Compositions containing milk fat triglycerides produced by microbial fermentation

Abstract

The present disclosure relates to microbial hosts and methods for the biosynthesis of compositions comprising milk fat triglycerides.

Description

[Background technology]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 316,521, filed March 4, 2022, which is incorporated herein by reference in its entirety.

[0002] Dairy milk and dairy products have been consumed worldwide for thousands of years. However, health issues such as lactose intolerance and milk allergies, ethical issues such as poor treatment of animals, and environmental issues such as land use, water use, and greenhouse gas emissions have increased the demand for alternative milk and dairy products worldwide. Alternatives derived from plant sources, such as soy milk, almond milk, oat milk, rice milk, cashew milk, and coconut milk, are abundantly available as non-dairy alternatives. However, apart from nutritional differences with dairy milk, non-dairy alternatives suffer from non-dairy taste and flavor (e.g., beany or nutty flavor). Because milk triglycerides (fats) have unique chemical properties, vegetable oils cannot match the physical and sensory properties of dairy milk. Therefore, a sustainable and scalable process for biosynthesizing compositions with similar chemical and sensory properties to dairy milk fats is desirable. Summary of the Invention

[0003] In various aspects, the disclosure provides methods for making compositions that are chemically similar to milk fat (eg, dairy milk fat), and host cells for use in these methods.

[0004] Thus, in one aspect, the disclosure provides engineered host cells (e.g., microbial host cells) for producing triglycerides characteristic of dairy milk by microbial fermentation or bioconversion. In some embodiments, the composition further comprises one or more lactones characteristic of dairy fat to provide a desired sensory profile. In some embodiments, the composition further comprises beta-carotene and vitamin A, which, among other things, provide a color of the composition that is more similar to a dairy product. The disclosure further provides methods of producing products containing the composition, including milk, cheese, and butter, among other products that typically require dairy milk. Such dairy fat-containing products can be produced at lower cost and in a more sustainable manner by the disclosure.

[0005] Thus, in some aspects, the invention provides a microbial cell for producing milk fat triglycerides. The microbial cell expresses a biosynthetic pathway comprising at least one heterologous enzyme, the biosynthetic pathway producing triglycerides having short chain fatty acids esterified at sn-3. For example, the cell produces triglycerides having at least about 5% C4 and C6 fatty acids esterified at sn-3 on a molar basis. In various embodiments, the cell produces triglycerides having about 25% to about 75% (on a molar basis) of the fatty acids esterified at sn-3 being C4 and C6 fatty acids. The triglycerides further comprise C12-C18 fatty acids esterified at sn-3. In various embodiments, the triglycerides comprise C4:0, C6:0, C8:0, and C10:0 fatty acids esterified at sn-3.

[0006] In various embodiments, the triglycerides have primarily C12-C18 fatty acids esterified at sn-1 and sn-2 (i.e., on a molar basis). For example, in some embodiments, the triglycerides include 12:0, 14:0, 16.0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-1. In some embodiments, the triglycerides include 12:0, 14:0, 16.0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-2. In some embodiments, the triglycerides include linoleic acid esterified at sn-2.

[0007] In some embodiments, a biosynthetic pathway that produces triglycerides comprises at least one heterologous fatty acyl-CoA synthetase (ACS) with specificity for short chain fatty acid substrates. In some embodiments, the biosynthetic pathway comprises at least one or at least two such heterologous ACS enzymes, which are optionally enzymes that comprise an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 1-7.

[0008] In some embodiments, the biosynthetic pathway that produces triglycerides comprises a heterologous diacylglycerol O-acyltransferase (DGA) that has specificity for short chain fatty acid CoA substrates. In some embodiments, the DGA comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from SEQ ID NOs:8-17.

[0009] In some embodiments, the microbial cell expresses a conjugated linoleic acid (CLA) isomerase. In some embodiments, the CLA isomerase comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from SEQ ID NO:19.

[0010] In some embodiments, the microbial cell comprises a modification that results in a reduction in the amount or activity of one or more endogenous diglyceride acyltransferases. In some embodiments, the microbial cell comprises a deletion(s) of a gene encoding one or more endogenous diglyceride acyltransferases. For example, in some embodiments, the microbial cell has a deletion, inactivation, or reduced expression of one or more of DGA1 and DGA2 (Y. lipolytica) or their orthologs.

[0011] In some embodiments, the microbial cell has a deletion, inactivation, or reduced expression or activity of one or more fatty acid desaturase or fatty acid elongase enzymes. In such embodiments, the composition of triacylglycerides can be further adjusted.

[0012] In some embodiments, the microbial cells further comprise a biosynthetic pathway to produce one or more lactones. In some embodiments, the microbial cells express one or more enzymes having fatty acid hydroxylase activity. In some embodiments, the enzymes are cytochrome P450 enzymes (such as CYP505 enzymes or derivatives thereof). In certain embodiments, the enzymes described herein are engineered to provide hydroxylase activity at a desired position of a desired fatty acid substrate to allow for the production of a desired lactone. In some embodiments, the cells further express a cytochrome P450 reductase. In some embodiments, the P450 enzyme comprises a domain having fatty acid hydroxylase activity and a CPR domain.

[0013] In some embodiments, the cells may further produce beta-carotene via a heterologous biosynthetic pathway, which may provide desirable color properties to products such as butter. For example, the microbial cells may express heterologous biosynthetic enzymes, such as phytoene desaturase and / or bifunctional lycopene cyclase / phytoene synthase. In some embodiments, the cells may further produce vitamin A (e.g., from beta-carotene substrate). In some embodiments, the microbial cells express beta-carotene 15,15'-monooxygenase, retinal dehydrogenase, and / or lecithin:retinol acyltransferase. Precursors of beta-carotene and vitamin A are provided by the mevalonate pathway (MVA) or the non-mevalonate pathway (MEP pathway), which may be complemented or engineered to improve productivity. The MVA and MEP pathways produce isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) precursors, which may be converted to farnesyl diphosphate.

[0014] In certain embodiments, fatty acids and / or fatty acid esters are fed to the cells to affect the fatty acid composition of the triacylglycerides, including the short and / or medium chain fatty acid composition of the triacylglycerides, as well as the saturated fatty acid composition, allowing the microbial cells to approach a desired fatty acid profile. For example, in some embodiments, the microbial cells are fed one or a combination of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, and C16:0.

[0015] In some embodiments, the microbial cell is a yeast cell or a fungal cell. In some embodiments, the yeast or fungal cell is Yarrowia lipolytica. In some embodiments, the yeast or fungal cell is Yarrowia phangngensis. In some embodiments, the microbial cell is a bacterial cell. In some embodiments, the microbial cell is a bacterium that accumulates large amounts of triacylglycerol.

[0016] In one aspect, the disclosure relates to a method for making a composition comprising milk fat triglycerides. In some embodiments, the method comprises culturing a microbial cell according to any of the embodiments disclosed herein in the presence of a fatty acid substrate. In some embodiments, the fatty acid substrate comprises one or a combination of C4:0, C6:0, C8:0, C10:0, C12:0, and C14:0 fatty acid substrates. The composition comprising milk fat triglycerides may be recovered from the culture. In some embodiments, the fatty acid substrate is added to the culture, optionally as an alkyl ester or glyceride. In some embodiments, the fatty acid substrate is synthesized by the cells. In some embodiments, the composition is recovered by separating the wet cell mass and purifying the composition from the wet cell mass. In some embodiments, the cells are disrupted mechanically or enzymatically. The host cells and methods are further suitable for commercial production of the composition, i.e., the cells and methods may be productive on a commercial scale.

[0017] In another aspect, the present disclosure provides a composition or product (e.g., a beverage or food product) comprising the milk fat triglycerides made according to the present disclosure.

[0018] In another aspect, the present disclosure provides a method for producing a product comprising milk fat triglycerides. The method includes incorporating a composition comprising the milk fat triglycerides of the present disclosure into a product (i.e., a food, beverage, flavoring, or food additive product). Examples of products in which the composition can be used include, but are not limited to, food, beverage, flavoring, and food additives. Exemplary products include milk, cheese, butter, yogurt, frozen yogurt, gelato, milk chocolate, cream (e.g., heavy cream, light cream, sour cream, etc.), ice cream, cream cheese, custard, anhydrous milk fat, condensed milk, milk powder, evaporated milk, etc.

[0019] Other aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief description of the drawings]

[0020] [Figure 1A] 1 shows the composition of milk. 2 shows a pie chart showing the composition of whole milk. [Figure 1B] Figure 1 shows the composition of milk and the major lipid classes present in milk. [Figure 1C] This shows the composition of milk. This shows the types of proteins present in milk. [Figure 2A] The fatty acid composition and sn-position in milk triglycerides are shown. The main fatty acids in milk triglycerides and vegetable oils are illustrated. The main fatty acids in milk fat triglycerides are highlighted in boxes. [Figure 2B] The fatty acid composition and sn-position in milk triglycerides are shown. The position distribution of the main fatty acids in milk triglycerides is shown. [Diagram 3] A comparison of milk triglycerides and vegetable triglycerides is shown. A shows the structure of a representative milk triglyceride. B shows the structure of a common vegetable triglyceride derived from vegetable oils (e.g., palm, soybean, corn, sunflower, and safflower). C shows the structure of a representative medium chain oil (e.g., coconut and palm kernel). [Figure 4A]1 shows a schematic diagram of metabolic engineering of yeast cells for the production of milk fat compositions. Carbon sources such as sugars and fatty acids (e.g., 4:0, 6:0, 8:0, 10:0, and 12:0 fatty acids) are used as substrates (these can be added to the culture). Carbon source(s) (e.g., sucrose, glucose, sugarcane, glycerol, starch, acetate) are converted to acetyl-CoA and malonyl-CoA via glycolysis and the TCA cycle. 14:0-CoA, 16:0-CoA, 18:0-CoA, 18:1-CoA, and 18:2-acyl-phospholipids (PL) are synthesized by native fatty acid synthesis followed by the sequential action of elongase, Δ9 desaturase, and Δ12 desaturase. These fatty acid acyl esters are converted to the indicated lysophosphatidic acid through the action of glycerol 3-phosphate sn-1 acyltransferase (SCT), which is converted to diacylglycerol through the action of 1-acyl-sn-glycerol-3-phosphate acyltransferase (SLC) and phosphatidic acid phosphatase (PAH). Free linoleic acid released from phospholipids via phospholipases can be further converted to conjugated linoleic acid (CLA) through the activity of heterologous conjugated linoleic acid isomerase and reincorporated into the pool of phospholipids and acyl esters. 8:0, 10:0, and 12:0 fatty acids can be converted to lactones such as δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octalactone through the activity of cytochrome P450 hydroxylases. The 4:0, 6:0, 8:0, 10:0, and 12:0 fatty acids are also converted to 4:0-CoA, 6:0-CoA, 8:0-CoA, 10:0-CoA, and 12:0-CoA, which are incorporated into the sn-3 triglyceride position via specialized diglycerol acyltransferases to generate milk fat triglycerides with the formula shown in the lower right rectangle. Finally, vitamin A and β-carotene can be biosynthesized by central metabolism via a farnesyl-pyrophosphate intermediate (see FIG. 4B). [Figure 4B]Schematic pathways for the biosynthesis of β-carotene and vitamin A (retinol and retinyl esters). ERG10, acetyl-CoA C-acetyltransferase, EC 2.3.1.9; ERG13, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, EC 2.3.3.10; HMG1 / HMG2, hydroxymethylglutaryl-CoA (HMG-CoA) reductase, EC 1.1.1.88; ERG12, mevalonate kinase, EC 2.7.1.36; ERG8, phosphomevalonate kinase, EC 2.7.4.2; IDI1, isopentenyl diphosphate:dimethylallyl diphosphate isomerase, EC 5.3.3.2; BTS1 / CrtE, geranylgeranyl diphosphate synthase, EC 2.5.1.1; CrtYB / CarRA / CarRP, bifunctional lycopene cyclase / phytoene synthase, EC 2.5.1.32;CrtI / CarB, phytoene desaturase, EC 1.3.99.31;BMCO, β-carotene 15,15'-monooxygenase, EC 1.13.11.63;RDH, retinal dehydrogenase, EC 1.2.1.36;LRAT, lecithin:retinol acyltransferase, EC 2.3.1.135. The following heterologous enzymes have been expressed in Yarrowia lipolytica to enable the production of β-carotene and vitamin A:CrtYB / CarRA / CarRP, CrtI / CarB, BMCO, RDH, and LRAT. [Diagram 5] 1 shows microscopic images of Y. lipolytica expressing heterologous DGAT enzymes. [Figure 6] GC-MS spectrum showing a peak corresponding to hexanoic acid methyl ester present in MY74 lipids and absent in MY27 lipids. [Figure 7A] 1 shows a downstream separation process for purification of a milk fat composition produced intracellularly in a microorganism. [Figure 7B] 1 shows a downstream separation process for purification of a milk fat composition produced intracellularly in a microorganism. [Figure 7C] 1 shows a downstream separation process for purification of a milk fat composition produced intracellularly in a microorganism. [Figure 7D] 1 shows a downstream separation process for purification of a milk fat composition produced intracellularly in a microorganism. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In various aspects, the disclosure provides methods for making compositions that are chemically similar to milk fat (eg, dairy milk fat), and host cells for use in these methods.

[0022] Dairy milk is an oil-in-water emulsion containing the macrocomponents shown in Figure 1A, but the actual composition of milk varies with mammalian species, breed, season, nutrition, and other factors. More than 98% of milk lipids can be triacylglycerides (Figure 1B). Unlike vegetable oils, milk triglycerides contain low molecular weight fatty acids. Figures 2A and 2B illustrate the main differences between vegetable oils and dairy milk triglycerides. Interestingly, low molecular weight fatty acids are not uniformly distributed in milk triglycerides. As shown in Figure 2B, unlike the sn-1 and sn-2 positions, which contain medium and long chain fatty acids, the sn-3 position has low molecular weight fatty acids such as butyric and caproic acids. The most common fatty acids at sn-1 are C16:0, C18:1, and C18:0, while the most common fatty acids at sn-2 are C16:0, C14:0, and C12:0 (Figures 2B and 3A).

[0023] Thus, in one aspect, the disclosure provides engineered host cells (e.g., microbial host cells) for producing triglycerides characteristic of dairy milk (e.g., cow's milk, sheep's milk, goat's milk, buffalo milk) by microbial fermentation or bioconversion. In some embodiments, the composition further comprises one or more lactones characteristic of dairy fat to provide a desired sensory profile. In some embodiments, the lactones comprise one or more of δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octalactone. In some embodiments, the composition further comprises β-carotene and vitamin A, which, among other things, provide the color of the composition more similar to a dairy milk product. The disclosure further provides methods of producing products containing the composition, including milk, cheese, and butter, among other products that typically require dairy milk. Such dairy fat-containing products may be produced at lower cost and in a more sustainable manner by the disclosure.

[0024] Thus, in some aspects, the present invention provides a microbial cell for producing milk fat triglycerides. The microbial cell expresses a biosynthetic pathway comprising at least one heterologous enzyme, the biosynthetic pathway producing triglycerides having short chain fatty acids esterified at sn-3. For example, the cell produces triglycerides having at least about 5% C4 and C6 fatty acids esterified at sn-3 on a molar basis. In some embodiments, the cell produces triglycerides having at least about 7%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40% C4 and C6 fatty acids esterified at sn-3 on a molar basis. In some embodiments, the cell produces triglycerides having at least about 45% or at least about 50% C4 and C6 fatty acids esterified at sn-3 on a molar basis. In various embodiments, the cells produce triglycerides in which about 25% to about 75% (on a molar basis) of the fatty acids esterified at sn-3 are C4 and C6 fatty acids (e.g., about 30% to about 70% or about 40% to about 60%).

[0025] The triglycerides further comprise C12-C18 fatty acids esterified at sn-3, hi various embodiments, the triglycerides comprise C4:0, C6:0, C8:0, and C10:0 fatty acids esterified at sn-3.

[0026] In various embodiments, the triglycerides have primarily C12-C18 fatty acids esterified at sn-1 and sn-2 (i.e., on a molar basis). For example, in some embodiments, the triglycerides include 12:0, 14:0, 16.0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-1. In some embodiments, the triglycerides include 12:0, 14:0, 16.0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-2. In some embodiments, the triglycerides include linoleic acid esterified at sn-2.

[0027] In some embodiments, a biosynthetic pathway that produces triglycerides comprises at least one heterologous fatty acyl-CoA synthetase (ACS) with specificity for short chain fatty acid substrates. In some embodiments, the biosynthetic pathway comprises at least one or at least two such heterologous ACS enzymes, which are optionally enzymes that comprise an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 1-7. In some embodiments, the ACS enzyme(s) comprises an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs: 1-7.

[0028] In some embodiments, the microbial cell expresses a heterologous ACS comprising an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the microbial cell expresses (or further expresses) a heterologous ACS comprising an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0029] In some embodiments, the biosynthetic pathway that produces triglycerides comprises a heterologous diacylglycerol O-acyltransferase (DGA) that has specificity for short chain fatty acid CoA substrates. In some embodiments, the DGA comprises an amino acid sequence that is at least about 70% identical to an amino acid sequence selected from SEQ ID NOs:8-17. In some embodiments, the DGA comprises an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs:8-17.

[0030] In some embodiments, the DGA comprises an amino acid sequence that is at least about 70% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the DGA comprises an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0031] In some embodiments, the microbial cell expresses a conjugated linoleic acid (CLA) isomerase. In some embodiments, the CLA isomerase comprises an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NO: 19.

[0032] In some embodiments, the microbial cell comprises a modification that results in a reduction in the amount or activity of one or more endogenous diglyceride acyltransferases. In some embodiments, the microbial cell comprises a deletion(s) of a gene encoding one or more endogenous diglyceride acyltransferases. For example, in some embodiments, the microbial cell has a deletion, inactivation, or reduced expression of one or more of DGA1 and DGA2 (Y. lipolytica) or their orthologs.

[0033] In some embodiments, the host cell has one or more genetic modifications that reduce C16:1 fatty acid production and / or increase C18:1 fatty acid production. In some embodiments, the microbial cell has a deletion, inactivation, or reduced expression or activity of one or more fatty acid desaturase enzymes. In some embodiments, the microbial cell has a deletion, inactivation, or reduced expression or activity of a delta-12 desaturase (e.g., the FAD2 delta-12 desaturase of Y. lipolytica or its orthologues). Alternatively or additionally, the microbial cell has a deletion, inactivation, or reduced expression or activity of one or more delta-9 desaturase enzymes (OLE1, YALI0C05951g or its orthologues). Alternatively or additionally, the microbial cell expresses a heterologous OLE1 that converts C18:0 to C18:1. See Sitepu IR.et al., Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species, Bioresource Technology, Vol.144,2013,360-369. Such a heterologous OLE1 may be selected from those that contain an amino acid sequence having at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity (or 100% sequence identity) to one of SEQ ID NOs:22-30.

[0034] In some embodiments, the microbial cell has a deletion, inactivation, or reduced expression or activity of one or more fatty acid elongase enzymes. In some embodiments, the microbial cell has a deletion, inactivation, or reduced expression or activity of ELO1 (e.g., Y. lipolytica YALI0F06754g or its orthologues). Alternatively or additionally, the microbial cell has a deletion, inactivation, or reduced expression or activity of ELO2 (e.g., YALI0F06754g or its orthologues).

[0035] In some embodiments, the microbial cell expresses one or more heterologous fatty acid synthases. For example, the heterologous fatty acid synthase may produce C10:0, C12:0, and C14:0 fatty acids. In some embodiments, the at least one heterologous fatty acid synthase comprises a subunit (FAS1) that comprises an amino acid sequence that is at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical (or has 100% sequence identity) to the amino acid sequence of SEQ ID NO:20. In some embodiments, the at least one heterologous fatty acid synthase comprises a subunit (FAS2) that comprises an amino acid sequence that is at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99% identical (or has 100% sequence identity) to the amino acid sequence of SEQ ID NO:21. These enzymes containing FAS1 and FAS2 subunits produce C10:0 fatty acids. In some embodiments, the microbial cells express modified Y. lipolytica FAS1 and / or FAS2 as described in Xu P. et al., Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals, PNAS, 2016, vol. 113, no. 39, 10848-10853; Rigouin C., et al., Production of Medium Chain Fatty Acids by Yarrowia lipolytica: Combining Molecular Design and TALEN to Engineer the Fatty Acid Synthase, ACS Synth. Biol. 2017, 6, 1870-1879.

[0036] In some embodiments, the microbial cells further comprise a biosynthetic pathway to produce one or more lactones. In some embodiments, the microbial cells express one or more enzymes having fatty acid hydroxylase activity. In some embodiments, the enzymes are cytochrome P450 enzymes (such as CYP505 enzymes or derivatives thereof). In certain embodiments, the enzymes described herein are engineered to provide hydroxylase activity at a desired position of a desired fatty acid substrate to allow for the production of a desired lactone. In some embodiments, the cells further express a cytochrome P450 reductase. In some embodiments, the P450 enzyme comprises a domain having fatty acid hydroxylase activity and a CPR domain.

[0037] CYP505E3 from Aspergillus terreus (SEQ ID NO: 18) is a self-contained P450 enzyme that contains a reductase domain and can catalyze intrachain hydroxylation involving the ω-7 position of alkanes, aliphatic alcohols, and fatty acids. In some embodiments, the P450 enzyme contains a domain having fatty acid hydroxylase activity, the domain having fatty acid hydroxylase activity containing an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or 100% sequence identity to amino acids 1-461 of SEQ ID NO: 18. In some embodiments, the P450 enzyme also contains a CPR domain, which is optionally the CPR domain of SEQ ID NO: 18 or a derivative thereof (i.e., containing an amino acid sequence that is at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 97% identical thereto), or optionally a heterologous CPR domain.

[0038] In some embodiments, the enzyme hydroxylates 8:0, 10:0, and 12:0 fatty acids, thereby forming one or more lactones selected from δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octalactone. In some embodiments, the microbial cell expresses a biosynthetic pathway that produces two or more lactones selected from δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octalactone when the cells are contacted with the corresponding fatty acid substrate (as described herein). In some embodiments, the microbial cell expresses a biosynthetic pathway that produces δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octalactone when the cells are contacted with the corresponding fatty acid substrate (as described herein).

[0039] In some embodiments, the cells may further produce beta-carotene via a heterologous biosynthetic pathway, which may provide desirable color properties to products such as butter. For example, the microbial cells may express heterologous biosynthetic enzymes, such as phytoene desaturase and / or bifunctional lycopene cyclase / phytoene synthase. In some embodiments, the cells may further produce vitamin A (e.g., from beta-carotene substrate). In some embodiments, the microbial cells express beta-carotene 15,15'-monooxygenase, retinal dehydrogenase, and / or lecithin:retinol acyltransferase. Precursors of beta-carotene and vitamin A are provided by the mevalonate pathway (MVA) or the non-mevalonate pathway (MEP pathway), which may be complemented or engineered to improve productivity. The MVA and MEP pathways produce isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) precursors, which may be converted to farnesyl diphosphate. The MVA pathway is shown in Figure 4B.

[0040] In some embodiments, microbial host cells are engineered to express or overexpress one or more enzymes of the MEP and / or MVA pathways to catalyze IPP and DMAPP biosynthesis from sugars, such as glucose, or other carbon sources.

[0041] In some embodiments, the microbial host cell is engineered to express or overexpress one or more enzymes of the MVA pathway. The MVA pathway refers to a biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway typically includes enzymes that catalyze the following steps: (a) condensation of two molecules of acetyl-CoA to acetoacetyl-CoA (e.g., by the action of acetoacetyl-CoA thiolase); (b) condensation of acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-coenzyme A (HMG-CoA) (e.g., by the action of HMG-CoA synthase (HMGS)); (c) conversion of HMG-CoA to mevalonate (e.g., by the action of HMG-CoA synthase (HMGS)). (d) phosphorylating mevalonate to mevalonate 5-phosphate (e.g., by the action of mevalonate kinase (MK)); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by the action of phosphomevalonate kinase (PMK)); and (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by the action of mevalonate pyrophosphate decarboxylase (MPD)). The MVA pathway, and the genes and enzymes that comprise the MVA pathway, are described in U.S. Pat. No. 7,667,017, which is incorporated herein by reference in its entirety. In some embodiments, the microbial host cell expresses or overexpresses one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, and MPD or engineered variants thereof, resulting in increased production of IPP and DMAPP. In some embodiments, FPP is produced, at least in part, by metabolic flux through the MVA pathway, and the microbial host cell has at least one additional gene copy of one or more of acetoacetyl-CoA thiolase, HMGS, HMGR, MK, PMK, MPD, or engineered variants thereof.

[0042] The MEP pathway (endogenous to bacterial hosts) and the genes and enzymes that make up the MEP pathway are described in U.S. Patent No. 8,512,988, which is incorporated herein by reference in its entirety. For example, genes that make up the MEP pathway include dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, and ispA. In some embodiments, the microbial host cell expresses or overexpresses one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof, resulting in increased production of IPP and DMAPP. In some embodiments, FPP is produced, at least in part, by metabolic flux through the MEP pathway, and the microbial host cell has at least one additional gene copy of one or more of dxs, ispC, ispD, ispE, ispF, ispG, ispH, idi, ispA, or modified variants thereof.

[0043] In some embodiments, the MEP pathway of the microbial host cell is engineered to increase the production of IPP and DMAPP from glucose, as described in U.S. Pat. No. 10,662,442 and / or U.S. Pat. No. 10,480,015, the contents of which are incorporated herein by reference in their entirety. For example, in some embodiments, the microbial host cell overexpresses MEP pathway enzymes, and balances the expression of push / pull carbon flux to IPP and DMAPP. In some embodiments, the microbial host cell is engineered to increase the availability or activity of Fe-S cluster proteins, thereby supporting higher activity of the Fe-S enzymes IspG and IspH. In some embodiments, the host cell is engineered to overexpress IspG and IspH, thereby increasing carbon flux to the 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) intermediate, but with balanced expression to prevent accumulation of HMBPP in amounts that reduce cell growth or viability or inhibit MEP pathway flux.

[0044] In alternative embodiments, the microbial host cells are not engineered to increase production of FPP from MEP or MVA pathway precursors, but FPP or precursor compounds are fed to the cells to provide FPP substrates for the production of beta-carotene or vitamin A. In yet another embodiment, the microbial cells express an isoprenol utilization pathway as described in US 11,034,980, which is incorporated herein by reference in its entirety. Such cells are capable of producing IPP and DMAPP precursors from prenol and / or isoprenol substrates provided to the culture.

[0045] In certain embodiments, fatty acids and / or fatty acid esters are fed to the cells to affect the fatty acid composition of the triacylglycerides, including the short and / or medium chain fatty acid composition of the triacylglycerides, as well as the saturated fatty acid composition, allowing the microbial cells to approach a desired fatty acid profile. For example, in some embodiments, the microbial cells are fed one or a combination of C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, and C16:0.

[0046] As discussed in more detail below, the microbial cell may be a yeast or fungal cell, or in some embodiments, a bacterial cell. Thus, when a gene is disrupted or inactivated according to an embodiment of the present disclosure, the gene that is disrupted or inactivated depends on the host species. For ease of understanding, unless otherwise stated, the gene name in this disclosure is Yarrowia lipolytica gene. Those skilled in the art will understand how to identify homologs, orthologs, or paralogs of different host species. Furthermore, the strain may be engineered to increase the expression of a particular gene (e.g., by gene complementation or editing of expression control sequences) or to decrease the activity of a particular gene (e.g., by loss-of-function mutation(s) or editing of expression control sequences), and such derivatives may generally comprise an amino acid sequence that is at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98% identical to the reference amino acid sequence.

[0047] The similarity or identity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined through sequence alignment. Such alignment can be performed by several algorithms known in the art, for example, by the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), by hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or by the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The grade of sequence identity (sequence matching) can be calculated, for example, using BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215:403-410. BLAST polynucleotide searches can be performed with the BLASTN program, score=100, wordlength=12.

[0048] BLAST protein searches can be performed with the BLASTP program (score=50, word length=3). To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis can be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov Random Fields.

[0049] Expression of enzymes can be adjusted for optimal activity, for example, using gene modules (e.g., operons) or independent expression of enzymes. For example, expression of genes can be regulated through the selection of promoters, such as inducible or constitutive promoters with different strengths (e.g., strong, medium, or weak). Furthermore, expression of genes can be controlled by manipulating the copy number of genes or operons in the cell. In some embodiments, expression of genes can be regulated by manipulating the order of genes in a module, with genes that are transcribed first in an operon generally being expressed at higher levels. In some embodiments, expression of genes is regulated by the integration of one or more genes into a chromosome.

[0050] Optimization of expression can also be achieved by selection of appropriate promoters and ribosome binding sites. In some embodiments, this can include selection of high copy number plasmids, or single, low, or medium copy number plasmids. The transcription termination step can also be targeted for regulation of gene expression by introduction or removal of structures such as stem loops.

[0051] Expression vectors that contain all the elements necessary for expression are commercially available and known to those skilled in the art.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.Cells are genetically modified by introducing heterologous DNA into cells.To allow the expression of heterologous DNA in host cells, the heterologous DNA is placed under the operable control of transcriptional elements.

[0052] In some embodiments, endogenous genes are edited, as opposed to gene complementation. Editing can modify endogenous promoters, ribosome binding sequences, or other expression control sequences, and / or in some embodiments, modify trans-acting and / or cis-acting factors in gene regulation. Genome editing can be performed using CRISPR / Cas genome editing techniques, or similar techniques using zinc finger nucleases and TALENs. In some embodiments, endogenous genes are replaced with heterologous genes by homologous recombination.

[0053] In some embodiments, the gene is at least partially overexpressed by controlling gene copy number.The copy number of the gene can be conveniently controlled using a plasmid with different copy numbers, but gene duplication and chromosomal integration can also be used.For example, the process of genetically stable tandem gene duplication is described in US2011 / 0236927, which is incorporated herein by reference in its entirety.

[0054] According to the present disclosure, when a gene is deleted, the gene may be deleted (i.e., inactivated) in whole or in part, which may include deletion of the coding sequence and / or expression control sequences.

[0055] In some embodiments, the microbial cell is a yeast cell or a fungal cell. In some embodiments, the yeast or fungal cell belongs to a genus selected from Aspergillus, Aurantiochytrium, Bastobotyrs, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Issatchenkia, Kluyveromyces, Kodamaea, Leucosporidiella, Linderna, Lipomyces, Mortierella, Myxozyma, Mucor, Occultifur, Ogataea, Penicillium, Phaffia, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Scheffersomyces, Schizosaccharomyces, Sporidiobolus, Sporobolomyces, Starmerella, Tremella, Trichosporon, Wickerhamomyces, Waltomyces, and Yarrowia.In some embodiments, the yeast or fungal cells are Yarrowia lipolytica, Yarrowia phangngensis, Pichia kudriavzevii, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Rhodosporidium toruloides, Sporidiobolus ruinenii, Sporidiobolus salmonicolor, Aspergillus oryzae, Mortierella isabellina, Waltomyces lipofer, Candida tropicalis, Candida boidinii, Scheffersomyces stipitis, Mucor circinelloides, Ashbya gossypii, Trichoderma harzianum, Pichia guilliermondii, Kodamaea ohmeri, Rhodotorula aurantiaca, Lindnera saturnus, Penicillium roqueforti, Lipomyces In some embodiments, the yeast or fungal cell is Yarrowia lipolytica. In some embodiments, the yeast or fungal cell is Yarrowia phangngensis.

[0056] In some embodiments, the microbial cell is a bacterial cell. In some embodiments, the microbial cell is a bacterium that accumulates large amounts of triacylglycerol. In some embodiments, the bacterial cell belongs to a genus selected from Acidovorax, Acinetobacter, Actinomyces, Alcanivorax, Arthrobacter, Brevibacterium, Bacillus, Clostridium, Corynebacterium, Dietzia, Escherichia, Gordonia, Marinobacter, Mycobacterium, Micrococcus, Micromonospora, Moraxella, Nocardia, Pseudomonas, Psychrobacter, Rhodococcus, Salmonella, Streptomyces, Thalassolituus, and Thermomonospora. In some embodiments, the bacterial cell belongs to a species selected from Rhodococcus opacus, Acinetobacter calcoaceticus, Streptomyces coelicolor, Rhodococcus jostii, and Acinetobacter baylyi.

[0057] In one aspect, the disclosure relates to a method for making a composition comprising milk fat triglycerides. In some embodiments, the method comprises culturing a microbial cell according to any of the embodiments disclosed herein in the presence of a fatty acid substrate. In some embodiments, the fatty acid substrate comprises C4:0, C6:0, C8:0, C10:0, C12:0, and C14:0 fatty acid substrates. The composition comprising milk fat triglycerides can be recovered from the culture. In some embodiments, the fatty acid substrate is added to the culture, optionally as an alkyl ester or glyceride. In some embodiments, the fatty acid substrate is synthesized by the cells.

[0058] In some embodiments, the composition is recovered by separating the wet cell mass and purifying the composition from the wet cell mass. In some embodiments, the cells are mechanically or enzymatically disrupted. In some embodiments, the composition is extracted using an organic solvent. An exemplary recovery process is shown in Figures 5A-D.

[0059] The host cells and methods are further suitable for commercial production of the compositions, i.e., the cells and methods can be productive on a commercial scale. In some embodiments, the size of the culture is at least about 100 L, or at least about 200 L, or at least about 500 L, or at least about 1,000 L, or at least about 10,000 L, or at least about 50,000 L, or at least about 100,000 L, or at least about 200,000 L, or at least about 500,000 L, or at least about 1,000,000 L. In various embodiments, the culture is performed in a batch culture.

[0060] In another aspect, the present disclosure provides a composition or product (e.g., a beverage or food product) comprising the milk fat triglycerides made according to the present disclosure.

[0061] In another aspect, the present disclosure provides a method for producing a product comprising milk fat triglycerides. The method includes incorporating a composition comprising the milk fat triglycerides of the present disclosure into a product (i.e., a food, beverage, flavoring, or food additive product). Examples of products in which the composition can be used include, but are not limited to, food, beverage, flavoring, and food additives. Exemplary products include milk, cheese, butter, yogurt, frozen yogurt, gelato, milk chocolate, cream (e.g., heavy cream, light cream, sour cream, etc.), ice cream, cream cheese, custard, anhydrous milk fat, condensed milk, milk powder, evaporated milk, etc.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0063] As used herein, the term "about" means ±10% of the associated numerical value, unless the context requires otherwise. EXAMPLES

[0064] A biosynthetic scheme for the metabolic engineering of microorganisms to produce components of dairy fat is shown in Figure 4. The following examples describe several non-limiting embodiments for the metabolic engineering and purification of fats produced by microorganisms.

[0065] Example 1. Manipulation of C4-C12 fatty acyl-CoA synthetase activity Yarrowia lipolytica converts free fatty acids to acyl-CoA via two endogenous acyl-CoA synthetases, FAA1 and FAT1. FAA1 is localized in the cytosol and is involved in the incorporation of fatty acids into larger biomolecules. FAT1 is localized in peroxisomes and is involved in the breakdown of fatty acids into carbon and energy. Both enzymes have a preference for long-chain fatty acids and have little activity with short- and medium-chain fatty acids.

[0066] The production of triglycerides with short chains at the sn-3 position requires a supply of short- and medium-chain acyl-CoAs in the cytosol. Therefore, we expressed heterologous acyl-CoA synthetases and screened them for activity with short- and medium-chain (C4–C12) fatty acid substrates.

[0067] Yarrowia lipolytica MY16 was transformed with plasmids carrying acyl-CoA synthetases (SEQ ID NOs: 1-7) and cell-free extracts were prepared. Acyl-CoA synthetase (ACS) activity was quantified by an in vitro assay that measures the conversion of CoA and carboxylic acids to acyl-CoA via quantification of the free CoA concentration remaining after incubation with cell-free extracts. The results of the assay are shown in Table 1. From this in vitro assay, it was found that when expressed in Y. lipolytica, the enzyme of SEQ ID NO: 2 was able to synthesize >20 μmol min−1 for C4, C5, C6, and C7 fatty acids. -1 mg protein -1 When expressed in Y. lipolytica MY16, the enzyme of SEQ ID NO:3 was found to have activity of >20 μmol min for C4, C5, C6, C7, C8, C9, C10, C11, and C12 fatty acids. -1 mg protein -1 and the enzyme of SEQ ID NO:6 had activity against C5, C6, C7, C8, C9, C10, and C11.

[0068] Example 2. Expression of PpLvaE and RpDcaA acyl-CoA synthetases in DGA1Δ DGA2Δ Yarrowia lipolytica strains The Yarrowia lipolytica diglyceride acyltransferases DGA1 (YALI0E3269g) and DGA2 (YALI0D07986g) were targeted for gene deletion. These native DGA enzymes are responsible for the majority of triacylglycerol synthesis but do not accept short-chain acyl-CoA substrates. The background of the DGA1Δ DGA2Δ Yarrowia lipolytica strain may be suitable for the expression of heterologous diglyceride acyltransferases capable of incorporating short- and medium-chain acyl-CoA substrates into the sn-3 position of triglycerides. DGA1 and DGA2 were deleted by homologous recombination in Y. lipolytica MY16, resulting in strain MY43, which contains DGA1Δ DGA2Δ and a hygromycin selection marker. The hygromycin marker was then removed, resulting in a DGA1Δ DGA2Δ strain designated MY54.

[0069] A vector co-expressing the acyl-CoA synthetase of SEQ ID NO:2 from the Y. phangngensis TEF1 promoter and the acyl-CoA synthetase of SEQ ID NO:3 from the Y. lipolytica TEF1 promoter was created using standard molecular biology techniques, designated pMY64, and transformed into MY43. Colonies were assayed for in vitro short- and medium-chain acyl-CoA synthetase activity as described in Example 1, and showed an average of 35 μmol min-1 activity with butyrate substrate. -1 mg protein -1 and 14 μmol min with octanoic acid substrate. -1 mg protein -1 It was found to have activity.

[0070] Example 3. Expression of heterologous DGAT enzymes in MY54 strains DNA sequences (codon optimized for expression in Y. lipolytica) were generated for the enzymes BtDGAT1, ChDGAT1, OaDGAT1, BbDGAT1, BtDGAT2, OaDGAT2, BtDGAT2L6, ChDGAT2L6, CaDGAT1, and EaDAcT (SEQ ID NOs: 8-17) and incorporated into a gene cassette driven by the Y. lipolytica TEF1 promoter in a Y. lipolytica expression vector. These vectors were named pY65 (BtDGAT1), pY66 (ChDGAT1), pY67 (OaDGAT1), pY68 (BbDGAT1), pY69 (BtDGAT2), pY70 (OaDGAT2), pY71 (BtDGAT2L6), pY72 (ChDGAT2L6), pY73 (CaDGAT1), and pY74 (EaDAcT) and transformed into MY54 strain. The isolated strains were grown in minimal glucose medium at 30°C and 900 rpm for 95 hours and observed by microscope for the presence of increased lipid body size. Images of these strains are shown in Figure 5.

[0071] Example 4: Yarrowia lipolytica strains accumulating lipids with 6:0 fatty acyl chains.

[0072] Yarrowia lipolytica strain MY54, carrying the genotype DGA1Δ DGA2Δ, was transformed with a non-targeted integration cassette containing the Bos taurus DGAT2 enzyme expressed from the Y. lipolytica TEF1 promoter region. Several colonies were reisolated into single colonies and incubated in defined minimal glucose medium at 30°C and 250 rpm for 72 hours. The cell cultures were then examined by microscopy for the presence of lipid bodies, and one culture with visible large lipid bodies was designated strain MY63. MY63 was then transformed with plasmid pY64, and a transformant was isolated and designated MY74. MY74 and the unmodified control strain MY27 were cultured in minimal medium containing glycerol and 4.8 mM hexanoic acid. After 94 hours of incubation, the cells were harvested and lipids were extracted from both samples by a modified Bligh-Dwyer method. Lipids were dissolved in methyl-tert-butyl ether (MBTE) at a concentration of 100 mg / mL and subjected to transesterification with a 100-fold volume excess of 0.5 N sodium methoxide and incubated at 50° C. for 30 min. A reference sample of ghee butter was included for analysis. Fatty acid methyl esters derived from yeast or ghee butter lipids were analyzed by gas chromatography-mass spectrometry. Compounds were identified by comparison to the NIST mass spectral database and by Supelco 37 FAME reference standard. The chromatogram of the MY74 lipid-derived sample contained a peak with the same retention time as hexanoic acid methyl ester and 93% match to the NIST mass spectral reference library. Figure 6. This peak was also present in ghee butter but not in the lipid sample from MY27.

[0073] Example 5. Altering triacylglyceride profiles by feeding engineered yeast short-, medium-, and saturated fatty acids The engineered Yarrowia lipolytica yeast strain MY167 was generated by transforming vector pY111 into the DGA1Δ DGA2Δ parent strain MY54. Vector pY111 contains a multigene expression cassette containing BtDGAT2, PplvaE, and RpDcaA expressed via separate constitutive promoters. To analyze triacylglyceride composition, cells were grown in 96-well plates with shaking at 30 °C in glucose minimal medium. Twenty-four hours after seeding, fatty acids provided as either free fatty acids (C6-C10) or ethyl esters (C12-C16) were added directly to the fermentation medium. Cells were continuously shaken for 70 hours after seeding, and then cell cultures were harvested, washed, dried, subjected to methanolic chloride-catalyzed transesterification, and analyzed by gas chromatography with a flame ionization detector to determine the composition of lipid acyl chains. The results of lipid characterization are shown in Table 2. The results show that the composition of short-, medium-, and saturated fatty acids in triacylglycerides can be altered by feeding the engineered yeast strains with similar species of fatty acids. This allows the engineered strains to approach the fatty acid profile of representative bovine milk fat. For example, adding C14:0 fatty acids to the fermentation medium increases the content of C14:0 triacylglycerides from 0.9% to 32.7%. The target for C14:0 in bovine milk fat is 12.0%.

[0074] Example 6. Changes in triacylglyceride profile by inactivation of FAD2 delta-12 desaturase and ELO1 fatty acid elongase Yarrowia lipolytica yeast strain MY167 was transformed with a gene disruption cassette targeting the FAD2 (YALI0B10153g) locus. The resulting strain MY168 was grown in a 96-well plate with glucose minimal medium at 30°C with shaking for 140 hours. The cell culture was then harvested, washed, dried, subjected to methanolic chloride-catalyzed transesterification, and analyzed by gas chromatography with a flame ionization detector to determine the composition of lipid acyl chains. The results of lipid characterization are shown in Table 3. The results show that the polyunsaturated fatty acid content can be reduced from 6.4% to 0.0% by deletion of the FAD2 gene. The content of polyunsaturated fat in a typical bovine milk fat is 2%.

[0075] Yarrowia lipolytica yeast strain MY168 was transformed with a gene disruption cassette targeting the ELO1 (YALI0F06754g) locus. The resulting strain MY174 was grown in 96-well plates with glucose minimal medium at 30°C with shaking for 140 hours. The cell culture was then harvested, washed, dried, subjected to methanolic chloride-catalyzed transesterification, and analyzed by gas chromatography with a flame ionization detector to determine the composition of lipid acyl chains. The results of lipid characterization are shown in Table 3. The results indicate that the ratio of C16 to C18 fatty acid species can be engineered from 0.33 (yeast MY168) to 1.01 (yeast MY14) by deletion of the ELO1 gene. The ratio of C16 to C18 fatty acid species in a representative bovine milk fat is 1.58.

[0076] Example 7. Purification of milk fat triglycerides from Yarrowia lipolytica Milk fat triglycerides are produced intracellularly and accumulate inside special organelles called lipid bodies. To separate the milk fat product from whole yeast cells, the following downstream separation processes can be used:

[0077] As shown in Figure 7A, the cell mass can be separated from the fermentation broth by centrifugation or filtration. The cell mass is dried and expeller pressed to create crude milk fat lipids and a cell pressate. The crude milk fat can be further processed by refining, bleaching, and deodorization to produce purified milk fat.

[0078] As shown in Figure 7B, the dried cell mass can be suspended in an organic solvent (e.g., hexane, chloroform, methanol, cyclopentyl methyl ether, isooctane, ethyl acetate, ethanol, acetone, isopropyl alcohol, or mixtures thereof) and ground in a bead mill to release intracellular lipids into the solvent phase. The milk fat-containing solvent is then separated from the remaining solids, and the solvent is evaporated to produce crude milk fat. The crude milk fat can be further processed into a refined milk fat product. This process is feasible for a wide range of lipid content, from 30% to >70% w / w of the total cell mass. In variations of this process, the grinding with a bead mill can be replaced with high pressure homogenization (French press), sonication, microwave, pulsed electric field, or other mechanical cell disruption methods.

[0079] As shown in Figure 7C, the wet cell mass may be used for aqueous phase extraction by the addition of a water-miscible organic solvent such as ethanol. The water-solvent-oil-cell mass mixture is mixed under process conditions that promote cell lysis, and then the oil is separated into an organic phase by decanter centrifugation. The addition of organic solvents inhibits the formation of stable oil-water emulsions. See U.S. Patent No. 5,928,696, the entire contents of which are incorporated herein by reference.

[0080] As shown in Figure 7D, cell lysis can be improved by the addition of cell wall degrading enzymes. Yeast and fungal cell walls are composed of the polysaccharide chitin as well as proteins. Cell wall degrading enzymes such as endochitinase, chitobiosidase, lytic polysaccharide monooxygenase, N-acetylglucosaminidase, and protease soften and remove the yeast cell wall, facilitating subsequent mechanical, chemical, or osmotic separation steps.

[0081] array Acyl-CoA synthetase enzyme SEQ ID NO:1 Escherichia coli FadK (ecFadK) MHPTGPHLGPDVLFRESNMKVTLTFNEQRRAAYRQQGLWGDASLADYWQQTARAMPDKIAVVDNHGASYTYSALDHAASCLANWMLAKGIESGDRIAFQLPGWCEFTVIYLACLKIGAVSVPLLPSWREAELVWVLNKCQA KMFFAPTLFKQTRPVDLILPLQNQLPQLQQIVGVDKLAPATSSLSLSQIIADNTSLTTAITTHGDELAAVLFTSGTEGLPKGVMLTHNNILASERAYCARLNLTWQDVFMMPAPLGHATGFLHGVTAPFLIGARSVLLDIFT PDACLALLEQQRCTCMLGATPFVYDLLNVLEKQPADLSALRFFLCGGTTIPKKVARECQQRGIKLLSVYGSTESSPHAVVNLDDPLSRFMHTDGYAAAGVEIKVVDDARKTLPPGCEGEEASRGPNVFMGYFDEPELTARA LDEEGWYYSGDLCRMDEAGYIKITGRKKDIIVRGGENISSREVEDILLQHPKIHDACVVAMSDERLGERSCAYVVLKAPHHSLSLEEVVAFFSRKRVAKYKYPEHIVVIEKLPRTTSGKIQKFLLRKDIMRRLTQDVCEEIE SEQ ID NO:2 Pseudomonas putida LvaE(ppLvaE) MMVPTLEHELAPNEANHVPLSPLSFLKRAAQVYPQRDAVIYGARRYSYRQLHERSRALASALERGVQPGERVAILAPNIPEMLEAHYGVPGAGAVLVCINIRLEGRSIAFILRHCAAKVLICDREFGAVANQALAM LDAPPLLVGIDDDQAERADLAHDLDYEAFLAQGDPARPLSAPQNEWQSIAINYTSGTTGDPKGVVLHHRGAYLNACAGALIFQLGPRSVYLWTLPMFHCNGWSHTWAVTLSGGTHVCLRKVQPDAINAIAEHAVTH LSAAPVVMSMLIHAEHASAPPVPVSVITGGAAPPSAVIAAMEARGFNITHAYGMTESYGPSTCLWQPGVDELPLEARAQFMSRQGVAHPLLEEATVLDTDTGRVPADGLTLGELVVRGNTVMKGYLHNPEATRAA LANGWLHTGDLAVLHLDGYVEIKDRAKDIIISGGENISSLEIEEVLYQHPEVVEAAVVARPDSRWGETPHAFVTLRADALASGDDLVRWCRERLAHFKAPRHVSLVDLPKTATGKIQKFVLREWARQQEAQIADAEH sequence number 3 Rhodopseudomonas palustris DcaA(rpDcaA) MSFAYFDWIAHHAEVRPERIAVVDLASSRKISYRAMDRLAAHLAALGVGRGDRVAVLALNAVETLEVQFACFRLGAIFVPNLNVRLTVHELSYIVGDAAPRVLAHDDELAPMAKELKAACSVPHLAFGAAYEAALAASPRLGASEPVTLDDVSTIMYTSGTTGKPKGAMITHGMTFINAVNLGIPAFISQRTVFLCVLPLFHTGGLNCYTNPVLHAGGTTLLMRAFDPGAALSIIGDPSVGLTHFFGVPS IYQFMCQHPAFAATDLSRLQIAGVGGAPMPVPLLKIWQERGCALVQGYGMTETSPAVLMLDADDAARKAGSAGKPVLHADLKIVGPDGDPVKPGEMGELWVKGPNITPGYWNRPDANRTSFTDGWLHTGDAARVDDEGFYYIVDRTKDMYISGGENVYPAEVEDVLYQLPEAEAAVIGAPDPQWGETGVAVVALKPGQELSEAKLLAHCRERLARFKCPQRVSFVEALPRNATGKVHKPTLRERILVRETADA sequence number 4 Rhodopseudomonas palustris DcaC(rpDcaC) MTSLEATGGVPGPGRIGRVAIGDILRKSARRFPDRVALTDGGSRSVTYTELERDANRFANALVARGLKPGAKISTVCNNSIEFVKALFGIHRAGLVWVPINTMLGPDDMGYILDHAGVKVAVIDDNLHGQPERRAALEARGIDLIAINGLAGKAADTGLPVFDQLIEGLSEIEPDVAFDDRLAMIIYTSGTTSRPKGAMHCHLAVTMAVMSNAIEMQLSRKDGITGQFPLFHCAAHVLLLSYLIVGGQMAIMRGFDPVACMEAIQRNKLTVFIGLPLMYQVILDHPRRKEFDLSSLRCCIYTMAPMPRPLLERAIAELCPTFVQPSGQTEMYPATTMSQPDRQLARFGNYWGESTLVNETAIMDDAGNLLPPGEVGEIVHRGPNVMLGYYKDPEATEAARKFGWHHTGDLALIDEHGEVLFLDRKKDMIKSGGENVASIKIEETLLAHPSVMNAAVVGLPHPQWGEAVSGFVKLKPGASATEAEIVEHCKKHLGGFQVPKLLRIVDEMPMTATGKLRKVELRNQFTDHFMLGQTG sequence number 5 Cannabis sativa AAE1(csAAE1) MGKNYKSLDSVVASDFIALGITSEVAETLHGRLAEIVCNYGAATPQTWINIANHILSPDLPFSLHQMLFYGCYKDFGPAPPAWIPDPEKVKSTNMGALLEKRGKEFLGVKYKDPISSFSHFQEFSVRNPEVYWRTVLMDEMKISFSKDPECILRRDDINNPGGSEWLPGGYLNSAKNCLNVNSNKKLNDTMIVWRDEGNDDLPLNKTLDQLRKRKRVWLVGYALEEMGLEKGCAIAIDMPMHVDAVVIYLAIVLAGYVVVSIADSFSAPEISTRLRLSKAKAIFTQDHIIRGKKRIPLYSRVVEAKSPMAIVIPCSGSNIGAELRDGDISWDYFLERAKEFKNCEFTAREQPVDAYTNIL FSSGTTGEPKAIPWTQATPLKAAADGWSHLDIRKGDVIVWPTNLGWMMGPWLVYASLLNGASIALYNGSPLVSGFAKFVQDAKVTMLGVVPSIVRSWKSTNCVSGYDWSTIRCFSSSGEASNVDEYLWLMGRANYKPVIEMCGGTEIGGAFSAGSFLQAQSLSSFSSQCMGCTLYILDKN GYPMPKNKPGIGELAGPVMFGASKTLNLGNHHDVYFKGMPTLNGEVLRRHGDIFELTSNGYYHAHGRADDTMNIGGIKISSIEIERVCNEVDDRVFETTAIGVPPLGGGPEQLVIFFVLKDSNDTTIDLNQLRLSFNLGLQKKLNPLFKVTRVVPLSSLPRTATNKIMRRVLRQQFSHFE sequence number 6 Arabidopsis thaliana ACS(atACS) MASEENDLVFPSKEFSGQALVSSPQQYMEMHKRSMDDPAAFWSDIASEFYWKQKWGDQVFSENLDVRKGPISIEWFKGGITNICYNCLDKNVEAGGLDGTKAIHWEGNELGVDASLTYSELLQRVCQLANYLKDNGVKKGDAVVIYLPMLMELPIAMLACARIGAV HSVVFAGFSADSLAQRIVDCKPNVILTCNAVKRGPKTINLKAIVDAALDQSSKDGVSVGICLTYDNSLATTRENTKWQNGRDVWWQDVISQYPTSCEVEWVDAEDPLFLLYTSGSTGKPKGVLHTTGGYMIYTATTFKYAFDYKSTDVYWCTADCGWIGGHSYVT YGPMLNGATVVVFEGAPNYPDPGRCWDIVDKYKVSIFYTAPTLVRSLMRDDDKFVTRHSRKSLRVLGSAGEPINPSAWRWFFNVVGDSRCPISDTGQTETGGFMITPLPAWPQKPGSATFPFFGVQPVIVDEKGNEIEGECSGYLCVKGSWPGAFRTLFGDHERYETTYFKPFAGYYFSGDGCSRDKDGYYWLTGRVDDVINVSGHRIGTAEVESALVLHPQCAEAAVVGIEHEVKGQGIYAFVTLLEGVPYSEELRKSLVLMVRNQIGAFAAPDRIHWAPGLPKTRSGKIMRRILRKIASRQLEELGDTSTLADPSVVDQLIALADV sequence number 7 Saccharomyces cerevisiae FAA2(scFAA2) MAAPDYALTDLIESDPRFESLKTRLAGYTKGSDEYIEELYSQLPLTSYPRYKTFLKKQAVAISNPDNEAGFSSIYRSSLSSENLVSCVDKNLRTAYDHFMFSARRWPQRDCLGSRPIDKATGTWEETFRFESYSTVSKRCHNIGSGILSLVNTKRKRPLEANDFVVAILSHNNPEWILTDLACQAY SLTNTALYETLGPNTSEYILNLTEAPILIFAKSNMYHVLKMVPDMKFVNTLVCMDELTHDELRMLNESLLPVKCNSLNEKITFFSLEQVEQVGCFNKIPAIPPTPDSLYTISFTSGTTGLPKGVEMSHRNIASGIAFAFSTFRIPPDKRNQQLYDMCFLPLAHIFERMVIAYDLAIGFGIGFLHKP DPTVLVEDLKILKPYAVALVPRILTRFEAGIKNALDKSTVQRNVANTILDSKSARFTARGGPDKSIMNFLVYHRVLIDKIRDSLGLSNNSFIITGSAPISKDTLLFLRSALDIGIRQGYGLTETFAGVCLSEPFEKDVGSCGAIGISAECRLKSVPEMGYHADKDLKGELQIRGPQVFERYFKNPN ETSKAVDQDGWFSTGDVAFIDGKGRISVIDRVKNFFKLAHGEYIAPEKIENIYLSSCPYITQIFVFGDPLKTFLVGIVGVDVDAAQPILAAKHPEVKTWTKEVLVENLNRNKKLRKEFLNKINKCTDGLQGFEKLHNIKVGLEPLTLEDDVVTPTFKIKRAKASKFFKDTLDQLYAEGSLVKTEKL Diacylglycerol O-acyltransferase enzyme SEQ ID NO:8 Bos taurus (cow) DGAT1 (BtDGAT1) MGDRGGAGGSRRRRTGSRPSIQGGSGPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFLKDPYSWPALCLVIVANIFAVAFQVEKRLAVGALTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMVYTILFLKLFSYRDVNLWCRERRAGAKAKAALAGKAANGGAAQRTVSYPDNLTYRDLYYFLAFTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKDMDYSRIVERLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESITYFWQNWNIPVHKWCIRHFYKPMLRRGSSKWAARTAVFLASAFFHEYLVSIPLRMFRWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQPVAVLMYVHDYYVLNREAPAAGT sequence number 9 Capra hircus(ヤギ)DGAT1(ChDGAT1) MGDRGGAGGSRRRRTGSRPSIQGGSRPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFL KDPYSWPALCLVIVANIFAVAAFQVEKRLAVGALTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMMVYTILFLKLFSYRDVNLWCRERRAGAKAKAALAGKKANGGAAQRTVSY PDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKDMDYSRIVERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWW NSESITYFWQNWNIPVHKWCLRHFYKPMLRRGSSKWAARTGVFLASAFFHEYLVSIPLRMFRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQPVAVLMYVHDYYVLNREAPTAGT SEQ ID NO:10 Ovis aries (sheep) DGAT1 (OaDGAT1) MGDRGGAGGSRRRRTGSRPSIQGGSRPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFSSDSGFSNYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFL KDPYSWPALCLVIVANIFAVAAFQVEKRLAVGALTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMMVYTILFLKLFSYRDVNLWCRERRAGAKAKAALAGKKANGGAAQRTVSY PDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKDMDYSRIVERLLKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWW NSESITYFWQNWNIPVHKWCLRHFYKPMLRRGSSKWAARTGVFLASAFFHEYLVSIPLRMFRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQPVAVLMYVHDYYVLNREAPTAGT SEQ ID NO:11 Bubalus bubalis (domestic water buffalo) DGAT1 (BbDGAT1) MGDRGGAGGSRRRRTGSRPSIQGGSGPAAAEEEVRDVGAGGDAPVRDTDKDGDVDVGSGHWDLRCHRLQDSLFSSDSGFSSYRGILNWCVVMLILSNARLFLENLIKYGILVDPIQVVSLFL KDPYSWPALCLVIVANIFAVAAFQVEKRLAVGALTEQAGLLLHGVNLATILCFPAAVAFLLESITPVGSVLALMVYTILFPKLFSYRDVNLWCRERRAGAKAKAALAGKKANGGAAQRTVSY PDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRLLEMLFLTQLQVGLIQQWMVPAIQNSMKPFKDMDYSRIVERLLKLAVPNHLIWLIFFYWLFHSCPNAVAELMQFGDREFYRDWW NSESITYFWLNWNIPVHKWCIRHFYKPMLRRGSSKWAARTAVFLASAFFHGYLVSIPLRMFRLWAFTGMMAQIPLAWIVGRFFRGNYGNAAVWLSLIIGQPVAVLMYVHDYYVLNREAPAAGT SEQ ID NO:12 Bos taurus (cow) DGAT2 (BtDGAT2) MKTLIAAYSGVLRGTGSSILSALQDLFSVTWLNRSKVEKQLQVISVLQWVLSFLVLGVACSVILMYTFCTDCWLIAVLYFTWLVFDWNTPKKGGRRSQWVRNWAVWRYFRDYFPIQLVKTHNLLTSRNYIFGYHPHGIMGLGAFCNFSTEATEVSKKFPGIRPYLATLAGNFRMPVLREY LMSGGICPVNRDTIDYLLSKNGSGNAIIIVVGGAAESLSSMPGKNAVTLRNRKGFVKLALRHGADLVPTYSFGENEVYKQVIFEEGSWGRWVQKKFQKYIGFAPCIFHGRGLFSSDTWGLVPYSKPITTVVGEPITIPRLERPTQQDIDLYHAMYVQALVKLFDQHKTKFGLPETEVLEVN SEQ ID NO:13 Ovis aries (sheep) DGAT2 (OaDGAT2) MKTLIAAYSGVLRGTGSSILSALQDLFSVTWLNRSKVEKQLQVISVLQWVLSFLVLGVACSAILMYAFCTDCWLIAVLYFTWLVFDWNTPKKGGRRSQWVRNWAVWRYFRDYFPIQLVKTHNLLTSRNYIFGYHPHGIMGLGAFCNFSTEATEVSKKFPGIRPYLATLAGNFRMPVLREY LMSGGICPVNRDTIDYLLSKNGSGNAIIIVVGGAAESLSSMPGKNAVTLRNRKGFVKLALRHGADLVPIYSFGENEVYKQVIFEEGSWGRWVQKKFQKYIGFAPCIFHGRGLFSSDTWGLVPYSKPITTVVGEPITIPKLEHPTQQDIDLYHAMYMEALVKLFDQHKTKFGLPETEVLEVN SEQ ID NO:14 Bos taurus (cow) DGAT2L6 (BtDGAT2L6) MAMAFLSQLNLQEILQTLSVLQWMPVYVFLGAIPIIVIPYFLVFTKFWMVSVLALAWLAYDWNTHSQGGRRSAWVRNWTIWKYFQNYFPIKLVKTHDLSPRHNYIIASHPHGVLPYGTFINFATETTGFARIFPGITPYVATLEGIFWIPIVREYVMSMGVCPVSELAL KYLLTQKGSGNAVVIMVGGGAEALLCHPGATTVLLKQRKGFVKVALETGAYLVPSYSFGQNEVHNQETFPEGTWKRFFQKALQDTLKKLLRLSVCTFHGRGLTRGSWGFLPFNHPITTVVGEPLPIPRIKKPNEETVDKYHALYINALQKLFDEHKVQYGLSETQELTII SEQ ID NO:15 Capra hircus(goat)DGAT2L6(ChDGAT2L6) MAMAFLSQLNLQEILQTLSVLQWVPVYYIFLGAIPIILIPYFLVFTKFWMVSVLALAWLAYDWNTHSQGGRRSAWVRNWTIWKYFRNYFPVMLVKTHDLSPRQNYIIASHPHGILPYGIFINFATETTGFARIFPAITPYIATLEGIFWIPIVREYVMSMGVCPVSELAL KYLLTQKGSGNAVVIVVGGAAEALLCHPGASTVFLKQRKGFVKVALKTGAYLVPSYSFGQNEVHNQETFPEGTWKRFFQKTLQNTFKKILGLNFCTHFHGRLIRGSWGFLPFNHPITTVVGEPLPIPRIKKPNKETVDKYHALYINALRKLFDEHKVQYGLSETQELTIV sequence number 16 Cuphea avigera var pulcherrima DGAT1(CaDGAT1) MAAHRTSSPVHLKLAESPLSSNRIFKQNHEGLFNLCMVTLVAVIIRLFLENLLKYGWLMKRDFWLSTFTAWPLFICSLGLPIFPLAAFVVEKLAQKNLLPEPIVLCSHVIITSASVLYPALVILRFDCALMSGIGLMLYSCALWLKLVSYAHTSYDMRCEAKSRLEGKSSADSKNGELPYRVNIKDLAYFMVAPTLCYQLSYPRTQFIRKFW VARQVLKLILVNVVMGFIIEQYMIPVMHNSKPPRRGYWLHFIERNLKLAVPSIGLWFCIFYSIFHLWLNIVAELLRFGDREFYKDWWNAKNMEEYWKMWNIPVHRWMVRHLYGPCMKRKLPRWVAISISFLLSAVLHEICVSVPCHVFQLWAFNGMMLQIPLVLSSKPLQKRFPSKAGNVFFWFLFCIYGQPNCVLMYYHALMERRGLRID sequence number 17 Euonymus alatus(ホウキギ)DAcT(EaDAcT) MMDAHQEIKNFIKVWVQAMVCLSYAYYFSSRLPKGLLRLLSLLPVLYLLLIAPLNISSFILSSITGFFLAWLTTFKVISFAFDQGPLYPLPQNLLHFISIACLPTIKRNPSKLKSTTNPSPISHLLKKAFMSFPSKVLFHWVIAHLYQYKKYMDPNVVLVIYCCHVYVMLDISLCAT LAEFLCGFDVEPQFKEPYLATSLQDFWGRRWNIIVSSVLRSTVYAPTRNIASYLIGSRWAYFPAIIATFVVSGVMHDVVYYVYMMHMYPKWDMTGHFVLHGICEALEVEMKCKRSRSDKWRRHPAVDWVMVMGFVMGTSVSLLFVPLLRDNVDQIVAEEYSILFNFVREKIVMLGTRFVCGN CYP505 enzyme sequence number 18 Aspergillus terreus CYP505E3_1 Conjugated Linoleic Acid (CLA) Isomerase SEQ ID NO:19 Cutibacterium (Propionibacterium) acnes CLA isomerase MSSKDSRAGAGAGAAGMYAGHDYTRTDHVGGKCHSNYHGRRYMGAMGVSYDTMDRTGDKVDGKRRHDGYVKDVRGVMAAVKGATKYGYDANGHYNKVHDMDANGCAARDWNTAGYGHDNVAAYVKYDVTMMSAKGDWTWADGTAM HNATHARNVDTRTRDGKVHHTTDWDRSDVVTVKDYSDADDDRYSKHYMVDACVKYTSGYVDNMRRGHVMVYYHRWADDHTTYRNHDYADKTCRMVDDMTGHVKTWYYHVSSDYKAGWYKVGMGRRNTYAGMSGNDVCHYSKDVTRV Fatty Acid Synthase Enzyme SEQ ID NO:20 Schizosaccharomyces japonicum FAS1 SEQ ID NO:21 Schizosaccharomyces japonicum FAS2 OLE1ホモLOG sequence no. 22 Rhodotorula toruloides MTASSALETSLPHSVGPEAATTTAKPPRAPLRMRHPDYTQTDVLDSSDSDAASDSEGETTAVDDGTYEDDNYVRKVLSKEKPLPPITWKNIHRNIQWISTLALTIVPLLAIYGAFTTPLKWQTAVWSVVYYYYTGLGITAGYHRWAHRSYTASLPLQYFLALGGSGAVEGSVKWWSRGHRAHHRYTDTDLDPYSAQKGFWWAHLGWMIVKPRRRPGVADVSDLNNNPVVKWQHRYYLPLILGMGFVFPTIVAGLGWGDFRGGFFFAGAARL LFVHHSTFCVNSLAHWLGETPFDDKHTPKDHWLTALATVGEGYHNFHHEFPSDYRNALRWWQYDPTKLFIWTMSKLGLASQLKTFPDNEIKKGQYAMTLKAVAREAENIEWPKSSNHLPVLTWDEFQDACKTRQLLVVAGFIHDVSTFIDQHPGGAGLIKTRLGRDATTAFYGGYYDHSNGAANLLAQYRVGVIEGGYEVEHMKKYSEVVENLKKHGADGVAGKSADLVKGPKQTSVIKGDPQLKSAPLETLAKPPTFSETNLGGLSLKVKA sequence no. 23 Rhodotorula graminis MRHPDYSQGDVVESSDSDREASDSEGETTAVDDGTYQDDNFVRKVLAKERPLPPITLKTLPQNINVISTLALTVVPALAIYGAFTTQIKWQTALWSVIYYFYTGLGITAGYHRLWAHRSYTASLPLQYF LALGGGSGAVEGSIKWWSRGHRAHHRYTDTDLDPYSAQKGFWWSHIGWMVVKPRRTPGVADVSDLSVNEVVKWQHRWYVYLIVGMGFVFPTLVAGLGWGDYRGGFFFAGAARLLFVHHSTFCVNSLAHWL GETPFDDKHSPRDHWITALVTVGEGYHNFHHEFPQDFRNAIQTFQYDPTKKWFIIVMHWLGLASQLKTFPDNEIRRGQYAMKLKAVAREADEIRWPKSSNHLPVLTWDEFQEACKTRQLMVISGYIHDVS TFIDEHPGGRALIKTRLGRDATNAFYGGYYDHSNGANNVLAQYRVGVIEGGYEVEHLKRFSKLIEDLKESGADGVAGKSADLHGGAKSSTRTTVIKGDPQLKSAPLADVAQPPTFKNVRLTGGLGHAIYA sequence no. 24 Rhodotorula diobovata MAAAQPTTAVNAALATALPHAVDSATTTAKPPRAPLRMRHPNYAEGDVVESSDSDREATDSEGETTAVDDGTYQDDNYVRKVLAKERPLPPITLKTLPQNINVISTLALTVVPALAIYGAFTTQIRWQTALWSVIYY FYTGLGITAGYHRLWAHRSYTASLPLQYFLALGGSGAVEGSIKWWSRGHRAHHRYTDTDLDPYSAQKGFWWSHIGWMIVKPRRTPGVADVSDLSANEVVKWQHRWYVYLIVGMGFVFPTLVAGLGWGDYRGGFFFAGA ARLLFVHHSTFCVNSLAHWLGETPFDDKHSPRDHWITALVTVGEGYHNFHHEFPQDFRNAIQTFQYDPTKWFIIVMHWLGLASQLKTFPDNEIRRGQYAMKLKAVAKEADEIRWPKDSNHLPVLTWDEFQEACKTRQLMVISGYIHDVSTFIDEHPGGRALIKTRLGRDATNAFYGGYYDHSNGANNVLAQYRVGVIEGGYEVEHLKRFSKLIEDLKESGADGVAGKNADLQSGPARVTVIKGDPQLKSAPLADVAKPPTFKNVRLTGGGLGHPIEA sequence no. 25 Rhodotorula mucilaginosa MAMAAEHAVTSALATSLPHSVGPEAAAAKPPRAPLRMRHPDMTNMPDSSDSDHDASDSEGETTAVDDGTYNDDNYVRKVLAKEKPLPPITLGNLYKNINVISTLALTVVPALAIYGAFTTPVMWQTALWAVAYYFYTGLGITAGYHRWAHRAYTASLPLQYFLALGGSGAVEGSIKWWSRGHRAHHRYTDTDLDPYSASKGFWWAHVGWMIVKPRRRPGVADVSDLSVNPVVQWQHRWYLPLIVGMGFVFPTVVAGLGWGDWRGGFFFAGAARLLFVHHSTFCVNSLAHWLGETPFDDKHSPKDHWVTALVTIGEGYHNFHHEFPQDYRNATKFYQWDPTKLFIRTASLLGLASQLKTFPDNEIRRGQFAMKLKAISRQAEEIQWPKDSNHLPVLTWDDFEAECKTRQLMVIGGFIHDVSTFIDEHPGGRALIKTRLGRDATTAFYGGVYDHSNGASNVLSRYRVGVISGGYEVEHMK sequence no. 26 Cryptococcus gattii MSAAVLPMTPPLEKEESELVHRRPSPADELTPPHTPEQNPCKSTNGKGKDPIDPSLLPSDRHIPDNYVSYTIANQKYLPPITWKNLIHNIQWISFLALTVTPSLAIYGIFTTTWNTKTAIWSVIYYFITGLGITAGYHRLWAHRAYN ASIPLQYVLATAGSGAVEGSIKWWCRGHRAHHRYTDTDLDPYSAEKGFFWSHVGWMLVKPRGKIGVADVSDLSKNRVVKWQHRNYIPLILGMGFVFPTVVAGGLWGDWRGGFFFAGAARLCFVHHSTFCVNSLAHWLGEQPFDNKHSP RDHIITALCTIGEGYHNFHHQFPQDFRNAIKWFQYDPTKKWFIWTMSTLGLASHLKRFPDNEVKKGQYTMKLQLLKEQADELQWPKSSNDLPVISWDDFKAEAKERSLVAIHGFIHDCSSFVEDHPGGAHLIKRAIGTDATTAFFGGV YDHSNAAHNLLAMMRVGILDGGMEVEHLKRRPAESAASSIINSPVSSASASSVDIQSLADDDFRLDQTQLNSQGPKPKAPFGQPQAQVADRWTLSVPPSEKLRIVQTVPEIRPGLLTHRSVGKLDKVTKADVGGEANEFSGNEPVAAA sequence no. 27 Cryptococcus neoformans MSAAVPPMTPPFEKEASELVHRRPSPVDQLDPAFTPPPHTPEQNPCKSTKGKGIDPIDPTLLASDRHIPDNYVSYTIANQKYLPPITWKNLIYNIQWISFLVLTVTPSLAIYGVFTTAWNTKTAIWSVIYYFITGLGITAGYHRLWAHR AYNAGIPLQFAFAIAGSGAVEGSIKWWCRGHRAHHRYTDTELDPYSAEKGFFWSHVGWMLVKPRGKIGVADVSDLSRNRVVKWQHRNYIPLILGMGFVVPTVVAGLGWGDWRGGFFFAGAARLCFVHHSTFCVNSLAHWLGEQPFDNKH SPRDHIITALCTIGEGYHNFHHQFPQDFRNAIKWFQYDPTKWFIWTMSQLGLASHLKRFPDNEVKKGQYTMKLQLLKEQADQLEWPKSSNDLPVISWDDFKAEAKERSLVAIHGFIHDCSSFVEDHPGGAHLIKRAIGTDATTAFFGG VYDHSNAAHNLLAMMRVGILDGGMEVEHLKRRPAESAASSVTNSPVSSASASSVDIQSLADDDFRLDQTQLNSQGPKPKAPFGQPQAQVADRWTLSVPPSEKLRIVQTVPEIRPGLLTHRQAGKLDKVTKADVGGEVIEFIGEKPVAAA SEQ ID NO:28 Vanrija humicola MSAAAVSQPPPKVVNELDPEADDFQTPDNYVTKTVQNQKRLPPITWRNLLSNIQWISTLALTVPPPALAIYGLMNYPLQWKTLVWSIVYYFITGLGITAGYHRLWAHRSYNASKPLQVALALAGAGSVQGSIKW WSRGHRAHHRYTDTKLDPYSAHEGFWWAHMGWMLVKPRGKIGVADISDLNKSKVVRWQHNNYVLIMVFMGLIFPTLVAGGLGWGDWAGGYFFAGAARLVFVHHSTFCVNSLAHWLGETPFDNKHTPKDHFITAL VTVGEGYHNFHHQFPMDFRNAIKWYQYDPTKWFIWTMSKLGLASHLKKFPDNEIKKGQYTMKLQVLQEQSKDIKWPVHSNDLPVISWEDFKAEAKERSLVAIHGFIHDVSSFIEDHPGGAHLIKKAIGTDATT AFFGGVYDHSNAAHNLLAMMRVGILDGGMEVEHLKLQGLRRNLSSASLESGVSSASSVSVQSILSEVENIENHKPEYQPVAMVSNPYTFSIPPSEKLRIIQSTPEVRPTVLSRLNSQANSAAPSRPISPPLSP sequence no. 29 Cutaneotrichosporon curvatum MSASTATAPPATAPAVANPTPAAASAAAAPAATKDKAETIDPESEHFVVSQNYVTRTVENMTMLPPPVTWSNLLQNIQWISFTALTVPPAMAIYGLCTLELQRKTVIWAIVYYFMTGLGITAG YHRLWAHRAYNASAPLQYFLALCGAGSVQGSIKWWSRGHRAHHRYTDTKLDPYSAHEGFWWAHVGWMLVKPRGKIGVADISDLSRNPVVKWQHNNYVMLMVLMGLVFPTLVAGLGWGDWKGGL LFAGAARLVFVHHSTFCVNSLAHWLGETPFDNKHTPKDHFITALVTVGEGYHNFHHQFPMDFRNAIKWYQYDPTKWFIWTMSNVGLASHLKKFPDNEIKKGQYTMKLQMLQEQSGSIQWPKHS NDLPVISWEDFQAEAKERSLVAIHGFIHDCSSFLEDHPGGIHLIKKAIGTDATTAFFGGVYDHSNAAHNLLAMMRVGILDGGMEVESLKLENLQRSMSVSSMESDAASSASSVSVSSISPRWPH SEQ ID NO:30 Filobasidium floriforme MADTAFAAMHRKAPTNPVPIPQEEKSETKLGVKPLEPTDIPSDLDVPDNYVTRTIETQKRLPPVTMANWHKNIQWVSFLALTVTPALTIYGLFNVKWNTYTAIWSVVYYFVTGLGITAGYHRLWAHRSYTASRPLEYILACAGAGAVQGSIKWWSRGHRAHHRYTDTSLDPYSAHEGFWWAHVGWMIFKPRTKIGVADISDLTRSKVVRWQHNNYLSLLLVMGLFFPMGVAGLGWGDWKGGFFFAGAARLLFVHHSTFCVNSLAHWLGEHTFDDKHTPRDHFITALVTIGEGYHNFHHQFPMDYRNAIKWNQYDPTKWFIATAQLLGLASHLKKFPDNEIKKGIYTMQLQKLAQAGEVIEWPTDSNHLPVVSWDDFVDESKNRALIAVHGFIHDVSSFMDDHPGGQHALKKFIGKDATTAFYGGVYDHSHAAQNLLAMMRVGCLEGGMEVEHLKQKVRERSLSGSSANSAFSSNEDVASLASYSSGRLSDGELSDVDFLQDLPKKEKPKHQPGCYVPDKFTLAIPPSEEFKIIRSAPRLRKTGGGHGSMASMTSLEDVSRADKIGDVAPLVAAAA

Table 1

Table 2

Table 3

Claims

1. A microbial cell for producing milk fat triglycerides, wherein the cell expresses a biosynthetic pathway comprising at least one heterologous enzyme, the biosynthetic pathway produces a triglyceride having sn-3 esterified short-chain fatty acids, and the cell produces a triglyceride having at least about 5% on a molar basis of sn-3 esterified C4 and C6 fatty acids.

2. The microbial cell according to claim 1, wherein the cell produces triglycerides having at least about 10% sn-3 esterified C4 and C6 fatty acids on a molar basis.

3. The microbial cell according to claim 1 or 2, wherein the triglyceride further comprises a C12-C18 fatty acid esterified at sn-3.

4. The microbial cell according to claim 1 or 2, wherein the triglyceride comprises sn-3 esterified C4:0, C6:0, C8:0, and C10:0 fatty acids, and / or the triglyceride mainly comprises sn-1 and sn-2 esterified C12-C18 fatty acids on a molar basis.

5. The microbial cell according to claim 4, wherein the triglyceride comprises 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-1, and / or the triglyceride comprises 12:0, 14:0, 16:0, 16:1, 18:0, 18:1, and 18:2 fatty acids esterified at sn-2.

6. The microbial cell according to claim 1 or 2, wherein the biosynthetic pathway comprises one or more heterologous fatty acid acyl-CoA synthetases (ACS) that are specific to short-chain fatty acid substrates.

7. The microbial cell according to claim 6, wherein at least one heterologous ACS comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 7.

8. The microbial cell according to claim 7, wherein at least one or at least two heterologous ACS enzymes include an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs: 1 to 7.

9. The microbial cell according to claim 1 or 2, wherein the biosynthetic pathway comprises a heterologous diacylglycerol O-acyltransferase (DGA) that has specificity for short-chain fatty acid CoA substrates.

10. The microbial cell according to claim 9, wherein the DGA comprises an amino acid sequence that is at least 70% identical to an amino acid sequence selected from SEQ ID NOs: 8 to 17, and optionally, the DGA comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NOs: 8 to 17.

11. The microbial cell according to claim 1 or 2, wherein the microbial cell expresses conjugated linoleic acid (CLA) isomerase.

12. The microbial cell according to claim 11, wherein the CLA isomerase comprises an amino acid sequence that is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence selected from SEQ ID NO:

19.

13. The microbial cell according to claim 1 or 2, wherein the microbial cell comprises a modification that results in a decrease in the amount or activity of one or more endogenous diglyceride acyltransferases.

14. The microbial cell according to claim 1 or 2, wherein the cell has one or more gene modifications that reduce C16:1 fatty acid production and / or increase C18:1 fatty acid production.

15. The microbial cell according to claim 14, wherein the microbial cell has the deletion, inactivation, or reduction of the expression or activity of one or more fatty acid desaturase enzymes, wherein the enzymes are optionally delta-12 desaturase and / or delta-9 desaturase.

16. The microbial cell according to claim 14, wherein the microbial cell expresses at least one heterologous OLE1 enzyme that converts C18:0 to C18:1, and the enzyme is optionally selected from enzymes comprising an amino acid sequence having at least 80% sequence identity with one of SEQ ID NOs: 22 to 30, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity (or 100% sequence identity).

17. The microbial cell according to claim 14, wherein the microbial cell has the deletion, inactivation, or reduction of the expression or activity of one or more fatty acid elongase enzymes.

18. The microbial cell according to claim 1 or 2, wherein the microbial cell expresses one or more heterologous fatty acid synthases (FAS), and optionally, the heterologous FAS enzyme produces one or more C10:0, C12:0, and C14:0 fatty acids.

19. The microbial cell according to claim 18, wherein at least one heterologous fatty acid synthase comprises a subunit (FAS1) having an amino acid sequence identical by at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of SEQ ID NO: 20, and a subunit (FAS2) having an amino acid sequence identical by at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of SEQ ID NO:

21.

20. A microbial cell according to claim 1 or 2, further comprising a biosynthetic pathway that produces one or more lactones, wherein the one or more lactones are optionally selected from δ-dodecalactone, δ-decalactone, γ-dodecalactone, γ-decalactone, and δ-octaractone.

21. The microbial cell according to claim 20, wherein the biosynthetic pathway that produces one or more lactones comprises one or more enzymes having fatty acid hydroxylase activity.

22. The microbial cell according to claim 21, wherein the fatty acid hydroxylase enzyme is a CYP505 enzyme or a derivative thereof, and optionally the CYP505 enzyme comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or 100% sequence identity with amino acids 1 to 461 of SEQ ID NO: 18, and optionally the CYP505 enzyme further comprises a cytochrome P450 reductase (CPR) domain.

23. The microbial cell according to claim 1 or 2, wherein the cell produces β-carotene via a heterologous biosynthetic pathway and optionally further produces vitamin A via a heterologous biosynthetic pathway.

24. The microbial cell according to claim 1 or 2, wherein the microbial cell is a yeast or fungal cell.

25. The microbial cell according to claim 24, wherein the yeast or fungal cell is Yarrowia lipolytica or Yarrowia phanggensis.

26. The microbial cell according to claim 1 or 2, wherein the microbial cell is a bacterial cell, and optionally, the bacterial cell is a bacterium that accumulates a large amount of triacylglycerol.

27. A method for preparing a composition containing milk fat triglycerides, comprising culturing microbial cells according to claim 1 or 2 in the presence of C4:0, C6:0, C8:0, C10:0, C12:0, and C14:0 fatty acid substrates, and recovering the composition from the culture.

28. The method according to claim 27, wherein the fatty acid substrate is optionally added to the culture as an alkyl ester or glyceride.

29. The method according to claim 27, wherein the fatty acid substrate is synthesized by the cells.

30. The method of claim 27, wherein the composition is recovered by separating a wet cell mass and purifying the composition from the wet cell mass, the cells are optionally mechanically or enzymatically disrupted, and / or the composition is extracted using an organic solvent.

31. A composition comprising milk fat triglycerides produced according to the method of claim 30.

Images