Engineered yeast and use for production of triacylglycerol

Abstract

An engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT) and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or a microorganism comprises (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT; or a microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT; or a microorganism comprising a specific LPAAT2, optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT, and use for the production of triacylglycerol.

Description

Leydig 5158431ENGINEERED YEAST AND USE FOR PRODUCTION OF TRIACYLGLYCEROLSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with Government support under Grant 1R43HD114503-01 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 723,014 fded on November 20, 2024, the entire disclosure of which is hereby incorporated by reference.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 293,325 Byte Extensible Markup Language (XML) file named "515843- SequenceListing.XML," created on November 19, 2025.BACKGROUND OF THE INVENTION

[0004] Infant formula is a manufactured food designed to substitute for human breast milk sourced mainly from plants. While vegetable-based fats can be blended to mimic the fatty acyl composition of human milk fat, plant-based fats have a different arrangement of acyl groups on the glycerol backbone of the triglyceride molecules. In human milk fat, more than 70% of the C 16:0 is present at the stereospecific numbering (sn) 2 position, with unsaturated fatty acyl groups (mainly Cl 8:1) occupying the outer sn-1 and sn-3 positions. By contrast, vegetable fats contain almost no saturated long chain fats at the sn-2 position, and saturated long-chain fatty acyl groups such as C16:0 occupy the sn-1 / 3 positions. Clinical trials suggest that the stereoisomeric structure of the fats is important for digestion of the infant formula. Thus, there remains a need for alternative methods of efficiently producing fats that mimic the structure of human milk fat.BRIEF SUMMARY OF THE INVENTION

[0005] Provided herein is an engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT) and (ii) a heterologousLeydig 5158432 nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT.

[0006] Also provided herein is an engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT.

[0007] Further provided herein is an engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT). and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT.

[0008] Also provided is a microorganism comprising a heterologous nucleic acid encoding Chlamydomonas moewusii LPAAT2, optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0009] FIG. 1 is an illustration of the triacylglycerol (TAG) production in wild t pe Yarrowia lipolytica yeast and Yarrowia lipolytica yeast strains engineered to modify the amount of 1,3- dioleoyl-2-palmitoyl glycerol (OPO) and 1,2,3-tri-oleoyl glycerol (OOO). GPAT represents glycerol-3-phosophate acyltransferase; AGP AT represents acylglycerolphosphate acyltransferase; Hs AGP ATI represents Homo sapiens acylglycerol phosphate acyltransferase 1, PA represents phosphatidic acid, DGAT represents diacylglycerol acyltransferase; the circles each represent a glycerol, and the circle with a “P" represents a phosphorylated glycerol.

[0010] FIG. 2 is a bar graph and associated data table showing the TAG profile of transgenic strains of Yarrowia lipolytica (T11301 and T1102) and the base strain SY001, as percent of l,3-dioleoyl-2-stearoyl glycerol (OSO), l,3-dioleoyl-2-palmitoleoyl glycerol (OPoO), 1,2,3- tri-oleoyl glycerol (OOO), and l,3-dioleoyl-2-linoleoyl- glycerol (OLO) of TAG content.

[0011] FIG. 3 is a bar graph and associated data table showing the TAG profile of transgenic strains of Yarrowia lipolytica (T13201,T13202, T13301, T13302, and T134) and the base strain SY005, as the percentage of l,3-dioleoyl-2-linoleoyl- glycerol (OLO), l-oleoyl-2- linoleoyl-3-palmitoyl glycerol (OLP), 1.2,3-tri-oleoyl glycerol (OOO), 1.2-dioleoyl-3-Leydig 5158433 palmitoyl glycerol (OOP), 1.3-dioleoyl-2-palmitoyl glycerol (OPO), l,3-dioleoyl-2- palmitoleoyl glycerol (OPoO), l,3-dipalmitoyl-2-oleoyl glycerol (POP), l,3-dipalmitoyl-2- linoleoyl glycerol (PLP), 1, 2, 3-tri -palmitoyl glycerol (PPP), and other TAGs of total TAG content.

[0012] FIG. 4 is a bar graph and associated data table showing the TAG profde of transgenic strains of Yarrowia lipofytica (T1390LT13902, T14001, T14002, T14101, and T14102) and the base strain SY006, as the percentage of l,3-dioleoyl-2-linoleoyl- glycerol (OLO), 1- oleoyl-2-linoleoyl-3 -palmitoyl glycerol (OLP), 1,2,3-tri-oleoyl glycerol (OOO), 1,2-dioleoyl- 3-palmitoyl glycerol (OOP), l,3-dioleoyl-2-palmitoyl glycerol (OPO), l,3-dioleoyl-2- palmitoleoyl glycerol (OPoO), l,3-dipalmitoyl-2-oleoyl glycerol (POP), l,3-dipalmitoyl-2- linoleoyl glycerol (PLP), 1,2,3-tri -palmitoyl glycerol (PPP), and other TAGs of total TAG content.

[0013] FIG. 5 is a bar graph and associated data table showing the TAG profde of transgenic strains of Yarrowia lipofytica (T 14201,T 14202, T14301, T14302, T14401, and T14402) and the base strain SY007, as the percentage of l,3-dioleoyl-2-linoleoyl- glycerol (OLO), 1- oleoyl-2-linoleoyl-3 -palmitoyl glycerol (OLP), 1,2,3-tri-oleoyl glycerol (OOO), 1,2-dioleoyl- 3-palmitoyl glycerol (OOP), l,3-dioleoyl-2-palmitoyl glycerol (OPO), l,3-dioleoyl-2- palmitoleoyl glycerol (OPoO). l,3-dipalmitoyl-2-oleoyl glycerol (POP), l,3-dipalmitoyl-2- linoleoyl glycerol (PLP), 1,2, 3-tri -palmitoyl glycerol (PPP), and other TAGs of total TAG content.

[0014] FIG. 6 is a bar graph and associated data table show ing the TAG profde of transgenic strains of Yarrowia lipofytica (T145, SY009, SY010, T14701,T14702, T14703, and T14704) and the base strains SY007, as the percentage of l,3-dioleoyl-2-linoleoyl- glycerol (OLO). 1- oleoyl-2-linoleoyl-3-palmitoyl glycerol (OLP), 1,2,3-tri-oleoyl glycerol (OOO), 1,2-dioleoyl- 3-palmitoyl glycerol (OOP), l,3-dioleoyl-2-palmitoyl glycerol (OPO), l,3-dioleoyl-2- palmitoleoyl glycerol (OPoO), l,3-dipalmitoyl-2-oleoyl glycerol (POP), l,3-dipalmitoyl-2- linoleoyl glycerol (PLP). 1,2,3-tri-palmitoyl glycerol (PPP), and other TAGs of total TAG content.

[0015] FIG. 7 is a bar graph and associated data table showing the l,3-dioleoyl-2-palmitoyl glycerol (OPO) content as the percentage of total TAG content for transgenic strains of Yarrowia lipofytica (SY011, T15201, T15202, T154, T155. T156, T15703. T15803, T15901, T16003, T16101, T161012, SY009, and SY010).Leydig 5158434

[0016] FIG. 8 is a bar graph and associated data table showing the TAG profde of transgenic strains of Yarrowia lipotytica (SY012, SY017, SY018, SY019, SY01 1, and SY010), as the percentage of l,3-dioleoyl-2-linoleoyl- glycerol (OLO), l-oleoyl-2-linoleoyl-3-palmitoyl glycerol (OLP), 1,2,3-tri-oleoyl glycerol (000), l,3-dioleoyl-2-palmitoyl glycerol (OPO), l,3-dioleoyl-2-palmitoleoyl glycerol (OPoO), 1.3-dipalmitoyl-2-oleoyl glycerol (POP), 1,3- dipalmitoyl-2-linoleoyl glycerol (PLP), and other TAGs of total TAG content.

[0017] FIG. 9 is a bar graph and associated data table showing the l,3-dioleoyl-2-palmitoyl glycerol (OPO) content as the percentage of total TAG content for transgenic strains of Yarrowia lipotytica (T17701, T17702. T17801, T17903, SY013, T18101, T18201, T1830I, T18401, T18402, T1850L T18601, T18702, T1880L T18902, T19003, and SY009).

[0018] FIG. 10 is a bar graph showing the l,3-dioleoyl-2-palmitoyl glycerol (OPO) content as the percentage of total TAG content for transgenic strains of Yarrowia lipotytica (SY014, T19202, T19203, T19301, T19302, T19303, SY012, T19401. T19402, T19403, T1950L SY015, T19503, SY019. and SY009).

[0019] FIG. 11 is a bar graph and associated data table showing the percentage of fatty acids in the middle position (sn2) of the glycerol backbone of TAGs that are palmitate (sn2- C16:0 / Total sn2-FA) and the percentage of palmitates in the glycerol backbone of TAGs that are at the middle position (sn2-C16:0 / Total C 16:0) in RBD oil 49-52 and Eastmond (Glucose).

[0020] FIG. 12 is a bar graph and associated data table showing the TAG profile for OPO producing strains (SY010, SY009, SY008, SY005, and SY002) where for each strain the outer left bar shows the percentage of l,3-dipalmitoyl-2-oleoyl glycerol (POP), 1,3- dipalmitoyl-2-linoleoyl glycerol (PLP), 1.3-dioleoyl-2-palmitoyl glycerol (OPO), 1,2,3-tri- oleoyl glycerol (000), l -oleoyl-2-linoleoyl-3-palmitoyl glycerol (OLP), 1 ,3-dioleoyl-2- linoleoyl- glycerol (OLO), and other TAGs of total TAG content; the inner left bar shows percentage of fatty acid methyl esters in the glycerol backbone of the TAGs that are methyl palmitate (C16:0), methyl stearate (C18:0), methyl oleate (C18: l), methyl linolenate / linoleate (Cl 8:2 n6), and other fatty acids; the inner right bar shows the percentage of fatty acids in the middle position of the glycerol backbone of TAGs that are palmitate (sn2-C16:0 in Total sn2- FA); and the outer right bar shows the percentage of palmitate in the glycerol backbone of TAGs that are at the middle position (sn2-C16:0 in Total C16:0).Leydig 5158435DETAILED DESCRIPTION OF THE INVENTION

[0021] Provided herein is an engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT). or a second LPAAT.

[0022] In some embodiments, the engineered microorganism is a fungi or a yeast. Examples of a yeast include, but are not limited to any yeast from the Yarrowia clade, such as Candida alimentaria, Yarrowia deformans, Candida galli, Candida hispaniensis, Candida hollandica, Candida oslonensis, Candida phangngensis, Yarrowia yakushimensis, and Yarrowia lipolytica. In some embodiments, the microorganism is Yarrowia lipolytica. In some embodiments, the engineered microorganism is a yeast, such as Yarrowia lipolytica, and does not comprise MatA, leu2-270, ura3-302, xpr2-322, and / or axp-2 modifications.

[0023] The engineered microorganism provided herein comprises a heterologous nucleic acid encoding a Lysophosphatidic acid acyltransferase (LPAAT). LPAATs acylate the sn-2 hydroxyl group of lysophosphatidic acid to form phosphatidic acid. In some embodiments, the LPAAT is a mammalian LPAAT, optionally a human, pig, camel, or horse LPAAT. In some embodiments, the LPAAT is a plant LPAAT, optionally a Brassica, Arabidopsis, Elaeis, Theobroma, or Shorea LPAAT. In some embodiments, the LPAAT is an algal LPAAT, optionally anAuxenochlorella. Chlamydomonas, Chlor ella, Edaphochlamys, Monoraphidium, Prototheca, Tetradesmis, or Vol vox LPAAT. In some embodiments, the first LPAAT is a bacterial LPAAT, optionally an Actinomycetota, Gordonia, Mycobacterium, Rhodococcus, or Speluncibacter LPAAT. In some embodiments, the LPAAT is a Homo sapiens acylglycerophosphate acyltransferase (HsAGPAT, such as HsAGPATl (SEQ ID NO: 34), HsAGPAT2 (SEQ ID NO: 44), HsAGPAT3 (SEQ ID NO: 46), or HsAGPAT4 (SEQ ID NO: 48)), Chlamydomonas moewusii LPAAT2 (CmLPAAT2) (SEQ ID NO: 36), Edaphochlamys debaryana LPAAT2 (EdLPAAT2) (SEQ ID NO: 38), Volvulina compacta LPAAT2 (VcLPAAT2) (SEQ ID NO: 40), Vitreochlamys sp. (VitrLPAAT2 (SEQ ID NO: 42), a Nannochloropsis oceanica LPAAT (NoLPAAT, such as NoLPAT4 (SEQ ID NO: 30) or tNoLPAT4 (SEQ ID NO: 32)), a Sus scrofa AGP AT (SsAGPAT, such as SsAGPAT2 (SEQ ID NO: 50) or SsAGPAT5 (SEQ ID NO: 52)). In some embodiments, the LPAAT is a Homo sapiens acylglycerophosphate acyltransferase (HsAGPAT, such as HsAGPATl (SEQ ID NO: 34), HsAGPAT2 (SEQ ID NO: 44). HsAGPAT3 (SEQ ID NO: 46), or HsAGPAT4 (SEQ ID NO: 48)), Chlamydomonas moewusii LPAAT2 (CmLPAAT2) (SEQ ID NO: 36),Leydig 5158436Edaphochlamys debaryana LPAAT2 (EdLPAAT2) (SEQ ID NO: 38), Volvulina compacta LPAAT2 (VcLPAAT2) (SEQ ID NO: 40), Vitreochlcimys sp. (VitrLPAAT2) (SEQ ID NO: 42).

[0024] In some embodiments, the engineered microorganism comprises multiple different heterologous nucleic acid sequences encoding multiple different LPAATs. Thus, for instance, the engineered microorganism can comprise a first heterologous nucleic acid encoding a first LPAAT, and a second heterologous nucleic acid encoding a second LPAAT. The first and second LPAATs can be any LPAATs, such as any of the LPAATs identified herein. In some embodiments, the first LPAAT is specific for a C16:0 substrate. In some embodiments, the first LPAAT preferentially acts at position sn-2 of a triacylglycerol as compared to sn-1 or sn-3. In some embodiments, the first LPAAT is a mammalian LPAAT, optionally a human, pig, camel, or horse LPAAT. In some embodiments, the first LPAAT is a plant LPAAT, optionally a Brassica. Arabidopsis, Elaeis, Theobroma. or Shorea LPAAT. In some embodiments, the first LPAAT is an algal LPAAT, optionally an Auxenochlorella. Chlamydomonas, Chlorella, Edaphochlamys, Monoraphidium, Prototheca, Tetradesmis, or Volvox LPAAT. In some embodiments, the first LPAAT is a bacterial LPAAT, optionally an Actinomycetota, Gordonia. Mycobacterium, Rhodococcus, or Speluncibacter LPAAT. In some embodiments, the first LPAAT is Homo sapiens acylglycerophosphate acyltransferase (HsAGPAT, such as HsAGPATl (SEQ ID NO: 34), HsAGPAT2 (SEQ ID NO: 44), HsAGPAT3 (SEQ ID NO: 46), or HsAGPAT4 (SEQ ID NO: 48)), Chlamydomonas moewusii LPAAT2 (CmLPAAT2) (SEQ ID NO: 36), Edaphochlamys debaryana LPAAT2 (EdLPAAT2) (SEQ ID NO: 38). Volvulina compacta LPAAT2 (VcLPAAT2) (SEQ ID NO: 40), Vitreochlcimys sp. (VitrLPAAT2) (SEQ ID NO: 42).

[0025] In some embodiments, the second LPAAT preferentially acts at sn- 1 or sn-3 positions of a triacylglycerol as compared to sn-2. In some embodiments, the second LPAAT is a bacterial, algal, yeast, fungus, or plant LPAAT. In some embodiments, the second LPAAT is aNannochloropsis oceanica LPAAT, optionally NoLPAT4 (SEQ ID NO: 30) or tNoLPAT4 (SEQ ID NO: 32).

[0026] The engineered microorganism can further comprise additional heterologous nucleic acids encoding additional LPAATs (e.g., a second LPAAT, third LPAAT, fourth LPAAT, and so on). The heterologous nucleic acids encoding the different LPAATs can be separate from one another (e g., separate nucleic acid molecules or nucleic acid constructs eachLeydig 5158437 encoding an LPAAT), or they can be provided as a single nucleic acid molecule or nucleic acid construct encoding multiple LPAATs (e.g., a single nucleic acid construct, such as an integrating or non-integrating vector or cassette, encoding multiple different LPAATs).

[0027] In some embodiments, the engineered microorganism comprising a heterologous nucleic acid encoding an LPAAT (or heterologous nucleic acid(s) encoding multiple LPAATs) further comprises a heterologous nucleic acid encoding a glycerol-3 -phosophate acyltransferase (GPAT). Glycerol-3-phosphate acyltransferase (GPAT) regulates esterification at the sn-1 position in the triacylglycerol synthesis. In some embodiments, the GPAT is a mammalian GPAT. including but not limited to a human, cow, goat, pig, camel, or horse GPAT. In some embodiments, the GPAT is a plant GPAT, including but not limited to an Arabidopsis, Brassica, Camellia, Elaeis, Panicum, Raphanus, or Zizipus GPAT.Examples of GPATs include those from Arabidopsis thaliana, Ziziphus jujube, and Haematococcus lacustris. In some embodiments, the GPAT is specific for a Cl 8: 1 substrate. An example of a GPAT specific for a C 18: 1 substrate includes GP AT9 from Arabidopsis (SEQ ID NO: 107). In some embodiments, the GPAT is specific for a C18:2 substrate. In some embodiments, the heterologous gene encoding a GPAT is a Homo sapiens GPAT (e.g., HsGPATl(SEQ ID NO: 72), HsGPAT2, HsGPAT3 (SEQ ID NO: 74), or HsGPAT4 (SEQ ID NO: 76), Arabidopsis GPAT (e.g. GPAT9 (SEQ ID NO: 106)), Chlamydomonas GPAT, Nannochloropsis oceanica GPAT (e.g., NoGPAT (SEQ ID NO: 78)), or Sus scrofa GPAT (e g., SsGPAT2 (SEQ ID NO: 80)).

[0028] The engineered microorganism can comprise additional heterologous nucleic acids encoding additional GPATs (e.g., a second GPAT, third GPAT, fourth GPAT, and so on). The heterologous nucleic acids encoding the different GPATs can be separate from one another (e.g., separate nucleic acid molecules or nucleic acid constructs each encoding a GPAT), or they can be provided as a single nucleic acid molecule or nucleic acid construct encoding multiple GPATs (e.g., a single nucleic acid construct, such as an integrating or nonintegrating vector or cassette, encoding multiple different GPATs).

[0029] In some embodiments, some embodiments, the engineered microorganism comprising a heterologous nucleic acid encoding an LPAAT (or heterologous nucleic acid(s) encoding multiple LPAATs) further comprises a heterologous nucleic acid encoding diacylglycerol acyltransferase (DGAT). Diacylglycerol acyltransferase (DGAT) is an enzyme that esterifies a diacylglyerol (DAG) with a fatty acid to form a triacylglycerol (TAG). In some embodiments, the DGAT is a mammalian DGAT, including but not limited to a human, cow.Leydig 5158438 goat, pig. camel, or horse DGAT. In some embodiments, the DGAT is a plant GPAT or DGAT, including but not limited to an Arabidopsis, Brassica, Camellia, Elaeis. Panicum, Raphanus, or Zizipus DGAT. Examples of DGATs include those from Brassica napus, Auxenochlorella prototheocoides , Camellia sinensis, and Raphanus sativus. In some embodiments, the DGAT is a yeast or fungi DGAT. including but not limited to a Candida, Cryptococcus. Geotrichum. Lipomyces, Polyspora, Rhodosporidium, Rhodotorula,Saccharomyces, Trichosporon, Yarrowia, Fusarium, Sarocladium, Mortierella, or Microsphaeropsis DGAT. In some embodiments, the DGAT is an algal DGAT, including but not limited to a Botryococcus, Chlamydomonas , Chlor ella, Auxenochlorella, Nannochlor opsis, or Volvox DGAT. In some embodiments, the DGAT is a cyanobacteria DGAT, including but not limited to an Anabaena, Synechococcus, Synechocystis, or Microcystis DGAT. In some embodiments, the DGAT is specific for a C 18: 1 substrate. An example of a DGAT specific for a C18: 1 substrate includes DGAT from Brassica napus (SEQ ID NO: 105). In some embodiments, the DGAT is specific for a Cl 8:2 substrate. In some embodiments, the heterologous gene encoding a DGAT is a Homo sapiens DGAT (e.g., HsDGAl (SEQ ID NO: 62) or HsDGA2 (SEQ ID NO: 64)), Candida DGAT (e.g., Candida tropicalis; CtDGA2 (SEQ ID NO: 56)), Ettlia oleoabundans DGAT (e.g., EtDGA2 (SEQ ID NO: 60)), Arabidopsis thaliana DGAT (e.g., AtDGA (SEQ ID NO: 54)), Elaeis guineensis DGAT (e,g„ EgDGA2 (SEQ ID NO: 58)), Sus scrofa DGAT (e g., SsDGAl (SEQ ID NO:66) or SsDGA2 SEQ ID NO: 68)), Thraustochytrium aureum DGAT (e.g., TaDGA2 (SEQ ID NO: 70)), or a Brassica napus DGAT (e.g. BnDGAT (SEQ ID NO: 104)).

[0030] The engineered microorganism can further comprise additional heterologous nucleic acids encoding additional DGATs (e.g., a second DGAT, third DGAT, fourth DGAT, and so on). The heterologous nucleic acids encoding the different DGATs can be separate from one another (e.g., separate nucleic acid molecules or nucleic acid constructs each encoding a DGAT), or they can be provided as a single nucleic acid molecule or nucleic acid construct encoding multiple DGATs (e.g., a single nucleic acid construct, such as an integrating or nonintegrating vector or cassette, encoding multiple different DGATs).

[0031] In some embodiments, the engineered microorganism comprises a heterologous nucleic acid encoding an LPAAT, and heterologous nucleic acid encoding a GPAT, and a heterologous nucleic acid encoding a DGAT. Furthermore, the engineered microorganism can comprise multiple heterologous nucleic acids encoding multiple different LPAATs, multiple different GPATs, and / or multiple different DGATs. Any of the heterologousLeydig 5158439 nucleic acids encoding the one or more various LPAATs. GPATs, or DGATs, can be separate from one another or single nucleic acid molecules or nucleic acid constructs encoding multiple different enzy mes (e.g., separate nucleic acid molecules or nucleic acid constructs each encoding one enzyme or a single nucleic acid construct encoding all or a subset of enzymes in any combination).

[0032] In some embodiments, the engineered microorganism comprises (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT. In other embodiments, the engineered microorganism comprises (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT.

[0033] In some embodiments, the engineered microorganism comprises (i) a heterologous nucleic acid encoding HsAGPATl HsAGPATl (SEQ ID NO: 34), CmLPAAT2 (SEQ ID NO: 36), or CrLPAAT2 (SEQ ID NO: 82), and (ii) a heterologous nucleic acid encoding NoLPAT4 (SEQ ID NO: 30) or tNoLPAT4 (SEQ ID NO: 32); optionally wherein the engineered microorganism further comprises a heterologous nucleic acid encoding EtDGATl (e g.. EtDGA2 (SEQ ID NO: 60)).

[0034] In another embodiment, the disclosure provides an engineered microorganism comprising a heterologous Chlamydomonas moewusii LPAAT2, optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT.

[0035] In any of the foregoing embodiments, the LPAAT, GPAT, and DGAT are as described above.

[0036] In some embodiments, the engineered microorganism further comprises a genetic modification that reduces the activity of an LPAAT native to the microorganism. For instance, the genetic modification can be a substitution, addition, or deletion mutation of a native gene encoding an LPAAT such that the activity' of native LPAAT is disrupted (reduced or eliminated) by reducing the expression of the functional native LPAAT enzy me. In some embodiments, the microorganism is Yarrowia and the engineered Yarrowia comprises a genetic modification that reduces or eliminates native AGP AT activity.Leydig 51584310

[0037] In some embodiments, the engineered microorganism produces more 1.3-dioleoyl-2- palmitoyl glycerol (OPO) (as measured by OPO per gram of biomass), or produces triacylglycerols (TAGs) with a higher proportion of OPO by weight of total TAG content (as determined by LC-MS / MS), as compared to a microorganism of the same type without the given genetic modifications of the engineered microorganism, such as a wild-type microorganism of the same type or a parent strain that has the same base genetics as the engineered microorganism but lacks the specific genetic modifications mentioned herein. In some embodiments, the engineered microorganism produces more OPO per gram of biomassThe genetic modifications can be any of those described in the foregoing embodiments.

[0038] Thus, for instance, in some embodiments the engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT) and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT, produces more OPO or produces TAGs with a higher proportion of OPO by weight, as compared to a microorganism of the same type without (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT) and without (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or as compared to a wild-type microorganism of the same type.

[0039] In some embodiments, the engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT, produces more OPO or produces TAGs with a higher proportion of OPO by weight as compared to a microorganism of the same type without (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, and (iii) a heterologous nucleic acid encoding a DGAT; or as compared to a wild-type microorganism of the same type.

[0040] Similarly, in some embodiments, the engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprisingLeydig 51584311(iii) a heterologous nucleic acid encoding a DGAT and / or GPAT produces more OPO or produces TAGs with a higher proportion of OPO by weight as compared to a microorganism of the same type without (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), (ii) a heterologous nucleic acid encoding a second LPAAT, and (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT; or as compared to a wild-type microorganism of the same type.

[0041] In still other embodiments, the engineered microorganism comprising a heterologous nucleic acid encoding Chlamydomonas moewusii LPAAT2, optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT, produces more OPO or produces TAGs with a higher proportion of OPO by weight as compared to a microorganism of the same type without a heterologous nucleic acid encoding Chlamydomonas moewusii LPAAT2, or a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or as compared to a wild-type microorganism of the same type.

[0042] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs), and at least about 10% of the total triacylglycerol (TAG) content (e.g., 10-90%, 10- 80%, or 10-70%), or at least about 20% (e.g., 20-90%, 20-80%. or 20-70%), or at least about 30% (e.g., 30-90%, 30-80%. or 30%-70%), or at least about 40% (e.g.. 40-90%, 40-80%, or 40%-70%), of the total TAG content of the engineered yeast is l,3-dioleoyl-2-palmitoyl glycerol (OPO), as determined by liquid chromatography -mass spectrometry7(LC-MS / MS) analysis. All statements of percent composition with respect to TAGs is by weight as a percentage of total TAG content unless otherwise specified.

[0043] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs), at least about 60% of the total TAG content, and optionally about 70-80% of the total palmitic acid (C16:0) content is esterified at the sn-2 position, as determined by sn-2 specific lipases analytical method.

[0044] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs), and the ratio of OPO:OOO by weight is about 1.5 or more (e.g., 1.5-20, 1.5-15, 1.5- 10, or 1.5-6), 2 or more (e.g., 2-20, 2-15, 2-10, 2-8, or 2-6), 3 or more (e.g., 3-20, 3-15, 3-10, 3-8, or 3-6), 4 or more (e.g., 4-20, 4-15, 4-10, 4-8, or 4-6), or 5 or more (e.g., 5-20. 5-15. 5- 10, or 5-8), as determined by liquid chromatography-mass spectrometry (LC-MS) analysis.Leydig 51584312

[0045] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs) wherein the ratio of OPO:OLO by weight is about 1 or more (e.g., 1-20, 1 -15, 1 -10, or 1-6), 2 or more (e.g., 2-20, 2-15, 2-10, 2-8, or 2-6), 3 or more (e.g., 3-20, 3-15, 3-10, 3-8, or 3-6), 4 or more (e.g., 4-20, 4-15, 4-10, 4-8, or 4-6), or 5 or more (e.g., 5-20, 5-15, 5-10, or 5-8), as determined by liquid chromatography -mass spectrometry (LC-MS) analysis.

[0046] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs) wherein the ratio of OPO:OSO by weight is about 1 or more (e.g., 1-20, 1-15, 1-10, or 1-6), 2 or more (e.g., 2-20, 2-15, 2-10, 2-8, or 2-6), 3 or more (e.g., 3-20, 3-15, 3-10, 3-8, or 3-6). 4 or more (e.g., 4-20, 4-15, 4-10, 4-8, or 4-6), or 5 or more (e.g., 5-20, 5-15, 5-10, or 5-8), as determined by liquid chromatography -mass spectrometry (LC-MS) analysis.

[0047] In some embodiments, the engineered microorganism produces triacylglycerols (TAGs), and the ratio of OPO:POP by weight is about 1 or more (e.g., 1-20, 1-15, 1-10, or 1- 6), 2 or more (e.g., 2-20, 2-15, 2-10, 2-8, or 2-6), 3 or more (e.g., 3-20, 3-15, 3-10, 3-8, or 3- 6), 4 or more (e.g.. 4-20. 4-15. 4-10. 4-8, or 4-6), or 5 or more (e.g., 5-20, 5-15, 5-10, or 5-8), as determined by liquid chromatography-mass spectrometry (LC-MS) analysis.

[0048] In some embodiments, the invention provides a method of producing l,3-dioleoyl-2- palmitoyl glycerol (OPO), the method comprising culturing any engineered microorganism disclosed herein. In some embodiments, the microorganism is cultured as described in Liu et al., "Yarrowia lipolytica as an Oleaginous Platform for the Production of Value-Added Fatty Acid-Based Bioproducts,” Frontiers in Microbiology, 11 (2021) and Park & Ledesma- Amaro, “What makes Yarrowia lipolytica well suited for industry ?” Trends Biotechnol, 41(2): 242-254 (2023).

[0049] In some embodiments, the culture media comprises a carbon source. Examples of carbon sources in the culture media include, but are not limited to one or more sugars, such as fermentable sugars, e.g. xylose, lactose, cellulose, glucose, fructose, sucrose, or hydrolysed lignocellulose; one or more fatty acids and / or fatty' acid esters, such as C16:0 fatty acids or fatty acid esters; one or more vegetable oils, such as palm oil; glycerol; acetate; methanol; or a CCh-derived feedstock, such as CCh-derived acetate or methanol.

[0050] The culture media can comprise any' suitable amount of the carbon source(s). In some embodiments, the culture media comprises 10 to 60 g / L of a sugar and / or glycerol. In some embodiments, the culture mediate comprises 1 to 5 g / L of fatty acids or fatty acid esters. In some embodiments, the culture media comprises 1 to 10 g / L of a vegetable oil.Leydig 51584313

[0051] A suitable pH range for the fermentation is typically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as the range for the initial growth conditions. The fermentation process includes media that may have a carbon composition typically ranging from 5g / L to lOOg / L, and a C / N ratio between 25 and 250. The fermentation may be conducted under aerobic or anaerobic conditions. In some embodiments, the microorganism is cultured aerobically. A two-stage fermentation process may be used in desired, whereby the first stage of the fermentation is dedicated to the generation and accumulation of cell mass and is characterized by rapid cell growth and cell division, and the second stage of the fermentation utilizes conditions (e.g., nitrogen deprivation) that promotes high levels of lipid accumulation. Production of lipid from a recombinant microbial host may be produced by a batch, fed-batch or continuous fermentation process.

[0052] In some embodiments, the method further comprises harvesting OPO (e.g., OPO- containing lipids) from the engineered microorganism culture. In some embodiments, the harvested OPO (e.g.. OPO-containing lipids) is refined, bleached, and deodorized. OPO, and other TAGs, can be isolated and purified from the culture by any suitable method, such as by using organic solvents, sonication, supercritical fluid extraction (e.g., using carbon dioxide), saponification and physical means such as presses, or combinations thereof. In some embodiments, the invention provides a triacylglycerol (TAG) composition made by any engineered microorganism disclosed herein or method of producing OPO disclosed herein. The TAG produced by the engineered microorganism is believed to be useful in food compositions for humans or animals, especially imitation dairy products, dietary supplements, and particularly human milk fat substitute compositions such as infant formula. Thus, also provided herein is a food composition, especially an infant formula composition, comprising the TAG, optionally combined with other nutrients (e.g., carbohydrates, fats, and / or proteins). Such a composition will typically comprise 3-5 wt.% of the TAG composition in liquid form, or 25-30 wt.% in dry powder form. The TAGs produced by the engineered microorganism can be isolated prior to combining with the other components of the food composition, or the TAGs can be used without isolation from the engineered microorganism, which can be used as a “whole cell” composition (optionally lysed) or fraction thereof including the TAGs and the other cellular components.

[0053] Genetic modifications to microorganisms as needed to make the engineered microorganisms provided herein can be accomplished by any suitable technique. For instance, heterologous nucleic acids (e.g., genes) to be introduced into the engineeredLeydig 51584314 microorganism can be delivered by way of a vector, such as a plasmid. The nucleic acids encoding desired proteins can be under the control of a suitable promoter, which can be heterologous or endogenous to the engineered microorganism. Furthermore, the vector can express the genes without integration into the engineered microorganism genome (e.g., selfpropagating plasmids), or the vector can integrate the target genes into the engineered microorganism’s genome. For example, the vector can comprise an expression cassette comprising the heterologous nucleic acid of interest along with any desired regulatory sequences (e.g., promoters, transcription termination sequences, etc.) and flanking sequences that will integrate into a target region of the engineered microorganism’s chromosome by homologous recombination). The heterologous nucleic acid to be expressed will be under control of a promoter. Promoters and other regulatory sequences can be native or non-native, and can be any typically used in yeast. Examples of native promoters include, but are not limited to TEF promoter (SEQ ID NO: 89), GAPDH promoter (SEQ ID NO: 90), FBA promoter (SEQ ID NO: 91), GPAT promoter, GPD promoter. GPM promoter, FBAIN promoter, GPDIN promoter, YAT1 promoter, and EXP1 promoter. Other promoters include, for instance, GPI promoter, PFK1 promoter, FBA1 promoter, TDH1 promoter, GPM1 promoter, PENO1 promoter, PPYK1 promoter, and PTPIl promoter. Termination regions also can be native or non-native. and can be any typically used in an engineered microorganism. Examples of termination regions include, but are not limited to approximately 100 bp of the 3' end of the Yarrowia lipolytica extracellular protease (XPR; GenBank Accession No. M17741 (SEQ ID NO: 92)); GAPDH terminator (3’UTR) (SEQ ID NO: 93); YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94); the acyl-coA oxidase (Aco3: GenBank Accession No. AJ001301 and No. CAA04661) terminator (SEQ ID NO: 95); the Pex20 (GenBank Accession No. AF054613) terminator (SEQ ID NO: 96); the Pexl 6 (GenBank Accession No. U75433) terminator (SEQ ID NO: 97); the Lipl (GenBank Accession No. Z50020) terminator (SEQ ID NO: 98); the Lip2 (GenBank Accession No. AJ012632) terminator (SEQ ID NO: 99); and the 3-oxoacyl-coA thiolase (OCT; GenBank Accession No. X69988) terminator (SEQ ID NO: 100). Transformation can be effected by any of several routine methods, such as lithium acetate, heat shock, or electroporation.

[0054] Genes can be disrupted or deleted in the engineered microorganism by any of several known techniques. For instance, genes can be disrupted or deleted by inserting a non-native nucleic acid (e.g., a selectable marker) into the native gene to interrupt the coding sequence and thereby functionally deactivate the gene. Deletion cassettes for this purpose can beLeydig 51584315 inserted into the gene targeted for deletion using, for example, homologous recombination. Other techniques for gene disruption also can be used, such as RNA-guided endonuclease (CRISPR / CAS) systems. Disruption or deletion of a given gene is accomplished if the expression of the gene after the modification is less than the expression of the gene before the modification (or as compared to the same microorganism without the modification).

[0055] Thus, also provided herein is a method of producing an engineered microorganism, such as an engineered microorganism that produces TAGs, particularly an engineered microorganism with increased production of OPO. The method can be used to produce any of the foregoing microorganisms described herein. In some embodiments, the method comprises introducing into a microorganism (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT. In some embodiments, the method comprises introducing into a microorganism (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3- phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT. In some embodiments, the method comprises introducing into a microorganism (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT. In some embodiments, the method comprises introducing into a microorganism a heterologous nucleic acid encoding Chlamydomonas moewusil LPAAT2 (SEQ ID NO: 36), optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT. The heterologous nucleic acids can be introduced into the microorganism by any suitable technique, such as by use of a vector (e.g., plasmid) comprising one or more of the heterologous nucleic acids. Furthermore, the vector can be engineered so that the heterologous nucleic acid integrates into the genome of the host cell at random locus (random integration) or a directed site. The heterologous nucleic acids can be introduced into any gene locus that does not affect the growth of the engineered microorganism and does not adversely affect production or accumulation of TAGs to any significant degree.

[0056] The method of producing an engineered microorganism as described in any of the foregoing embodiments can further comprise a step of disrupting the activity of an LPAATLeydig 51584316 native to the microorganism. Disruption of the activity of a native LPAAT can be accomplished, for instance, by deletion, substitution, or addition of nucleotides into a gene encoding a native LPAAT, thereby reducing the expression of a functional LPAAT enzy me. In some embodiments, the heterologous nucleic acid encoding an LPAAT, DGAT or GPAT is integrated at a locus that disrupts a native gene encoding a native LPAAT.

[0057] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.EXAMPLES

[0058] In the examples, the procedure described by Tsakraklides et al., “Targeted Integration Through Transformation of Hydroxyurea- Arrested Cells," Methods Mol Biol., 2307: 139-145 (2021) was used to transform all Yarrowia lipolytica transgenic strains unless otherwise noted. Cells were grown overnight and tranformed with DNA constructs including the gene of interest and the selection cassette as described above. The cells were plated onto selective plates after w ashing with 1 mL of sterile water. The expression cassettes within the transgenic strains were verified by single-colony PCR and sequenced.

[0059] In the examples, fatty acid methyl ester (FAME) analysis was performed in accordance with AOCS Official Method Ce lj-07 (American Oil Chemists’ Society (2017), AOCS Official Method Ce lj-07: cis-, trans-, Saturated, Monounsaturated and Polyunsaturated Fatty Acids in Extracted Fats by Capillary GLC, Official Methods and Recommended Practices of the AOCS, 7 th ed. AOAC International). This is referenced in the Examples as the LC-MS / MS method. All results presented as percent composition (%) of various TAGs refer to % by weight of the total TAG content of the tested sample.

[0060] The following strains of Yarrowia lipolytica and plasmid expression cassettes are referenced in the Examples:Yarrowia StrainsLeydig 51584317Expression CassettesLeydig 51584318

[0061] The expression cassetes referenced above are described in detail in the section following the examples.EXAMPLE 1

[0062] This example illustrates the production of Yarrowia lipolytica strains engineered to express the heterologous Nannochloropsis oceanica LPAT4 (NoLPAT4).

[0063] A plasmid vector, expression cassete 1 (SEQ ID NO: 1), was constructed to introduce NoLPAT4 into a parent Yarrowia lipolytica strain (SY001) via random integration.

[0064] To determine the effects of NoLPAT4 (SEQ ID NO: 31) on the TAG profile, two transgenic strains (T11301 and T1 1302) were generated by introducing cassette 1 into strain SY001. Strains were grown for 72 hours in 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and pelleted. The TAG profile data w as generated using LC-MS / MS method (FIG. 2 and Table 1). The TAG profiles of T11301 and T11302 demonstrated that NoLPAT4 (SEQ ID NO: 31) was successfully integrated into Yarrowia lipolytica strain SY001 and increased expression of OLO, OOO, OPoP, and OSO TAGs compared to the Yarrowia lipolytica base strain (SY001).Table 1 : TAG profile from transgenic strains expressing NoLPAT4.EXAMPLE 2

[0065] This example illustrates the production of strains comprising multiple LPAATs and shows the expression of heterologous NoLPAT4 in Yarrowia lipolytica strains SY005, SY006, SY007, and SY008, with and without native SLC1 (AGP AT) disrupted and the effect on oil production.Leydig 51584319

[0066] Plasmid vectors with expression cassetes 2 (SEQ ID NO: 2) and 3 (SEQ ID NO: 3) were designed to introduce NoLPAT4 expression via random integration (cassete 2) or SLCl-targeted integration (cassette 3) respectively. Plasmid vector comprising cassete 4 (SEQ ID NO: 4) was used to introduce tNoLPAT4* (SEQ ID NO: 33) expression (truncated version of NoLPAT4) by targeted integration of at SLC1 (SEQ ID NO: 84), which reduces native AGP AT expression. Plasmid vector comprising cassete 5 was designed for targeted disruption of SLC1 (AGP AT) without additional expression of a functional metabolic enzyme.

[0067] SY 005 is a Yarrowia lipolytica strain engineered to express CrLPAAT2 via a randomly integrating cassete encoding CrLPAAT2. To determine the effects of expressing NoLPAT4 (SEQ ID NO: 31) and tNoLPAT4* (SEQ ID NO: 33) on the TAG profile of Yarrowia lipolytica strain SY005, five transgenic strains were generated (T13201, T13202, T13301, T13302, and T134) by introducing plasmid vectors containing cassetes 2, 3, and 5 into base strain SY005 (see Table 2). Strains were grown for 72 hours in a 50-mL conical tube at 250 rpm at 28 °C in lipid medium. Biomass was harvested and a TAG profile was generated using an LC-MS / MS method. The results are shown in FIG. 3 and Table 2.Table 2: TAG profile from transgenic strains with (a) native AGP AT (SEQ ID NO: 84) disrupted, (b) expressing NoLPAT4 (SEQ ID NO: 31), or (c) both engineering combined.Leydig 51584320

[0068] Yarrowia lipolytica strains SY006. SY007, and SY008 are strains previously engineered to express CrLPAAT2 via a cassette integrated at Gsyl (SEQ ID NO: 85) (SY006), or to express HsAGPATl via targeted Gsyl (SEQ ID NO: 85) integration (SY007) or random integration (SY008) of a cassette encoding HsAGPATl. To determine the effects of NoLPAT4 (SEQ ID NO: 31) and SLC1 (SEQ ID NO: 84) knockout on the TAG profile of Yarrowia lipolytica strains SY007, SY007, and SY008, nineteen transgenic strains were generated by introducing cassettes 2, 3, 4, and 5 into base strains SY006, SY007, and SY008. Strains were grown for 72 hours in 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and pelleted. The TAG profile data was generated using LC-MS / MS method. The results are shown in FIGs. 4-5 and Table 3.Table 3: TAG profile from transgenic strains with native AGP AT (SEQ ID NO: 84) disrupted, expressing NoLPAT4 (SEQ ID NO: 31), or both engineering combined.Leydig 51584321EXAMPLE 3

[0069] This example illustrates the production of engineered Y. lipolyticci expressing Chlamydomonas moewusii LPAAT2 (CmLPAAT2) (SEQ ID NO: 37), Edaphochlamys debaryana LPAAT2 (EdLPAAT2) (SEQ ID NO: 39), Volvulina compacta LPAAT2 (VcLPAAT2) (SEQ ID NO: 41), Vilreochlamys sp. LPAAT2 (VitrLPAAT2) (SEQ ID NO: 43), Homo sapiens AGPAT2 (HsAGPAT2) (SEQ ID NO: 45), Homo sapiens AGPAT3 (HsAGPAT3) (SEQ ID NO: 47), Homo sapiens AGPAT4 (HsAGPAT4) (SEQ ID NO: 49), Sus scrofa AGPAT2 (SsAGPAT2) (SEQ ID NO: 51), Sus scrofa AGPAT5 (SsAGPAT5) (SEQ ID NO: 53), wAHomo sapiens AGP ATI (HsAGPATl) (SEQ ID NO: 35) with and without disrupted native SLC1 (AGP AT) (SEQ ID NO: 84).

[0070] CmLPAAT2 (SEQ ID NO: 37), EdLPAAT2 (SEQ ID NO: 39), VcLPAAT2 (SEQ ID NO: 41), VitrLPAAT2 (SEQ ID NO: 43), HsAGPATl (SEQ ID NO: 35), HsAGPAT2 (SEQ ID NO: 45), HsAGPAT3 (SEQ ID NO: 47), HsAGPAT4 (SEQ ID NO: 49), SsAGPAT2 (SEQ ID NO: 51), and SsAGPAT5 (SEQ ID NO: 53) were introduced into wild-type Y. lipolytica strain SY002 using plasmid expression cassettes 6-15. Expression cassettes 6-14 were designed for random integration, whereas expression cassette 15 was designed for targeted integration at SLC 1 (SEQ ID NO: 84), which disrupts expression of native AGP AT.

[0071] To determine the effects of NoLPAT4 (SEQ ID NO: 31) on the OPO production of Yarrowia lipolytica strain SY002, twelve transgenic strains were generated by introducing cassettes 6-15 into SY002. Strains SY009 and SY010 (example 2) were included in the study for comparison. Strains were grown for 72 hours in a 50-mL conical tube at 250 rpm at 28Leydig 51584322°C in lipid production medium. Biomass was harvested and a TAG profile was generated using LC-MS / MS method. The results are shown in FIG. 7 and Table 4. OPO content for Y. lipolytica base strain SY002 served as a non-transgenic control was not shown.Table 4: OPO production from transgenic strains expressing CmLPAAT2 (SEQ ID NO: 37), EdLPAAT2 (SEQ ID NO: 39), VcLPAAT2 (SEQ ID NO: 41), VitrLPAAT2 (SEQ ID NO: 43), HsAGPAT2 (SEQ ID NO: 45), HsAGPAT3 (SEQ ID NO: 47), HsAGPAT4 (SEQ ID NO: 49), SsAGPAT2 (SEQ ID NO: 51), SsAGPAT5 (SEQ ID NO: 53), and HsAGPATl(SEQ ID NO: 35) with native AGP AT (SEQ ID NO: 84) disrupted.EXAMPLE 4

[0072] This example illustrates the expression ofNoLPAT4 (SEQ ID NO: 31) and tNoLPAT4* (SEQ ID NO: 33) in Yarrowia lipolytica strain SY011 expressing CmLPAAT2 with native SLC1 (SEQ ID NO: 84) (AGP AT) disrupted.

[0073] SY011 is a strain previously engineered to express CmLPAAT2 via randomly integrating plasmid cassette. NoLPAT4 (SEQ ID NO: 31) and tNoLPAT4* (SEQ ID NO: 33) were introduced into strain SY011 using the expression cassettes 3 and 4, which are designed to integrate at SLC1 (SEQ ID NO: 84) and disrupt expression of native AGP AT.Leydig 51584323Engineered Y. lipolytica strain SY010 (Example 2) was included in the study for comparison. Strains were grown for 72 hours in a 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and TAG profile data was generated using LC- MS / MS method. The results are shown in FIG. 8 and Table 5.Table 5: TAG profile from transgenic strains with native AGP AT (SEQ ID NO: 84) disrupted, expressing NoLPAT4 (SEQ ID NO: 31), or tNoLPAT4 (SEQ ID NO: 33)EXAMPLE 5

[0074] The example demonstrates the expression of Arabidopsis thaliana DGA (AtDGA) (SEQ ID NO: 55), Candida tropicalis DGA2 (CtDGA2) (SEQ ID NO: 57). Elaeis guineensis DGA2 (EgDGA2) (SEQ ID NO: 59), Ettlia oleoabundans DGA2 (EtDGA2) (SEQ ID NO: 61), Homo sapiens DGA1 (HsDGAl) (SEQ ID NO: 63), Homo sapiens DGA2 (HsDGA2) (SEQ ID NO: 65), Sus scrofa DGA1 (SsDGAl) (SEQ ID NO: 67), Sus scrofa DGA2 (SsDGA2) (SEQ ID NO: 69), Thraustochytrium aureum DGA2 (TaDGA2) (SEQ ID NO: 71), Homo sapiens GPAT1 (HsGPATl) (SEQ ID NO: 73), Homo sapiens GPAT3 (HsGPAT3) (SEQ ID NO: 75), Homo sapiens GPAT4 (HsGPAT4) (SEQ ID NO: 77), Nannochlor opsis oceanica GPAT (NoGPAT) (SEQ ID NO: 79), and Sus scrofa GPAT2 (SsGPAT2) (SEQ ID NO: 81) genes in Yarrowia lipolytica strain SY009. SY009 is a strainLeydig 51584324 previously engineered to express HsAGPATl (SEQ ID NO: 35) and tNoLPAT (SEQ ID NO: 31) with native (SLC1 (SEQ ID NO: 84); AGP AT) disruption (see Example 2).

[0075] To determine the effects of AtDGA(SEQ ID NO: 55), CtDGA2 (SEQ ID NO: 57), EgDGA2 (SEQ ID NO: 59), EtDGA2 (SEQ ID NO: 61), HsDGAl (SEQ ID NO: 63), HsDGA2 (SEQ ID NO: 65), SsDGAl (SEQ ID NO: 67), SsDGA2 (SEQ ID NO: 69), TaDGA2 (SEQ ID NO: 71), HsGPATl (SEQ ID NO: 73), HsGPAT3)(SEQ ID NO: 75), HsGPAT4 (SEQ ID NO: 77), NoGPAT (SEQ ID NO: 79), and SsGPAT2 (SEQ ID NO: 81) on OPO production of Yarrow ia lipolytica strain SY009 sixteen transgenic strains were generated using cassettes 16-29. Strains were grown for 72 hours in a 50-mL conical tube at 250 rpm at 28 °C in lipid production medium. Biomass was harvested and TAG profile data was generated using LC-MS / MS method. The results with respect to OPO production are shown in FIG. 9 and Table 6. Expressing AtDGA(SEQ ID NO: 55), CtDGA2 (SEQ ID NO: 57), EgDGA2 (SEQ ID NO: 59), EtDGA2 (SEQ ID NO: 61), HsDGAl (SEQ ID NO: 63), HsDGA2 (SEQ ID NO: 65). SsDGAl (SEQ ID NO: 67), SsDGA2 (SEQ ID NO: 69), and TaDGA2 (SEQ ID NO: 71) genes also increased the neutral lipid production in the Y. lipolytica strain SY009 (data not shown).Table 6: OPO production from transgenic strains expressing AtDGA(SEQ ID NO: 55). CtDGA2 (SEQ ID NO: 57). EgDGA2 (SEQ ID NO: 59). EtDGA2 (SEQ ID NO: 61), HsDGAl (SEQ ID NO: 63), HsDGA2 (SEQ ID NO: 65), SsDGAl (SEQ ID NO: 67), SsDGA2 (SEQ ID NO: 69), TaDGA2 (SEQ ID NO: 71), HsGPATl (SEQ ID NO: 73), HsGPAT3)(SEQ ID NO: 75), HsGPAT4 (SEQ ID NO: 77), NoGPAT (SEQ ID NO: 79), and SsGPAT2 (SEQ ID NO: 81).Leydig 51584325EXAMPLE 6

[0076] This example demonstrates the expression of Ettlia oleoabundcins EtDGA2 in Yarrowia lipolytica strains SY012 and SY019 from Example 4.

[0077] To determine the effects of EtDGA2 (SEQ ID NO: 61) on the TAG profile of strains SY012 and SY019, twelve transgenic strains were generated by introducing cassette 19 into strains SY012 and SY019. SY009, which is a strain engineered to express Hs AGP ATI (SEQ ID NO: 31) and tNoLPAT (SEQ ID NO: 33) with native (SLC1 (SEQ ID NO: 84) ; AGP AT) disruption (see Example 2), was included as an additional comparison. Strains were grown for 72 hours in a 50-mL conical tube at 250 rpm at 28 °C in lipid production medium.Biomass was harvested and the TAG profile data was generated using LC-MS / MS method. Results with respect to OPO production are provided in FIG. 10 and Table 7. Expressing EIDGA2 (SEQ ID NO: 61) also increased the neutral lipid production in Y. lipolytica strains SY012 and SY019 (data not shown).Table 7: OPO production from transgenic strains expressing EtDGA2.Leydig 51584326EXAMPLE 7

[0078] The example demonstrates the sn2 analysis of dow nstream processed oil samples that were produced by engineered Yarrowia lipolytica strains.

[0079] To determine the effects of NoLPAT4 (SEQ ID NO: 31) and tNoLPAT4* (SEQ ID NO: 33) on Yarrowia lipolytica strains analyses of the sn2 fatty' acid compositions were performed. 10 L fermentations using Y. lipolytica strains SY009 (Example 2), SY013 (Example 5). and SY012 (Example 4) were conducted. The oil was extracted, refined and bleached C RB " oil), or refined, bleached, and deodorized C’RBD” oil). The sn2 fatty acid compositions for RBD or RB oil samples were analyzed using lipase-based method. Table 8 shows the sn2-C16:0 content of oil samples from SY009, SY012, and SY013, and FIG. 11 shows the same for RBD Oil 49-52 (derived from fermentation of strain SY009) and Eastmond (Glucose).Table 8: sn2-C16:0 analysis of oil samplesEXAMPLE 8

[0080] A complete TAG profile was generated for Yarrowia lipolytica strains SY010 and SY009, SY008, SY005, and SY002. The results are provided in FIG. 12.DETAILED DESCRIPTION OF PLASMID EXPRESSION CASSETTES USED IN THE EXAMPLES

[0081] Cassette 1 illustrated in the appendix can be described as TEFin-NoLPAT4-XPRt- GAPDHp-NAT-GAPDHt. The Yarrowia lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics,Leydig 51584327 driving the expression of NoLPAT4 (SEQ ID NO: 30). The start codon ATG and stop codon TAG for NoLPAT4 (SEQ ID NO: 30) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Yarrowia lipolyticci XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Yarrowia lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Yarrowia lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, and underlined italics.

[0082] Cassette 2 (SEQ ID NO: 2) can be described as TEFin-NoLPAT4-XPRt-GAPDHp- ShBle-GAPDHt. The cassette is designed for random integration of NoLPAT4 (SEQ ID NO: 30). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in low ercase, underlined italics, driving the expression of NoLPAT4 (SEQ ID NO: 30). The start codon ATG and stop codon TAG for NoLPAT4 (SEQ ID NO: 30) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated byuppercase, bold italics, driving the expression of selection marker ShBle (zeocin resistance gene) (SEQ ID NO: 102), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3 -UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0083] Cassette 3 (SEQ ID NO: 3) can be described as 5’ SLCl ::TEFin-NoLPAT4-XPRt- GAPDHp-ShBle-GAPDHt::3’ SLC1 . Proceeding in the 5’ to 3’ direction lowercase sequences represent genomic DNA from SY002-based trans genics strains that permit targeted integration at SLC1 locus (5’) via homologous recombination. The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of NoLPAT4 (SEQ ID NO: 30). The start codon ATG and stop codon TAG for NoLPAT4 (SEQ ID NO: 30) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker ShBle (zeocin resistance gene) (SEQ ID NO: 102) whichLeydig 51584328 is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics, followed by the SLC1 (SEQ ID NO: 84) genomic region (3’) indicated by lowercase text.

[0084] Cassette 4 (SEQ ID NO: 4) can be described as 5’ SLCl::TEFin-tNoLPAT4*-XPRt- GAPDHp-ShBle-GAPDHt::3’ SLC1. Proceeding in the 5’ to 3’ direction lowercase sequences represent genomic DNA from SY002-based transgenics strains that permit targeted integration at SLC1 (SEQ ID NO: 84) locus (5’) via homologous recombination. The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of tNoLPAT4* (truncated version of NoLPAT4) (SEQ ID NO: 32). The start codon ATG and stop codon TAG for tNoLPAT4* (SEQ ID NO: 32) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker ShBle (zeocin resistance gene) (SEQ ID NO: 102), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics, followed by the SLC1 (SEQ ID NO: 84) genomic region (3’) indicated by lowercase text.

[0085] Cassette 5 (SEQ ID NO: 5) can be described as 5’ SLCl::GAPDHp-ShBle- GAPDHt::3’ SLC1. Proceeding in the 5’ to 3’ direction lowercase sequences represent genomic DNA from SY002 -based transgenics strains that permit targeted integration at SLC1 (SEQ ID NO: 84) locus (5’) via homologous recombination to achieve native AGP AT KO. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker ShBle (zeocin resistance gene) (SEQ ID NO: 102), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics, followed by the SLC1 (SEQ ID NO: 84) genomic region (3’) indicated by lowercase text.

[0086] Cassette 6 (SEQ ID NO: 6) can be described as TEFin-CmLPAAT2-XPRt-GAPDHp- NAT-GAPDHt. The cassette is designed for random integration of CmLPAAT2 (SEQ ID NO: 36). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase,Leydig 51584329 underlined text, with its intron shown in lowercase, underlined italics, driving the expression of CmLPAAT2 (SEQ ID NO: 36). The start codon ATG and stop codon TGA for CmLPAAT2 (SEQ ID NO: 36) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG show n in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3'-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0087] Cassette 7 (SEQ ID NO: 7) can be described as TEFin-EdLPAAT2-XPRt-GAPDHp- NAT-GAPDHt. The cassette is designed for random integration of EdLPAAT2 (SEQ ID NO: 38). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of EdLPAAT2 (SEQ ID NO: 38). The start codon ATG and stop codon TGA for EdLPAAT2 (SEQ ID NO: 38) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG show n in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3'-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0088] Cassette 8 (SEQ ID NO: 8) can be described as TEFin-VcLPAAT2-XPRt-GAPDHp- NAT-GAPDHt. The cassette is designed for random integration of VcLPAAT2 (SEQ ID NO: 40). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of VcLPAAT2 (SEQ ID NO: 40). The start codon ATG and stop codon TGA for VcLPAAT2 (SEQ ID NO: 40) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase,Leydig 51584330 bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0089] Cassette 9 (SEQ ID NO: 9) can be described as TEFin-VitrLPAAT2-XPRt-GAPDHp- NAT-GAPDHt. The cassette is designed for random integration of VitrLPAAT2 (SEQ ID NO: 42). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of VitrLPAAT2 (SEQ ID NO: 42). The start codon ATG and stop codon TGA for VitrLPAAT2 (SEQ ID NO: 42) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0090] Cassette 10 (SEQ ID NO: 10) can be described as TEFin-HsAGPAT2-XPRt- GAPDHp-NAT-GAPDHt. The cassette is designed for random integration of HsAGPAT2 (SEQ ID NO: 44. The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsAGPAT2 (SEQ ID NO: 44). The start codon ATG and stop codon TGA for HsAGPAT2 (SEQ ID NO: 44) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0091] Cassette 11 (SEQ ID NO: 11) can be described as TEFin-HsAGPAT3-XPRt- GAPDHp-NAT-GAPDHt. The cassette is designed for random integration of HsAGPAT3 (SEQ ID NO: 46). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving theLeydig 51584331 expression of HsAGPAT3 (SEQ ID NO: 46). The start codon ATG and stop codon TGA for HsAGPAT3 (SEQ ID NO: 46) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0092] Cassette 12 (SEQ ID NO: 12) can be described as TEFin-HsAGPAT4-XPRt- GAPDHp-NAT-GAPDHt. The cassette is designed for random integration of HsAGPAT4 (SEQ ID NO: 48). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsAGPAT4 (SEQ ID NO: 48). The start codon ATG and stop codon TGA for HsAGPAT4 (SEQ ID NO: 48) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0093] Cassette 13 (SEQ ID NO: 13) can be described as TEFin-SsAGPAT2-XPRt- GAPDHp-NAT-GAPDHt. The cassette is designed for random integration of SsAGPAT2 (SEQ ID NO: 50). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of SsAGPAT2 (SEQ ID NO: 50). The start codon ATG and stop codon TGA for SsAGPAT2 (SEQ ID NO: 50) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold,Leydig 51584332 underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0094] Cassette 14 (SEQ ID NO: 14) can be described as TEFin-SsAGPAT5-XPRt- GAPDHp-NAT-GAPDHt. The cassette is designed for random integration of SsAGPAT5 (SEQ ID NO: 52). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of SsAGPAT5 (SEQ ID NO: 52). The start codon ATG and stop codon TGA for SsAGPAT5 (SEQ ID NO: 52) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics.

[0095] Cassette 15 (SEQ ID NO: 15) can be described as 5’ SLC l::TEFin-HsAGPATl- XPRt-GAPDHp-NAT-GAPDHt::3’ SLC1. Proceeding in the 5’ to 3' direction lowercase sequences represent genomic DNA from SY002-based trans genics strains that permit targeted integration at SLC1 (SEQ ID NO: 84) locus (5’) via homologous recombination. The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsAGPATl (SEQ ID NO: 34). The start codon ATG and stop codon TAG for HsAGPATl (SEQ ID NO: 34) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica GAPDH promoter (SEQ ID NO: 90) is indicated by uppercase, bold italics, driving the expression of selection marker NAT (nourseothricin N-acetyl transferase) (SEQ ID NO: 101), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TAG shown in lowercase, bold, underlined italics. The Y. lipolytica GAPDH terminator (3’-UTR) (SEQ ID NO: 93) is indicated by uppercase, bold, underlined italics, followed by the SLC1 genomic region (3’) indicated by lowercase text.

[0096] Cassette 16 (SEQ ID NO: 16) can be described as TEFin-AtDGA-XPRt-FBAp-DsdA- E10659t. The cassette is designed for random integration of AtDGA (SEQ ID NO: 54). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, withLeydig 51584333 its intron shown in lowercase, underlined italics, driving the expression of AtDGA (SEQ ID NO: 54). The start codon ATG and stop codon TAA for AtDGA are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0097] Cassette 17 (SEQ ID NO: 17) can be described as TEFin-CtDGA2-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of CtDGA2 (SEQ ID NO: 56). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in low ercase, underlined italics, driving the expression of CtDGA2 The start codon ATG and stop codon TGA for CtDGA2 (SEQ ID NO: 56) are indicated byuppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0098] Cassette 18 (SEQ ID NO: 18) can be described as TEFin-EgDGA2-XPRt-FBAp- DsdA-E 106591. The cassette is designed for random integration of EgDGA2. The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron showm in low ercase, underlined italics, driving the expression of EgDGA2 (SEQ ID NO: 58). The start codon ATG and stop codon TAA for EgDGA2 (SEQ ID NO: 58) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.Leydig 51584334

[0099] Cassete 19 (SEQ ID NO: 19) can be described as TEFin-EtDGA2-XPRt-FBAp- DsdA-El 06591. The cassete is designed for random integration of Ettlia oleoabundans EtDGA2 (SEQ ID NO: 60). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of EtDGA2. The start codon ATG and stop codon TAA for EtDGA2 (SEQ ID NO: 60) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0100] Cassete 20 (SEQ ID NO: 20) can be described as TEFin-HsDGAl-XPRt- FBAp-DsdA-E10659t. The cassete is designed for random integration of HsDGAl (SEQ ID NO: 62). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsDGAl (SEQ ID NO: 62). The start codon ATG and stop codon TAA for HsDGAl (SEQ ID NO: 62) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0101] Cassete 21 (SEQ ID NO: 21) can be described as TEFin-HsDGA2-XPRt- FBAp-DsdA-E10659t. The cassete is designed for random integration of HsDGA2 (SEQ ID NO: 64). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsDGA2 (SEQ ID NO: 64). The start codon ATG and stop codon TAA for HsDGA2 (SEQ ID NO: 64) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicatedLeydig 51584335 by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0100] Cassette 22 (SEQ ID NO: 22) can be described as TEFin-SsDGAl-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of SsDGAl (SEQ ID NO: 66). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of SsDGAl (SEQ ID NO: 66). The start codon ATG and stop codon TAA for SsDGAl (SEQ ID NO: 66) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3'-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0101] Cassette 23 (SEQ ID NO: 23) can be descnbed as TEFin-SsDGA2-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of SsDGA2 (SEQ ID NO: 68). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in low ercase, underlined italics, driving the expression of SsDGA2 (SEQ ID NO: 68). The start codon ATG and stop codon TAA for SsDGA2 (SEQ ID NO: 68) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by low ercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0102] Cassette 24 (SEQ ID NO: 24) can be described as TEFin-TaDGA2-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of TaDGA2 (SEQ ID NO: 70). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlinedLeydig 51584336 text, with its intron shown in lowercase, underlined italics, driving the expression of TaDGA2 (SEQ ID NO: 70). The start codon ATG and stop codon TGA for TaDGA2 (SEQ ID NO: 70) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3'-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0103] Cassette 25 (SEQ ID NO: 25) can be descnbed as TEFin-HsGPATl-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of HsGPATl (SEQ ID NO: 72). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsGPATl (SEQ ID NO: 72). The start codon ATG and stop codon TGA for HsGPATl (SEQ ID NO: 72) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated byuppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0104] Cassette 26 (SEQ ID NO: 26) can be described as TEFin-HsGPAT3-XPRt-FBAp- DsdA-E10659t. The cassette is designed for random integration of HsGPAT3 (SEQ ID NO: 74). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in low ercase, underlined italics, driving the expression of HsGPAT3 (SEQ ID NO: 74). The start codon ATG and stop codon TAA for HsGPAT3 (SEQ ID NO: 74) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3 -UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated byuppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y.Leydig 51584337 lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0105] Cassette 27 can be described as TEFin-HsGPAT4-XPRt-FBAp-DsdA-E10659t. The cassette is designed for random integration of HsGPAT4 (SEQ ID NO: 76). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of HsGPAT4 (SEQ ID NO: 76). The start codon ATG and stop codon TAA for HsGPAT4 (SEQ ID NO: 76) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3'-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0106] Cassette 28 can be described as TEFin-NoGPATl-XPRt-FBAp-DsdA-E10659t. The cassette is designed for random integration of NoGPATl (SEQ ID NO: 78). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of NoGPATl (SEQ ID NO: 78). The start codon ATG and stop codon TAA for NoGPATl (SEQ ID NO: 78) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics. The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_El 0659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.

[0107] Cassette 29 can be described as TEFin-SsGPAT2-XPRt-FBAp-DsdA-E10659t. The cassette is designed for random integration of SsGPAT2 (SEQ ID NO: 80). The Y. lipolytica TEF promoter (SEQ ID NO: 89) is indicated by uppercase, underlined text, with its intron shown in lowercase, underlined italics, driving the expression of SsGPAT2 (SEQ ID NO: 80). The start codon ATG and stop codon TAA for SsGPAT2 (SEQ ID NO: 80) are indicated by uppercase, bold text while the coding region is indicated by uppercase text. The Y. lipolytica XPR terminator (3’-UTR) (SEQ ID NO: 92) is indicated by lowercase italics.Leydig 51584338The Y. lipolytica FBA promoter (SEQ ID NO: 91) is indicated by uppercase, bold italics, driving the expression of selection marker MDsdA (D-serine deaminase) (SEQ ID NO: 103), which is indicated by lowercase, bold italic, with the start codon ATG and stop codon TGA shown in lowercase, bold, underlined italics. The Y. lipolytica YALI0_E10659g terminator (3’-UTR) (SEQ ID NO: 94) is indicated by uppercase, bold, underlined italics.Nucleotide sequence for the expression cassette encoding NoLPAT4.Leydig 51584339Leydig 51584340Sequences for cassettes encoding NoLPAT4.Leydig 51584341Leydig 51584342Leydig 51584343Leydig 51584344Leydig 51584345Leydig 51584346Sequences for the cassette encoding tNoLPAT4*Leydig 51584347Leydig 51584348Sequences for the cassete for native AGP AT disruption.Leydig 51584349Leydig 51584350Sequences for cassetes encoding CmLPAAT2, EdLPAAT2, VcLPAAT2, and VitrLPAAT2.Leydig 51584351Leydig 51584352Leydig 51584353Leydig 51584354Leydig 51584355Leydig 51584356Leydig 51584357Leydig 51584358Leydig 51584359Leydig 51584360Leydig 51584361Leydig 51584362Sequences for the cassette encoding tNoLPAT4*.Leydig 51584363Leydig 51584364Sequences for cassettes encoding AtDGA, QDGA2, EgDGA2, EtDGA2, HsDGAl,HsDGA2, SsDGAl, SsDGA2, SsDGA3, TaDGA2, HsGPATl, HsGPAT3, HsGPAT4,NoGPAT, and SsGPAT2Leydig 51584365Leydig 51584366Leydig 51584367Leydig 51584368Leydig 51584369Leydig 51584370Leydig 51584371Leydig 51584372Leydig 51584373Leydig 51584374Leydig 51584375Leydig 51584376Leydig 51584377Leydig 51584378Leydig 51584379Leydig 51584380Leydig 51584381Leydig 51584382Leydig 51584383Leydig 51584384Leydig 51584385Leydig 51584386Leydig 51584387Leydig 51584388Leydig 51584389Leydig 51584390Leydig 51584391Leydig 51584392Leydig 51584393Sequences for LPAATs.Leydig 51584394Leydig 51584395Leydig 51584396Leydig 51584397Leydig 51584398Sequences for DGATs.Leydig 51584399Leydig 515843100Leydig 515843101Leydig 515843102Leydig 515843103Sequences for GPATs.Leydig 515843104Leydig 515843105Leydig 515843106Leydig 515843107Nucleotide sequence for SLC1 from the approximately 3 kb region of the genome containing AGP AT locus from Yarrowia lipolytica W29.Leydig 515843108Nucleotide sequence for GSY1 from the approximately 5 kb region of the genome containing GSY1 locus from Yarrowia lipolyticci W29.Leydig 515843109Nucel otide sequences of Yarrowia lipolytica promoters.Leydig 515843110Nucleotide sequences of Yarrowia lipolytica terminators.Leydig 515843IllNucleotide sequences of selection markers.Leydig 515843112

[0108] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0109] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary- language (e.g., “such as”) provided herein, is intended merely to better illuminate theLeydig 515843113 invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0110] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherw ise indicated herein or otherwise clearly contradicted by context.

Claims

Leydig 515843114CLAIM(S):

1. An engineered microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or a microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT) or a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT; or a microorganism comprising (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a second LPAAT, optionally further comprising (iii) a heterologous nucleic acid encoding a DGAT and / or GPAT; or a microorganism comprising a heterologous nucleic acid encoding Chlamydomonas moewusii LPAAT2 (SEQ ID NO: 36), optionally further comprising a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT.

2. The engineered microorganism of claim 1, wherein the microorganism is a yeast.

3. The engineered microorganism of claim 1 or 2, wherein the microorganism is Yarrowia lipolytica.

4. The engineered microorganism of any of claims 1-3, wherein the GPAT or DGAT is a mammalian GPAT or DGAT, optionally a human, cow, goat, pig, camel, or horse DGAT or GPAT.

5. The engineered microorganism of any of claims 1-3, wherein the GPAT or DGAT is a plant GPAT or DGAT. optionally an Arabidopsis. Brassica. Camellia, Elaeis, Panicum, Raphanus, or Zizipus GPAT or DGAT.

6. The engineered microorganism of any of claims 1-3, wherein the GPAT or DGAT is a yeast or fungi GPAT or DGAT, optionally a Candida, Cryptococcus, Geotrichum, Lipomyces, Polyspora, Rhodosporidium, Rhodotorula,Saccharomyces , Trichosporon, Yarrowia, Fusarium. Sarocladium, Mortierella, or Microsphaeropsis GPAT or DGAT.Leydig 5158431157. The engineered microorganism of any of claims 1-3, wherein the GPAT or DGAT is an algal GPAT or DGAT, optionally a Botryococcus, Chlamydomonas, Chlorella, Auxenochlorella, Nannochlor opsis, or Volvox GPAT or DGAT.

8. The engineered microorganism of any of claims 1-3. wherein the GPAT or DGAT is a cyanobacteria GPAT or DGAT, optionally an Anabaena, Synechococcus. Synechocystis, or Microcystis GPAT or DGAT .

9. The engineered microorganism of any of claims 1-3, wherein the GPAT is specific for a Cl 8: 1 substrate.

10. The engineered microorganism of any of claims 1-3, wherein the GPAT is specific for a Cl 8:2 substrate.

11. The engineered microorganism of any of claims 1-3, wherein the heterologous gene encoding a GPAT is a Homo sapiens GPAT, Arabidopsis GPAT, or Chlamydomonas GPAT.

12. The engineered microorganism of any of claims 1-3, wherein the DGAT is specific for a Cl 8: 1 substrate.

13. The engineered microorganism of any of claims 1-3, wherein the DGAT is specific for a Cl 8:2 substrate.

14. The engineered microorganism of any of claims 1-3. wherein the heterologous gene encoding a DGAT is a Homo sapiens DGAT, Candida DGAT, or Elaeis DGAT.

15. The engineered microorganism of any of claims 1-14, wherein the second LPAAT preferentially acts at sn-1 or sn-3 positions of a triacylglycerol as compared to sn-2.

16. The engineered microorganism of claim 15, wherein the second LPAAT is a bacterial, algal, yeast, fungus, or plant LPAAT.

17. The engineered microorganism of claim 16, wherein the second LPAAT is a Nannochloropsis LPAAT, optionally NoLPAT4 (SEQ ID NO: 31) or tNoLPAT4 (SEQ ID NO: 33).

18. The engineered microorganism of any of claims 1-17, wherein the first LPAAT is specific for a Cl 6:0 substrate.

19. The engineered microorganism of any of claims 1-18, wherein the first LPAAT preferentially acts at position sn-2 of a triacylglycerol as compared to sn-1 or sn-3.

20. The engineered microorganism of any of claims 1-19, wherein the first LPAAT is a mammalian LPAAT, optionally a human, pig, camel, or horse LPAAT.Leydig 51584311621. The engineered microorganism of any of claims 1-19, wherein the first LPAAT is a plant LPAAT, optionally a Brassica, Arabidopsis, Elaeis, Theobroma, or Shorea LPAAT.

22. The engineered microorganism of any of claims 1-19, wherein the first LPAAT is an algal LPAAT, optionally an Auxenochlorella, Chlamydomonas, Chlorella, Edaphochlamys, Monoraphidium, Prototheca, Tetradesmis, or Volvox LPAAT.

23. The engineered microorganism of any of claims 1-19, wherein the first LPAAT is a bacterial LPAAT, optionally aa Actinomycetota, Gordonia, Mycobacterium, Rhodococcus, or Speluncibacter LPAAT.

24. The engineered microorganism of any of claims 1-19, wherein the first LPAAT is a Homo sapiens AGP AT (HsAGPAT), Chlamydomonas moewusii LPAAT2 (CmLPAAT2) (SEQ ID NO: 37), Edaphochlamys debaryana LPAAT2 (EdLPAAT2) (SEQ ID NO: 9), Volvulina compacta LPAAT2 (VcLPAAT2) (SEQ ID NO: 41), or Vitreochlamys sp. (VitrLPAAT2) (SEQ ID NO: 43).

25. The engineered microorganism of any of claims 1-3, wherein the engineered microorganism comprises (i) a heterologous gene encoding HsAGPATl (SEQ ID NO: 34), CmLPAAT2 (SEQ ID NO: 36), or CrLPAAT2 (SEQ ID NO: 82), and (ii) a heterologous gene encoding NoLPAT4 (SEQ ID NO: 30) or tNoLPAT4 (SEQ ID NO: 32); optionally wherein the engineered microorganism further comprises a heterologous gene encoding EtDGATl (SEQ ID NO: 60).

26. The engineered microorganism of any of claims 1-25, further comprising a genetic modification that reduces the activity of an LPAAT native to the microorganism, optionally wherein the microorganism is Yarrowia comprising a genetic modification that reduces native AGP AT activity.

27. The engineered microorganism of any of claims 1-26, further comprising a heterologous gene encoding DGAT1 or DGAT2.

28. The engineered microorganism of any of claims 1-27, wherein the microorganism is Yarrowia lipolytica, and does not comprise MatA, leu2-270, ura3-302, xpr2-322, and / or axp-2 modifications.

29. The engineered microorganism of any of claims 1-28, wherein the engineered microorganism produces more l,3-dioleoyl-2-palmitoyl glycerol (OPO) as compared to a microorganism of the same type without (i) the heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) the heterologous nucleic acidLeydig 515843117 encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or as compared to a wild-type microorganism of the same type; or wherein the engineered microorganism produces triacylglycerols (TAGs) in which OPO forms a greater proportion of total TAG content as compared to a microorganism of the same ty pe without (i) the heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) the heterologous nucleic acid encoding a glycerol-3- phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT; or as compared to a wild-type microorganism of the same type.

30. The engineered microorganism of any of claims 1-29, wherein the microorganism produces triacylglycerols (TAGs), and at least about 10% of the total triacylglycerol (TAG) content, optionally about 10%-70% or 40%-70%, of the total TAG content, of the engineered yeast is 1.3 -dioleoy 1-2 -palmitoyl glycerol (OPO), as determined by liquid chromatography -mass spectrometry (LC-MS) analysis.

31. The engineered microorganism of any of claims 1-30, wherein the microorganism produces triacylglycerols (TAGs), and at least about 60% of the total TAG content, and optionally about 70-80% of the total palmitic acid (C1 :0) content is esterified at the sn-2 position.

32. The engineered microorganism of any of claims 1-31, wherein the microorganism produces triacylglycerols (TAGs), and the ratio of OPO:OOO is about 1.5 or more, or about 3 or more, as determined by liquid chromatography-mass spectrometry (LC- MS) analysis.

33. The engineered microorganism of any of claims 1-32, wherein the microorganism produces triacylglycerols (TAGs), and the ratio of OPO:OLO is about 1 or more, or about 5 or more, as determined by liquid chromatography-mass spectrometry (LC- MS) analysis.

34. The engineered microorganism of any of claims 1-33, wherein the microorganism produces triacylglycerols (TAGs), and the ratio of OPO:OSO is about 2 or more as determined by liquid chromatography-mass spectrometry (LC-MS) analysis.

35. The engineered microorganism of any of claims 1-34, wherein the microorganism produces triacylglycerols (TAGs). and the ratio of OPO:POP is about 1 or more, or about 3 or more.Leydig 51584311836. A method of producing l,3-dioleoyl-2-palmitoyl glycerol (OPO), the method comprising culturing the engineered microorganism of any of claims 1-35.

37. The method of claim 36, wherein the microorganism is aerobically cultured .

38. The method of claim 37, wherein the method further comprises harvesting OPO from the yeast culture, and wherein the harvested OPO is refined, bleached and deodorized.

39. A triacylglycerol (TAG) composition made by the engineered microorganism of any of claims 1-35 or the method of any of claims 36-38.

40. A method of producing an engineered microorganism of any of claims 1-35, the method comprising introducing into a microorganism (i) a heterologous nucleic acid encoding a first lysophosphatidic acid acyltransferase (LPAAT), and (ii) a heterologous nucleic acid encoding a glycerol-3-phosophate acyltransferase (GPAT), a diacylglycerol acyltransferase (DGAT), or a second LPAAT.

41. The method of claim 40, further comprising disrupting the activity of an LPAAT native to the microorganism.

42. The method of claim 40 or 41, wherein the heterologous gene of (i) or (ii) is introduced by random integration.

43. The method of any of claims 40-42, wherein at least (i) or (ii) is introduced at a locus that disrupts the activity of a native LPAAT, native GPAT, or native DGAT.

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