ENGINEERED MICROBES AND METHODS FOR MICROBIAL OIL

OVERPRODUCTION FROM CELLULOSIC MATERIALS

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. DE-AR0000059 awarded by the Department of Energy. The government has certain rights in this invention.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to United States provisional patent application serial number 61/663,391, filed June 22, 2012, the entire contents of which are incorporated herein by reference.

BACKGROUND

Sustainably produced biofuels are an alternative to fossil fuels and may help to alleviate the depletion of easily accessible fossil fuel stocks, such as cellulosic biomass, while avoiding fossil fuel- associated pollution and greenhouse gas emission, thus satisfying a rising demand for affordable energy in a sustainable way. The development of methods and oil- producing organisms suitable for the efficient conversion of carbon sources to lipids is prerequisite for widespread implementation of microbial biofuel production.

SUMMARY OF CERTAIN ASPECTS OF THE INVENTION

Microbial oil production by heterotrophic organisms is a most promising path for the cost-effective production of biofuels from renewable resources provided high conversion yields can be achieved. The key to cost-effective microbial oil production from renewable feedstocks is a high carbohydrate to oil conversion yield. Additionally, the use of available and abundant cellulosic biomass feedstocks for biofuel production is currently limited by the high cost and energy associated with processing such sources. Metabolic engineering has emerged as the enabling technology applied to this end and numerous examples exist of successful pathway engineering that markedly improved the performance of microbial biocatalysts in the synthesis of chemical, pharmaceutical and fuel products.

Prior efforts at engineering microbes for oil production have focused on amplifying presumed rate-controlling steps in the fatty acid synthesis pathway, using traditional carbon sources such as glucose. Significant drawbacks of such approaches include the high cost of a glucose-based feedstock, and that increasing carbon flux into fatty acid synthesis pathways increases the level of saturated fatty acids in the cell, which activate a potent negative feedback loop of fatty acid biosynthesis.

Some aspects of this disclosure provide a strategy for microbe engineering that combines the utilization of nontraditional carbon sources, such as those obtained from cellulosic materials, including xylose, with amplification of upstream (metabolite-forming pathways, also referred to herein as "push") and downstream (product- sequestering pathways, also referred to herein as "pull") metabolic pathways. Some aspects of this invention provide that a balanced combination of push-and-pull modifications in a microbe results in large carbon flux amplifications into lipid synthesis pathways without significant departures of the concentrations of intermediate metabolites from their homeostatic physiological levels, thus avoiding feedback inhibition of lipid synthesis.

Some aspects of this disclosure provide engineered microbes, and methods of use thereof, that can utilize carbon sources from cellulosic biomass that are not typically or efficiently metabolized for lipid synthesis. In some aspects, such a carbon source in cellulosic biomass is xylose.

According to one aspect of the invention, isolated oleaginous cells are provided. The cells include a genetic modification that increases expression of: a) a xylose reductase (XYL1) gene product and a xylitol dehydrogenase (XYL2) gene product; and/or b) a xylose isomerase (XYLA) gene product. In some embodiments, the cells also include a genetic modification that increases expression of a xylulokinase (XYL3) gene product. In some embodiments, the cells also include a genetic modification that increases expression of a diacylglycerol acyltransferase (DGA) gene product, an acetyl-coA carboxylase (ACC) gene product, a stearoyl-CoA-desaturase (SCD) gene product, and/or an ATP-citrate lyase (ACL) gene product.

In some embodiments, the genetic modification includes a nucleic acid construct that increases the expression of the gene product, the nucleic acid construct comprising (a) an expression cassette comprising a nucleic acid sequence encoding the gene product under the control of a suitable homologous or heterologous promoter, and/or (b) a nucleic acid sequence that modulates the level of expression of the gene product when inserted into the genome of the cell. In certain embodiments, the promoter is an inducible or a constitutive promoter. In some embodiments, the promoter is a TEF promoter. In some embodiments, the expression construct further comprises an intron. In certain embodiments, the intron is downstream of the transcription initiation site. In some preferred embodiments, the intron is within the nucleic acid sequence encoding the gene product.

In some embodiments, the nucleic acid construct inhibits or disrupts the natural regulation of a native gene encoding the gene product resulting in overexpression of the native gene. In certain embodiments, inhibition or disruption of the natural regulation of the native gene is mediated by deletion, disruption, mutation and/or substitution of a regulatory region, or a part of a regulatory region regulating expression of the gene.

In some embodiments, the gene product is a transcript. In other embodiments, the gene product is a protein.

In some embodiments, the nucleic acid construct is inserted into the genome of the cell.

In some embodiments, the increased expression of the gene product confers a beneficial phenotype for the conversion of a carbon source to a fatty acid, fatty acid derivative and/or triacylglycerol (TAG) to the cell. In certain embodiments, the beneficial phenotype is a modified fatty acid profile, a modified TAG profile, an increased fatty acid and/or triacylglycerol synthesis rate, an increase conversion yield, an increased

triacylglycerol accumulation in the cell, and/or an increased triacylglycerol accumulation in a lipid body of the cell. Increased in this context means increased relative to cells that do not have increased expression of the gene product. In some embodiments, the synthesis rate, yield or accumulation of a fatty acid or a TAG of the cell is at least 2-fold increased as compared to unmodified cells of the same cell type. In certain embodiments, the synthesis rate, yield or accumulation of a fatty acid or a TAG of the cell is at least 5-fold increased as compared to unmodified cells of the same cell type. In some embodiments, the synthesis rate, yield or accumulation of a fatty acid or a TAG of the cell is at least 10-fold increased as compared to unmodified cells of the same cell type.

In some embodiments, the cell converts a carbon source to a fatty acid or a TAG at a conversion rate within the range of about 0.025g/g to about 0.32g/g (g TAG produced/ g Glucose consumed). In some embodiments, the cell converts a carbon source to a fatty acid or a TAG at a conversion rate of at least about 0.1 lg/g. In some embodiments, the cell converts a carbon source to a fatty acid or a TAG at a conversion rate of at least about 0.195g/g. In some embodiments, the cell converts a carbon source to a fatty acid or a TAG at a conversion rate of at least about 0.27g/g.

In some embodiments, the cell comprises a lipid body or vacuole.

In some embodiments, the cell is a bacterial cell, an algal cell, a fungal cell, or a yeast cell. In certain embodiments, the cell is an oleaginous yeast cell. In preferred embodiments, the cell is a Y. lipolytica cell.

According to another aspect of the invention, cultures are provided that include the oleaginous cells described herein. In some embodiments, the culture also includes a carbon source. In some embodiments, the carbon source comprises a fermentable sugar. In certain embodiments, the fermentable sugar is a C5 and/or a C6 sugar. In some embodiments, the carbon source includes glucose. In some embodiments, the carbon source includes xylose. In certain embodiments, the xylose is at a concentration of about 8% wt./vol.. In some embodiments, the carbon source includes arabitol.

In some embodiments, the carbon source includes glycerol. In certain embodiments, the glycerol is at a concentration of about 2% wt./vol.

In some embodiments, the culture includes a carbon/nitrogen (C/N) ratio of about

100.

According to another aspect of the invention, methods are provided. The methods includes contacting a carbon source with an isolated oleaginous cell as described herein and incubating the carbon source contacted with the cell under conditions suitable for at least partial conversion of the carbon source into a fatty acid or a triacylglycerol by the cell.

In some embodiments, the carbon source comprises a fermentable sugar. In certain embodiments, the fermentable sugar is a C5 and/or a C6 sugar. In some embodiments, the carbon source includes glucose. In some embodiments, the carbon source includes xylose. In certain embodiments, the xylose is at a concentration of about 8% wt./vol.. In some embodiments, the carbon source includes arabitol.

In some embodiments, the carbon source includes glycerol. In certain embodiments, the glycerol is at a concentration of about 2% wt./vol.

In some embodiments, the method includes a carbon/nitrogen (C/N) ratio of about 100.

In some embodiments, the carbon source contacted with the isolated oleaginous cell is incubated in a reactor. In some embodiments, the carbon source is contacted with the isolated oleaginous cell and incubated for conversion of the carbon source to a fatty acid or a triacylglycerol in a fed batch process. In other embodiments, the carbon source is contacted with the isolated oleaginous cell and incubated for conversion of the carbon source to a fatty acid or a triacylglycerol in a continuous process.

In some embodiments, the fatty acid or the triacylglycerol is extracted from the carbon source contacted with the isolated oleaginous cell by solvent extraction. In certain embodiments, the solvent extraction is a chloroform methanol extraction. In other embodiments, the solvent extraction is a hexane extraction.

In some embodiments, the fatty acid or the triacylglycerol is separated from the carbon source contacted with the isolated oleaginous cell and subsequently refined by transesterification.

According to another aspect of the invention, methods for increasing productivity of production of fatty acid or triacylglycerol by an oleaginous cell are provided. The methods include culturing an oleaginous cell as described herein or a culture as described herein with at least two types of carbon sources, wherein the first type of carbon source contains or is xylose, and wherein the second type of carbon source is a carbon source other than xylose. In such methods the productivity of production of fatty acid or triacylglycerol by an oleaginous cell is improved relative to culturing the cell or the culture without the second type of carbon source.

In some embodiments, the second type of carbon source contains or is a C2 carbon source, a C3 carbon source, a C5 carbon source other than xylose or a C6 carbon source.

In some embodiments, the methods also include culturing the oleaginous cell or the culture and the at least two types of carbon sources under conditions suitable for at least partial conversion of the carbon source into a fatty acid or a triacylglycerol by the cell or the culture.

In some embodiments, the xylose is at a concentration of about 8% wt./vol.

In some embodiments, the second type of carbon source includes glucose. In some embodiments, the second type of carbon source includes arabitol. In some embodiments, the second type of carbon source includes glycerol. In certain embodiments, the glycerol is at a concentration of about 2% wt./vol. In some embodiments, the second type of carbon source comprises cellulosic material. In some embodiments, the method comprises a carbon/nitrogen (C/N) ratio of about

100.

The subject matter of this application may involve, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of a single system or article.

Other advantages, features, and uses of the invention will be apparent from the detailed description of certain non-limiting embodiments, the drawings, which are schematic and not intended to be drawn to scale, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Diagnosing the functionality of endogenous xylose utilization genes. (A) Diagram of utilization pathways for xylose, xylitol, and D-arabitol. (B) Shake flask experiments with control strain MTYL038 grown on these substrates demonstrate growth on D-arabitol, poor growth on xylitol, and no growth on xylose.

Figure 2. (A) Growth of adapted Y. lipolytica strain MTYL081 on xylose as sole carbon source in minimal media shake flask, compared to unadapted MTYL081 and control strain MTYL038 that underwent the adaptation protocol. (B) Transcriptional comparison of the xylose utilization pathway of an adapted Y. lipolytica strain and an unadapted strain. psXYLl and psXYL2 are heterologously expressed from S. stipitis, while ylXYLl, ylXYL2, ylXYL3 are the endogenous putative xylose utilization pathway.

Figure 3. Cofermentation of xylose with glucose (A) , glycerol (B), or D-arabitol (C).

Cultures were grown on 20 g/L xylose and 4 g/L of the secondary substrate.

Figure 4. 2-L bioreactor fermentation of strain MTYL081 on glycerol and xylose. C/N ratio was adjusted to 100, with 20 g/L of glycerol and 80 g/L of xylose. Samples were taken in triplicate.

Figure 5. Comparison of mRNA levels of genes responsible for energy production during xylose cofermentation with a secondary substrate: glucose, glycerol, arabitol. The comparison is between two time points during the cofermentation: when primarily the secondary substrate is being consumed vs. when the secondary substrate is depleted and only xylose is being consumed. Transcript levels that did not change significantly are shown in white boxes. Transcript levels that increased more than two-fold after transitioning to xylose utilization are shown in green boxes. Transcript levels that decreased more than two-fold after transitioning to xylose are shown in red boxes. Numbers inside of each box indicate the ratio of transcripts during the xylose-only phase vs. secondary substrate phase. Numbers greater than 1.0 signify up-regulation of the gene when transitioning from secondary substrate to xylose, while numbers less than 1.0 signify downregulation. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Liquid biofuels are a promising alternative to fossil fuels that can help ease concerns about climate change and smoothen supply uncertainties(i). Biodiesel, jet oil and other oil- derived fuels in particular are necessary for aviation and heavy vehicle transport. They are presently produced exclusively from vegetable oils, which is a costly and unsustainable path(2). An attractive possibility is the non-photo synthetic conversion of renewable carbohydrate feedstocks to oil(J). For biodiesel, a transition from vegetable oil to microbial oil production for the oil feedstock presents numerous additional advantages: adaptability to diverse feedstocks, flexibility in land requirements, efficient process cycle turnover, and ease of scale-up(4). In the search for improved feedstocks, the push towards cellulosic biofuels is a clear choice. Cellulosic biomass mitigates the need to compete with food crop production; an estimated 1.3+ billion dry tons per year of biomass is potentially available in the US alone (Perlack 2005). Additionally, cellulosic materials can be more efficiently grown and more stably produced compared to sugar crops. However cellulosic materials are not naturally consumable by most biofuel-producing organisms, and thus cellulose requires pretreatment and hydrolysis to break the material down into monomeric sugar. The resulting hydrolysate can then be used as a sugar rich feedstock. Since hydrolysis of lignocellulosic biomass results in 20-30% carbohydrates in the form of xylose, utilization of pentose sugars is one of the first steps toward efficiently using cellulosic materials.

Another factor in a cost-effective microbial technology for the conversion of carbohydrates to oils is a high carbohydrate to oil conversion yield. Metabolic engineering has emerged as the enabling technology applied to this end and numerous examples exist of successful pathway engineering that markedly improved the performance of microbial biocatalysts in the synthesis of chemical, pharmaceutical and fuel products. Prior efforts at engineering microbes with high lipid synthesis have focused on amplifying presumed rate- controlling steps in the fatty acid synthesis pathway. These efforts, however, have produced mixed results, presumably because modulating fatty acid flux gave rise to the levels of saturated fatty acids, which are potent allosteric inhibitors of fatty acid biosynthetic enzymes providing a negative feedback loop for the fatty acid biosynthesis. Certain aspects of this disclosure describe an approach that combines the introduction of xylose metabolic genes to utilize xylose as a carbon source, with the amplification of upstream, metabolite-forming pathways in the lipid synthesis pathway, with a similar increase in the flux of downstream, metabolite-consuming pathways. Combining the utilization of xylose as a carbon source with a push-and-pull strategy can achieve large flux amplifications without significant departures of the concentrations of intermediate metabolites from their homeostatic physiological levels, while growing the cells on a renewable cellulosic carbohydrate feedstock.

The oleaginous yeast Yarrowia lipolytica is an attractive candidate for microbial oil production, which has also demonstrated usefulness in a wide range of other industrial applications: citric acid production, protein production (e.g., proteases and lipases), and bioremediation. With a fully sequenced genome and a growing body of genetic engineering tools, engineering of Y. lipolytica can be achieved with relative ease. Y. lipolytica also has been found to be robust in culture, able to grow on a variety of substrates, and has been used for lipid production on agro-industrial residues, industrial glycerol, and industrial fats. It has excellent lipid accumulation capacity, commonly accumulating up to 36% of its dry cell weight (DCW) in lipids.

The metabolic pathways for de novo lipid synthesis in Y. lipolytica are beginning to be fully mapped out. Glucose entering glycolysis enters the mitochondria as pyruvate for use in the TCA cycle; however, excess acetyl-coA is transported from the mitochondria to the cytosol via the citrate shuttle. Cytosolic acetyl-CoA is then converted into malonyl-CoA by acetyl-CoA carboxylase (ACC) as the first step of fatty acid synthesis. After fatty acid synthesis, triacylglycerol (TAG) synthesis follows the Kennedy pathway, which occurs in the endoplasmic reticulum (ER) and lipid bodies. Acyl-CoA is the precursor used for acylation to the glycerol-3-phosphate backbone to form lysophosphatidic acid (LPA), which is further acylated to form phosphatidic acid (PA). PA is then dephosphorylated to form diacylglycerol (DAG) and then a final acylation occurs by diacylglycerol acyltransferase (DGA) to produce TAG.

Transport of acetyl-CoA from the mitochondria to the cytosol is carried out by the ATP-citrate lyase (ACL) -mediated cleavage of citrate via the citrate shuttle yielding Acetyl- CoA and Oxaloacetate (OAA). Acetyl-CoA carboxylase (ACC) then catalyzes the first committed step towards lipid biosynthesis, converting cytosolic acetyl-CoA into malonyl- CoA, which is the primary precursor for fatty acid elongation. Completed fatty acyl-CoA chains are then transported to the endoplasmic reticulum (ER) or lipid body membranes for the final assembly of triacylglycerol (TAG) via the Kennedy pathway. Over 80% of the storage lipids produced in Y. lipolytica are in the form of TAG. Cytosolic OAA is converted to malate by malic dehydrogenase and transported back into the mitochondria to complete the citrate shuttle cycle. Reducing equivalents in the form of NADPH is provided either by the pentose phosphate pathway (PPP) or by malic enzyme in the transhydrogenase cycle. In Y. lipolytica, high PPP flux and ineffectual malic enzyme overexpression suggest that the former is the primary source for NADPH.

Instead of utilizing glucose as a carbon source, the metabolic conversion of xylose to lipids is a favorable alternative for reasons described herein. Xylose enters the cell and can be catabolized through a redox pathway, whereby xylose reductase (XD or XYL1) converts xylose to xylitol using NADPH as a reducing equivalent. Xylitol is then converted to xylulose through the action of xylitol dehydrogenase (XDH or XYL2) using NAD+ as an electron acceptor. Xylulokinase (XK or XYL3) then phosphorylates xylulose to form xylulose-5-P. Alternatively, the xylose isomerase (XYLA) enzyme bypasses the requirement of reducing equivalents, producing xylulose directly from xylose, which is then converted to xylulose-5-P by XYL3. Xylulose-5-P can then enter central metabolism through the non- oxidative pathway of the PPP where it ultimately produces glyceraldehyde- 3 -phosphate

(G3P) and fructose-6-phosphate (F6P). These two products can then enter the rest of central metabolism, going through glycolysis to enter the TCA cycle. Production of lipids occurs normally through the transport of mitochondrial citrate into the cytosol, where it is cleaved by ATP citrate lyase into oxaloacetate and cytosolic acetyl-coA. The acetyl-coA can then enter the fatty acid synthesis pathway through the enzymatic activity of acetyl-coA carboxylase. Acyl-CoA generated from the fatty acid synthase complex are transferred to a glycerol-3- phosphate backbone and ultimately sequestered within lipid bodies as triacylglycerol (TAG). Intracellular lipid accumulation can occur via two methods: de novo lipid synthesis or ex novo incorporation of exogenous fatty acids and lipids. Lipid accumulation most commonly occurs when nutrient supply is exhausted in the presence of excess carbon. In culture, this state typically coincides with the onset of the stationary phase. In practice, the most commonly used limiting-nutrient is nitrogen, as it is easily controllable in media compositions. Despite these inducible conditions, lipid synthesis pathways are highly regulated in order for the organism to balance cell growth with energy storage. For example, ACC alone is regulated at multiple levels and by multiple factors.

This tight regulation was circumvented in certain cases. By eliminating peroxisomal oxidation pathways and engineering glycerol metabolism, Y. lipolytica was able to achieve 40 -70 lipids through ex novo lipid accumulation. Coexpression of Δ6- and Δ12- desaturase genes allowed for significant production of γ-linolenic acid (GLA) (20). However, engineering lipid biosynthesis pathways in Y. lipolytica is still relatively unexplored and strategies are still being developed for effective engineering of the lipid production pathways to maximize output.

Some aspects of this disclosure provide engineered microbes for the production of biofuel or biofuel precursor. The term "biofuel" refers to a fuel that is derived from a biological source, such as a living cell, microbe, fungus, or plant. The term includes, for example, fuel directly obtained from a biological source, for example, by conventional extraction, distillation, or refining methods, and fuel produced by processing a biofuel precursor obtained from a biological source, for example by chemical modification, such as transesterification procedures. Examples of biofuels that are directly obtainable are alcohols such as ethanol, propanol, and butanol, fat, and oil. Examples of biofuels that are obtained by processing of a biofuel precursor (e.g., a lipid), are biodiesel (e.g., produced by

transesterification of a lipid), and green diesel/modified oil fuels (e.g., produced by hydrogenation of an oil). Biodiesel, also referred to as fatty acid methyl (or ethyl) ester, is one of the economically most important biofuels today and can be produced on an industrial scale by transesterification of lipids, in which sodium hydroxide and methanol (or ethanol) reacts with a lipid, for example, a triacylglycerol, to produce biodiesel and glycerol.

Feedstocks for industrial- scale production of biodiesel include animal fats, vegetable oils, palm oil, hemp, soy, rapeseed, flax, sunflower, and oleaginous algae. In other approaches, biomass is converted by a microbe into a biofuel precursor, for example, a lipid, that is subsequently extracted and further processed to yield a biofuel. The term "biomass" refers to material produced by growth and/or propagation of a living cell or organism, for example, a microbe. Biomass may contain cells, microbes and/or intracellular contents, for example cellular fatty acids and TAGS, as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by a cell, for example, secreted fatty acids or TAGs. Important types of biomass for biofuel production are algal biomass and plant-derived biomass, for example, corn stover and wood fiber. In some embodiments, biomass for biofuel or biofuel precursor production may comprise plant derived sugars, for example, sugarcane or corn derived sugars.

Some aspects of this disclosure relate to the engineering and development of a microbial source of lipids, useful, for example, for economically viable, industrial- scale biodiesel production. The term "lipid" refers to fatty acids and their derivatives.

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

Some aspects of this invention relate to engineered microbes for biofuel or biofuel precursor production. In some embodiments, the microbes provided herein are engineered to use 5C sugars as a carbon source, for example xylose. In some embodiments, the microbes provided herein also are engineered to optimize their lipid metabolism for lipid production. The term "lipid metabolism" refers to the molecular processes that involve the creation or degradation of lipids. Fatty acid synthesis, fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAG storage and TAG degradation are examples of processes that are part of the lipid metabolism of a cell. Accordingly, the term "fatty acid metabolism" refers to all cellular or organismic processes that involve the synthesis, creation, transformation or degradation of fatty acids. Fatty acid synthesis, fatty acid oxidation, TAG synthesis, and TAG degradation are examples of processes are part of the fatty acid metabolism of a cell. The term "triacylglycerol" (TAG, sometimes also referred to as triglyceride) refers to a molecule comprising a single molecule of glycerol covalently bound to three fatty acid molecules, aliphatic monocarboxylic acids, via ester bonds, one on each of the glycerol molecule's three hydroxyl (OH) groups. Triacylglycerols are highly concentrated stores of metabolic energy because of their reduced, anhydrous nature, and are a suitable feedstock for biodiesel production.

Many cells and organisms store metabolic energy in the form of fatty acids and fatty acid derivatives, such as TAGs. Fatty acids and their derivatives, such as TAGs, provide an ideal form to store metabolic energy. The energy contained in the C-C bonds can be efficiently released by β-oxidation, a reaction formally equivalent to the reverse of fatty acid biosynthesis, but mediated and regulated by different enzymes constituting a different molecular pathway. Microbes can derive fatty acids from external supply, endogenous turnover, and de novo synthesis. Some aspects of this invention relate to the identification of a microbe for biofuel or biofuel precursor production based on the microbe's ability to synthesize and store fatty acids or fatty acid derivatives, such as TAGs, efficiently from an externally supplied carbon source.

Natural fatty acid molecules commonly have an unbranched, aliphatic chain, or tail, of 4 to 28 carbon atoms. Fatty acids are referred to as "saturated", if all carbon atoms of the aliphatic chain are connected via a C-C single bond, or as "unsaturated", if two or more carbon atoms are connected via a C-C double bond. Unsaturated fatty acids play important roles in the regulation of membrane fluidity, cellular activity, metabolism and nuclear events governing gene transcription.

The spectrum of fatty acids in yeast consists mostly of C16 and C18 fatty acids, for example palmitic acid (CI 6), palmitoleic acid (CI 6), stearic acid (CI 8) and oleic acid (CI 8). Palmitic acid is an unbranched, saturated fatty acid, with an aliphatic chain of 16 carbon atoms (carbon atoms/unsaturated bonds: 16.0). Stearic acid is an unbranched, saturated fatty acid with an aliphatic chain of 18 carbon atoms (18.0). Palmitoleic acid is a monounsaturated fatty acid with an aliphatic chain of 16 carbon atoms (16.1). Oleic acid is a monounsaturated fatty acid with an aliphatic chain of 18 carbon atoms (18.1). Minor fatty acid species in yeast include C14 and C26 fatty acids, which play essential functions in protein modification or as components of sphingolipids and GPI anchors, respectively. De novo synthesis of fatty acids utilizes substantial amounts of metabolites, acetyl- CoA, ATP and NADPH, and thus competes with other cellular processes that are dependent on these compounds. NADPH is required for two reduction steps in the fatty acid elongation cycle, linking fatty acid synthesis to the metabolic state of the cell and results in fatty acid synthesis being restricted to conditions of high energy load of the cells, indicated by increased ATP/ AMP ratio, elevated reduction equivalents and elevated acetyl-CoA pool. Almost all subcellular organelles are involved in fatty acid metabolism, indicating that maintenance of fatty acid homeostasis requires regulation at multiple levels. Lipid synthesis steps that generate metabolites, acetyl-CoA, ATP, or NADPH for lipid biosynthesis are sometimes referred to herein as "push steps" of lipid synthesis. The amplification of a process that increases the production of a metabolites, acetyl-CoA, ATP, or NADPH for lipid synthesis in a cell, for example, by overexpressing a gene product mediating such a metabolite-producing process, is sometimes referred to herein as a "push modification."

Most organisms, including yeast, are able to synthesize fatty acids de novo from a variety of carbon sources. In an initial step, acetyl-CoA is carboxylated by the addition of C0 ₂ to malonyl-CoA, by the enzyme acetyl-CoA carboxylase (ACC; encoded by ACC1 and HFA1 in yeast). Biotin is an essential cof actor in this reaction, and is covalently attached to the ACC apoprotein, by the enzyme biotin:apoprotein ligase (encoded by BPL1/ACC2 in yeast). ACC is a trifunctional enzyme, harboring a biotin carboxyl carrier protein (BCCP) domain, a biotin-carboxylase (BC) domain, and a carboxyl-transferase (CT) domain. In most bacteria, these domains are expressed as individual polypeptides and assembled into a heteromeric complex. In contrast, eukaryotic ACC, including mitochondrial ACC variants (Hfal in yeast) harbor these functions on a single polypeptide. Malonyl-CoA produced by ACC serves as a two carbon donor in a cyclic series of reactions catalyzed by fatty acid synthase, FAS, and elongases.

The immediate product of de novo fatty acid synthesis are saturated fatty acids. Saturated fatty acids are known to be the precursors of unsaturated fatty acids in eukaryotes, including yeast. Unsaturated fatty acids are generally produced by desaturation of C-C single bonds in saturated fatty acids by specialized enzymes, called desaturases. The control mechanisms that govern the conversion of saturated fatty acids to unsaturated fatty acids are not well understood. In eukaryotes, unsaturated fatty acids play important roles in the regulation of membrane fluidity, cellular activity, metabolism and nuclear events that govern gene transcription. Typically, about 80% of yeast fatty acids are monounsaturated, meaning that they contain one unsaturated bond in their aliphatic chain.

Fatty acids are potent inhibitors of fatty acid synthesis and the feedback inhibition of fatty acid synthesis by fatty acids is a major obstacle in engineering microbes for oil production. Some aspects of this disclosure are based on the recognition that while push modifications of lipid synthesis are typically unable to override fatty acid-mediated feedback inhibition of lipid synthesis, a combination of a push modification (e.g., ACC1

overexpression) with a pull modification (e.g., DGA1 overexpression), can efficiently bypass the feedback inhibition, thus fully realizing the increased carbon flux to the lipid synthesis pathway, for example, in TGAs stored in a lipid body or vacuole of the cell

Engineering the capacity for 5C sugar utilization and increased lipid synthesis in oleaginous microbes

Some aspects of this disclosure provide strategies for engineering microbes for oil production. In some embodiments, such strategies employ genetic engineering of oleaginous microbes, for example, Y. lipolytica, to utilize five carbon (5C) sugars, such as xylose, as a carbon source for lipid synthesis.

Some aspects of this disclosure are based on the surprising discovery, described herein, that oleaginous microbes, such as Y. lipolytica, which are unable to metabolize xylose for lipid synthesis, can be engineered to be able to utilize five carbon (5C) sugars as feedstocks or in feedstocks. Some aspects of this disclosure relate to the engineering of oleaginous microbes to utilize 5C sugars, such as xylose, through the introduction of exogenous xylose metabolism genes or the amplification or modification of endogenous xylose metabolism genes. Some aspects of this disclosure relate to the discovery that an oleaginous microbe such as Y. lipolytica has within its genome a copy of an XYL3 gene that produces a functional gene product. Some aspects of this disclosure are related to the heterologous overexpression of xylose metabolism genes, such as XYLl and XYLl, or XYLA, in an oleaginous microbe such as Y. lipolytica, which enables the microbe to utilize xylose as a sole carbon source in the production TAGs.

Some aspects of this disclosure provide strategies for additional engineering of 5C- utilizing microbes for oil production. In some embodiments, such strategies employ genetic engineering of oleaginous microbes, for example Y. lipolytica, to simultaneously amplify a push- and a pull- step of lipid synthesis. Significant increases of lipid production in oleaginous yeast host cells can be achieved using these strategies.

According to some aspects of this invention, modifying the lipid metabolism in a microbe in accordance with methods provided herein, for example by simultaneously overexpressing a gene product mediating a metabolite-generating (push) step and a gene product mediating a product-sequestering (pull) step of lipid synthesis, allows for the generation of a microbe optimized for use in biofuel or biofuel precursor production processes. Some aspects of this invention provide strategies and methods for engineering the fatty acid metabolism in a microbe by simultaneously amplifying a push step and a pull step of lipid biosynthesis, resulting in increased synthesis rate and accumulation of fatty acids and fatty acid derivatives in the microbe.

Some aspects of this invention provide methods that include genetic modifications resulting in the modulation of the expression and/or activity of gene products regulating the lipid metabolism of microbes for biofuel or biofuel precursor production. Such genetic modifications according to some aspects of this invention are targeted to increase

carbohydrate to fatty acid and/or TAG conversion in order to optimize the modified microbe for large-scale production of lipids from a carbon source, for example, a carbohydrate source such as a 5C sugar, e.g., xylose. Some modifications provided according to some aspects of this invention, for example, overexpression, knockout, knock-down, activation and/or inhibition of specific gene products, may be effected alone or in combination, and/or in combination with other modifications known to those of skill in the art. The term

"modification" refers to both genetic manipulation, for example, overexpression, knockout, knock-down, activation and/or inhibition of specific gene products, and non-genetic manipulation, for example, manipulation of the growth media, substrate, substrate pretreatment, pH, temperature, conversion process, etc.

A modification of gene expression, also referred to herein as a modulation of gene expression, can be a disruption or inhibition of the natural regulation of expression, an overexpression, an inhibition of expression, or a complete abolishment of expression of a given gene. The insertion of a heterologous promoter upstream of a native gene sequence, for example the native DGA1 or ACC1 gene sequence, or the deletion of regulatory sequences within a promoter, for example regulatory sequences that mediate the feedback inhibition of the DGA1 or ACC1 gene by saturated fatty acids, are examples of a disruption or inhibition of the natural regulation of expression. Strategies for the modulation of gene expression may include genetic alterations, for example by recombinant technologies, such as gene targeting or viral transductions, or non-genetic alterations, for example environmental alterations known to result in the up- or down-regulation of gene expression, or transient delivery of modulators, for example drugs or small RNA molecules to the target cells. Methods for genetic and non-genetic alterations of microbes are well known to those of skill in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194),

Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351 , Academic Press; 1st edition (July 9, 2002); Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998); and Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), all of which are incorporated by reference herein.

The term "overexpression", as used herein, refers to an increased level of expression of a given gene product in a given cell, cell type or cell state, as compared to a reference cell, for example, a wild type cell of the same cell type or a cell of the same cell type but lacking a specific modification, for example, a genetic modification. Forced, continuous expression of the DGA1 and/or ACC1 gene in Y. lipolytica cells exhibiting concentrations of saturated fatty acids that would inhibit DGA1 or ACC1 gene expression in wild- type cells is an example of gene overexpression.

Some aspects of this invention provide a method for the manipulation of the activity of a xylose reductase (XD or XYL1) gene product in a microbe, including for biofuel or biofuel precursor production. The XYL1 gene encodes a reductase that reduces xylose to xylitol, the initial step of metabolizing xylose as required for entry into the PPP pathway. XYL1 uses NADPH as a reducing equivalent, generating xylitol and NADP+. Xylitol is then acted upon by XYL2 as described herein. In some embodiments, the manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a XYL1 gene product, for example, an XD protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for a XYL1 gene product comprises the coding sequence of SEQ ID NO: 1. In some embodiments, the XYL1 is Scheffersomyces stipitis XYL1, for example, S. stipitis XYL1 comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the microbe is Y. lipolytica. In some embodiments, manipulation of the activity of a XYL1 gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, using xylose as the carbohydrate source. XYL1 gene and gene product sequences are well known to those of skill in the art.

Exemplary, representative gene and gene product sequences can be found under entry XM_001385144 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYL1 nucleic acid and protein sequences are provided below. Additional suitable XYL1 sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

Xylose Reductase

XYL1 DNA {Scheffersomyces stipitis)

XM_001385144 TACAACTATACTACAATGCCTTCTATTAAGTTGAACTCTGGTTACGACATGCCAGCCGTC GGTTTCGGCTGTTGG AAAGTCGACGTCGACACCTGTTCTGAACAGATCTACCGTGCTATCAAGACCGGTTACAGA TTGTTCGACGGTGCC GAAGATTACGCCAACGAAAAGTTAGTTGGTGCCGGTGTCAAGAAGGCCATTGACGAAGGT ATCGTCAAGCGTGAA G AC T T G T T C C T T AC C T C C AAG T T G T GG AAC AAC T AC C AC C AC C C AG AC AAC G T C G AAAAGGC C T T G AAC AG AAC C CTTTCTGACTTGCAAGTTGACTACGTTGACTTGTTCTTGATCCACTTCCCAGTCACCTTC AAGTTCGTTCCATTA G AAG AAAAG T AC C C AC C AGG AT T C T AC T G T GG T AAGGG T G AC AAC T T C G AC T AC G AAG AT GTTCCAATTT TAG AG ACCTGGAAGGCTCTTGAAAAGTTGGTCAAGGCCGGTAAGATCAGATCTATCGGTGTTTCT AACTTCCCAGGTGCT TTGCTCTTGGACTTGTTGAGAGGTGCTACCATCAAGCCATCTGTCTTGCAAGTTGAACAC CACCCATACTTGCAA CAACCAAGATTGATCGAATTCGCTCAATCCCGTGGTATTGCTGTCACCGCTTACTCTTCG TTCGGTCCTCAATCT TTCGTTGAATTGAACCAAGGTAGAGCTTTGAACACTTCTCCATTGTTCGAGAACGAAACT ATCAAGGCTATCGCT GCTAAGCACGGTAAGTCTCCAGCTCAAGTCTTGTTGAGATGGTCTTCCCAAAGAGGCATT GCCATCATTCCAAAG TCCAACACTGTCCCAAGATTGTTGGAAAACAAGGACGTCAACAGCTTCGACTTGGACGAA CAAGATTTCGCTGAC ATTGCCAAGTTGGACATCAACTTGAGATTCAACGACCCATGGGACTGGGACAAGATTCCT ATCTTCGTCTAAGAA GGTTGCTTTATAGAGAGGAAATAAAACCTAATATACATTGATTGTACATTT ( SEQ I D NO : 1 )

Xylose Reductase

XYL1 Protein (Scheffersomyces stipitis)

XP_001385181 MPS IKL SGYDMPAVGFGCWKVDVDTCSEQI YRAIKTGYRLFDGAEDYANEKLVGAGVKKAI DEGIVKREDLFLT SKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFL IHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVP I LETWKAL EKLVKAGKIRS IGVSNFPGALLLDLLRGAT IKPSVLQVEHHPYLQQPRL IEFAQSRGIAVTAYS SFGPQSFVELN QGRALNTSPLFENET IKAIAAKHGKSPAQVLLRWS SQRGIAI IPKSNTVPRLLENKDV SFDLDEQDFADIAKLD INLRFNDPWDWDKIP IFV ( SEQ I D NO : 2 )

Some aspects of this invention provide a method for the manipulation of the activity of a xylitol dehydrogenase (XDH or XYL2) gene product in a microbe for biofuel or biofuel precursor production. As described herein, this manipulation may be made in combination with manipulation of XYL1. The XYL2 gene encodes a dehydrogenase that dehydrogenates xylitol to xylulose, the second step of metabolizing xylose as required for entry into the PPP. XYL2 uses NAD+ as an electron acceptor, generating xylulose and NADH. Xylulose is then acted upon by XYL3 as described herein. In some embodiments, the manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a XYL2 gene product, for example, an XDH protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for a XYL2 gene product comprises the coding sequence of SEQ ID NO: 3. In some embodiments, the XYL2 is Scheffersomyces stipitis XYL2, for example, S. stipitis XYL2 comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the microbe is Y. lipolytica. In some embodiments, manipulation of the activity of a XYL2 gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, using xylose as the carbohydrate source. XYL2 gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under entry XM_001386945 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYL2 nucleic acid and protein sequences are provided below. Additional suitable XYL2 sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

Xylitol dehydrogenase

XYL2 DNA (Scheffersomyces stipitis)

XM_001386945

CCTCACTTTAGTTTGTTTCAATCACCCCTAATACTCTTCACACAATTAAAATGACTG CTAACCCTTCCTTGGTGT TGAACAAGATCGACGACATTTCGTTCGAAACTTACGATGCCCCAGAAATCTCTGAACCTA CCGATGTCCTCGTCC AGGTCAAGAAAACCGGTATCTGTGGTTCCGACATCCACTTCTACGCCCATGGTAGAATCG GTAACTTCGTTTTGA CCAAGCCAATGGTCTTGGGTCACGAATCCGCCGGTACTGTTGTCCAGGTTGGTAAGGGTG TCACCTCTCTTAAGG TTGGTGACAACGTCGCTATCGAACCAGGTATTCCATCCAGATTCTCCGACGAATACAAGA GCGGTCACTACAACT TGTGTCCTCACATGGCCTTCGCCGCTACTCCTAACTCCAAGGAAGGCGAACCAAACCCAC CAGGTACCTTATGTA AGTACTTCAAGTCGCCAGAAGACTTCTTGGTCAAGTTGCCAGACCACGTCAGCTTGGAAC TCGGTGCTCTTGTTG AGCCATTGTCTGTTGGTGTCCACGCCTCTAAGTTGGGTTCCGTTGCTTTCGGCGACTACG TTGCCGTCTTTGGTG CTGGTCCTGTTGGTCTTTTGGCTGCTGCTGTCGCCAAGACCTTCGGTGCTAAGGGTGTCA TCGTCGTTGACATTT TCGACAACAAGTTGAAGATGGCCAAGGACATTGGTGCTGCTACTCACACCTTCAACTCCA AGACCGGTGGTTCTG AAGAATTGATCAAGGCTTTCGGTGGTAACGTGCCAAACGTCGTTTTGGAATGTACTGGTG CTGAACCTTGTATCA AGTTGGGTGTTGACGCCATTGCCCCAGGTGGTCGTTTCGTTCAAGTCGGTAACGCTGCTG GTCCAGTCAGCTTCC CAATCACCGTTTTCGCCATGAAGGAATTGACTTTGTTCGGTTCTTTCAGATACGGATTCA ACGACTACAAGACTG CTGTTGGAATCTTTGACACTAACTACCAAAACGGTAGAGAAAATGCTCCAATTGACTTTG AACAATTGATCACCC ACAGATACAAGTTCAAGGACGCTATTGAAGCCTACGACTTGGTCAGAGCCGGTAAGGGTG CTGTCAAGTGTCTCA TTGACGGCCCTGAGTAAGTCAACCGCTTGGCTGGCCCAAAGTGAACCAGAAACGAAAATG ATTATCAAATAGCTT TATAGACCTTTATCCAAATTTATGTAAACTAATAG (SEQ ID NO: 3)

Xylitol Dehydrogenase

XYL2 Protein {Scheffersomyces stipitis)

XP_001386982

MTANPSLVLNKIDDI SFETYDAPEI SEPTDVLVQVKKTGICGSDIHFYAHGRIGNFVLTKPMVLGHESAGTVVQV GKGVTSLKVGDNVAIEPGIPSRFSDEYKSGHYNLCPHMAFAATPNSKEGEPNPPGTLCKY FKSPEDFLVKLPDHV SLELGALVEPLSVGVHASKLGSVAFGDYVAVFGAGPVGLLAAAVAKTFGAKGVIVVDIFD NKLKMAKDIGAATHT FNSKTGGSEELIKAFGGNVPNVVLECTGAEPCIKLGVDAIAPGGRFVQVGNAAGPVSFPI TVFAMKELTLFGSFR YGFNDYKTAVGIFDTNYQNGRENAPIDFEQLITHRYKFKDAIEAYDLVRAGKGAVKCLID GPE (SEQ ID NO: 4) Some aspects of this invention provide a method for the manipulation of the activity of a xylulokinase (XK or XYL3) gene product in a microbe for biofuel or biofuel precursor production. As described herein, this manipulation may be made in combination with manipulation of XYL1 and XYL2. The XYL3 gene encodes a kinase that uses ATP as a phosphate donor, phosphorylating xylulose to form xylulose-5-P adnADP, the final step of metabolizing xylose as required for entry into the PPP. Xylulose-5-P enters the PPP where it ultimately produces glyceraldehyde- 3 -phosphate (G3P) and fructose-6-phosphate (F6P). These two products can then enter the rest of central metabolism, going through glycolysis to enter the TCA cycle. Production of lipids occurs normally through pathways described herein. In some embodiments, the manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a XYL3 gene product, for example, an XK protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for a XYL3 gene product comprises the coding sequence of SEQ ID NO: 5. In some embodiments, the XYL3 is Y. lipolytica XYL2, for example, Y. lipolytica XYL2 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the microbe is Y.

lipolytica. In some embodiments, manipulation of the activity of a XYL3 gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, using xylose as the carbohydrate source. XYL3 gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under entry XM_505266 in the NCBI database

(www . ncbi .nlm. nih . go v) .

Non-limiting examples of suitable sequences of XYL3 nucleic acid and protein sequences are provided below. Additional suitable XYL3 sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

Xylulokinase XYL3 DNA (Yarrowia Upolytica)

XM_505266

ATGTATCTCGGACTGGATCTTTCGACTCAACAGCTCAAGGGCATCATTCTGGACACA AAAACGCTGGACACGGTC ACACAAGTCCATGTGGACTTTGAGGACGACTTGCCGCAGTTCAACACCGAAAAGGGCGTC TTTCACAGCTCTACA GTGGCCGGAGAAATCAATGCTCCTGTGGCAATGTGGGGGGCAGCTGTGGACTTGCTGATA GAGCGTCTGTCAAAG GAAATAGACCTTTCCACGATCAAGTTTGTGTCGGGCTCGTGCCAGCAACACGGCTCTGTT TATCTCAACAGCAGC TACAAGGAGGGCCTGGGTTCTCTGGACAAACACAAAGACTTGTCTACAGGAGTGTCATCC TTACTGGCGCTCGAA GTCAGCCCCAATTGGCAGGATGCAAGCACGGAGAAGGAGTGTGCGCAGTTTGAGGCTGCA GTCGGCGGTCCCGAG CAGCTGGCTGAGATCACTGGCTCTCGAGCACATACTCGTTTCACCGGGCCCCAGATTCTC AAGGTCAAGGAACGC AACCCCAAGGTATTCAAGGCCACGTCACGGGTCCAGCTCATATCCAACTTTCTAGCATCT CTGTTTGCCGGCAAG GCGTGCCCCTTTGATCTTGCTGACGCCTGTGGAATGAATCTGTGGGACATCCAGAATGGC CAGTGGTGCAAGAAA CTCACAGATCTCATCACCGATGACACCCACTCGGTCGAGTCCCTCCTTGGAGACGTGGAA ACAGACCCCAAGGCT CTACTGGGCAAAATCTCGCCCTATTTCGTCTCCAAGGGCTTCTCTCCCTCTTGTCAGGTG GCACAGTTCACAGGC GACAACCCAGGCACTATGCTGGCTCTCCCCTTACAGGCCAATGACGTGATTGTGTCTTTG GGAACATCTACGACC GCCCTCGTCGTAACAAACAAGTACATGCCCGACCCCGGATACCATGTGTTCAACCACCCC ATGGAGGGATACATG GGCATGCTGTGCTACTGCAACGGAGGTCTAGCACGAGAGAAGATCCGAGACGAGCTTGGA GGCTGGGACGAGTTT AATGAGGCGGCCGAGACCACCAACACAGTGTCTGCTGACGATGTCCATGTTGGCATCTAC TTTCCACTACGAGAA ATCCTTCCTCGAGCAGGTCCCTTTGAACGACGTTTCATCTACAACAGACAAAGTGAACAG CTTACAGAGATGGCT TCTCCAGAGGACTCACTGGCAACCGAACACAAACCGCAGGCTCAAAATCTCAAGGACACG TGGCCGCCACAAATG GACGCCACTGCCATCATTCAAAGCCAGGCCCTCAGTATCAAAATGAGACTCCAACGCATG ATGCATGGCGATATT GGAAAGGTGTATTTTGTGGGAGGCGCCTCGGTCAACACTGCTATCTGCAGCGTAATGTCT GCCATCTTAAAACCA ACAAAGGGCGCTTGGAGATGTGGTCTGGAAATGGCAAACGCTTGTGCCATTGGAAGTGCC CATCACGCCTGGCTT TGCGACCCCAACAAGACAGGCCAGGTACAGGTTCACGAAGAAGAGGTCAAATACAAGAAT GTGGACACAGACGTG CTACTCAAGGCGTTCAAGCTGGCCGAAAACGCCTGCCTGGAGAAATAA (SEQ ID NO: 5)

Xylulokinase

XYL3 Protein (Yarrowia Upolytica)

XP_505266

MYLGLDLSTQQLKGI ILDTKTLDTVTQVHVDFEDDLPQFNTEKGVFHSSTVAGEINAPVAMWGAAVDLLIERLSK EIDLSTIKFVSGSCQQHGSVYLNSSYKEGLGSLDKHKDLSTGVSSLLALEVSPNWQDAST EKECAQFEAAVGGPE QLAEITGSRAHTRFTGPQILKVKERNPKVFKATSRVQLI SNFLASLFAGKACPFDLADACGMNLWDIQNGQWCKK LTDLITDDTHSVESLLGDVETDPKALLGKI SPYFVSKGFSPSCQVAQFTGDNPGTMLALPLQANDVIVSLGTSTT ALVVTNKYMPDPGYHVFNHPMEGYMGMLCYCNGGLAREKIRDELGGWDEFNEAAETTNTV SADDVHVGIYFPLRE ILPRAGPFERRFIYNRQSEQLTEMASPEDSLATEHKPQAQNLKDTWPPQMDATAI IQSQALS IKMRLQRMMHGDI GKVYFVGGASVNTAICSVMSAILKPTKGAWRCGLEMANACAIGSAHHAWLCDPNKTGQVQ VHEEEVKYKNVDTDV LLKAFKLAENACLEK (SEQ ID NO: 6)

Some aspects of this invention provide a method for the manipulation of the activity of a xylose isomerase (XYLA) gene product in a microbe for biofuel or biofuel precursor production. The XYLA gene encodes an isomerase that converts xylose directly to xylulose without the requirement of reducing equivalents, effectively eliminating one step as described herein with the redox pathway (XYL1/XYL2). Xylulose may then be acted upon by XYL3 to form xylulose-5-P, the final step of metabolizing xylose as required for entry into the PPP, as described herein. In some embodiments, the manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a XYLA gene product, for example, a XYLA protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for a XYLA gene product comprises the coding sequence of SEQ ID NO: 7. In some embodiments, the XYLA is Piromyces sp. E2 XYLA, for example, Piromyces sp. E2 XYLA comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the microbe is Y. lipolytica. In some embodiments, manipulation of the activity of a XYLA gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, using xylose as the carbohydrate source. XYLA gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under GenBank entries HV445113, FW568191, and HC036431 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of XYLA nucleic acid and protein sequences are provided below. Additional suitable XYLA sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

Piromyces sp E2 Xylose isomerase

DNA sequence

ATGGCTAAAGAGTACTTCCCACAGATTCAGAAGATAAAGTTCGAGGGCAAAGATTCT AAAAACCCTTTGGCTTTC CACTACTATGATGCAGAGAAGGAAGTCATGGGAAAGAAAATGAAGGATTGGTTGAGATTT GCTATGGCTTGGTGG CATACTTTGTGTGCTGAAGGTGCAGACCAGTTCGGCGGTGGCACTAAGTCTTTTCCTTGG AATGAGGGTACTGAT GCCATTGAAATCGCCAAACAAAAGGTAGACGCTGGTTTTGAGATCATGCAGAAGTTGGGC ATCCCTTATTACTGT TTTCACGATGTCGATTTGGTGAGTGAAGGCAATAGTATAGAGGAATACGAGTCTAACTTA AAGGCAGTCGTTGCC TATTTGAAGGAGAAGCAAAAGGAAACTGGTATCAAATTGTTGTGGAGTACTGCTAACGTC TTCGGCCACAAAAGA TACATGAACGGTGCTTCTACTAATCCAGACTTTGATGTAGTCGCTAGAGCTATAGTCCAG ATTAAGAATGCTATC GACGCCGGAATTGAGTTGGGAGCTGAGAACTATGTTTTTTGGGGAGGTAGGGAAGGCTAT ATGTCTTTGTTGAAT ACTGACCAGAAGAGAGAGAAAGAACACATGGCAACAATGTTAACTATGGCAAGAGATTAC GCAAGGAGTAAGGGC TTTAAGGGCACTTTTTTGATTGAACCTAAGCCTATGGAACCAACTAAACACCAATATGAT GTTGACACTGAAACA GCCATCGGTTTCTTGAAGGCCCACAACTTGGATAAAGATTTTAAGGTAAACATTGAGGTC AATCACGCCACCTTG GCCGGTCACACTTTCGAACATGAATTGGCTTGTGCTGTTGATGCTGGAATGTTGGGTTCT ATTGATGCAAATAGA GGCGATTATCAGAATGGTTGGGATACTGATCAATTTCCAATCGACCAATACGAATTGGTT CAAGCCTGGATGGAA ATCATAAGAGGTGGTGGCTTTGTAACTGGTGGAACTAACTTCGATGCCAAAACAAGAAGA AACTCCACTGACTTG GAGGATATCATTATTGCTCACGTTTCCGGTATGGATGCAATGGCCAGGGCCTTGGAGAAC GCTGCTAAGTTGTTA CAAGAATCCCCCTACACTAAGATGAAGAAAGAGAGGTACGCATCATTCGATTCTGGAATC GGCAAGGATTTTGAG GACGGAAAGTTGACTTTAGAGCAGGTTTATGAGTACGGTAAAAAGAATGGCGAGCCTAAA CAAACCTCTGGTAAG CAGGAATTGTACGAAGCTATTGTCGCAATGTATCAATAA (SEQ ID NO: 7)

Piromyces sp E2

Xylose Isomerase

Protein Sequence

MAKEYFPQIQKIKFEGKDSKNPLAFHYYDAEKEVMGKKMKDWLRFAMAWWHTLCAEG ADQFGGGTKSFPWNEGTD AIEIAKQKVDAGFEIMQKLGIPYYCFHDVDLVSEGNS IEEYESNLKAVVAYLKEKQKETGIKLLWSTANVFGHKR YMNGASTNPDFDVVARAIVQIKNAIDAGIELGAENYVFWGGREGYMSLLNTDQKREKEHM ATMLTMARDYARSKG FKGTFLIEPKPMEPTKHQYDVDTETAIGFLKAHNLDKDFKV IEV HATLAGHTFEHELACAVDAGMLGS IDANR GDYQNGWDTDQFPIDQYELVQAWMEI IRGGGFVTGGTNFDAKTRRNSTDLEDI I IAHVSGMDAMARALENAAKLL QESPYTKMKKERYASFDSGIGKDFEDGKLTLEQVYEYGKKNGEPKQTSGKQELYEAIVAM YQ (SEQ ID NO: 8)

Some aspects of this invention provide a method for the manipulation of the activity of a diacylglycerol acyltransf erase 1 (DGA1) gene product in a microbe for biofuel or biofuel precursor production. The DGA1 gene encodes an acyltransferase that catalyzes the terminal step of triacylglycerol (TAG) formation, acylating diacylglycerol using acyl-CoA as an acyl donor. The result of this acyltransferase reaction are triacylglycerols, which do not exhibit the same inhibitory feedback effect on fatty acid synthesis as fatty acids themselves. TAGs are typically stored in lipid bodies or vacuoles in lipid producing cells. In some embodiments, the manipulation is an overexpression. In some embodiments, the

manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a DGA1 gene product, for example, a DGAT2 protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for a DGA1 gene product comprises the coding sequence of SEQ ID NO: 9. In some

embodiments, the DGA1 is Y. lipolytica DGA1, for example, Y. lipolytica DGA1 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the microbe is Y.

lipolytica. In some embodiments, manipulation of the activity of a DGA1 gene product in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, for example increased lipid synthesis rate, increased carbohydrate to lipid conversion efficiency, increased lipid storage and, increased growth rate, increased tolerance to elevated concentrations of a carbon source or a lipid product. DGA1 gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under entry XM_504700 in the NCBI database (www . ncbi .nlm. nih . go v) .

Non-limiting examples of suitable sequences of DGA1 nucleic acid and protein sequences are provided below. Additional suitable DGA1 sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

>gil50554582lreflXM_504700.1l Yarrowia lipolytica YALI0E32769p (YALI0E32769g) mRNA, complete cds

ATGACTATCGACTCACAATACTACAAGTCGCGAGACAAAAACGACACGGCACCCAAAATC GCGGGAATCCGATAT GCCCCGCTATCGACACCATTACTCAACCGATGTGAGACCTTCTCTCTGGTCTGGCACATT TTCAGCATTCCCACT TTCCTCACAATTTTCATGCTATGCTGCGCAATTCCACTGCTCTGGCCATTTGTGATTGCG TATGTAGTGTACGCT GTTAAAGACGACTCCCCGTCCAACGGAGGAGTGGTCAAGCGATACTCGCCTATTTCAAGA AACTTCTTCATCTGG AAGCTCTTTGGCCGCTACTTCCCCATAACTCTGCACAAGACGGTGGATCTGGAGCCCACG CACACATACTACCCT CTGGACGTCCAGGAGTATCACCTGATTGCTGAGAGATACTGGCCGCAGAACAAGTACCTC CGAGCAATCATCTCC ACCATCGAGTACTTTCTGCCCGCCTTCATGAAACGGTCTCTTTCTATCAACGAGCAGGAG CAGCCTGCCGAGCGA GATCCTCTCCTGTCTCCCGTTTCTCCCAGCTCTCCGGGTTCTCAACCTGACAAGTGGATT AACCACGACAGCAGA TATAGCCGTGGAGAATCATCTGGCTCCAACGGCCACGCCTCGGGCTCCGAACTTAACGGC AACGGCAACAATGGC ACCACTAACCGACGACCTTTGTCGTCCGCCTCTGCTGGCTCCACTGCATCTGATTCCACG CTTCTTAACGGGTCC CTCAACTCCTACGCCAACCAGATCATTGGCGAAAACGACCCACAGCTGTCGCCCACAAAA CTCAAGCCCACTGGC AGAAAATACATCTTCGGCTACCACCCCCACGGCATTATCGGCATGGGAGCCTTTGGTGGA ATTGCCACCGAGGGA GCTGGATGGTCCAAGCTCTTTCCGGGCATCCCTGTTTCTCTTATGACTCTCACCAACAAC TTCCGAGTGCCTCTC TACAGAGAGTACCTCATGAGTCTGGGAGTCGCTTCTGTCTCCAAGAAGTCCTGCAAGGCC CTCCTCAAGCGAAAC CAGTCTATCTGCATTGTCGTTGGTGGAGCACAGGAAAGTCTTCTGGCCAGACCCGGTGTC ATGGACCTGGTGCTA CTCAAGCGAAAGGGTTTTGTTCGACTTGGTATGGAGGTCGGAAATGTCGCCCTTGTTCCC ATCATGGCCTTTGGT GAGAACGACCTCTATGACCAGGTTAGCAACGACAAGTCGTCCAAGCTGTACCGATTCCAG CAGTTTGTCAAGAAC TTCCTTGGATTCACCCTTCCTTTGATGCATGCCCGAGGCGTCTTCAACTACGATGTCGGT CTTGTCCCCTACAGG CGACCCGTCAACATTGTGGTTGGTTCCCCCATTGACTTGCCTTATCTCCCACACCCCACC GACGAAGAAGTGTCC GAATACCACGACCGATACATCGCCGAGCTGCAGCGAATCTACAACGAGCACAAGGATGAA TATTTCATCGATTGG ACCGAGGAGGGCAAAGGAGCCCCAGAGTTCCGAATGATTGAGTAA (SEQ ID NO: 9)

>gil50554583lreflXP_504700.1l YALI0E32769p [Yarrowia lipolytica]

MTIDSQYYKSRDKNDTAPKIAGIRYAPLSTPLLNRCETFSLVWHIFS IPTFLTIFMLCCAIPLLWPFVIAYVVYA VKDDSPSNGGVVKRYSPI SRNFFIWKLFGRYFPITLHKTVDLEPTHTYYPLDVQEYHLIAERYWPQNKYLRAI I S TIEYFLPAFMKRSLS INEQEQPAERDPLLSPVSPSSPGSQPDKWINHDSRYSRGESSGSNGHASGSELNGNGNNG TTNRRPLSSASAGSTASDSTLLNGSLNSYANQI IGENDPQLSPTKLKPTGRKYIFGYHPHGI IGMGAFGGIATEG AGWSKLFPGIPVSLMTLTNNFRVPLYREYLMSLGVASVSKKSCKALLKRNQS ICIVVGGAQESLLARPGVMDLVL LKRKGFVRLGMEVGNVALVPIMAFGENDLYDQVSNDKSSKLYRFQQFVKNFLGFTLPLMH ARGVFNYDVGLVPYR RPV IVVGSPIDLPYLPHPTDEEVSEYHDRYIAELQRIYNEHKDEYFIDWTEEGKGAPEFRMIE ( SEQ ID NO: 10)

Some aspects of this invention provide a method for the manipulation of an acetyl- CoA carboxylase (ACC) gene product in a microbe for biofuel or biofuel precursor production, for example, in Y. lipolytica. ACC gene products mediate the conversion of acetyl-CoA, the main C2 -precursor in fatty acid synthesis, to malonyl-CoA, which is considered the first committed step in fatty acid synthesis and has been suggested to also be the rate-limiting step in fatty acid synthesis (see Cao Y, Yang J, Xian M, Xu X, Liu W. Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Appl Microbiol Biotechnol. 2010). In some embodiments, ACC activity manipulation is ACC overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for an ACC gene product, for example, an ACC1 protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for an ACC gene product comprises the coding sequence of SEQ ID NO: 11. In some embodiments, the ACC gene product is an ACCl protein comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, ACC overexpression in a microbe increases fatty acid synthesis rate and/or confers a beneficial phenotype for large-scale carbohydrate to biofuel or biofuel precursor conversion, for example increased lipid synthesis rate, increased carbohydrate to lipid conversion efficiency, increased lipid storage and, increased growth rate, increased tolerance to concentrations of a substance, e.g. a carbon source, a biofuel or biofuel precursor, or a toxic substance. ACC gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under the entry for GenelDs: 855750 and 2909424, or under the entry NC_006069 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of ACC nucleic acid and protein sequences are provided below. Additional suitable ACC sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

ACC encoding nucleic acid sequence:

ATGCGACTGCAATTGAGGACACTAACACGTCGGTTTTTCAGGTGAGTAAACGACGGTGGC CGTGGCCACGACAGC CGAGGCGTCACGATGGGCCAGACGAGCACATTCTCGCCGCCACAACCTCGCCAGCACAAG AAACTAACCCAGTAT GGCTTCAGGATCTTCAACGCCAGATGTGGCTCCCTTGGTGGACCCCAACATTCACAAAGG TCTCGCCTCTCATTT CTTTGGACTCAATTCTGTCCACACAGCCAAGCCCTCAAAAGTCAAGGAGTTTGTGGCTTC TCACGGAGGTCATAC AGTTATCAACAAGGTGAGTATTTGACGTTTAGACTGTATAACAGGCGGCCGCAGTGCAAC AACGACCAAAAAGGG TCGAAAAAGGGTCGAAAACGGACACAAAAGCTGGAAAACAAGAGTGTAATACATTCTTAC ACGTCCAATTGTTAG ACAAACACGGCTGTTCGGTCCCAAAACCACCAGTATCACCTATTTTCCACTTGTGTCTCG GATCTGATCATAATC T G AT C T C AAG AT GAAAT T T AC GC C AC C G AC AT GATATTGTGATTTTCGGATTCTC C AG AC C G AGC AG AT T C C AGC AATACCACCACTTGCCCACCTTCAGCGGCCTCTCGGCGCGATTCGCCACTTTCCCCAACG AGTGTTACTAACCCA GGTCCTCATCGCTAACAACGGTATTGCCGCAGTAAAGGAGATCCGTTCAGTACGAAAATG GGCCTACGAGACCTT TGGCGACGAGCGAGCAATCTCGTTCACCGTCATGGCCACCCCCGAAGATCTCGCTGCCAA CGCCGACTACATTAG AATGGCCGATCAGTACGTCGAGGTGCCCGGAGGAACCAACAACAACAACTACGCCAACGT CGAGCTGATTGTCGA CGTGGCTGAGCGATTCGGCGTCGATGCCGTGTGGGCCGGATGGGGCCATGCCAGTGAAAA TCCCCTGCTCCCCGA GTCGCTAGCGGCCTCTCCCCGCAAGATTGTCTTCATCGGCCCTCCCGGAGCTGCCATGAG ATCTCTGGGAGACAA AATTTCTTCTACCATTGTGGCCCAGCACGCAAAGGTCCCGTGTATCCCGTGGTCTGGAAC CGGAGTGGACGAGGT TGTGGTTGACAAGAGCACCAACCTCGTGTCCGTGTCCGAGGAGGTGTACACCAAGGGCTG CACCACCGGTCCCAA GCAGGGTCTGGAGAAGGCTAAGCAGATTGGATTCCCCGTGATGATCAAGGCTTCCGAGGG AGGAGGAGGAAAGGG TATTCGAAAGGTTGAGCGAGAGGAGGACTTCGAGGCTGCTTACCACCAGGTCGAGGGAGA GATCCCCGGCTCGCC CATCTTCATTATGCAGCTTGCAGGCAATGCCCGGCATTTGGAGGTGCAGCTTCTGGCTGA TCAGTACGGCAACAA TATTTCACTGTTTGGTCGAGATTGTTCGGTTCAGCGACGGCATCAAAAGATTATTGAGGA GGCTCCTGTGACTGT GGCTGGCCAGCAGACCTTCACTGCCATGGAGAAGGCTGCCGTGCGACTCGGTAAGCTTGT CGGATATGTCTCTGC AGGTACCGTTGAATATCTGTATTCCCATGAGGACGACAAGTTCTACTTCTTGGAGCTGAA TCCTCGTCTTCAGGT CGAACATCCTACCACCGAGATGGTCACCGGTGTCAACCTGCCCGCTGCCCAGCTTCAGAT CGCCATGGGTATCCC CCTCGATCGAATCAAGGACATTCGTCTCTTTTACGGTGTTAACCCTCACACCACCACTCC AATTGATTTCGACTT CTCGGGCGAGGATGCTGATAAGACACAGCGACGTCCCGTCCCCCGAGGTCACACCACTGC TTGCCGAATCACATC CGAGGACCCTGGAGAGGGTTTCAAGCCCTCCGGAGGTACTATGCACGAGCTCAACTTCCG ATCCTCGTCCAACGT GTGGGGTTACTTCTCCGTTGGTAACCAGGGAGGTATCCATTCGTTCTCGGATTCGCAGTT TGGTCACATCTTCGC CTTCGGTGAGAACCGAAGTGCGTCTCGAAAGCACATGGTTGTTGCTTTGAAGGAACTATC TATTCGAGGTGACTT CCGAACCACCGTCGAGTACCTCATCAAGCTGCTGGAGACACCGGACTTCGAGGACAACAC CATCACCACCGGCTG GCTGGATGAGCTTATCTCCAACAAGCTGACTGCCGAGCGACCCGACTCGTTCCTCGCTGT TGTTTGTGGTGCTGC TACCAAGGCCCATCGAGCTTCCGAGGACTCTATTGCCACCTACATGGCTTCGCTAGAGAA GGGCCAGGTCCCTGC TCGAGACATTCTCAAGACCCTTTTCCCCGTTGACTTCATCTACGAGGGCCAGCGGTACAA GTTCACCGCCACCCG GTCGTCTGAGGACTCTTACACGCTGTTCATCAACGGTTCTCGATGCGACATTGGAGTTAG ACCTCTTTCTGACGG TGGTATTCTGTGTCTTGTAGGTGGGAGATCCCACAATGTCTACTGGAAGGAGGAGGTTGG AGCCACGCGACTGTC TGTTGACTCCAAGACCTGCCTTCTCGAGGTGGAGAACGACCCCACTCAGCTTCGATCTCC CTCTCCCGGTAAGCT GGTTAAGTTCCTGGTCGAGAACGGCGACCACGTGCGAGCCAACCAGCCCTATGCCGAGAT TGAGGTCATGAAGAT GTACATGACTCTCACTGCTCAGGAGGACGGTATTGTCCAGCTGATGAAGCAGCCCGGTTC CACCATCGAGGCTGG CGACATCCTCGGTATCTTGGCCCTTGATGATCCTTCCAAGGTCAAGCATGCCAAGCCCTT TGAGGGCCAGCTTCC CGAGCTTGGACCCCCCACTCTCAGCGGTAACAAGCCTCATCAGCGATACGAGCACTGCCA GAACGTGCTCCATAA CATTCTGCTTGGTTTCGATAACCAGGTGGTGATGAAGTCCACTCTTCAGGAGATGGTTGG TCTGCTCCGAAACCC TGAGCTTCCTTATCTCCAGTGGGCTCATCAGGTGTCTTCTCTGCACACCCGAATGAGCGC CAAGCTGGATGCTAC TCTTGCTGGTCTCATTGACAAGGCCAAGCAGCGAGGTGGCGAGTTTCCTGCCAAGCAGCT TCTGCGAGCCCTTGA GAAGGAGGCGAGCTCTGGCGAGGTCGATGCGCTCTTCCAGCAAACTCTTGCTCCTCTGTT TGACCTTGCTCGAGA GTACCAGGACGGTCTTGCTATCCACGAGCTTCAGGTTGCTGCAGGCCTTCTGCAGGCCTA CTACGACTCTGAGGC CCGGTTCTGCGGACCCAACGTACGTGACGAGGATGTCATTCTCAAGCTTCGAGAGGAGAA CCGAGATTCTCTTCG AAAGGTTGTGATGGCCCAGCTGTCTCATTCTCGAGTCGGAGCCAAGAACAACCTTGTGCT GGCCCTTCTCGATGA ATACAAGGTGGCCGACCAGGCTGGCACCGACTCTCCTGCCTCCAACGTGCACGTTGCAAA GTACTTGCGACCTGT GCTGCGAAAGATTGTGGAGCTGGAATCTCGAGCTTCTGCCAAGGTATCTCTGAAAGCCCG AGAGATTCTCATCCA GTGCGCTCTGCCCTCTCTAAAGGAGCGAACTGACCAGCTTGAGCACATTCTGCGATCTTC TGTCGTCGAGTCTCG ATACGGAGAGGTTGGTCTGGAGCACCGAACTCCCCGAGCCGATATTCTCAAGGAGGTTGT CGACTCCAAGTACAT TGTCTTTGATGTGCTTGCCCAGTTCTTTGCCCACGATGATCCCTGGATCGTCCTTGCTGC CCTGGAGCTGTACAT CCGACGAGCTTGCAAGGCCTACTCCATCCTGGACATCAACTACCACCAGGACTCGGACCT GCCTCCCGTCATCTC GTGGCGATTTAGACTGCCTACCATGTCGTCTGCTTTGTACAACTCAGTAGTGTCTTCTGG CTCCAAAACCCCCAC TTCCCCCTCGGTGTCTCGAGCTGATTCCGTCTCCGACTTTTCGTACACCGTTGAGCGAGA CTCTGCTCCCGCTCG AACCGGAGCGATTGTTGCCGTGCCTCATCTGGATGATCTGGAGGATGCTCTGACTCGTGT TCTGGAGAACCTGCC CAAACGGGGCGCTGGTCTTGCCATCTCTGTTGGTGCTAGCAACAAGAGTGCCGCTGCTTC TGCTCGTGACGCTGC TGCTGCTGCCGCTTCATCCGTTGACACTGGCCTGTCCAACATTTGCAACGTTATGATTGG TCGGGTTGATGAGTC TGATGACGACGACACTCTGATTGCCCGAATCTCCCAGGTCATTGAGGACTTTAAGGAGGA CTTTGAGGCCTGTTC TCTGCGACGAATCACCTTCTCCTTCGGCAACTCCCGAGGTACTTATCCCAAGTATTTCAC GTTCCGAGGCCCCGC ATACGAGGAGGACCCCACTATCCGACACATTGAGCCTGCTCTGGCCTTCCAGCTGGAGCT CGCCCGTCTGTCCAA CTTCGACATCAAGCCTGTCCACACCGACAACCGAAACATCCACGTGTACGAGGCTACTGG CAAGAACGCTGCTTC CGACAAGCGGTTCTTCACCCGAGGTATCGTACGACCTGGTCGTCTTCGAGAGAACATCCC CACCTCGGAGTATCT CATTTCCGAGGCTGACCGGCTCATGAGCGATATTTTGGACGCTCTAGAGGTGATTGGAAC CACCAACTCGGATCT CAACCACATTTTCATCAACTTCTCAGCCGTCTTTGCTCTGAAGCCCGAGGAGGTTGAAGC TGCCTTTGGCGGTTT CCTGGAGCGATTTGGCCGACGTCTGTGGCGACTTCGAGTCACCGGTGCCGAGATCCGAAT GATGGTATCCGACCC CGAAACTGGCTCTGCTTTCCCTCTGCGAGCAATGATCAACAACGTCTCTGGTTACGTTGT GCAGTCTGAGCTGTA CGCTGAGGCCAAGAACGACAAGGGCCAGTGGATTTTCAAGTCTCTGGGCAAGCCCGGCTC CATGCACATGCGGTC TATCAACACTCCCTACCCCACCAAGGAGTGGCTGCAGCCCAAGCGGTACAAGGCCCATCT GATGGGTACCACCTA CTGCTATGACTTCCCCGAGCTGTTCCGACAGTCCATTGAGTCGGACTGGAAGAAGTATGA CGGCAAGGCTCCCGA CGATCTCATGACTTGCAACGAGCTGATTCTCGATGAGGACTCTGGCGAGCTGCAGGAGGT GAACCGAGAGCCCGG CGCCAACAACGTCGGTATGGTTGCGTGGAAGTTTGAGGCCAAGACCCCCGAGTACCCTCG AGGCCGATCTTTCAT CGTGGTGGCCAACGATATCACCTTCCAGATTGGTTCGTTTGGCCCTGCTGAGGACCAGTT CTTCTTCAAGGTGAC GGAGCTGGCTCGAAAGCTCGGTATTCCTCGAATCTATCTGTCTGCCAACTCTGGTGCTCG AATCGGCATTGCTGA CGAGCTCGTTGGCAAGTACAAGGTTGCGTGGAACGACGAGACTGACCCCTCCAAGGGCTT CAAGTACCTTTACTT CACCCCTGAGTCTCTTGCCACCCTCAAGCCCGACACTGTTGTCACCACTGAGATTGAGGA GGAGGGTCCCAACGG CGTGGAGAAGCGTCATGTGATCGACTACATTGTCGGAGAGAAGGACGGTCTCGGAGTCGA GTGTCTGCGGGGCTC TGGTCTCATTGCAGGCGCCACTTCTCGAGCCTACAAGGATATCTTCACTCTCACTCTTGT CACCTGTCGATCCGT TGGTATCGGTGCTTACCTTGTTCGTCTTGGTCAACGAGCCATCCAGATTGAGGGCCAGCC CATCATTCTCACTGG TGCCCCCGCCATCAACAAGCTGCTTGGTCGAGAGGTCTACTCTTCCAACTTGCAGCTTGG TGGTACTCAGATCAT GTACAACAACGGTGTGTCTCATCTGACTGCCCGAGATGATCTCAACGGTGTCCACAAGAT CATGCAGTGGCTGTC ATACATCCCTGCTTCTCGAGGTCTTCCAGTGCCTGTTCTCCCTCACAAGACCGATGTGTG GGATCGAGACGTGAC GTTCCAGCCTGTCCGAGGCGAGCAGTACGATGTTAGATGGCTTATTTCTGGCCGAACTCT CGAGGATGGTGCTTT CGAGTCTGGTCTCTTTGACAAGGACTCTTTCCAGGAGACTCTGTCTGGCTGGGCCAAGGG TGTTGTTGTTGGTCG AGCTCGTCTTGGCGGCATTCCCTTCGGTGTCATTGGTGTCGAGACTGCGACCGTCGACAA TACTACCCCTGCCGA TCCCGCCAACCCGGACTCTATTGAGATGAGCACCTCTGAAGCCGGCCAGGTTTGGTACCC CAACTCGGCCTTCAA GACCTCTCAGGCCATCAACGACTTCAACCATGGTGAGGCGCTTCCTCTCATGATTCTTGC TAACTGGCGAGGCTT TTCTGGTGGTCAGCGAGACATGTACAATGAGGTTCTCAAGTACGGATCTTTCATTGTTGA TGCTCTGGTTGACTA CAAGCAGCCCATCATGGTGTACATCCCTCCCACCGGTGAGCTGCGAGGTGGTTCTTGGGT TGTGGTTGACCCCAC CATCAACTCGGACATGATGGAGATGTACGCTGACGTCGAGTCTCGAGGTGGTGTGCTGGA GCCCGAGGGAATGGT CGGTATCAAGTACCGACGAGACAAGCTACTGGACACCATGGCTCGTCTGGATCCCGAGTA CTCCTCTCTCAAGAA GCAGCTTGAGGAGTCTCCCGATTCTGAGGAGCTCAAGGTCAAGCTCAGCGTGCGAGAGAA GTCTCTCATGCCCAT CTACCAGCAGATCTCCGTGCAGTTTGCCGACTTGCATGACCGAGCTGGCCGAATGGAGGC CAAGGGTGTCATTCG TGAGGCTCTTGTGTGGAAGGATGCTCGTCGATTCTTCTTCTGGCGAATCCGACGACGATT AGTCGAGGAGTACCT CATTACCAAGATCAATAGCATTCTGCCCTCTTGCACTCGGCTTGAGTGTCTGGCTCGAAT CAAGTCGTGGAAGCC TGCCACTCTTGATCAGGGCTCTGACCGGGGTGTTGCCGAGTGGTTTGACGAGAACTCTGA TGCCGTCTCTGCTCG ACTCAGCGAGCTCAAGAAGGACGCTTCTGCCCAGTCGTTTGCTTCTCAACTGAGAAAGGA CCGACAGGGTACTCT CCAGGGCATGAAGCAGGCTCTCGCTTCTCTTTCTGAGGCTGAGCGGGCTGAGCTGCTCAA GGGGTTGTGA (SEQ ID NO: 11) >gil50548503lreflXP_501721.1l YALI0C11407p [Yarrowia lipolytica]

MRLQLRTLTRRFFSMASGSSTPDVAPLVDPNIHKGLASHFFGLNSVHTAKPSKVKEFVAS HGGHTVINKVLIANN GIAAVKEIRSVRKWAYETFGDERAI SFTVMATPEDLAANADYIRMADQYVEVPGGTNNNNYANVELIVDVAERFG VDAVWAGWGHASENPLLPESLAASPRKIVFIGPPGAAMRSLGDKI SSTIVAQHAKVPCIPWSGTGVDEVVVDKST NLVSVSEEVYTKGCTTGPKQGLEKAKQIGFPVMIKASEGGGGKGIRKVEREEDFEAAYHQ VEGEIPGSPIFIMQL AGNARHLEVQLLADQYGN I SLFGRDCSVQRRHQKI IEEAPVTVAGQQTFTAMEKAAVRLGKLVGYVSAGTVEYL YSHEDDKFYFLELNPRLQVEHPTTEMVTGVNLPAAQLQIAMGIPLDRIKDIRLFYGVNPH TTTPIDFDFSGEDAD KTQRRPVPRGHTTACRITSEDPGEGFKPSGGTMHELNFRSSSNVWGYFSVGNQGGIHSFS DSQFGHIFAFGENRS ASRKHMVVALKELS IRGDFRTTVEYLIKLLETPDFEDNTITTGWLDELI SNKLTAERPDSFLAVVCGAATKAHRA SEDSIATYMASLEKGQVPARDILKTLFPVDFIYEGQRYKFTATRSSEDSYTLFINGSRCD IGVRPLSDGGILCLV GGRSHNVYWKEEVGATRLSVDSKTCLLEVENDPTQLRSPSPGKLVKFLVENGDHVRANQP YAEIEVMKMYMTLTA QEDGIVQLMKQPGSTIEAGDILGILALDDPSKVKHAKPFEGQLPELGPPTLSGNKPHQRY EHCQNVLHNILLGFD NQVVMKSTLQEMVGLLRNPELPYLQWAHQVSSLHTRMSAKLDATLAGLIDKAKQRGGEFP AKQLLRALEKEASSG EVDALFQQTLAPLFDLAREYQDGLAIHELQVAAGLLQAYYDSEARFCGPNVRDEDVILKL REENRDSLRKVVMAQ LSHSRVGAKNNLVLALLDEYKVADQAGTDSPASNVHVAKYLRPVLRKIVELESRASAKVS LKAREILIQCALPSL KERTDQLEHILRSSVVESRYGEVGLEHRTPRADILKEVVDSKYIVFDVLAQFFAHDDPWI VLAALELYIRRACKA YS ILDINYHQDSDLPPVI SWRFRLPTMSSALYNSVVSSGSKTPTSPSVSRADSVSDFSYTVERDSAPARTGAIVA VPHLDDLEDALTRVLENLPKRGAGLAI SVGASNKSAAASARDAAAAAASSVDTGLS ICNVMIGRVDESDDDDTL IARI SQVIEDFKEDFEACSLRRITFSFG SRGTYPKYFTFRGPAYEEDPTIRHIEPALAFQLELARLSNFDIKPV HTDNR IHVYEATGKNAASDKRFFTRGIVRPGRLRE IPTSEYLI SEADRLMSDILDALEVIGTTNSDLNHIFIN FSAVFALKPEEVEAAFGGFLERFGRRLWRLRVTGAEIRMMVSDPETGSAFPLRAMINNVS GYVVQSELYAEAKND KGQWIFKSLGKPGSMHMRS INTPYPTKEWLQPKRYKAHLMGTTYCYDFPELFRQS IESDWKKYDGKAPDDLMTCN ELILDEDSGELQEVNREPGANNVGMVAWKFEAKTPEYPRGRSFIVVANDITFQIGSFGPA EDQFFFKVTELARKL GIPRIYLSANSGARIGIADELVGKYKVAWNDETDPSKGFKYLYFTPESLATLKPDTVVTT EIEEEGPNGVEKRHV IDYIVGEKDGLGVECLRGSGLIAGATSRAYKDIFTLTLVTCRSVGIGAYLVRLGQRAIQI EGQPI ILTGAPAINK LLGREVYSSNLQLGGTQIMYNNGVSHLTARDDLNGVHKIMQWLSYIPASRGLPVPVLPHK TDVWDRDVTFQPVRG EQYDVRWLI SGRTLEDGAFESGLFDKDSFQETLSGWAKGVVVGRARLGGIPFGVIGVETATVDNTTPAD PANPDS IEMSTSEAGQVWYPNSAFKTSQAINDFNHGEALPLMILANWRGFSGGQRDMYNEVLKYGS FIVDALVDYKQPIMV YIPPTGELRGGSWVVVDPTINSDMMEMYADVESRGGVLEPEGMVGIKYRRDKLLDTMARL DPEYSSLKKQLEESP DSEELKVKLSVREKSLMPIYQQI SVQFADLHDRAGRMEAKGVIREALVWKDARRFFFWRIRRRLVEEYLITKINS ILPSCTRLECLARIKSWKPATLDQGSDRGVAEWFDENSDAVSARLSELKKDASAQSFASQ LRKDRQGTLQGMKQA LASLSEAERAELLKGL (SEQ ID NO: 12) .

Some aspects of this invention provide a method for the manipulation of the activity of a stearoyl-CoA-desaturase (SCD) in a microbe for biofuel or biofuel precursor production. SCD is a Δ9 desaturase that inserts a double bond between C9 and CIO of stearic acid coupled to CoA, a key step in the generation of desaturated fatty acids and their derivatives, as described in more detail elsewhere herein. In some embodiments, the manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for a SCD gene product, for example, a SCD protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for an SCD gene product comprises the coding sequence of SEQ ID NO: 13. In some embodiments, the SCD is Y. lipolytica SCD, for example, Y. lipolytica SCD comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the microbe is Y. lipolytica. In some embodiments, manipulation of the activity of a SCD in a microbe is effected to confer a beneficial phenotype for large-scale

carbohydrate to lipid conversion, for example increased lipid synthesis rate, increased carbohydrate to lipid conversion efficiency, increased lipid storage and, increased growth rate, increased tolerance to elevated concentrations of a carbon source or a lipid product. Stearoyl-CoA Desaturase gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under the entry for GenelD: 852825 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of SCD nucleic acid and protein sequences are provided below. Additional suitable SCD sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

>gil50548052lreflXM_501496.1l Yarrowia lipolytica YALI0C05951p (YALI0C05951g) mRNA, complete cds

ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGA GATGTCAACTACAAG GTCAAGTACACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGGGCCGCCAC ATTTCCGAGCAGCCC TTCACCTGGGCCAACTGGCACCAGCACATCAACTGGCTCAACTTCATTCTGGTGATTGCG CTGCCTCTGTCGTCC TTTGCTGCCGCTCCCTTCGTCTCCTTCAACTGGAAGACCGCCGCGTTTGCTGTCGGCTAT TACATGTGCACCGGT CTCGGTATCACCGCCGGCTACCACCGAATGTGGGCCCATCGAGCCTACAAGGCCGCTCTG CCCGTTCGAATCATC CTTGCTCTGTTTGGAGGAGGAGCTGTCGAGGGCTCCATCCGATGGTGGGCCTCGTCTCAC CGAGTCCACCACCGA TGGACCGACTCCAACAAGGACCCTTACGACGCCCGAAAGGGATTCTGGTTCTCCCACTTT GGCTGGATGCTGCTT GTGCCCAACCCCAAGAACAAGGGCCGAACTGACATTTCTGACCTCAACAACGACTGGGTT GTCCGACTCCAGCAC AAGTACTACGTTTACGTTCTCGTCTTCATGGCCATTGTTCTGCCCACCCTCGTCTGTGGC TTTGGCTGGGGCGAC TGGAAGGGAGGTCTTGTCTACGCCGGTATCATGCGATACACCTTTGTGCAGCAGGTGACT TTCTGTGTCAACTCC CTTGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTCCCCGAGACCACGCT CTTACCGCCCTGGTC ACCTTTGGAGAGGGCTACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGCC CTCATCTGGTACCAG TACGACCCCACCAAGTGGCTCATCTGGACCCTCAAGCAGGTTGGTCTCGCCTGGGACCTC CAGACCTTCTCCCAG AACGCCATCGAGCAGGGTCTCGTGCAGCAGCGACAGAAGAAGCTGGACAAGTGGCGAAAC AACCTCAACTGGGGT ATCCCCATTGAGCAGCTGCCTGTCATTGAGTTTGAGGAGTTCCAAGAGCAGGCCAAGACC CGAGATCTGGTTCTC ATTTCTGGCATTGTCCACGACGTGTCTGCCTTTGTCGAGCACCACCCTGGTGGAAAGGCC CTCATTATGAGCGCC GTCGGCAAGGACGGTACCGCTGTCTTCAACGGAGGTGTCTACCGACACTCCAACGCTGGC CACAACCTGCTTGCC ACCATGCGAGTTTCGGTCATTCGAGGCGGCATGGAGGTTGAGGTGTGGAAGACTGCCCAG AACGAAAAGAAGGAC CAGAACATTGTCTCCGATGAGAGTGGAAACCGAATCCACCGAGCTGGTCTCCAGGCCACC CGGGTCGAGAACCCC GGTATGTCTGGCATGGCTGCTTAG (SEQ ID NO: 13)

>gil50548053 Iref IXP_501496.11 YALI0C05951p [Yarrowia lipolytica]

MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKMSQGAYDDKGRHI SEQPFTWANWHQHINWLN ILVIALPLSS FAAAPFVSFNWKTAAFAVGYYMCTGLGITAGYHRMWAHRAYKAALPVRI ILALFGGGAVEGS IRWWASSHRVHHR WTDSNKDPYDARKGFWFSHFGWMLLVPNPKNKGRTDI SDLNNDWVVRLQHKYYVYVLVFMAIVLPTLVCGFGWGD WKGGLVYAGIMRYTFVQQVTFCVNSLAHWIGEQPFDDRRTPRDHALTALVTFGEGYHNFH HEFPSDYRNALIWYQ YDPTKWLIWTLKQVGLAWDLQTFSQNAIEQGLVQQRQKKLDKWRNNLNWGIPIEQLPVIE FEEFQEQAKTRDLVL I SGIVHDVSAFVEHHPGGKALIMSAVGKDGTAVFNGGVYRHSNAGHNLLATMRVSVIRGGM EVEVWKTAQNEKKD QNIVSDESGNRIHRAGLQATRVENPGMSGMAA (SEQ ID NO : 1 ) Some aspects of this invention provide a method for the manipulation of the activity of an ATP-citrate lyase (ACL) in a microbe for biofuel or biofuel precursor production. ACL provides cytosolic acetyl-CoA by cleaving citrate which is shuttled out of the mitochondria as a product of the TCA cycle. Cleaving citrate into oxaloacetate and acetyl-CoA, ACL gene products provide an acetyl-CoA substrate for ACC, which then mediates the conversion of acetyl-CoA, the main C2 -precursor in fatty acid synthesis, to malonyl-CoA, which is considered the first committed step in fatty acid synthesis, as described in more detail elsewhere herein. In some embodiments, an ACL gene product is a protein composed of two subunits encoded by separate genes. In some embodiments, an ACL gene product is composed of two subunits encoded by the same gene. In some embodiments, the

manipulation is an overexpression. In some embodiments, the manipulation is effected by contacting a microbe for biofuel or biofuel precursor production with an expression construct comprising a nucleic acid coding for an ACL gene product, for example, an ACL protein, operably linked to a heterologous promoter, for example, a constitutive or an inducible promoter. In some embodiments, the nucleic acid coding for an ACL gene product comprises the coding sequences of SEQ ID NO: 15 and SEQ ID NO: 17. In some embodiments, the ACL is Y. Upolytica ACL, for example, Y. Upolytica ACL comprising the amino acid sequences of SEQ ID NO: 16 and SEQ ID NO: 18. In some embodiments, the microbe is Y. Upolytica. In some embodiments, manipulation of the activity of a ACL in a microbe is effected to confer a beneficial phenotype for large-scale carbohydrate to lipid conversion, for example increased lipid synthesis rate, increased carbohydrate to lipid conversion efficiency, increased lipid storage and, increased growth rate, increased tolerance to elevated

concentrations of a carbon source or a lipid product. ATP-citrate lyase gene and gene product sequences are well known to those of skill in the art. Exemplary, representative gene and gene product sequences can be found under the entry for GenelD: 2912101 and 2910381 in the NCBI database (www.ncbi.nlm.nih.gov).

Non-limiting examples of suitable sequences of ACL nucleic acid and protein sequences are provided below. Additional suitable ACL sequences, including sequences from other species, will be apparent to those of skill in the art, and the invention is not limited in this respect.

ATP Citrate Lyase (Yarrowia Upolytica) subunit 1, ACL1 DNA

YALI0E34793g

XM_504787

ATGTCTGCCAACGAGAACATCTCCCGATTCGACGCCCCTGTGGGCAAGGAGCACCCCGCC TACGAGCTCTTCCAT AACCACACACGATCTTTCGTCTATGGTCTCCAGCCTCGAGCCTGCCAGGGTATGCTGGAC TTCGACTTCATCTGT AAGCGAGAGAACCCCTCCGTGGCCGGTGTCATCTATCCCTTCGGCGGCCAGTTCGTCACC AAGATGTACTGGGGC ACCAAGGAGACTCTTCTCCCTGTCTACCAGCAGGTCGAGAAGGCCGCTGCCAAGCACCCC GAGGTCGATGTCGTG GTCAACTTTGCCTCCTCTCGATCCGTCTACTCCTCTACCATGGAGCTGCTCGAGTACCCC CAGTTCCGAACCATC GCCATTATTGCCGAGGGTGTCCCCGAGCGACGAGCCCGAGAGATCCTCCACAAGGCCCAG AAGAAGGGTGTGACC ATCATTGGTCCCGCTACCGTCGGAGGTATCAAGCCCGGTTGCTTCAAGGTTGGAAACACC GGAGGTATGATGGAC AACATTGTCGCCTCCAAGCTCTACCGACCCGGCTCCGTTGCCTACGTCTCCAAGTCCGGA GGAATGTCCAACGAG CTGAACAACATTATCTCTCACACCACCGACGGTGTCTACGAGGGTATTGCTATTGGTGGT GACCGATACCCTGGT ACTACCTTCATTGACCATATCCTGCGATACGAGGCCGACCCCAAGTGTAAGATCATCGTC CTCCTTGGTGAGGTT GGTGGTGTTGAGGAGTACCGAGTCATCGAGGCTGTTAAGAACGGCCAGATCAAGAAGCCC ATCGTCGCTTGGGCC ATTGGTACTTGTGCCTCCATGTTCAAGACTGAGGTTCAGTTCGGCCACGCCGGCTCCATG GCCAACTCCGACCTG GAGACTGCCAAGGCTAAGAACGCCGCCATGAAGTCTGCTGGCTTCTACGTCCCCGATACC TTCGAGGACATGCCC GAGGTCCTTGCCGAGCTCTACGAGAAGATGGTCGCCAAGGGCGAGCTGTCTCGAATCTCT GAGCCTGAGGTCCCC AAGATCCCCATTGACTACTCTTGGGCCCAGGAGCTTGGTCTTATCCGAAAGCCCGCTGCT TTCATCTCCACTATT TCCGATGACCGAGGCCAGGAGCTTCTGTACGCTGGCATGCCCATTTCCGAGGTTTTCAAG GAGGACATTGGTATC GGCGGTGTCATGTCTCTGCTGTGGTTCCGACGACGACTCCCCGACTACGCCTCCAAGTTT CTTGAGATGGTTCTC ATGCTTACTGCTGACCACGGTCCCGCCGTATCCGGTGCCATGAACACCATTATCACCACC CGAGCTGGTAAGGAT CTCATTTCTTCCCTGGTTGCTGGTCTCCTGACCATTGGTACCCGATTCGGAGGTGCTCTT GACGGTGCTGCCACC GAGTTCACCACTGCCTACGACAAGGGTCTGTCCCCCCGACAGTTCGTTGATACCATGCGA AAGCAGAACAAGCTG ATTCCTGGTATTGGCCATCGAGTCAAGTCTCGAAACAACCCCGATTTCCGAGTCGAGCTT GTCAAGGACTTTGTT AAGAAGAACTTCCCCTCCACCCAGCTGCTCGACTACGCCCTTGCTGTCGAGGAGGTCACC ACCTCCAAGAAGGAC AACCTGATTCTGAACGTTGACGGTGCTATTGCTGTTTCTTTTGTCGATCTCATGCGATCT TGCGGTGCCTTTACT GTGGAGGAGACTGAGGACTACCTCAAGAACGGTGTTCTCAACGGTCTGTTCGTTCTCGGT CGATCCATTGGTCTC ATTGCCCACCATCTCGATCAGAAGCGACTCAAGACCGGTCTGTACCGACATCCTTGGGAC GATATCACCTACCTG GTTGGCCAGGAGGCTATCCAGAAGAAGCGAGTCGAGATCAGCGCCGGCGACGTTTCCAAG GCCAAGACTCGATCA TAG (SEQ ID NO: 15)

ATP Citrate Lyase (Yarrowia lipolytica) subunit 1, ACL1 Protein

YALI0E34793p

XP_504787

MSANE I SR DAPVGKEHPAYELFHNHTRS VYGLQPRACQGMLD D ICKRENPSVAGVIYPFGGQFVTKMYWG TKETLLPVYQQVEKAAAKHPEVDVVVNFASSRSVYSSTMELLEYPQFRTIAI IAEGVPERRAREILHKAQKKGVT I IGPATVGGIKPGCFKVGNTGGMMDNIVASKLYRPGSVAYVSKSGGMSNELNNI I SHTTDGVYEGIAIGGDRYPG TTFIDHILRYEADPKCKI IVLLGEVGGVEEYRVIEAVKNGQIKKPIVAWAIGTCASMFKTEVQFGHAGSMA SDL ETAKAKNAAMKSAGFYVPDTFEDMPEVLAELYEKMVAKGELSRISEPEVPKIPIDYSWAQ ELGLIRKPAAFISTI SDDRGQELLYAGMPI SEVFKEDIGIGGVMSLLWFRRRLPDYASKFLEMVLMLTADHGPAVSGAMNTI ITTRAGKD LI SSLVAGLLTIGTRFGGALDGAATEFTTAYDKGLSPRQFVDTMRKQNKLIPGIGHRVKSRN NPDFRVELVKDFV KKNFPSTQLLDYALAVEEVTTSKKDNLILNVDGAIAVSFVDLMRSCGAFTVEETEDYLKN GVLNGLFVLGRS IGL IAHHLDQKRLKTGLYRHPWDDITYLVGQEAIQKKRVEI SAGDVSKAKTRS (SEQ ID NO: 16)

ATP Citrate lyase {Yarrowia lipolytica) subunit 2, ACL2 DNA

YALI0D24431g

XM_503231

ATGTCAGCGAAATCCATTCACGAGGCCGACGGCAAGGCCCTGCTCGCACACTTTCTGTCC AAGGCGCCCGTGTGG GCCGAGCAGCAGCCCATCAACACGTTTGAAATGGGCACACCCAAGCTGGCGTCTCTGACG TTCGAGGACGGCGTG GCCCCCGAGCAGATCTTCGCCGCCGCTGAAAAGACCTACCCCTGGCTGCTGGAGTCCGGC GCCAAGTTTGTGGCC AAGCCCGACCAGCTCATCAAGCGACGAGGCAAGGCCGGCCTGCTGGTACTCAACAAGTCG TGGGAGGAGTGCAAG CCCTGGATCGCCGAGCGGGCCGCCAAGCCCATCAACGTGGAGGGCATTGACGGAGTGCTG CGAACGTTCCTGGTC GAGCCCTTTGTGCCCCACGACCAGAAGCACGAGTACTACATCAACATCCACTCCGTGCGA GAGGGCGACTGGATC CTCTTCTACCACGAGGGAGGAGTCGACGTCGGCGACGTGGACGCCAAGGCCGCCAAGATC CTCATCCCCGTTGAC ATTGAGAACGAGTACCCCTCCAACGCCACGCTCACCAAGGAGCTGCTGGCACACGTGCCC GAGGACCAGCACCAG ACCCTGCTCGACTTCATCAACCGGCTCTACGCCGTCTACGTCGATCTGCAGTTTACGTAT CTGGAGATCAACCCC CTGGTCGTGATCCCCACCGCCCAGGGCGTCGAGGTCCACTACCTGGATCTTGCCGGCAAG CTCGACCAGACCGCA GAGTTTGAGTGCGGCCCCAAGTGGGCTGCTGCGCGGTCCCCCGCCGCTCTGGGCCAGGTC GTCACCATTGACGCC GGCTCCACCAAGGTGTCCATCGACGCCGGCCCCGCCATGGTCTTCCCCGCTCCTTTCGGT CGAGAGCTGTCCAAG

GAGGAGGCGTACATTGCGGAGCTCGATTCCAAGACCGGAGCTTCTCTGAAGCTGACT GTTCTCAATGCCAAGGGC

CGAATCTGGACCCTTGTGGCTGGTGGAGGAGCCTCCGTCGTCTACGCCGACGCCATT GCGTCTGCCGGCTTTGCT

GACGAGCTCGCCAACTACGGCGAGTACTCTGGCGCTCCCAACGAGACCCAGACCTAC GAGTACGCCAAAACCGTA

CTGGATCTCATGACCCGGGGCGACGCTCACCCCGAGGGCAAGGTACTGTTCATTGGC GGAGGAATCGCCAACTTC

ACCCAGGTTGGATCCACCTTCAAGGGCATCATCCGGGCCTTCCGGGACTACCAGTCT TCTCTGCACAACCACAAG

GTGAAGATTTACGTGCGACGAGGCGGTCCCAACTGGCAGGAGGGTCTGCGGTTGATC AAGTCGGCTGGCGACGAG

CTGAATCTGCCCATGGAGATTTACGGCCCCGACATGCACGTGTCGGGTATTGTTCCT TTGGCTCTGCTTGGAAAG

CGGCCCAAGAATGTCAAGCCTTTTGGCACCGGACCTTCTACTGAGGCTTCCACTCCT CTCGGAGTTTAA (SEQ

ID NO: 17)

ATP Citrate lyase (Yarwwia lipolytica) subunit 2, ACL2 Protein

YALI0D24431p

XP_503231

MSAKS IHEADGKALLAHFLSKAPVWAEQQPI TFEMGTPKLASLTFEDGVAPEQIFAAAEKTYPWLLESGAKFVA KPDQLIKRRGKAGLLVLNKSWEECKPWIAERAAKPINVEGIDGVLRTFLVEPFVPHDQKH EYYINIHSVREGDWI LFYHEGGVDVGDVDAKAAKILIPVDIENEYPSNATLTKELLAHVPEDQHQTLLDFINRLY AVYVDLQFTYLEINP LVVIPTAQGVEVHYLDLAGKLDQTAEFECGPKWAAARSPAALGQVVTIDAGSTKVS IDAGPAMVFPAPFGRELSK EEAYIAELDSKTGASLKLTVLNAKGRIWTLVAGGGASVVYADAIASAGFADELANYGEYS GAPNETQTYEYAKTV LDLMTRGDAHPEGKVLFIGGGIANFTQVGSTFKGI IRAFRDYQSSLHNHKVKIYVRRGGPNWQEGLRLIKSAGDE LNLPMEIYGPDMHVSGIVPLALLGKRPKNVKPFGTGPSTEASTPLGV (SEQ ID NO: 18)

Some aspects of this invention provide oleaginous microbes for oil production comprising any of the modifications described herein, for example, in combination with modification of XYL1/XYL2 (and optionally XYL3) or XYLA: a DGA1 modification as described herein, an ACC1 modification as described herein, and/or an SCD modification as described herein. In some embodiments, a modified oleaginous microbe is provided that comprises a push modification as described herein and a pull modification as described herein. In some embodiments, the push modification comprises overexpression of an ACC1 gene product. In some embodiments, the pull modification comprises overexpression of a DGA1 and/or an SCD gene product.

Some aspects of this invention provide nucleic acids coding for a gene product conferring a required and/or desired phenotype for biofuel or biofuel precursor production to a microbe, for example, Y. lipolytica. In some embodiments, the nucleic acid encodes an XYL1 gene product, for example, an XYL1 protein. In some embodiments, the nucleic acid encodes an XYL2 gene product, for example, an XYL2 protein. In some embodiments, the nucleic acid encodes an XYL3 gene product, for example, an XYL3 protein. In some embodiments, the nucleic acid encodes an XYLA gene product, for example, an XYLA protein. In some embodiments, the nucleic acid is a nucleic acid derived from Y. lipolytica. In some embodiments, the nucleic acid encodes a DGA1 gene product, for example, a DGA1 protein. In some embodiments, the nucleic acid encodes an ACC1 gene product, for example, an ACC1 protein. In some embodiments, the nucleic acid encodes a desaturase, for example a Δ9 desaturase. In some embodiments, the nucleic acid encodes Y. lipolytica Δ9 desaturase (SCD). In some embodiments, a nucleic acid is provided that encodes a combination of gene products, for example in multiple cistrons, comprising a gene product the overexpression of which represents a push modification of lipid biosynthesis (e.g., an ACC1 gene product), and a gene product the overexpression of which represents a pull modification of lipid biosynthesis (e.g., a DGA1 and/or SCD gene product).

The term "nucleic acid" refers to a molecule comprising multiple linked nucleotides. "Nucleic acid" and "nucleic acid molecule" are used interchangeably and refer to

oligoribonucleotides as well as oligodeoxyribonucleotides. The terms also include polynucleosides (i.e., a polynucleotide minus a phosphate) and any other organic base containing nucleic acid. The organic bases include adenine, uracil, guanine, thymine, cytosine and inosine. The nucleic acids may be single or double stranded. The nucleic acid may be naturally or non-naturally occurring. Nucleic acids can be obtained from natural sources, or can be synthesized using a nucleic acid synthesizer (i.e., synthetic). Isolation of nucleic acids are routinely performed in the art and suitable methods can be found in standard molecular biology textbooks. (See, for example, Maniatis' Handbook of Molecular Biology.) The nucleic acid may be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA, cDNA, rRNA, miRNA, PNA or LNA, or a combination thereof, as described herein. Non- naturally occurring nucleic acids such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) can also be used in accordance with some aspects of this invention.

Some aspects of this invention relate to the use of nucleic acid derivatives. The use of certain nucleic acid derivatives may increase the stability of the nucleic acids of the invention by preventing their digestion, particularly when they are exposed to biological samples that may contain nucleases. As used herein, a nucleic acid derivative is a non-naturally occurring nucleic acid or a unit thereof. Nucleic acid derivatives may contain non-naturally occurring elements such as non-naturally occurring nucleotides and non-naturally occurring backbone linkages. Nucleic acid derivatives according to some aspects of this invention may contain backbone modifications such as but not limited to phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. The backbone composition of the nucleic acids may be homogeneous or heterogeneous.

Nucleic acid derivatives according to some aspects of this invention may contain substitutions or modifications in the sugars and/or bases. For example, some nucleic acid derivatives may include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position (e.g., an 2'-0-alkylated ribose group). Nucleic acid derivatives may include non-ribose sugars such as arabinose. Nucleic acid derivatives may contain substituted purines and pyrimidines such as C-5 propyne modified bases, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-thiouracil and pseudoisocytosine.

In some embodiments, a nucleic acid may comprise a peptide nucleic acid (PNA), a locked nucleic acid (LNA), DNA, RNA, or a co-nucleic acids of the above such as DNA- LNA co-nucleic acid.

As used herein the term "isolated nucleic acid molecule" refers to a nucleic acid that is not in its natural environment, for example a nucleic acid that has been (i) extracted and/or purified from a cell or microbe, for example, a bacteria or yeast, by methods known in the art, for example, by alkaline lysis of the host cell and subsequent purification of the nucleic acid, for example, by a silica adsorption procedure; (ii) amplified in vitro, for example, by polymerase chain reaction (PCR); (iii) recombinantly produced by cloning, for example, a nucleic acid cloned into an expression vector; (iv) fragmented and size separated, for example, by enzymatic digest in vitro or by shearing and subsequent gel separation; or (v) synthesized by, for example, chemical synthesis. In some embodiments, the term "isolated nucleic acid molecule" refers to (vi) an nucleic acid that is chemically markedly different from any naturally occurring nucleic acid. In some embodiments, an isolated nucleic acid can readily be manipulated by recombinant DNA techniques well known in the art.

Accordingly, a nucleic acid cloned into a vector, or a nucleic acid delivered to a host cell and integrated into the host genome is considered isolated but a nucleic acid in its native state in its natural host, for example, in the genome of the host, is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a small percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein.

Some aspects of this invention relate to nucleic acids encoding a gene product conferring a required or desirable phenotype to a microbe for biofuel or biofuel precursor production which are linked to a promoter or other transcription activating element. In some embodiments, the nucleic acid encoding the gene product and linked to a promoter is comprised in an expression vector or expression construct. As used herein, the terms

"expression vector" or "expression construct" refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host microbe, for example, an oleaginous yeast. In some embodiments, the expression vector may be part of a plasmid, virus, or nucleic acid fragment. In some embodiments, the expression vector includes the coding nucleic acid to be transcribed operably linked to a promoter. A promoter is a nucleic acid element that facilitates transcription of a nucleic acid to be transcribed. A promoter is typically located on the same strand and upstream (or 5') of the nucleic acid sequence the transcription of which it controls. In some embodiments, the expression vector includes the coding nucleic acid to be transcribed operably linked to a heterologous promoter. A heterologous promoter is a promoter not naturally operably linked to a given nucleic acid sequence. For example, the DGA1 gene in Y. lipolytica is naturally operably linked to the Y. lipolytica DGA1 gene promoter. Any promoter other than the wildtype Y. lipolytica DGA1 gene promoter operably linked to the DGA1 gene, or parts thereof, for example in an expression construct, would, therefore, be a heterologous promoter in this context. For example, a TEF1 promoter linked to a nucleic acid encoding a DGA1 gene product is a heterologous promoter in the DGA1 context.

In some embodiments, the expression vector includes a coding nucleic acid, for example, a nucleic acid encoding a XYL1 and XYL2 (and optionally XYL3) gene product, or a XYLA gene product, and optionally a DGA1, ACC1, and/or SCD gene product, operably linked to a constitutive promoter. The term "constitutive promoter" refers to a promoter that allows for continual transcription of its associated gene. In some embodiments, the expression vector includes a coding nucleic acid, for example, a nucleic acid encoding a XYL1 and XYL2 (and optionally XYL3) gene product, or a XYLA gene product, and optionally a DGA1, ACC1, and/or SCD gene product, operably linked to an inducible promoter. The term "inducible promoter", interchangeably used herein with the term

"conditional promoter", refers to a promoter that allows for transcription of its associated gene only in the presence or absence of biotic or abiotic factors. Drug-inducible promoters, for example tetracycline/doxycycline inducible promoters, tamoxifen-inducible promoters, as well as promoters that depend on a recombination event in order to be active, for example the cre-mediated recombination of loxP sites, are examples of inducible promoters that are well known in the art.

Some aspects of this disclosure relate to the surprising discovery that overexpression of a given gene product from a heterologous promoter in oleaginous microbes can be significantly enhanced by including an intron in the respective expression construct. Some aspects of this disclosure provide an intron-enhanced constitutive promoter for gene overexpression in oleaginous microbes and expression constructs and vectors comprising this intron-enhanced promoter. In some embodiments, an intron-enhanced TEF promoter is provided, that comprises a TEF promoter sequence, a transcription start site, an intronic sequence downstream of the transcription start site, and a coding nucleic acid sequence, for example, a nucleic acid sequence encoding a XYL1 and XYL2 (and optionally XYL3) gene product, or a XYLA gene product, and optionally a DGA1, ACC1 and/or SCD gene product. In some embodiments, the intron is positioned downstream of the translation start site, yet within the open reading frame of the gene sequence, e.g., after the start codon, but before the termination site of the nucleic acid sequence encoding the gene product. In some

embodiments, the intron is positioned immediately downstream of the translation start site, e.g., an ATG start codon, yet upstream of the remainder of the coding sequence. For illustration purposes, a non-limiting, exemplary structure of an intron-enhanced expression construct is provided as follows: 5'— TEF promoter— transcription start site— intron— XYL1 coding sequence— 3' . Another non-limiting, exemplary structure of an intron-enhanced expression construct is provided as follows:

5'— TEF promoter— transcription start site - start codon— intron— XYL1 coding sequence— stop codon - 3'. Expression constructs for XYL2, XYL3, XYLA, DGA1, ACC1 and SCD gene products would have the XYL1 coding sequence substituted by an XYL2, XYL3, XYLA, DGA1, ACC or SCD coding sequence, respectively.

Suitable TEF promoter sequences as well as suitable intron sequences will be apparent to those of skill in the art. Some intron-less TEF promoter sequences are disclosed, for example, in U.S. Patent #6,265,185. Some exemplary, representative sequences are provided below. However, it will be understood that the invention is not limited in this respect.

Exemplary TEF promoter sequence:

agagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaatt gaccccaaattgacccagtagcgggcccaa ccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccg ttaagggcgtagggtactgcagtctgga atctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgc cggacgcaaaatagactactgaaaatttttttgc tttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcct cttcttttctctctctccttgtcaactcacaccc gaaatcgttaagcatttccttctgagtataagaatcattcaaa (SEQ ID NO: 19)

Exemplary intron sequence:

gtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtgg tcccattcccatcgacacaagacgccacgt catccgaccagcactttttgcagtactaaccgcag (SEQ ID NO: 20)

Exemplary TEF promoter-intron sequence comprising a start codon (ATG) between the promoter and the intron sequences:

agagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaatt gaccccaaattgacccagtagcgggcccaa ccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccg ttaagggcgtagggtactgcagtctgga atctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgc cggacgcaaaatagactactgaaaatttttttgc tttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcct cttcttttctctctctccttgtcaactcacaccc gaaatcgttaagcatttccttctgagtataagaatcattcaaaATGgtgagtttcagagg cagcagcaattgccacgggctttgagca cacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcact ttttgcagtactaaccgcag (SEQ ID NO: 21)

Methods to deliver expression vectors or expression constructs into microbes, for example, into yeast cells, are well known to those of skill in the art. Nucleic acids, including expression vectors, can be delivered to prokaryotic and eukaryotic microbes by various methods well known to those of skill in the relevant biological arts. Methods for the delivery of nucleic acids to a microbe in accordance to some aspects of this invention, include, but are not limited to, different chemical, electrochemical and biological approaches, for example, heat shock transformation, electroporation, transfection, for example liposome-mediated transfection, DEAE-Dextran-mediated transfection or calcium phosphate transfection. In some embodiments, a nucleic acid construct, for example an expression construct comprising a combination of XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, and/or SCD encoding nucleic acid sequences, is introduced into the host microbe using a vehicle, or vector, for transferring genetic material. Vectors for transferring genetic material to microbes are well known to those of skill in the art and include, for example, plasmids, artificial chromosomes, and viral vectors. Methods for the construction of nucleic acid constructs, including expression constructs comprising constitutive or inducible heterologous promoters, knockout and knockdown constructs, as well as methods and vectors for the delivery of a nucleic acid or nucleic acid construct to a microbe are well known to those of skill in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194),

Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351 , Academic Press; 1st edition (July 9, 2002); Gregory N. Stephanopoulos, Aristos A. Aristidou and Jens Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press; 1 edition (October 16, 1998); and Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press; 1 edition (July 28, 2009), all of which are incorporated by reference herein.

In some embodiments, the native promoter of a gene encoding a gene product conferring a required or desirable phenotype to a microbe, for example, the native XYL1, XYL2, XYL3, XYLA, DGAl, ACCl, or SCD promoter, is modified in the microbe to alter the regulation of its transcriptional activity. In some embodiment, the modified promoter exhibits an increased transcriptional activity as compared to its unmodified counterpart. The term "modified promoter", as used herein, refers to a promoter the nucleotide sequence of which has been artificially altered. Nucleotide deletion(s), insertion(s) or mutation(s), alone or in combination, are examples of such artificial alterations. Artificial promoter alterations can be effected in a targeted fashion, for example by homologous recombination approaches, such as gene targeting, knockout, knock in, site-directed mutagenesis, or artificial zinc finger nuclease-mediated strategies. Alternatively, such alterations may be effected by a random or quasi-random event, such as irradiation or non-targeted nucleotide integration and subsequent selection. Promoter modifications, in general, are fashioned in order to modulate the transcriptional activation properties of the respective promoter. For example, the disruption or deletion of a regulatory element mediating the repression of a XYL1, XYL2, XYL3, XYLA, DGAl, ACCl, or SCD promoter in response to elevated intracellular fatty acid levels would lead to continued transcriptional activation of the respective gene even under conditions of elevated intracellular fatty acid levels. Similarly, the insertion of a

constitutively active transcriptional activator element into a conditional promoter region may effect overexpression of the respective gene under normally inhibitive conditions. Methods for the targeted disruption of a native promoter, for example, a native XYL1, XYL2, XYL3, XYLA, DGAl, ACCl, or SCD promoter, in a microbe, for example, for targeted disruption resulting in an increased transcription rate, are well known to those of skill in the art.

Some aspects of this invention relate to engineering of a microbe, for example, Y. lipolytica, to exhibit a required and/or desirable phenotype for large-scale production of a biofuel or biofuel precursor. Some aspects of this invention relate to the metabolic engineering of the lipid synthesis pathway in order to yield a microbe optimized for biofuel production. Some aspects of this invention relate to metabolic engineering that comprises a combination of genetic modifications modulating the expression of genes regulating carbon flux into a lipid synthesis pathway in order to yield a microbe optimized for biofuel production. In some embodiments, the combination of genetic modifications includes a push modification and a pull modification. In some embodiments, the push modification comprises a genetic modification that increases the level of metabolites, acetyl-CoA, ATP, or NADPH for lipid synthesis in a cell, for example, overexpression of an ACCl gene product. In some embodiments, the pull modification is a genetic modification that decreases the level of a product or intermediary of lipid synthesis that exhibits a feedback inhibitory function, for example, a fatty acid. In some embodiments, the pull modification comprises overexpression of a DGA1 and/or an SCD gene product.

Engineered microbes for biofuel production

Some aspects of this invention relate to a microbe engineered and/or optimized for large-scale biofuel or biofuel precursor production. . In some embodiments, an engineered microbe is provided that has been manipulated by a method or using a nucleic acid or protein provided by some aspects of this invention, for example, an expression construct or a combination of expression constructs as provided herein, resulting in the overexpression of a gene product or a combination of gene products mediating the metabolism of a 5C sugar such as xylose, such as XYL1 and XYL2, and optionally XYL3, or XYLA. In some

embodiments, an engineered microbe is provided that has been manipulated by a method or using a nucleic acid or protein provided by some aspects of this invention, for example, an expression construct or a combination of expression constructs as provided herein, resulting in the overexpression of a combination of a gene product mediating a push process of lipid synthesis (e.g., an ACC1 product), and a gene product mediating a pull process of lipid synthesis (e.g., a DGA1 and/or SCD gene product). In some embodiments, an engineered microbe is provided, that overexpresses a push-and-pull combination of gene products that, according to some aspects of this invention, confers a required and/or desirable phenotype for biofuel or biofuel precursor production to the microbe. In some embodiments, a microbe comprising an increased XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, SCD, or ACL gene product activity is provided. In some embodiments, the microbe exhibits an increased fatty acid synthesis rate, an increased TAG storage, and/or an additional required or desirable trait.

In some embodiments, the engineered microbe is an oleaginous yeast, for example, Y. lipolytica. In some embodiments, an engineered yeast provided by this invention exhibits one or more highly desirable and unexpected phenotypic characteristics, for example: increased carbon to oil conversion rate or efficiency, increased lipid accumulation in a lipid body.

In some embodiments, the engineered microbe, for example, the engineered yeast, provided by aspects of this invention exhibits a carbon to oil conversion rate within the range of about 0.02g/g (g oil, lipid, or TAG produced/ g Glucose consumed) to about 0.3 g/g. In some embodiments, the engineered microbe, for example, the engineered yeast, provided by aspects of this invention exhibits a carbon to oil conversion of about O.OlOg/g (g TAG produced/ g Glucose consumed), about 0.02g/g, about 0.025g/g, about 0.03g/g, about 0.04g/g, about 0.05g/g, about 0.06g/g, about 0.07g/g, about 0.075g/g, about 0.08g/g, about 0.09g/g, about O. lg/g, about O.l lg/g, about 0.12g/g, about 0.13g/g, about 0.14g/g, about 0.15g/g, about 0.16g/g, about 0.17g/g, about 0.18g/g, about 0.19g/g, about 0.2g/g, about 0.21g/g, about 0.22g/g, about 0.23g/g, about 0.24g/g, about 0.25g/g, about 0.26g/g, about 0.27 g/g, about 0.28g/g, about 0.29g/g, about 0.3g/g, about 0.31g/g, about 0.32g/g, or approaching theoretical values. In some embodiments, the engineered microbe, for example, the engineered yeast, provided by aspects of this invention exhibits a carbon to oil conversion rate of at least about O.OlOg/g (g TAG produced/ g Glucose consumed), at least about 0.02g/g, at least about 0.025g/g, at least about 0.03g/g, at least about 0.04g/g, at least about 0.05g/g, at least about 0.06g/g, at least about 0.07g/g, at least about 0.075g/g, at least about 0.08g/g, at least about 0.09g/g, at least about O.lg/g, at least about O. l lg/g, at least about 0.12g/g, at least about 0.13g/g, at least about 0.14g/g, at least about 0.15g/g, at least about 0.16g/g, at least about 0.17g/g, at least about 0.18g/g, at least about 0.19g/g, at least about 0.2g/g, at least about 0.21g/g, at least about 0.22g/g, at least about 0.23g/g, at least about 0.24g/g, at least about 0.25g/g, at least about 0.26g/g, at least about 0.27 g/g, at least about 0.28g/g, at least about 0.29g/g, at least about 0.3g/g, at least about 0.31g/g, at least about 0.32g/g, or approaching theoretical values.

Some aspects of this invention provide engineered microbes for oil production that can use a variety of carbon sources, including, but not limited to fermentable sugars, for example, C5 sugars, such as xylose; C6 sugars, such as glucose; organic acids, e.g., acetic acid, and/or their salts, e.g., acetate; polyol compounds, such as glycerol; and sugar alcohols, such as arabitol.

Microbial cultures for biofuel production

Some aspects of this invention relate to cultures of genetically modified microbes provided herein. In some embodiments, the culture comprises a genetically modified microbe provided herein and a medium, for example, a liquid medium. In some embodiments, the culture comprises a genetically modified microbe provided herein and a carbon source, for example, a fermentable carbohydrate source, or an organic acid or salt thereof. In some embodiments, the culture comprises a genetically modified microbe provided herein and a salt and/or buffer establishing conditions of salinity, osmolarity, and pH, that are amenable to survival, growth, and/or carbohydrate to biofuel or biofuel precursor conversion by the microbe. In some embodiments, the culture comprises an additional component, for example, an additive. Non-limiting examples of additives are nutrients, enzymes, amino acids, albumin, growth factors, enzyme inhibitors (for example protease inhibitors), fatty acids, lipids, hormones (e.g., dexamethasone and gibberellic acid), trace elements, inorganic compounds (e.g., reducing agents, such as manganese), redox-regulators (e.g., antioxidants), stabilizing agents (e.g., dimethylsulfoxide), polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g., NaCl), chelating agents (e.g., EDTA, EGTA), and enzymes (e.g., cellulase, dispase, hyaluronidase, or DNase). In some

embodiments, the culture may comprise a drug inducing or inhibiting transcription from a conditional or inducible promoter, for example doxicycline, tetracycline, tamoxifen, IPTG, hormones, or metal ions.

While the specific culture conditions, for example, the concentration of the carbon source, will depend upon the respective engineered microorganism to be cultured, general methods and culture conditions for the generation of microbial cultures are well known to those of skill in the art, and are described, for example, in J. Sambrook and D. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001); David C. Amberg, Daniel J. Burke; and Jeffrey N. Strathern, Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005); John N. Abelson, Melvin I. Simon, Christine Guthrie, and Gerald R. Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194

(Methods in Enzymology Series, 194), Academic Press (March 11, 2004); Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B,

Volume 350 (Methods in Enzymology, Vol 350), Academic Press; 1st edition (July 2, 2002); and Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press; 1st edition (July 9, 2002), all of which are incorporated by reference herein. For oil production, the cultures of engineered microbes described herein are cultured under conditions suitable for oil accumulation, as known in the art. In some embodiments, the genetically modified microbe exhibits a growth advantage over wild type microbes of the same kind and/or over other microbes, for example, microbes commonly found to contaminate microbial cultures for carbon source to biofuel or biofuel precursor conversion. In some embodiments, the growth and/or proliferation advantage of an engineered microbe provided by aspects of this invention translates into the possibility of using non- sterile culturing and fermentation conditions for biofuel or biofuel precursor production, because the problem of culture overgrowth by contaminating microbes is mitigated or completely abolished. In some embodiments, an engineered microbe provided by aspects of this invention is cultured under non- sterile conditions for biofuel or biofuel precursor production. For example, in some embodiments, non- sterilized feedstock, non- sterilized culture media, non- sterilized supplements, or a non- sterilized bioreactor (e.g. an open reactor under non-sterile conditions) is used for biofuel or biofuel precursor production.

A variety of different microbes can be genetically modified according to some aspects of this invention and used for industrial- scale biofuel or biofuel precursor production, for example, microbes from various sources of yeast, such as oleaginous yeast, bacteria, algae and fungi. Non-limiting examples of suitable yeast cells are cells from Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Saccharomyces cerevisiae, S. bayanus, S. K. lactis, Waltomyces lipofer. Mortierella alpine, Mortierella isabellina, Hansenula polymorpha., Mucor rouxii, Trichosporon cutaneu, Rhodotorula glutinis Saccharomyces diastasicus, Schwanniomyces occidentalis, S. cerevisiae, Pichia stipitis, and Schizosaccharomyces pombe. Non-limiting examples of suitable bacteria are Bacillus subtilis, Salmonella, Escherichia coli, Vibrio cholerae, Streptomyces, Pseudomonas fluorescens, Pseudomonas putida,

Pseudomonas sp, Rhodococcus sp, Streptomyces sp, and Alcaligenes sp. Non-limiting examples of suitable fungal cells can, for example, be cultured from species such as

Aspergillus shiwusamii, Aspergillus niger and Trichoderma reesei. Non-limiting examples of suitable algal cells are cells from Neochloris oleoabundans, Scenedesmus obliquus,

Nannochloropsis sp., Dunaliella tertiolecta, Chlorella vulgaris, Chlorella emersonii, and Spirulina maxima.

Methods for biofuel production/feedstock/bioreactors

Some aspects of this invention provide methods for the production of biofuel or biofuel precursors using genetically modified microbes provided herein. In some embodiments, methods for biofuel or biofuel precursor production on an industrial scale are provided.

A variety of carbon sources can be converted into a biofuel or biofuel precursor using a method and/or a genetically modified microbe provided herein. In some embodiments, the carbon source comprises a carbohydrate. Sugars, starches, and fibers are non-limiting examples of carbohydrate sources suitable for conversion methods provided herein.

According to some aspects of this invention, a carbohydrate source may comprise a refined and/or unrefined sugar, starch, and/or fiber, or a combination of any of these. Non-limiting examples of sugars are fermentable sugars, such as , xylose, glucose, fructose, sucrose and lactose. Non-limiting examples of starches are amylase and amylopectin. Non-limiting examples of fibers are plant fibers, such as cellulose, hemicellulose and wood fibers. Some aspects of this invention relate to the use of industrial byproducts, intermediates, or waste products, for example raw plant extracts, molasses, stover, or sewage as a carbon source. In some embodiments, the carbon source is derived from algae. In some embodiments, algal biomass is produced specifically for use as a carbon source in microbe-mediated biofuel or biofuel precursor production.

In some embodiments, methods for the production of biofuel or biofuel precursor are provided that include the use of a cheap, abundant, and readily available carbon source feedstock as the carbon source. In some embodiments, cellulose or hemicellulose is used as the carbon source. In some embodiments, the cellulose or hemicellulose is derived from industrial by- or waste products. In some embodiments, the cellulose or hemicellulose is derived directly from plant or algal biomass. Plant or algal biomass is one of the most abundant feedstocks and comprises a significant amount of non-fermentable sugars and fibers, for example, cellulose and hemi-cellulose. In some embodiments, biomass feedstock is pretreated to convert a non-fermentable sugar or fiber into a fermentable sugar, thus making them available for microbe growth and microbe-mediated biofuel or biofuel precursor production. In some embodiments, the pretreatment of biomass feedstock includes depolymerizing cellulose and/or hemicellulose components to monomeric sugars using a pretreatment method known to those of skill in the art, for example, a dilute acid or ammonia fiber expansion (AFEX) method (see, e.g., Yang B, Wyman CE. Dilute acid and

autohydrolysis pretreatment. Methods Mol Biol. 2009;581: 103-14; Balan V, Bals B, Chundawat SP, Marshall D, Dale BE, Lignocellulosic biomass pretreatment using AFEX Methods Mol Biol. 2009;581:61-77). Other methods for depolymerization of biomass polymers to monomeric sugars are well known to those of skill in the art and are

contemplated to be used in some embodiments of this invention.

In some embodiments, a biomass feedstock containing non-fermentable sugars is pretreated using a dilute acid method to depolymerize a non-fermentable sugar to a monomeric, fermentable sugar. In some embodiments, biomass is treated with dilute sulphuric acid at moderately mild temperatures for a defined period of time. For example, in some embodiments, the biomass is treated with about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, or about 6% sulphuric acid. In some embodiments, the biomass is treated at about 30°C, at about 37°C, at about 40°C, at about 50°C, at about 60°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, at about 110°C, at about 120°C, at about 130°C, at about 140°C, at about 150°C, at about 175°C, at about 200°C, or at above about 200°C.

In some embodiments, the resulting hydrolysate contains insoluble lignin and solubilized cellulosic and hemicellulosic polymers. The latter products can be further treated to generate hexose and pentose sugars such as glucose and xylose monomers by methods well known to those of skill in the art, for example, by treatment with cellulase or other hydrolyzing enzymes. In some embodiments, the pretreatment of non-fermentable sugars with dilute acid results in the generation of by-products that include toxic compounds which inhibit growth, decrease viability, and/or inhibit biofuel or biofuel precursor production of microbes not engineered according to aspects of this invention. In some embodiments, the pre-treated feedstock is washed, supplemented with media supporting microbial growth and biofuel or biofuel precursor production, and/or over-limed for detoxification.

In some embodiments, a biomass feedstock containing non-fermentable sugars is pretreated using an AFEX method to depolymerize a non-fermentable sugar to a monomeric, fermentable sugar. In some embodiments, biomass is treated with liquid ammonia at high temperature and pressure for a defined period of time. In some embodiments, biomass is treated for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, or longer. In some embodiments, biomass is treated at about 30°C, at about 37°C, at about 40°C, at about 50°C, at about 60°C, at about 70°C, at about 80°C, at about 90°C, at about 100°C, at about 110°C, at about 120°C, at about 130°C, at about 140°C, at about 150°C, at about 175°C, at about 200°C, or at above about 200°C. In some embodiments, the AFEX pretreatment results in the conversion of crystalline cellulose contained in the feedstock into an amorphous, fermentable form. In some embodiments, the AFEX pre-treated biomass feedstock does not contain significant amounts of toxic byproducts that inhibit microbial growth and/or biofuel or biofuel precursor production, and is used without prior

detoxification for microbial biofuel or biofuel precursor production.

In some embodiments, biomass feedstock, with or without pre-treatment, is treated with an enzyme that hydrolyzes or depolymerizes sugar polymers, for example, with a cellulase or hemicellulase enzyme. In some embodiments, the feedstock is contacted with the enzyme in a liquid phase and incubated at a temperature allowing for the enzyme to catalyze a depolymerization or hydrolyzation reaction for a time sufficient to hydrolyze or depolymerize a significant amount of the non-fermentable sugar or fiber in the biomass feedstock. In some embodiments, the liquid phase of the feedstock contacted with the enzyme, which contains the soluble, fermentable sugar fraction, is separated from the solid phase, including non-fermentable sugars and fibers, after incubation for hydrolyzation and depolymerization, for example, by centrifugation. In some embodiments, the liquid fraction of the feedstock is subsequently contacted with a microbe, for example, a microbe provided by aspects of this invention, for conversion to biofuel or biofuel precursor. In some embodiments, enzymatic conversion of non-fermentable sugars or fiber occurs in a consolidated bioprocess, for example, at the same time and/or in the same reactor as microbial conversion of the produced fermentable sugars to biofuel or biofuel precursor. In some embodiments, the enzymatic conversion is performed first, and the feedstock contacted with enzyme is subsequently contacted with the microbe for biofuel or biofuel precursor production. In some embodiments, enzymatic and microbial conversion are performed at the same time and in the same reactor.

In some embodiments, an engineered microbe as provided herein, for example, a Yarrowia lipolytica overexpressing a XYL1, XYL2, XYL3, XYLA, DGA1, ACC1, SCD, or ACL gene product, is grown on glycerol. In some embodiments, the genetically modified microbes are intermittently contacted with glycerol. In some embodiments, the microbes are continuously or semi-continuously contacted with glycerol. In some embodiments, the microbes are contacted with glycerol at a concentration of about 0.5%, about 1%, about 2%, about 3%, about 4%, or about 5% vol/vol. Contacting the engineered microbes provided herein with glycerol provides metabolites for the production of TAGs, as well as reducing moieties for the production of fatty acids from carbohydrates. In some embodiments, glycerol spiking or use is performed in biofuel or biofuel precursor production methods in combination with any other carbon source described herein.

In some embodiments, fermentation processes for large-scale microbe-mediated carbohydrate to lipid conversion may be carried out in bioreactors. As used herein, the terms "bioreactor" and "fermentor," which are interchangeably used, refer to an enclosure, or partial enclosure, in which a biological and/or chemical reaction takes place, at least part of which involves a living organism or part of a living organism. A "large-scale bioreactor" or "industrial-scale bioreactor" is a bioreactor that is used to generate a product, for example a biofuel or biofuel precursor, for example a fatty acid and/or TAG, on a commercial or quasi- commercial scale. Large scale bioreactors typically have volumes in the range of liters, hundreds of liters, thousands of liters, or more.

A bioreactor in accordance with aspects of this invention may comprise a microbe or a microbe culture. In some embodiments, a bioreactor may comprise a spore and/or any kind of dormant cell type of any isolated microbe provided by aspects of this invention, for example, in a dry state. In some embodiments, addition of a suitable carbohydrate source to such bioreactors may lead to activation of the dormant cell, for example to germination of a yeast spore, and subsequent conversion of the carbohydrate source, at least in part, to a biofuel or biofuel precursor.

Some bioreactors according to aspects of this invention may include cell culture systems where microbes are in contact with moving liquids and/or gas bubbles. Microbes or microbe cultures in accordance with aspects of this invention may be grown in suspension or attached to solid phase carriers. Non-limiting examples of carrier systems include

microcarriers (e.g., polymer spheres, microbeads, and microdisks that can be porous or non- porous), cross-linked beads (e.g., dextran) charged with specific chemical groups (e.g., tertiary amine groups), 2D microcarriers including cells trapped in nonporous polymer fibers, 3D carriers (e.g., carrier fibers, hollow fibers, multicartridge reactors, and semi-permeable membranes that can comprising porous fibers), microcarriers having reduced ion exchange capacity, encapsulation cells, capillaries, and aggregates. Carriers can be fabricated from materials such as dextran, gelatin, glass, and cellulose. Industrial- scale carbohydrate to lipid conversion processes in accordance with aspects of this invention may be operated in continuous, semi-continuous or non-continuous modes. Non-limiting examples of operation modes in accordance with this invention are batch, fed batch, extended batch, repetitive batch, draw/fill, rotating- wall, spinning flask, and/or perfusion mode of operation.

In some embodiments, bioreactors may be used that allow continuous or semi- continuous replenishment of the substrate stock, for example a carbohydrate source and/or continuous or semi-continuous separation of the product, for example a secreted lipid, an organic phase comprising a lipid, and/or cells exhibiting a desired lipid content, from the reactor.

Non-limiting examples of bioreactors in accordance with this invention are: stirred tank fermentors, bioreactors agitated by rotating mixing devices, chemostats, bioreactors agitated by shaking devices, airlift fermentors, packed-bed reactors, fixed-bed reactors, fluidized bed bioreactors, bioreactors employing wave induced agitation, centrifugal bioreactors, roller bottles, and hollow fiber bioreactors, roller apparatuses (for example benchtop, cart-mounted, and/or automated varieties), vertically- stacked plates, spinner flasks, stirring or rocking flasks, shaken multiwell plates, MD bottles, T-flasks, Roux bottles, multiple-surface tissue culture propagators, modified fermentors, and coated beads (e.g., beads coated with serum proteins, nitrocellulose, or carboxymethyl cellulose to prevent cell attachment).

Bioreactors and fermentors according to aspects of this invention may, optionally, comprise a sensor and/or a control system to measure and/or adjust reaction parameters. Non- limiting examples of reaction parameters are: biological parameters, for example growth rate, cell size, cell number, cell density, cell type, or cell state, chemical parameters, for example pH, redox-potential, concentration of reaction substrate and/or product, concentration of dissolved gases, such as oxygen concentration and C02 concentration, nutrient

concentrations, metabolite concentrations, glucose concentration, glutamine concentration, pyruvate concentration, apatite concentration, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products, physical/mechanical parameters, for example density, conductivity, degree of agitation, pressure, and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, as well as thermodynamic parameters, such as temperature, light intensity/quality etc.

Sensors able to measure parameters as described herein are well known to those of skill in the relevant mechanical and electronic arts. Control systems able to adjust the parameters in a bioreactor based on the inputs from a sensor as described herein are well known to those of skill in the art of bioreactor engineering.

The type of carbon source to be employed for conversion to a biofuel or biofuel precursor according to aspects of this invention depends on the specific microbe employed. Some microbes provided by aspects of this invention may be able to efficiently convert a specific carbohydrate source, while a different carbohydrate source may not be processed by the same microbe at high efficiency or at all. According to aspects of this invention, the modified oleaginous yeast Y. lipolytica, for example, can efficiently convert sugars, such as xylose, glucose, fructose, sucrose, and/or lactose, and carbohydrate sources high in sugars, for example molasses, other carbon sources such as glycerol and arabitol, and plant fibers into fatty acids and their derivatives.

In some embodiments, a biofuel or biofuel precursor, for example, a fatty acid or a triacylglycerol, generated from a carbon source feedstock is secreted, at least partially, by a microbe provided by aspects of this invention, for example, an oleaginous yeast, such as a Y. lipolytica cell. In some embodiments, a microbe provided by aspects of this invention is contacted with a carbohydrate source in an aqueous solution in a bioreactor, and secreted biofuel or biofuel precursor forms an organic phase that can be separated from the aqueous phase. The term organic phase, as used herein, refers to a liquid phase comprising a non- polar, organic compound, for example a fatty acid, TAG, and/or other non-polar lipid. And organic phase in accordance to this invention might further contain a microbe, a

carbohydrate, or other compound found in other phases found in a respective bioreactor. Methods useful for industrial scale phase separation are well known to those of ordinary skill in the art. In some embodiments, the organic phase is continuously or semi-continuously siphoned off. In some embodiments, a bioreactor is employed, comprising a separator, which continuously or semi-continuously extracts the organic phase.

In some embodiments, a biofuel or biofuel precursor is accumulated in cells according to aspects of this invention. In some embodiments, cells that have accumulated a desirable amount of biofuel or biofuel precursor, are separated continuously or semi-continuously from a bioreactor, for example, by centrifugation, sedimentation, or filtration. Cell separation can further be effected, for example, based on a change in physical cell characteristics, such as cell size or density, by methods well known to those skilled in the art. The accumulated biofuel or biofuel precursor can subsequently be extracted from the respective cells using standard methods of extraction well known to those skilled in the art, for example, solvent hexane extraction. In some embodiments, microbial cells are collected and extracted with 3 times the collected cell volume of hexane. In some embodiments, the extracted biofuel or biofuel precursor are further refined. In some embodiments, a biofuel precursor, for example a triacylglycerol is converted to a biofuel, for example, biodiesel, using a method well known to those of skill in the art, for example, a transesterification procedure.

The function and advantage of these and other embodiments of the present invention will be more fully understood from the examples below. The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention. Accordingly, it will be understood that the example section is not meant to limit the scope of the invention.

EXAMPLES Example 1. Engineering Xylose Utilization in the Oleaginous Yeast Yarrowia lipolytica for Biofuel Production

Introduction

In the search for improved feedstocks, the push towards cellulosic biofuels is a clear choice. Cellulosic biomass mitigates the need to compete with food crop production; an estimated 1.3+ billion dry tons per year of biomass is potentially available in the US alone (Perlack 2005). Additionally, cellulosic materials can be more efficiently grown and more stably produced compared to sugar crops. However cellulosic materials are not naturally consumable by most biofuel-producing organisms, and thus cellulose requires pretreatment and hydrolysis to break the material down into monomeric sugar. The resulting hydrolysate can then be used as a sugar rich feedstock.

Since hydrolysis of lignocellulosic biomass results in 20-30% carbohydrates in the form of xylose, utilization of pentose sugars is one of the first steps toward efficiently using cellulosic materials. Saccharomyces cerevisiae, the most productive of ethanologenic organisms, cannot ferment xylose; it lacks the ability to convert xylose into xylulose, which can then enter the pentose phosphate pathway (PPP). Transferring the xylose reductase (XR or XYL1) and xylitol dehydrogenase (XDH or XYL2) enzymes from Scheffersomyces stipitis (formerly Pichia stipitis) has been shown to enable growth of the yeast on xylose for production of ethanol (Jeffries 2006). The addition of xylulokinase (XK or XYL3) can also be used to further improve utilization, although S. cerevisiae already carries an endogenous version of this gene. A secondary pathway, using xylose isomerase (XYLA), can be used to convert xylose into xylulose. Compared to the XR/XDH redox pathway, which uses NADPH and NAD+ cofactors for shuttling of reducing equivalents, the isomerase pathway requires no cofactors. Nonetheless the redox pathway is much more prevalent in nature, and likewise in literature (Jeffries 2006; Matsushika et al. 2009).

Instead of ethanol production, it may also be advantageous to produce yeast oil for biodiesel from cellulosic feedstocks. As a robust lipid producing organism, Yarrowia lipolytica appears to be an attractive platform for the production of cellulosic biodiesel. By leveraging the knowledge and resources developed for xylose metabolic engineering in S. cerevisiae, xylose utilization in Y. lipolytica enables robust production of yeast oils from cellulosic materials. Because theoretical yields of lipid production from xylose are very similar to that of glucose (0.34 g/g compared to 0.32 g/g), the consumption of xylose represents an attractive and worthwhile opportunity in a developing cellulosic biodiesel microbial bioprocess (Ratledge 1988). Furthermore, Y. lipolytica has a very high relative PPP flux (Blank et al. 2005), a phenotype advantageous for growth on xylose since all flux must pass through the PPP. Upregulation of the PPP pathway is a commonly engineered aspect in xylose utilizing S. cerevisiae strains (Walfridsson et al. 1995).

For the metabolic conversion of xylose to lipids, xylose enters the cell and can be catabolized either through the redox (XR/XDH) pathway or the isomerase (XYLA) pathway, producing xylulose. It can then enter central metabolism through the non-oxidative pathway of the PPP where it ultimately produces glyceraldehyde-3-phosphate (G3P) and fructose-6- phosphate (F6P). These two products can then enter the rest of central metabolism, going through glycolysis to enter the TCA cycle. Production of lipids occurs normally through the transport of mitochondrial citrate into the cytosol, where it is cleaved by ATP citrate lyase into oxaloacetate and cytosolic acetyl-coA. The acetyl-coA can then enter the fatty acid synthesis pathway through the enzymatic activity of acetyl-coA carboxylase. Acyl-coA generated from the fatty acid synthase complex are transferred to a glycerol- 3 -phosphate backbone and ultimately sequestered within lipid bodies as triacylglycerol (TAG).

Here we describe the analysis of Y. lipolytica for its natural xylose utilization and the metabolic engineering of the organism enabling utilization of xylose for the production of lipids. By incorporation of XR/XDH genes we are able to enable growth on xylose as sole carbon source, and open up opportunities for the production of lipids from cofermentations. Next we study the performance of our engineered strain through the use of cofermentations to analyze for catabolite repression and response, and evaluate the performance of the strain in a scaled-up 2-L bioreactor glycerol-xylose cofermentation with respect to lipid production.

Finally we perform transcription analysis to observe the respiratory responses of the organism during cofermentation.

Methods

Yeast strains, growth, and culture conditions

The Y. lipolytica strains used in this study were derived from the wild-type Y.

lipolytica W29 strain (ATCC20460). The auxotrophic Polg (Leu-) used in all transformations was obtained from Yeastern Biotech Company (Taipei, Taiwan). All strains used in this study are listed in Table 1. Constructed plasmids were linearized with SacII and chromosomally integrated into Polg according to the one- step lithium acetate transformation method described by Chen et al. (Chen et al., 1997). MTYL transformants were named after the numbering of their corresponding integrated plasmids. Transformants were plated on selective media and verified by PCR of prepared genomic DNA. Verified transformants were then stored as frozen glycerol stocks at -80°C and on selective YNB plates at 4°C.

Media and growth conditions for Escherichia coli have been previously described by

Sambrook et al. (Sambrook and Russell 2001), and those for Y. lipolytica have been described by Barth and Gaillardin (Barth and Gaillardin 1997). Rich medium (YPD) was prepared with 20 g/L Bacto peptone (Difco Laboratories, Detroit, MI), 10 g/L yeast extract (Difco), 20 g/L glucose (Sigma- Aldrich, St. Louis, MO). YNB medium was made with 1.7 g/L yeast nitrogen base (without amino acids) (Difco), 0.69 g/L CSM-Leu (MP Biomedicals, Solon, OH), and 20 g/L glucose. Selective YNB plates contained 1.7 g/L yeast nitrogen base (without amino acids), 0.69 g/L CSM-Leu, 20 g/L glucose, and 15 g/L Bacto agar (Difco). Shake flask experiments were carried out using the following medium: 1.7 g/L yeast nitrogen base (without amino acids), 1.5 g/L yeast extract, and 50 g/L glucose. From frozen stocks, precultures were inoculated into YNB medium (5 mL in Falcon tube, 200 rpm, 28°C, 24 hr). Overnight cultures grown in YPD were centrifuged, washed, and reinoculated into 50 mL of media in 250 mL Erlenmeyer shake flask (200 rpm, 28°C). OD, biomass and sugar content were taken periodically and analyzed.

For adaptation of strains on xylose, verified transformants were inoculated into shake flasks containing minimal media and 20g/L xylose. The cultures were incubated at 30°C for at least 10 days, waiting for growth to occur, before reinoculation into fresh media. This process was repeated until the final OD of the culture reached at least 20, indicating adaptation to xylose. The culture was then stored as frozen stock in 15% glycerol at -80°C for subsequent use.

Bioreactor scale fermentation was carried out in a 2-liter baffled stirred-tank bioreactor. The medium used contained 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate), 2 g/L ammonium sulfate, 1 g/L yeast extract, and 90 g/L glucose. From a selective plate, an initial preculture was inoculated into YPD medium (40 mL in 250 mL Erlenmeyer flask, 200 rpm, 28 °C, 24 hr). Exponentially growing cells from the overnight preculture were transferred into the bioreactor to an optical density (A600) of 0.1 in the 2-L reactor (2.5 vvm aeration, pH 6.8, 28°C, 250 rpm agitation). Time point samples were stored at -20°C for subsequent lipid analysis. Sugar organic acid content was determined by HPLC. Biomass was determined by determined gravimetrically from samples washed and dried at 60°C for two nights. Lipid content was analyzed by direct transesterification.

Plasmid construction

Standard molecular genetic techniques were used throughout this study (Sambrook and Russell 2001). Restriction enzymes and Phusion High-Fidelity DNA polymerase used in cloning were obtained from New England Biolabs (Ipswich, MA). Genomic DNA from yeast transformants was prepared using Yeastar Genomic DNA kit (Zymo Research, Irvine, CA). All constructed plasmids were verified by sequencing. PCR products and DNA fragments were purified with PCR Purification Kit or QIAEX II kit (Qiagen, Valencia, CA). Plasmids used are described in Table 1. Primers used are described in Table 2. Plasmid pMT041 was constructed by amplifying the xylose reductase gene (XYL1; Accession Number: XM_001385144) from S. stipitis genomic DNA (ATCC 58376) using the primers MT243 and MT244 and inserting it between the Pmll and BamHI sites of pINA1269. Plasmid pMT044 was constructed by amplifying the xylitol dehydrogenase gene (XYL2; Accession Number: XM_001386945) using the primers MT233 and MT234 and inserting it between the Pmll and BamHI sites of pINA1269. XYL1 and XYL2 are both genes originally from the xylose utilizing yeast, S. stipitis.

Plasmid pMT059 was constructed by amplifying the XYL1 gene from pMT041 using the primers MT281 and MT282. The amplicon was then inserted into the TEFin expression plasmid, pMT015 between the sites SnaBI and Kpnl.

For the expression of multiple genes on a single plasmid, the promoter-gene- terminator cassette can be amplified from a parent vector using primers MT220 and MT265. The cassette can then be inserted into the receiving vector between the restriction sites Nrul and Asel, resulting in a tandem gene construct. The Asel restriction site was selected to facilitate selection, as it resides within the ampicillin resistance marker of the plasmid.

Because Nrul is a blunt end restriction site, insertion of the amplicon does not increase the total number of Nrul sites that helps facilitate progressive insertions. Plasmid pMT081 was constructed by amplifying the XYL2 cassette from pMT044 and inserting it into the plasmid pMT059, containing XYL1. Plasmid pMT085 was constructed by amplifying the DGA cassette from pMT053 and inserting it into the plasmid pMT081, which contains XYL12.

RNA isolation and transcript quantification

Shake flask cultures grown for 42 hrs were collected and centrifuged for 5 min at 10,000g. Each pellet was resuspended in 1.0 ml of Trizol reagent (Invitrogen) and 100 μΐ _^ of acid- washed glass beads were added (Sigma- Aldrich). Tubes were vortexed for 15 min at 4°C for cell lysis to occur. The tubes were then centrifuged for 10 min at 12,000g at 4°C and the supernatant was collected in a fresh 2-mL tube. 200 μΐ _^ chloroform was then added and tubes were shaken by hand for 10 seconds. The tubes were again centrifuged for 10 min at 12,000g at 4°C. 400 μΐ _^ of the upper aqueous phase was transferred to a new tube, and an equal volume of phenol-chloroform-isoamyl alcohol (pH 4.7) (Ambion, Austin, TX) was added. Tubes were again shaken by hand for 10 seconds and centrifuged for 10 min at 12,000g at 4°C. 250 μΐ _^ of the upper phase was transferred to a new tube with an equal volume of cold ethanol and 1/lOth volume sodium acetate (pH 5.2). Tubes were chilled at -20°C for thirty minutes to promote precipitation. Tubes were then centrifuged for 5 min at 12,000g, washed twice with 70% ethanol, dried in a 60°C oven and finally resuspended in RNAse free water. RNA quantity was analyzed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and samples were stored in -80°C freezer. qRT-PCR analyses were carried out using iScript One- step RT-PCR Kit with SYBR Green (Bio-Rad, Hercules, CA) using the Bio-Rad iCycler iQ Real-Time PCR Detection System. Fluorescence results were analyzed using Real-time PCR Miner and relative quantification and statistical analysis was determined with REST 2009 (Qiagen) using actin as the reference gene and MTYL038 as the reference strain (Zhao and Fernald 2005). Samples were analyzed in quadruplicate.

Table 1. Strains and plasmids used in this study

Strains (host Genotype or plasmid Source

strain)

E. coli

DH5a fhuA2 A(argF-lacZ)U169 phoA glnV44 Φ80 A(lacZ)M15 gyrA96 Invitrogen recAl relAl endAl thi-1 hsdR17

pINA1269 JMP62-LEU Yeastern pMT015 pINA1269 php4d::TEFin This Example pMT041 hp4d-XYLl This Example pMT044 hp4d-XYL2 This Example pMT053 YTEFin-DGAl This Example pMT059 TEFin-XYLl This Example pMT081 TEFin-XYLl + hp4d-XYL2 This Example pMT085 TEFin-XYLl + hp4d-XYL2 + TEFin-DGA This Example

Y. lipolytica

Polg MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 Yeastern

MTYL038 MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 TEF-LacZ- This Example

LEU2

MTYL053 MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 TEFin-DGA 1- This Example

LEU2

MTYL081 MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 TEFin-LacZ- This Example

LEU2

MTYL085 MATa, leu2-270, ura3-302::URA3, xpr2-332, axp-2 hp4d-ACCi- This Example

LEU2 Table 2. Primers used in this study. Relevant restriction sites are in bold.

Primer Description SEQ ID Sequence

NO

PCR

MT233 XYL2 22 AATGACTGCTAACCCTTCCTTGGTGT

MT234 XYL2 23 CTGGTCTAGGTGGATCCTTACTCAGGGCCGTCAATGAGAC

MT243 XYL1 24 AA TGCCTTCTA TTAAGTTGAA CTCTGGTTA C

MT244 XYL1 25 CTA GGTCTTA CTGGA TCCTTA GA CGAA GA TA GGAA TCTTGTCCCA

MT281 XYL1 26 TAA CCGCAGCA TCA TCA CCA TCA CCA CCCTTCTA TTAAGTTGAA C

TCTGGTTA CGA C

MT282 XYL1 27 CTTACAGGTACCTTAGACGAAGATAGGAATCTTGTCCCAG

RT-PCR

MTR001 Actin 28 TCCAGGCCGTCCTCTCCC

MTR002 Actin 29 GGCCAGCCATATCGAGTCGCA

MTR017 ylXYLl 30 AAGGAGTGGGCTGGATGGA

MTR018 ylXYLl 31 GGTCTCTCGGGTAGGGATCTTG

MTR019 ylXYL2 32 ATGGAGGAATCGGCGACTT

MTR020 ylXYL2 33 A CCA CCTCTCCGG CA CTTT

MTR031 DGA 34 AACGGAGGAGTGGTCAAGCGA

MTR032 DGA 35 TTATGGGGAAGTAGCGGCCAA

MTR051 psXYL2 36 CTCCAAGTTGGGTTCCGTTGC

MTR052 psXYL2 37 GCGACAGCAGCAGCCAAAAGA

MTR053 psXYLl 38 AGGCTATCGCTGCTAAGCACGG

MTR054 psXYLl 39 TTTGGAA TGA TGGCAA TGCCTC

MTR055 ylXYL3 40 CAGCTCAAGGGCATCATTCTGG

MTR056 ylXYL3 41 TGCGGCAAGTCGTCCTCAAA

MTR060 IDH1 42 CTTCGAACCGCCTACCTGGCTA

MTR061 IDH1 43 TGGGCTGGAA CA TGGTTCGA

MTR064 ACOl 44 CA CCGCTTTCGCCA TTGCT

MTR065 ACOl 45 GGGCTCCTTGAGCTTGAACTCC

MTR066 PDB 1 46 CTGTGGTGTCGTCAACGACTCC

MTR067 PDB 1 47 GCTCAATGGCGTAAGGAGTGG

MTR072 ICL 48 TACTCTCCCGAGGACATTGCC

MTR073 ICL 49 CAGCTTGAAGAGCTTGTCAGCC

Direct transestenfication

For routine lipid quantification to determine relative lipid accumulation, a method for direct transesterification of cell biomass was used, adapted from the two-step base-then-acid- catalyzed direct transesterification method developed by Griffiths et al. (Griffiths et al. 2010). A normalized quantity of cell culture was centrifuged and the media supernatant was

removed. Samples were then stored in -20 °C freezer or directly transesterified. The cell was then resuspended with the addition of 100 of hexane containing 10 mg/mL methyl tridecanoate internal standard. 500 μΐ _^ 0.5 N sodium methoxide, prepared by the addition of sodium hydroxide to methanol, was then added to the sample. The sample was then vortexed for 1 hour at room temperature. Next 40 μΐ _^ of sulfuric acid was carefully added to the sample, followed by the addition of 500 μΐ _^ of neat hexane. The sample was again vortexed at room temperature for another 30 minutes. 300 μΐ _^ of the upper hexane layer was then transferred into a glass vial and run using the GC-FID, under standard operating conditions. Total lipid content was calculated as the sum of total fatty acid content for the five primary FAMEs identified. Results & Discussion

Elucidating endogenous functionality of the xylose utilization pathway in Y. lipolytica

Within the literature, there are conflicting reports about the ability for Y. lipolytica to naturally consume xylose. In most reports, growth on xylose has not been observed (Pan et al. 2009; Ruiz-Herrera and Sentandreu 2002). However, there are reports of Y. lipolytica positively growing on xylose: strain Polg was found to consume xylose in a cane hydrolysate fermentation (Tsigie et al. 2011), and two strains of Y. lipolytica were grown on xylose to measure xylulose-5-phosphate phosphoketolase activity (Evans and Ratledge 1984). Beyond these incidences, there is otherwise very little reported evidence of using Y. lipolytica for growth on xylose, despite the volume of research of using the organism grow on other alternative and residual substrate sources (Papanikolaou et al. 2002; Papanikolaou et al. 2003; Scioli and Vollaro 1997). Table 3 lists putative XR/XDH/XK genes within the genome of Y. lipolytica from a BLAST comparison to known functional pathway genes. While the amino acid identity is only 40-52%, the expect value indicates significant likelihood of similarity, and Y. lipolytica often manages only 40-60% amino acid identity with orthologous genes from S. cerevisiae, due to distal phylogeny. Nonetheless, the low homology calls into question the potential functional characteristics of these genes, which further adds to the controversy. Table 3. BLAST results for endogenous xylose utilization pathway in Y. Upolytica. Amino acid identity is indicated in comparison with the parent sequence (organism indicated in parentheses). Expect value is the statistical false-positive rate.

To test the ability for Y. Upolytica to utilize its endogenous putative XYLl 23 pathway in laboratory conditions, control strain MTYL038 was grown in minimal media on three different substrates: xylose, xylitol, arabitol. As seen in Figure 1A, these three substrates can be used to diagnose the functionality of the three XYL123 genes. For example, growth on xylitol will demonstrate that XYL2 and XYL3 are functional, while growth on arabitol demonstrates that XYL3 is functional. Figure IB depicts the growth curves of MTYL038 on the various substrates, with a shake flask with no carbon substrate as the control. While it was found that the strain did not grow on xylose, it was found to grow weakly on xylitol and quite robustly on arabitol. This suggests that while XYLl, and most likely XYL2, are not naturally expressed or functional in Y. Upolytica in the presence of their respective substrates, XYL3 is expressed and the organism can grow utilizing this pathway as its primary catabolic pathway. Expression of XYL12 enables growth on xylose

With the knowledge that the endogenous xylulokinase is functional in Y. Upolytica, the remaining elements of the xylose utilization pathway were integrated to enable growth on xylose. The XYLl and XYL2 genes from S. stipitis cloned into Y. Upolytica expression cassettes. XYLl was cloned under the control of the stronger TEFin promoter, while the XYL2 gene was cloned under the control of hp4d. The XYL2 expression cassette was inserted into the XYLl plasmid, creating plasmid pMT081, expressing both XYLl and XYL2. Transformation of this plasmid into background strain Polg yielded the strain MTYL081.

Numerous experiments working with S. cerevisiae and the xylose utilization pathway have discovered that it is often necessary to include periods of adaptation - where serial dilution in xylose media is performed - for development of stable xylose utilization (Jeffries 2006; Kuyper et al. 2004; Tomas-Pejo et al. 2010). This was similarly found to be the case in Y. lipolytica - the verified transformant MTYL081 initially did not grow on xylose. It was grown in minimal xylose media in a shake flask for 10 days before reinoculating in fresh media. This serial dilution was repeated until there was an observed increase in maximum OD to above 15. Figure 2 A shows the growth curve on the third serial dilution compared to the original unadapted strain and a control strain that underwent serial dilution in xylose media. Lack of growth from the latter two strains shows that adaptation is necessary for xylose utilization and adaptation does not occur in strains lacking the heterologous XYL12 genes. Adapted growth was found to be steady and roughly exponential, with the maximum OD of 38 being reached after 130 hours. The doubling time is roughly 25 hrs, which is significantly lower than rates typically observed on glucose but comparable to that on arabitol (see Figure IB).

To explore the underlying adaptations that improved the xylose-utilizing phenotype, RT-PCR was performed comparing the expression of heterologously expressed XYL12 and endogenous XYL123 genes in the adapted and unadapted strains. Figure 2B shows the relative change in transcription level of the genes after adaptation. The heterologously expressed XYL1 was overexpressed 300-fold compared to the unadapted strain, while XYL2 was upregulated 17-fold. Within the adapted strain, XYL1 was expressed 6-fold greater than XYL2, which is in agreement with the expression expected from the promoters used.

Endogenous XYL123 was not significantly upregulated both in adapted MTYL081 and the control strain that underwent serial dilution, indicating that the observed adaptation to xylose was not an activation of the putative native xylose pathway. The strong upregulation of XYL1 and XYL2 has been similarly observed in metabolic engineering of S. cerevisiae, as the utilization pathway, being both heterologously expressed and potentially the rate-limiting step, requires strong overexpression for sufficient growth (Karhumaa et al. 2005; Karhumaa et al. 2007). This seems to likewise be the case in Y. lipolytica, as the two XYL12 steps achieve very strong overexpression and yet still only achieve a relatively low growth rate. However, it may also be that with the adapted XYL12 expression, new rate-limiting steps appear to hinder specific growth on xylose, such as PPP activity or pentose transport

(Karhumaa et al. 2005). The normal combined activity of XYL1 and XYL2 consumes one NADPH and generates one NADH. Without suitable means to regenerate NADPH from NADH, this can lead to cofactor imbalances and has been seen as a significant challenge in metabolic engineering of S. cerevisiae (Matsushika et al. 2009). However, with a potential cofactor imbalance, one would expect early cessation of growth and large accumulation of xylitol due to complete depletion on NADPH. In our shake flask cultures we observed only < 0.5 g/L xylitol formation after consumption of 32 g/L of xylose, while the maximum OD was very higher compared to what is typically observed in shake flasks, suggesting that cofactor balance may not be an issue in this situation. While this does not remove the possibility of rate-limiting steps in the exchange of NADPH to NADH, thus slowing but not stopping growth, in the presence of oxygen, mitochondrial function actively controls and maintains the NADPH/NADH equilibrium and exchange fluxes (Singh and Mishra 1995).

Cofermentation of two substrates for improved productivity

While metabolic engineering allowed growth on xylose in Y. lipolytica, growth was dramatically slower than on glucose. Possible factors contributing to the limited growth and productivity are the lack of dedicated pentose transporters, low PPP flux, and inability for the cell to identify xylose as a fermentable sugar (Jeffries 2006; Jin et al. 2004; Matsushika et al. 2009). To improve productivities with the limited specific growth on xylose, experiments were performed using two-substrate cofermentations. Cellulosic materials typically consist of a blend of both hexose and pentose sugars, and rarely consist of pure pentose (Lee et al. 2007). Furthermore, substrates like glycerol are a byproduct of biodiesel production, and may be recycled back into the process. First it was necessary to characterize and determine which cofermentation combinations are ideal for lipid production. Xylose was combined with a helper substrate - glucose, glycerol, or arabitol - and grown in shake flasks to determine growth characteristics and observe catabolite repression effects in the cofermentation system. Catabolite repression is the preferential uptake of one substrate through the repression of the utilization pathway of secondary substrates, and can be seen in a wide range of

cofermentations in Y. lipolytica (Morgunov and Kamzolova 2011). The strain MTYL085 was used, which contains the XYL12 pathway as well as DGA overexpression. DGA

overexpression is capable of improving lipid accumulation and was found to be a strong contributor to engineered lipid overproduction (Kamisaka et al. 2007). By combining both the xylose utilization pathway and elements for lipid overproduction, we may be able to direct flux from xylose towards lipids for a cellulosic biodiesel platform.

Figure 3 depicts the growth characteristics and depletion of both substrates for the three cofermentation combinations. For glycerol (Figure 3B), diauxic shift is clearly observed, with glycerol being consumed rapidly before any xylose is depleted. For glucose (Figure 3A), diauxic shift was less observable, as it is possible that at very low concentrations of glucose, catabolite repression is weak (Morgunov and Kamzolova 2011). At higher glucose concentrations, diauxic shift was clearly observable (data not shown). While all three cultures began with 4 g/L of the helper substrate, glycerol was converted into the most biomass after it was completely depleted, achieving an OD of 8 within 24 hrs. Glycerol has been known to be a highly preferred substrate for Y. lipolytica, and unlike S. cerevisiae, there is no loss in specific growth rate when growing on glycerol compared to glucose (Taccari et al. 2012). It is also Crabtree-negative, an effect that eschews the respiration-dependent nature of glycerol metabolism found in S. cerevisiae (De Deken 1966). As a result, MTYL085 is able to consume slightly more xylose by the end of the culture. The evidence of diauxic shift also indicates that while the xylose uptake rate may be constant when grown solely on xylose, other factors must be at play in repressing the utilization, most conspicuously pentose transport. There is a growing body of evidence that pentose transport is a key rate-limiting step in xylose utilization and may also be a strong contributing factor towards diauxic shift (Young et al. 2012).

The cofermentation of xylose and arabitol exhibits a much different response (Figure 3C). Since arabitol shares the same catabolic route for all but the initial pathway, it is likely the arabitol response will be most similar to the xylose growth phenotype. Furthermore, xylose depletion begins well before arabitol is consumed, exhibiting simultaneous utilization of both substrates. The smooth growth profile in this case is in contrast to the two-phase growth seen in glucose or glycerol - a product of diauxic growth. Nonetheless the overall growth rate and productivity is significantly lower than glucose or glycerol. Additionally, arabitol is not a common substrate in cellulosic material and would thus be a prohibitive cost to supplement as a feedstock.

Lipid production in xylose and glycerol cofermentation Because glycerol showed the greatest promise for increased productivity, a scale-up cofermentation was performed using glycerol and xylose as substrates. A 2-L bioreactor was initially charged with 20 g/L glycerol and 80 g/L xylose. The C/N ratio of the reactor was adjusted to be 100, which results nitrogen-limited conditions favorable for lipid

accumulation. The results of the fermentation are found in Figure 4. Over the course of 230 hrs, all the carbon substrate was consumed, with glycerol being depleted within the first 24 hrs. Diauxic shift can clearly be observed, as no xylose is consumed until after all the glycerol has been depleted. The 20 g/L of glycerol was able to generate 13 g/L of biomass. Lipid accumulation steadily occurred between 70 and 230 hours, with a majority of the biomass generated on xylose being accounted as lipids. The culture finally achieved a biomass concentration of 18 g/L with 7.64 g/L lipids, or 42% of total biomass. The overall productivity was 0.033 g lipids/L/hr. Strain MTYL085 was able to convert xylose into lipids at quantities similar to other Y. lipolytica fermentations (Beopoulos et al. 2009; Papanikolaou and Aggelis 2002). The yield of lipid production, however, was very low. Of the 80 g/L of xylose consumed, only 6.08 g/L of lipids was generated, for a yield of 0.074 g lipids/g xylose. This is only 21.7% of the theoretical yield. This low yield may be due to

overrespiration of the carbon substrate, as high aeration on a foreign substrate may lead to strong flux through the TCA cycle. Furthermore, 9.13 g/L citrate was also generated, which actually accounts for a significant yield from the 100 g/L of carbon substrate initially charged. It is possible that the C/N ratio was too high, as extreme C/N ratios in Y. lipolytica fermentations can tend to produce citrate instead of lipids, likely due to limited ability to generate sufficient ATP for fatty acid synthesis (Beopoulos et al. 2009). Despite these low yields, the vast majority (80%) of the lipids were produced after glycerol depletion and during the xylose-only phase, indicating successful conversion of xylose-to-lipids using Y. lipolytica, a first step in developing a cellulosic biodiesel platform.

Transcriptional expression affected by secondary substrate

To further investigate the response of Y. lipolytica during cofermentations with xylose and the overrespiration observed on glycerol-xylose, transcriptional analysis was performed on genes within the TCA cycle. Xylose consumption in S. cerevisiae elicits a non- fermentative response and general upregulation of the TCA cycle (Jin et al. 2004; Salusjarvi et al. 2006). This results in lower efficiencies in xylose utilization for ethanol production as downregulation of the TCA cycle is necessary to divert carbon flux towards ethanol fermentation, whether via anaerobic environmental conditions or activity of the Crabtree effect. In our cofermentation system, the response of Y. lipolytica when transitioning from the helper substrate to xylose was examined. An initial RNA extraction was performed during the cofermentation while still growing on glucose, glycerol or arabitol, and a second RNA extraction was performed after the helper substrate was depleted and the strain was exhibiting growth on xylose as sole carbon substrate. RT-PCR primers used in this study are listed in Table 2. From this we can identify if a similar respiratory response is observed on xylose. Figure 5 depicts the fold-change in transcripts for pyruvate dehydrogenase (PDB1, Accession Number: XM_504448), Aconitase (ACOl, Accession Number: XM_502616), isocitrate lyase (ICLl, Accession Number: XM_501923), and isocitrate dehydrogenase (IDH1, Accession Number: XM_503571). These genes represent key enzymatic steps for the utilization of TCA cycle intermediates: PDB 1, entrance into the TCA cycle; ACOl, diverting citrate to the TCA cycle instead of the cytosol; ICLl, diverting isocitrate through the glyoxylate shunt; IDH1, committed step into oxidative respiration.

In all three cases, PDB1 is significantly upregulated, suggesting that there is a stronger driving force towards the TCA cycle in xylose than any other substrate. Aconitase overexpression was not observed in the glucose-to-xylose transition, but was dramatically increased 50-fold in the glycerol-to-xylose transition. This was mostly due to very low transcription levels observed of ACOl on glycerol rather than extraordinarily high expression of ACOl on xylose. ACOl was upregulated in the transition from arabitol to xylose as well. For ICLl, significant increase in expression was observed during the glycerol-to-xylose transition and the arabitol-to-xylose transition, but not on glucose. In most organisms, ICLl is normally not expressed due to strong catabolite repression; however, Y. lipolytica seems to exhibit constitutive expression of the pathway (Flores and Gancedo 2005). Indeed, the magnitude of changes in expression of ICLl suggests significant expression prior to the transition. Finally, IDH1 expression is not significantly changed in glucose and arabitol, but is actually downregulated on glycerol, indicating that respiration is much more strongly upregulated on glycerol than xylose.

The upregulation of PDB 1 and ACOl in the glycerol fermentation demonstrate an elevated respiratory response when transitioning from glycerol to xylose utilization. While IDH1 is downregulated, the upstream regulation may be enough to result in the overrespiration observed in the bioreactor. It is unclear why ACOl is downregulated so dramatically when growing on glycerol, but any previous regulation on this enzyme must surely be alleviated. On the other hand, glucose-xylose cofermentation resulted in few significant changes in transcription. This may indicate that glucose-xylose cofermentation may yield better results at larger scales despite the stronger preference for glycerol by Y. lipolytica.

Conclusion

Pentose utilization represents a pressing need in the development of sustainable biofuel production, as the push and advantages for cellulosic feedstocks begin to outweigh the technical challenges. The oleaginous yeast Y. lipolytica is an example of a robust platform for the production of yeast oil that can be converted into biodiesel. Through metabolic engineering, the robust lipid production capabilities established in Y. lipolytica can be expanded to include xylose utilization, enabling further opportunities for microbial cellulosic biodiesel production. By testing native growth on a variety of substrates we showed that the endogenous XYL3 is functional in minimal media, while the putative XYL12 genes are not. Through heterologous expression of XYL1 and XYL2 genes from S. stipitis we enabled xylose utilization in Y. lipolytica after an adaptation period. Through cofermentation we are able to eliminate lag phases and increase growth and productivity on xylose, ultimately achieving 42% lipid accumulation in a strain that is metabolically engineered in both xylose utilization and lipid accumulation pathways. By observing that the TCA cycle response, we also observed variation between cofermentation substrates, suggesting a transcriptional regulatory basis for overrespiration. By leveraging the knowledge base developed from the study of xylose utilization in S. cerevisiae, these results establish a framework for studying and engineering the oleaginous yeast Y. lipolytica for xylose utilization and the production of cellulosic biodiesel.

References

Barth G, Gaillardin C. 1997. Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol. Rev. 19(4):219-237. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM. 2009. Yarrowia lipolytica as a model for bio-oil production. Progress in Lipid Research 48(6):375-387.

Blank LM, Lehmbeck F, Sauer U. 2005. Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts. FEMS Yeast Res. 5(6-7):545-558.

De Deken R. 1966. The Crabtree effect and its relation to the petite mutation. Journal of general microbiology 44(2): 157.

Evans CT, Ratledge C. 1984. Induction of xylulose- 5 -phosphate phosphoketolase in a variety of yeasts grown on d-xylose: the key to efficient xylose metabolism. Arch. Microbiol. 139(l):48-52.

Flores C-L, Gancedo C. 2005. Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase

Activity Show an Unusual Growth Phenotype. Eukaryotic Cell 4(2):356-364.

Griffiths MJ, van Hille RP, Harrison STL. 2010. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids 45(11): 1053-1060. Jeffries TW. 2006. Engineering yeasts for xylose metabolism. Curr. Opin. Biotechnol.

17(3):320-326.

Jin Y-S, Laplaza JM, Jeffries TW. 2004. Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response. Appl. Environ. Microbiol. 70(11):6816- 6825.

Kamisaka Y, Tomita N, Kimura K, Kainou K, Uemura H. 2007. DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Asnf2 disruptant of Saccharomyces cerevisiae. Biochemal Journal 408(l):61-68.

Karhumaa K, Hahn-Hagerdal B, Gorwa-Grauslund MF. 2005. Investigation of limiting

metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 22(5):359-368.

Karhumaa K, Sanchez R, Hahn-Hagerdal B, Gorwa-Grauslund M-F. 2007. Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microbial Cell Factories 6(1):5. Kuyper M, Winkler AA, van Dijken JP, Pronk JT. 2004. Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res. 4(6):655-664. Lee D, Owens VN, Boe A, Jeranyama P. 2007. Composition of herbaceous biomass feedstocks: South Dakota State University.

Matsushika A, Inoue H, Kodaki T, Sawayama S. 2009. Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Appl.

Microbiol. Biotechnol. 84(l):37-53.

Morgunov IG, Kamzolova SV. 2011. Yarrowia Lipolytica Yeast Possesses An Atypical

Catabolite Repression. Albany 2011: Conversation 17.

Pan LX, Yang DF, Li S, Wei L, Chen GG, Liang ZQ. 2009. Isolation of the Oleaginous

Yeasts from the Soil and Studies of Their Lipid-Producing Capacities. Food Technology and Biotechnology 47(2):215-220.

Papanikolaou S, Aggelis G. 2002. Lipid production by Yarrowia lipolytica growing on

industrial glycerol in a single-stage continuous culture. Bioresour. Technol. 82(l):43-49. Papanikolaou S, Chevalot I, Komaitis M, Marc I, Aggelis G. 2002. Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures. Appl. Microbiol. Biotechnol. 58(3):308-312.

Papanikolaou S, Muniglia L, Chevalot I, Aggelis G, Marc I. 2003. Accumulation of a cocoa- butter-like lipid by Yarrowia lipolytica cultivated on agro-industrial residues. Curr.

Microbiol. 46(2): 124-130.

Perlack RD. 2005. Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasability of a Billion-Ton Annual Supply. Oak Ridge National Lab.

Ratledge C. Single Cell Oil; 1988. Longman Scientific & Technical, p 33-70.

Ruiz-Herrera Je, Sentandreu R. 2002. Different effectors of dimorphism in Yarrowia

lipolytica. Arch Microbiol 178(6):477-483.

Salusjarvi L, Pitkanen JP, Aristidou A, Ruohonen L, Penttila M. 2006. Transcription analysis of recombinant Saccharomyces cerevisiae reveals novel responses to xylose. Appl.

Biochem. Biotechnol. 128(3):237-261.

Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual: CSHL press.

Scioli C, Vollaro L. 1997. The use of Yarrowia lipolytica to reduce pollution in olive mill wastewaters. Water Res. 31(10):2520-2524.

Singh A, Mishra P. 1995. Microbial Pentose Utilization: Current Applications in

Biotechnology: Elsevier Science. Taccari M, Canonico L, Comitini F, Mannazzu I, Ciani M. 2012. Screening of yeasts for growth on crude glycerol and optimization of biomass production. Bioresource

Technology 1: 1.

Tomas-Pejo E, Ballesteros M, Oliva J, Olsson L. 2010. Adaptation of the xylose fermenting yeast Saccharomyces cerevisiae F12 for improving ethanol production in different fed- batch SSF processes. Journal of Industrial Microbiology & Biotechnology 37(11): 1211- 1220.

Tsigie YA, Wang C-Y, Truong C-T, Ju Y-H. 2011. Lipid production from Yarrowia

lipolytica Polg grown in sugarcane bagasse hydrolysate. Bioresour. Technol.

102(19):9216-9222.

Walfridsson M, Hallborn J, Penttila M, Keranen S, Hahn-Hagerdal B. 1995. Xylose- metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TALI genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase. Applied and environmental microbiology 61(12):4184-4190.

Young EM, Comer AD, Huang H, Alper HS. 2012. A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae. Metab. Eng. 1: 1.

Zhao S, Fernald RD. 2005. Comprehensive Algorithm for Quantitative Real-Time

Polymerase Chain Reaction. Journal of Computational Biology 12(8): 1047- 1064. EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. , from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a

composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the

understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.