molybdenum, 2.28×10−5 M to 6.84×10−5 nickel, 3.60×10−5 M to 1.08×10−4 iron, or 4.70×10−5 M to 1.41×10−4 copper.

6. The method of claim 1, wherein said genetically modified yeast cell comprises a recombinant Lipid synthesis regulator (MGA2) gene, a genetically modified Lipid synthesis regulator (MGA2) gene, a recombinant Leucine Biosynthesis gene (LEU2), a genetically modified multifunctional enzyme (MFEI) gene, a genetically modified PEX10 Transcription Factor (PEX10) gene or a recombinant AMP Deaminase (AMPD) gene.

7. The method of claim 1, wherein said genetically modified yeast cell comprises a genetically modified multifunctional enzyme (MFE1) gene and a genetically modified PEX10 Transcription Factor (PEX10) gene.

8. The method of claim 1, wherein said genetically modified yeast cell comprises a recombinant Leucine Biosynthesis gene (LEU2), a genetically modified multifunctional enzyme (MFEI) gene and a genetically modified PEX10 Transcription Factor (PEX10) gene.

9. The method of claim 1, wherein said genetically modified yeast cell comprises a genetically modified multifunctional enzyme (MFEI) gene, a genetically modified PEX10 Transcription Factor (PEX10) gene and a recombinant AMP Deaminase (AMPD) gene.

10. The method of claim 1, wherein said genetically modified yeast cell comprises a recombinant Leucine Biosynthesis gene (LEU2), a genetically modified multifunctional enzyme (MFEI) gene, a genetically modified PEX10 Transcription Factor (PEX10) gene and a recombinant AMP Deaminase (AMPD)) gene.