Region-specific incorporation of fatty acids in triglyceride oils

Abstract

Provided herein are triglyceride oil compositions that are enriched in 1, 3-dioleoyl-2-palmitoyl glycerol (OPO). Also provided herein are methods of producing OPO-rich non-naturally occurring triglyceride oil compositions from non-naturally occurring microorganisms and their use in a variety of end products, including, for example, polyols and nutritional supplements.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 336,767, filed on April 29, 2022, and U.S. Provisional Application No. 63 / 420,188, filed on October 28, 2022, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on April 27, 2023, is named 50727-729_601_SL.xml, and is 480,759 bytes in size.

[0005] background

[0006] Oleaginous microorganism has the ability that carbon substrate is converted into oil (comprising triacylglycerol (TAG) and lipid) and accumulates these oils in the cell.Some microorganisms can have the ability of accumulating lipid with the amount of up to 80% dry weight.Therefore, oleaginous microorganism, comprises little algae, bacterium, fungi and yeast, can be used as the productive source of bio-based oil production.Hereditary and non-genetic modification technology can allow the generation of non-natural oil with specific fatty acid profile.

[0007] Incorporated by Reference

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments utilizing the principles of the present invention, wherein:

[0011] Figure 1 Two TAG species with the same molecular weight showing different daughter ion ratios are shown. Panel A shows OOP and Panel B shows OPO.

[0012] Figure 2Shown are mass spectra of OOP / OPO triglycerides from oils produced by various microalgae strains provided herein. Panel A shows CHK22, D552-1; Panel B shows CHK22, D552-3; Panel C shows CHK22, D552-4; Panel D shows non-transgenic strain CHK22.

[0013] Figure 3 , Figures A-C show an amino acid sequence alignment of the LPAATs described herein. EcPlsC (SEQ ID NO:22), OlLPAAT2 (SEQ ID NO:63), PmLPAAT2 (SEQ ID NO:24), CosLPAAT2 (SEQ ID NO:64), CrLPAAT2 (SEQ ID NO:13), ChsLPAAT2 (SEQ ID NO:14), CiLPAAT2 (SEQ ID NO:15), VaLPAAT2 (SEQ ID NO:21), VcLPAAT2 (SEQ ID NO:20), NoLPAT4 (SEQ ID NO:17), NoLPAT3 (SEQ ID NO:16), PmLPAAT1 (SEQ ID NO:23), Sll1848 (SEQ ID NO:19), BnBAT2 (SEQ ID NO:18), and CrLPAAT1 (SEQ ID NO:12).

[0014] Figure 4 A schematic diagram showing the domain structure of the LPAATs described herein is shown.

[0015] Figure 5 is a bar graph showing the fatty acid composition of oils from various algal strains, including strains that have been transformed with pCHK385, at the sn-2 position.

[0016] Figure 6 LC-MS results for oils from CHK22 and strain D552-3 are shown.

[0017] Figure 7 Shown are OPO-rich TAG oil and downstream TAG intermediates generated by the chemical conversion process.

[0018] Figure 8 Exemplary diols produced from OPO-rich TAG oil are shown.

[0019] Fig. 9 An overview of an exemplary classical strain improvement strategy for generating mutants derived from CHK22 (UTEX 1533) is shown.

[0020] Fig.10The strain improvement strategy for CHK22 to produce CHK100 is shown.

[0021] Fig.11 The transmembrane domain of the LPAAT disclosed herein is shown.

[0022] Overview

[0023] In some embodiments, the present invention provides a non-naturally occurring oil, the naturally occurring oil comprising: at least 10 mg ergosterol per 100 g oil; and a triacylglycerol (TAG) component, wherein at least 40% of the TAGs in the TAG component are TAG species with saturated fatty acids at the sn-2 position, wherein at least 50% of the acyl chains in the TAG component are C18: 1. The saturated fatty acid may be C16: 0. At least 50% of the TAGs in the TAG component may be TAG species with saturated fatty acids at the sn-2 position. At least 50% of the acyl chains in the TAG component may be C18: 1. The TAG species may comprise C18: 1 at the sn-1 and sn-3 positions. The TAG species may comprise C16: 0 at the sn-2 position. The TAG species may comprise or consist of: 1,3-dioleoyl-2-palmitoylglycerol (OPO). At least 50% of the acyl chains in the TAG component can be C18: 1, and at least 20% of the acyl chains in the TAG component can be C16: 0. At least 60% of the acyl chains in the TAG component can be C18: 1, and at least 30% of the acyl chains in the TAG component can be C16: 0. In some embodiments, 50%-67% or 60%-67% of the acyl chains in the TAG component can be C18: 1, and 20%-33% of the acyl chains in the TAG component can be C16: 0. The oil can have an OP: OO m / z ratio of at least 1.6, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil can have an OP: OO m / z ratio of at least 2, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil can have an OP:OO m / z ratio of at least 3, as determined by the abundance of DAG ions produced by the mass spectrometry fragmentation of the TAG component in the oil. The oil can have an OP:OO m / z ratio of at least 4, as determined by the abundance of DAG ions produced by the mass spectrometry fragmentation of the TAG component in the oil. The oil can contain at least 50mg ergosterol per 100g oil. The oil can contain at least 100mg ergosterol per 100g oil. The oil can contain 100-200mg ergosterol per 100g oil. The oil can also contain no more than 5mg campesterol, no more than 5mg β-sitosterol or no more than 5mg stigmasterol per 100g oil. For example, the oil can contain no more than 5mg campesterol per 100g oil. In some embodiments, the oil does not contain campesterol. For example, the oil can contain no more than 5mg stigmasterol per 100g oil. In some embodiments, the oil does not contain stigmasterol. For example, the oil may comprise no more than 5 mg beta-sitosterol per 100 g of oil. In some embodiments, the oil does not comprise beta-sitosterol.The oil may also contain one or more of ergosta-5,9-dien-3-ol, (3β)-; 5.xi.-ergosta-7-ene-3β-ol; 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)-; and ergosta-7,22-dien-3-ol, (3β). The oil may contain at least 1 mg of ergosta-5,9-dien-3-ol, (3β)- per 100 g of oil. The oil may contain 1-50 mg of ergosta-5,9-dien-3-ol, (3β)- per 100 g of oil. The oil may contain at least 15 mg of 5.xi.-ergosta-7-ene-3β-ol per 100 g of oil. The oil may contain 1-50 mg of 5.xi.-ergosta-7-ene-3β-ol per 100 g of oil. The oil may contain at least 20 mg of 5.xi.-ergost-7-ene-3β-ol per 100 g of oil. The oil may contain at least 5 mg of 9,19-cyclolanostane-3-ol, 24-methylene-, (3β)- per 100 g of oil. The oil may be an algae oil. The oil may be a genetically modified algae oil. The oil may be produced by algae cells. The oil may be produced by genetically modified algae cells. The oil may be produced by Prototheca cells.

[0024] Disclosed herein are compositions comprising oil and one or more excipients. The composition can be a nutritional supplement. The composition can be an infant formula. The infant formula can include one or more of whey, casein, lactose, vitamin D, human milk oligosaccharides (HMO), vegetable oils and antibodies. Non-limiting examples of vegetable oils include soybean oil, rapeseed oil, sunflower oil, coconut oil, palm oil and palm kernel oil. Infant formula can include soy protein. Disclosed herein are TAG polyols produced by oil.

[0025] Disclosed herein is a method for producing TAG polyols, the method comprising: subjecting an oil described herein to epoxidation to generate an epoxidized oil; and ring-opening the epoxidized oil in the presence of an alcohol, an acid, or hydrogen and a suitable catalyst to generate a TAG polyol. The epoxidized oil can be ring-opened in the presence of an alcohol. The epoxidized oil can be ring-opened in the presence of hydrogen and a suitable catalyst. The epoxidized oil can be ring-opened in the presence of an acid. Disclosed herein is a TAG polyol produced by the method described herein.

[0026] Disclosed herein is a method for producing TAG polyols, the method comprising: subjecting an oil described herein to hydroformylation to produce a hydroformylated oil; and reducing the hydroformylated oil in the presence of hydrogen and a suitable catalyst to produce a TAG polyol. Disclosed herein is a TAG polyol prepared by the method described herein.

[0027] Disclosed herein is a microalgae cell comprising an exogenous gene encoding an enzyme having lysophosphatidic acid acyltransferase activity, wherein the cell produces an oil comprising a TAG component, wherein at least 40% of the TAGs in the TAG component may be TAG species having a saturated fatty acid at the sn-2 position, wherein at least 50% of the acyl chains in the TAG component may be C18: 1. The enzyme may be a lysophosphatidic acid acyltransferase (LPAAT). The enzyme may be an algal lysophosphatidic acid acyltransferase (LPAAT).

[0028] In some embodiments, the enzyme disclosed herein may comprise a sequence motif selected from any one of (I) NHXXXXD (or NHX4D); (II) (F / Y)XXR; (III) EGXR; and (IV) proline, wherein X is any amino acid.

[0029] In some embodiments, the enzyme may comprise NHXXXXD (or NHX4D), wherein X is any amino acid. In some embodiments, the enzyme may comprise a sequence having at least 70%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 107-109.

[0030] In some embodiments, the enzyme may comprise (F / Y)XXR, wherein X is any amino acid. In some embodiments, the enzyme may comprise a sequence having at least 60%, at least 75%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 110-119.

[0031] In some embodiments, the enzyme may comprise EGXR, wherein X is any amino acid. In some embodiments, the enzyme may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 120-134.

[0032] In some embodiments, the enzyme may comprise a sequence having a conserved proline. In some embodiments, the enzyme may comprise a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90% or 100% sequence identity to any one of SEQ ID NOs: 135-154.

[0033] The enzyme may be Chlamydomonas reinhardtii lysophosphatidic acid acyltransferase (CrLPAAT1). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0034] The enzyme may be Chlamydomonas incerta lysophosphatidic acid acyltransferase (CiLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

[0035] The enzyme may be a Chlamydomonas schloesseri lysophosphatidic acid acyltransferase (ChsLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0036] The enzyme may be Volvox africanus lysophosphatidic acid acyltransferase (VaLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0037] The enzyme may be Nannochloropsis oceanica lysophosphatidic acid acyltransferase (NoLPAT3). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0038] The enzyme may be Synechocystis sp. 1-acyl-sn-glycerol-3-phosphate acyltransferase (Sll1848). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0039] The enzyme may be Chlamydomonas reinhardtii lysophosphatidic acid acyltransferase (CrLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

[0040] The enzyme may be Escherichia coli 1-acyl-sn-glycerol-3-phosphate acyltransferase (EcPlsC). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 22. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

[0041] The enzyme may be Volvox carteri lysophosphatidic acid acyltransferase (VcLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:25.

[0042] The enzyme may be a Brassica napus acyltransferase (BnBAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 18. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3. The enzyme may be a marine Nannochloropsis algae lysophosphatidic acid acyltransferase (NoLPAT4). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0043] The enzyme may be Astrephomene gubernaculifera lysophosphatidic acid acyltransferase (AgLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0044] The enzyme may be Edaphochlamys debaryana lysophosphatidic acid acyltransferase (EdLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 42. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27.

[0045] The enzyme may be Dunaliella salina lysophosphatidic acid acyltransferase (DsLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0046] The enzyme may be Scenedesmus sp. lysophosphatidic acid acyltransferase (SceLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0047] The enzyme may be Micractinium conductrix lysophosphatidic acid acyltransferase (McLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.

[0048] The enzyme may be Chlorella sorokiniana lysophosphatidic acid acyltransferase (ChsoLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31.

[0049] The enzyme may be Chlorella variabilis lysophosphatidic acid acyltransferase (ChvaLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 47. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.

[0050] The enzyme may be Raphidocelis subcapitata lysophosphatidic acid acyltransferase (RsLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 48. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0051] The enzyme may be Chlorella desiccata lysophosphatidic acid acyltransferase (ChdeLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0052] The enzyme may be Auxenochlorella protothecoides lysophosphatidic acid acyltransferase (ApLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35.

[0053] The enzyme may be a Chloropicon primus lysophosphatidic acid acyltransferase (ChprPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 51. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36.

[0054] The enzyme may be Homo sapiens lysophosphatidic acid acyltransferase (AGPAT1). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 52. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37.

[0055] The enzyme may be Chlamydomonas eustigma lysophosphatidic acid acyltransferase (CeLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0056] The enzyme may be a Pedinophyceae lysophosphatidic acid acyltransferase (PedLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 54. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.

[0057] The enzyme may be Volvox reticuliferus lysophosphatidic acid acyltransferase (VrLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 55. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57.

[0058] The enzyme may be Chlorella vulgaris lysophosphatidic acid acyltransferase (ChvuLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 56. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58.

[0059] The enzyme may be Volvulina compacta lysophosphatidic acid acyltransferase (VcomLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 83. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

[0060] The enzyme may be Vitreochlamys sp. CL-2021 lysophosphatidic acid acyltransferase (VitrLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 84. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 66.

[0061] The enzyme may be a Colemanosphaera charkowiensis lysophosphatidic acid acyltransferase (CchaLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67.

[0062] The enzyme may be Pleodorina japonica lysophosphatidic acid acyltransferase (PjapLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 86. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 68.

[0063] The enzyme may be Volvulina boldii lysophosphatidic acid acyltransferase (VbolLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 87. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 69.

[0064] The enzyme may be Pandorina morum lysophosphatidic acid acyltransferase (PmorLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 88. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 70.

[0065] The enzyme may be Volvox carteri f. weismannia lysophosphatidic acid acyltransferase (VcarfLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71.

[0066] The enzyme may be Eudorina cylindrica lysophosphatidic acid acyltransferase (EcylLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 90. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 72.

[0067] The enzyme may be Gonium multicoccum lysophosphatidic acid acyltransferase (GmulLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 91. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 73.

[0068] The enzyme may be Gonium viridistellatum lysophosphatidic acid acyltransferase (GvirLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 92. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

[0069] The enzyme may be Volvox ferrisii lysophosphatidic acid acyltransferase (VferLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 93. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 75.

[0070] The enzyme may be Vitreochlamys aulata lysophosphatidic acid acyltransferase (VaulLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 94. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 76.

[0071] The enzyme may be Chlamydomonas sp. CCAC2762_B lysophosphatidic acid acyltransferase (Ch_CCAC2762_LPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 95. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.

[0072] The enzyme may be Dunaliella salina lysophosphatidic acid acyltransferase (DsalLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 96. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0073] The enzyme may be Microglena sp. YARC lysophosphatidic acid acyltransferase (MyarcLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 97. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.

[0074] The enzyme may be Chlamydomonas sp. UWO_241 lysophosphatidic acid acyltransferase (CuwoLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 98. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

[0075] The enzyme may be Chlamydomonas moewusii lysophosphatidic acid acyltransferase (CmoeLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 99. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0076] The enzyme may be Oophila amblystomatis lysophosphatidic acid acyltransferase (OambLPAAT2). The enzyme may comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 100. The cell may comprise a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0077] Exogenous genes can be codon optimized for expression in Prototheca strains. In some embodiments, the cell does not contain an exogenous glycerol-3-phosphate acyltransferase (GPAT1). The cell can be from a Prototheca base strain. The cell can be from a mulberry-shaped Prototheca moriformis base strain UTEX1533. The cell can be derived from a classically improved strain of a mulberry-shaped Prototheca base strain UTEX 1533. The cell can be from a non-genetically modified Prototheca base strain producing an oil with a fatty acid profile of at least 50% oleic acid. The cell can be from a non-genetically modified Prototheca base strain producing an oil with a fatty acid profile of at least 30% palmitic acid. The Prototheca base strain can be Prototheca wickerhamii. The Prototheca base strain can be Prototheca moriformis. Microalgae cells can produce at least 60% lipid by dry cell weight. Microalgae cells can produce at least 50% lipid by dry cell weight. Oil can include triacylglyceride (TAG) component, wherein at least 40% TAG in the TAG component can be TAG species with saturated fatty acid at the sn-2 position, wherein at least 50% acyl chain in the TAG component can be C18:1. Oil can include at least 10mg ergosterol per 100g oil. Saturated fatty acid can be C16:0. At least 50% TAG in the TAG component can be TAG species with saturated fatty acid at the sn-2 position. At least 50% acyl chain in the TAG component can be C18:1. TAG species can include C18:1 at sn-1 and sn-3 positions. TAG species can include C16:0 at the sn-2 position. The TAG species may comprise or consist of 1,3-dioleoyl-2-palmitoylglycerol (OPO). At least 50% of the acyl chains in the TAG component may be C18:1, and at least 20% of the acyl chains in the TAG component may be C16:0. At least 60% of the acyl chains in the TAG component may be C18:1, and at least 30% of the acyl chains in the TAG component may be C16:0. In some embodiments, 50%-67% or 60%-67% of the acyl chains in the TAG component may be C18:1, and 20%-33% of the acyl chains in the TAG component may be C16:0. The oil may have an OP:OO m / z ratio of at least 1.6, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil may have an OP:OO m / z ratio of at least 2, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil may have an OP:OO m / z ratio of at least 3, as determined by the abundance of DAG ions produced by mass spectrometric fragmentation of the TAG component in the oil. The oil may comprise at least 50 mg ergosterol per 100 g of oil.The oil may contain at least 100 mg of ergosterol per 100 g of oil. The oil may also contain one or more of 5.xi.-ergost-7-ene-3β-ol; 9,19-cyclolanostane-3-ol, 24-methylene-, (3β)-; and ergosta-7,22-diene-3-ol, (3β). The oil may contain at least 20 mg of 5.xi.-ergost-7-ene-3β-ol per 100 g of oil. The oil may contain at least 5 mg of 9,19-cyclolanostane-3-ol, 24-methylene-, (3β)- per 100 g of oil. The oil may be an algae oil. The oil may be a genetically modified algae oil. The oil may be produced by algae cells. The oil may be produced by genetically modified algae cells. The oil may be produced by Prototheca cells.

[0078] Disclosed herein is a method for producing non-naturally occurring oil, the method comprising culturing microalgae cells in a culture medium, wherein the oil comprises a TAG component, wherein at least 40% of the TAG species in the TAG component have saturated fatty acids at the sn-2 position, wherein at least 50% of the acyl chains in the TAG component can be C18:1. The method can also include separating the oil composition from the culture medium. The method can also include expressing an exogenous enzyme with lysophosphatidic acid acyltransferase activity in the microalgae cell. The microalgae cell can be a microalgae cell described herein. The oil can comprise a triacylglyceride (TAG) component, wherein at least 40% of the TAG in the TAG component can be a TAG species with saturated fatty acids at the sn-2 position, wherein at least 50% of the acyl chains in the TAG component can be C18:1. The oil can comprise at least 10 mg ergosterol per 100 g of oil. The saturated fatty acids can be C16:0. At least 50% of the TAG in the TAG component can be a TAG species with saturated fatty acids at the sn-2 position. At least 50% of the acyl chains in the TAG component may be C18:1. The TAG species may comprise C18:1 at the sn-1 and sn-3 positions. The TAG species may comprise C16:0 at the sn-2 position. The TAG species may comprise or consist of: 1,3-dioleoyl-2-palmitoylglycerol (OPO). At least 50% of the acyl chains in the TAG component may be C18:1, and at least 20% of the acyl chains in the TAG component may be C16:0. At least 60% of the acyl chains in the TAG component may be C18:1, and at least 30% of the acyl chains in the TAG component may be C16:0. 50%-67% or 60%-67% of the acyl chains in the TAG component may be C18:1, and 20%-33% of the acyl chains in the TAG component may be C16:0. The oil may have an OP:OO m / z ratio of at least 1.6, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil may have an OP:OO m / z ratio of at least 2, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil may have an OP:OO m / z ratio of at least 3, as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. The oil may contain at least 50 mg of ergosterol per 100 g of oil. The oil may contain at least 100 mg of ergosterol per 100 g of oil. The oil may also contain one or more of 5.xi.-ergost-7-ene-3β-ol; 9,19-cyclolanostane-3-ol, 24-methylene-, (3β)-; and ergosta-7,22-diene-3-ol, (3β). The oil may contain at least 20 mg 5.xi.-ergost-7-en-3β-ol per 100 g of oil. The oil may contain at least 5 mg 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)- per 100 g of oil. The oil may be an algae oil.The oil may be a genetically modified algae oil. The oil may be produced by algae cells. The oil may be produced by genetically modified algae cells. The oil may be produced by Protocystis cells.

[0079] Details

[0080] Provided herein is an oil composition comprising the TAG rich in 1,3-dioleoyl-2-palmitoyl glycerol (OPO), its preparation method, preparation and application.Oil composition provided herein can be produced by the microorganism of genetic modification provided herein such as the microalgae cell provided herein.Produce and cultivate the method for the microorganism of oil composition provided herein.Provided herein also is a bioreactor comprising the non-natural existence provided herein that has been modified to produce the microorganism of oil composition provided herein.

[0081] definition

[0082] As used herein, the term "microbial oil" refers to oil produced or extracted from a microorganism (microbe), such as an oleaginous, unicellular, eukaryotic or prokaryotic microorganism, including but not limited to microalgae, yeast, bacteria, and fungi.

[0083] As used herein, the term "triacylglycerol," "triglyceride," or "TAG" refers to an ester between glycerol and three fatty acids.

[0084] As used herein, the term "OPO" or "1,3-dioleoyl-2-palmitoylglycerol" refers to a TAG species having palmitate (C16:0) at the sn-2 position of the TAG and oleate (C18:1) at the sn-1 and sn-3 positions of the TAG.

[0085] As used herein, the term "OOP" or "1,2-dioleoyl-3-palmitoylglycerol" refers to a TAG species having palmitate (C16:0) at the sn-3 position of the TAG and oleate (C18:1) at the sn-1 and sn-2 positions of the TAG.

[0086] As used herein, the term "polyol" refers to a triglyceride or fatty acid alcohol containing a hydroxyl functional group. As used herein, the term "polyol derived from TAG oil" generally refers to a polyol obtained from the chemical conversion of TAG oil, such as via epoxidation and ring opening, ozonolysis and reduction, or hydroformylation and reduction.

[0087] As used herein, the term "polyurethane," "PU," or "urethane" refers to a class of polymers that include carbamate (urethane) linkages formed between polyol and isocyanate moieties.

[0088] As used herein, the term "high oleic" may refer to greater than 60% oleic acid, greater than 70% oleic acid, greater than 80% oleic acid, or greater than 90% oleic acid.

[0089] As used herein, the term "about" refers to ± 10% of the provided value.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the present teachings, some exemplary methods and materials are described herein.

[0091] Human breast milk

[0092] Human breast milk is the primary source of nutrition for newborns. Breast milk includes fats, proteins, carbohydrates, and a variety of vitamins and minerals necessary to support healthy growth and development. Breast milk also contains factors that are important for protecting infants from infection and inflammation, while also contributing to the healthy development of the immune system and gut microbiome.

[0093] Breast milk contains about 3%-55% fat, and 98% of it is in the form of TAGs containing over 200 different fatty acids. Breast milk contains about 17%-25% palmitic acid, and 70%-75% of it is uniquely distributed in the stereochemical number 2 (sn-2) position of the TAG, while unsaturated fatty acids such as oleic acid and linoleic acid are mainly distributed in the sn-1 and sn-3 positions. The high content of palmitic acid in the sn-2 position may help enhance the absorption of calcium and essential fats in the infant intestine, as well as overall digestive health. The key TAG in human breast milk is 1,3-dioleoyl-2-palmitoylglycerol, also known as sn-2 palmitate, oleic acid-palmitic acid-oleic acid, or OPO. OPO is present in human breast milk, but is not generally present in bovine milk.

[0094] In view of the complexity of human breast milk, infant formula manufacturers have been working hard to reproduce even a small portion of the characteristics present in this extraordinary natural food. Some attention has been paid to achieve the relative ratio of the desired fatty acid in infant formula to reproduce the components of breast milk. Products that can be combined with other fat parts to simulate the TAG of human breast milk that are rich in OPO have also been produced. Human milk fat substitute (HMFS) is a class of structured lipids widely used as the ingredient of infant formula. Like human milk fat, HMFS is characterized in that it is rich in palmitoyl (C16:0) groups, particularly in the middle (sn-2 or β) position on the glycerol backbone. However, there is no fat substitute from a single source that can provide all the demonstrated benefits of natural products.

[0095] Although the fat used in most infant formula products comes from plants, many plants esterify saturated fatty acids to sn-1 and sn-3 positions, but do not include palmitic acid at the sn-2 position. In contrast, engineered oilseed crops and / or oil-producing microorganisms can be used to accumulate high levels of TAGs rich in saturated fatty acids at the sn-2 position, such as OPO. Therefore, alternative sources of these TAGs can better simulate the stereoisomeric structure of human milk fat and provide an improved HMF source for infant nutrition.

[0096] TAG biosynthesis

[0097] Lysophosphatidic acid acyltransferase (LPAAT) is a membrane-bound o-acyltransferase (MBOAT) encoded by a multigene family and is the rate-limiting enzyme in the Kennedy pathway of higher plants. The Kennedy pathway converts glycerol-3-phosphate to TAG using glycerol-3-phosphate acyltransferase (GPAT), LPAAT, phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT). In this pathway, LPAAT controls the production of the key intermediate phosphatidic acid (PA).

[0098] LPAAT genes are present in prokaryotes and eukaryotes. In mammals, LPAAT has been characterized as the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. For example, the Homo sapiens AGPAT gene is AGPAT1. AGPAT1 is located in the endoplasmic reticulum. In some embodiments, the amino acid sequence of AGPAT1 is SEQ ID NO:52. In some embodiments, AGPAT1 is expressed in cells by transforming with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:37. Plants also generally have several LPAAT genes; for example, Arabidopsis thaliana has 5 LPAAT genes. In most microalgae and higher plants, TAG biosynthesis occurs in chloroplasts and endoplasmic reticulum (ER), and the substrate preference of LPAAT enzymes is usually oleoyl-CoA. However, in other eukaryotes, including animals and fungi, the major organelle for TAG biosynthesis is the endoplasmic reticulum (ER), and similarly, the substrate preference of LPAAT enzymes is generally oleoyl-CoA.

[0099] An enzyme having lysophosphatidic acid acyltransferase activity may comprise four catalytic motifs: (I) NHXXXXD (or NHX4D); (II) (F / Y) XXR; (III) EGXR; and (IV) proline, wherein X is any amino acid. Motif I may comprise a conserved NHXXXXD (or NHX4D) sequence, wherein the residue after histidine (H) is usually hydrophilic, and the residue before aspartic acid (D) is almost always hydrophobic. Motif II may comprise a (F / Y) XXR sequence, wherein the first residue is phenylalanine (F) or tyrosine (Y), followed by two residues and a conserved arginine (R). The residue before the R may be hydrophilic, and the residue before the hydrophilic residue may be hydrophobic. Motif III may comprise a conserved EGXR sequence. Motif IV may comprise a conserved proline (P). The enzyme described herein may comprise a sequence having one, two, three, or four of these catalytic motifs. The cells described herein may contain an exogenous gene containing a sequence encoding an enzyme having one, two, three, or four of these catalytic motifs.

[0100] In some embodiments, the enzyme may comprise a sequence having at least 70%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 107-109. In some embodiments, the cell may comprise an exogenous gene comprising a sequence encoding an enzyme having at least 70%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 107-109.

[0101] In some embodiments, the enzyme may comprise a sequence having at least 60%, at least 75%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 110-119. In some embodiments, the cell may comprise an exogenous gene comprising a sequence encoding an enzyme having at least 60%, at least 75%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 110-119.

[0102] In some embodiments, the enzyme may comprise EGHR or EGTR. In some embodiments, the enzyme may comprise a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 120-134. In some embodiments, the cell may comprise an exogenous gene comprising a sequence encoding an enzyme having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 120-134.

[0103] In some embodiments, the enzymes described herein may comprise catalytic motif IV: conserved proline. In some embodiments, the enzymes described herein may comprise a third domain having a sequence of conserved proline. In some embodiments, the enzyme may comprise a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 135-154. In some embodiments, the cell may comprise an exogenous gene comprising a sequence encoding an enzyme having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 135-154.

[0104] Chlamydomonas reinhardtii is a unicellular green algae that contains two LPAAT genes: CrLPAAT1 and CrLPAAT2. The green algae-specific CrLPAAT2 enzyme is localized to the ER membrane. However, unlike typical ER-localized LPAATs, CrLPAAT2 prefers palmitoyl-CoA rather than oleoyl-CoA as an acyl donor substrate. In some embodiments, the amino acid sequence of CrLPAAT1 is SEQ ID NO: 12. In some embodiments, CrLPAAT1 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence of CrLPAAT2 is SEQ ID NO: 13. In some embodiments, CrLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. The related species Chlamydomonas schloesseri, Chlamydomonas incerta, and Chlamydomonas eustigma have similar genes ChsLPAAT2, CiLPAAT2, and CeLPAAT2, respectively. In some embodiments, the amino acid sequence of ChsLPAAT2 is SEQ ID NO: 14. In some embodiments, ChsLPAAT1 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the amino acid sequence of CiLPAAT2 is SEQ ID NO: 15. In some embodiments, CiLPAAT1 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the amino acid sequence of CeLPAAT2 is SEQ ID NO: 53. In some embodiments, CeLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38.

[0105] The marine microalgae genus Nannochloropsis has 4 LPAAT genes. Two of these genes, NoLPAT3 and NoLPAT4, are essential for TAG biosynthesis. NoLPAT3 and NoLPAT4 are usually present around the periphery of lipid droplets, although these two genes are not related to other lipid droplet-related proteins. NoLPAT3 and NoLPAT4 enhance the accumulation of TAGs at the position of lipid droplet periphery, especially during nutrient deprivation. In some embodiments, the amino acid sequence of NoLPAT3 is SEQ ID NO:16. In some embodiments, NoLPAT3 is expressed in cells by transforming with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of NoLPAT4 is SEQ ID NO 17. In some embodiments, NoLPAT4 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0106] In oilseed rape Brassica napus, one of the LPAAT genes involved in TAG biosynthesis is BnBAT2. BnBAT2 is a prokaryotic LPAAT gene. In some embodiments, the amino acid sequence of BnBAT2 is SEQ ID NO: 18. In some embodiments, BnBAT2 is expressed in cells by transforming with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3.

[0107] The cyanobacteria Synechocystis species LPAAT gene is referred to as Sll1848. Sll1848 incorporates C18:0 and C18:1 in the sn-2 position and is the major LPAAT specific for C16:0 in Synechocystis species. In some embodiments, the amino acid sequence of Sll1848 is SEQ ID NO: 19. In some embodiments, Sll1848 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0108] Species of green algae such as Volvox carterii, Volvox africanus, and Volvox reticularis also have similar LPAAT genes - VcLPAAT2, VaLPAAT2, and VrLPAAT2, respectively. In some embodiments, the amino acid sequence of VcLPAAT2 is SEQ ID NO: 20 or SEQ ID NO: 40. In some embodiments, VcLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 25. In some embodiments, the amino acid sequence of VaLPAAT2 is SEQ ID NO: 21. In some embodiments, VaLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the amino acid sequence of VrLPAAT2 is SEQ ID NO: 55. In some embodiments, VrLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57.

[0109] The LPAAT gene in Escherichia coli is referred to as the PlsC gene. In Escherichia coli (E. coli), the EcPlsC mutant strain is characterized by lacking LPAAT activity. The strain is also temperature sensitive and cannot grow at 42°C. In some embodiments, the amino acid sequence of EcPlsC is SEQ ID NO:22. In some embodiments, EcPlsC is expressed in cells by transforming with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQID NO:6.

[0110] The LPAAT of the microalga Prosporangium moriformis exhibits a strong preference for inserting unsaturated fatty acids at the sn-2 position, and rarely inserts saturated fatty acids. In some embodiments, the amino acid sequence of PmLPAAT1 is SEQ ID NO: 23. In some embodiments, the amino acid sequence of PmLPAAT2 is SEQ ID NO: 24.

[0111] Species of green algae such as Chlorella sorokinensis, Chlorella variabilis, Chlorella sicca and Chlorella vulgaris also have similar LPAAT genes - ChsoLPAAT2, ChvaLPAAT2, ChdeLPAAT2 and ChvuLPAAT2, respectively. In some embodiments, the amino acid sequence of ChsoLPAAT2 is SEQ ID NO: 46. In some embodiments, ChsoLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the amino acid sequence of ChvaLPAAT2 is SEQ ID NO: 47. In some embodiments, ChvaLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the amino acid sequence of ChdeLPAAT2 is SEQ ID NO: 49. In some embodiments, ChdeLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the amino acid sequence of ChvuLPAAT2 is SEQ ID NO: 56. In some embodiments, ChvuLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:58.

[0112] The multicellular green alga Astrephomene gubernaculifera comprises a LPAAT gene called AgLPAAT2. In some embodiments, the amino acid sequence of AgLPAAT2 is SEQ ID NO: 41. In some embodiments, AgLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0113] The green alga Edaphochlamys debaryana comprises a LPAAT gene called EdLPAAT2. In some embodiments, the amino acid sequence of EdLPAAT2 is SEQ ID NO: 42. In some embodiments, EdLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27.

[0114] The unicellular green algae Dunaliella salina contains an LPAAT gene called DsLPAAT2. In some embodiments, the amino acid sequence of DsLPAAT2 is SEQ ID NO: 43. In some embodiments, DsLPAAT2 is expressed in cells by transformation with a plasmid containing a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0115] The colony green alga Scenedesmus species comprises a LPAAT gene called SceLPAAT2. In some embodiments, the amino acid sequence of SceLPAAT2 is SEQ ID NO: 44. In some embodiments, SceLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29.

[0116] The colony green alga Volvulina compacta comprises a LPAAT gene called VcomLPAAT2. In some embodiments, the amino acid sequence of VcomLPAAT2 is SEQ ID NO: 83. In some embodiments, VcomLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

[0117] The colony green algae Heterozoites japonicus comprises an LPAAT gene called PjapLPAAT2. In some embodiments, the amino acid sequence of PjapLPAAT2 is SEQ ID NO: 86. In some embodiments, PjapLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 68.

[0118] The colony green alga Volvulina boldii comprises a LPAAT gene called VbolLPAAT2. In some embodiments, the amino acid sequence of VbolLPAAT2 is SEQ ID NO: 87. In some embodiments, VbolLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0119] The colony green alga Gonium multicoccum comprises a LPAAT gene called GmulLPAAT2. In some embodiments, the amino acid sequence of GmulLPAAT2 is SEQ ID NO: 91. In some embodiments, GmulLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 73.

[0120] The colony green alga Gonium viridistellatum comprises a LPAAT gene called GvirLPAAT2. In some embodiments, the amino acid sequence of GvirLPAAT2 is SEQ ID NO: 92. In some embodiments, GvirLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 74.

[0121] The unicellular green alga Micractinium conductrix comprises a LPAAT gene called McLPAAT2. In some embodiments, the amino acid sequence of McLPAAT2 is SEQ ID NO: 45. In some embodiments, McLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.

[0122] The unicellular green algae Dunaliella salina comprises a LPAAT gene called DsalLPAAT2. In some embodiments, the amino acid sequence of DsalLPAAT2 is SEQ ID NO: 96. In some embodiments, DsalLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 78.

[0123] The microalgae Raphidocelis subcapita comprises an LPAAT gene called RsLPAAT2. In some embodiments, the amino acid sequence of RsLPAAT2 is SEQ ID NO: 48. In some embodiments, RsLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0124] The heterotrophic green algae Chlorella protothecoides contains a LPAAT gene called ApLPAAT2. In some embodiments, the amino acid sequence of ApLPAAT2 is SEQ ID NO: 50. In some embodiments, ApLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 35.

[0125] The green alga Chloropicon primus comprises a LPAAT gene called ChprLPAAT2. In some embodiments, the amino acid sequence of ChprLPAAT2 is SEQ ID NO: 51. In some embodiments, ChprLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36.

[0126] Chlorophyceae and PedLPAAT2 contain an LPAAT gene called PedLPAAT2. In some embodiments, the amino acid sequence of PedLPAAT2 is SEQ ID NO: 54. In some embodiments, PedLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, PedLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 59. In some embodiments, PedLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 60. In some embodiments, PedLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 61. In some embodiments, PedLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 62.

[0127] The green algae Vitreochlamys species CL-2021 comprises an LPAAT gene called VitrLPAAT2. In some embodiments, the amino acid sequence of VitrLPAAT2 is SEQ ID NO: 84. In some embodiments, VitrLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 66.

[0128] The green alga Colemanosphaera charkowiensis comprises a LPAAT gene called CchaLPAAT2. In some embodiments, the amino acid sequence of CchaLPAAT2 is SEQ ID NO: 85. In some embodiments, CchaLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 67.

[0129] The green alga Volvulina boldii comprises a LPAAT gene called VbolLPAAT2. In some embodiments, the amino acid sequence of VbolLPAAT2 is SEQ ID NO: 87. In some embodiments, VbolLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69.

[0130] The green algae Chlorella contains a LPAAT gene called PmorLPAAT2. In some embodiments, the amino acid sequence of PmorLPAAT2 is SEQ ID NO: 88. In some embodiments, PmorLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 70.

[0131] The green alga Volvox f. weismannia comprises a LPAAT gene called VcarfLPAAT2. In some embodiments, the amino acid sequence of VcarfLPAAT2 is SEQ ID NO: 89. In some embodiments, VcarfLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 71.

[0132] The green alga Eudorina cylindrica comprises a LPAAT gene called EcylLPAAT2. In some embodiments, the amino acid sequence of EcylLPAAT2 is SEQ ID NO: 90. In some embodiments, EcylLPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 72.

[0133] The green alga Volvox ferrisii comprises a LPAAT gene called VferLPAAT2. In some embodiments, the amino acid sequence of VferLPAAT2 is SEQ ID NO: 93. In some embodiments, VferLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 75.

[0134] The green alga Vitreochlamys aulata comprises a LPAAT gene called VaulLPAAT2. In some embodiments, the amino acid sequence of VaulLPAAT2 is SEQ ID NO: 94. In some embodiments, VaulLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 76.

[0135] The green alga Chlamydomonas species CCAC2762_B comprises a LPAAT gene designated Ch_CCAC2762_LPAAT2. In some embodiments, the amino acid sequence of Ch_CCAC2762_LPAAT2 is SEQ ID NO: 95. In some embodiments, Ch_CCAC2762_LPAAT2 is expressed in a cell by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 77.

[0136] The green algae Echinops species YARC comprises a LPAAT gene called MyarcLPAAT2. In some embodiments, the amino acid sequence of MyarcLPAAT2 is SEQ ID NO: 97. In some embodiments, MyarcLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 79.

[0137] The green alga Chlamydomonas species UWO_241 comprises a LPAAT gene called CuwoLPAAT2. In some embodiments, the amino acid sequence of CuwoLPAAT2 is SEQ ID NO: 98. In some embodiments, CuwoLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80.

[0138] The green alga Chlamydomonas moewusii comprises a LPAAT gene called CmoeLPAAT2. In some embodiments, the amino acid sequence of CmoeLPAAT2 is SEQ ID NO: 99. In some embodiments, CmoeLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 81.

[0139] The green alga Oophila amblystomatis comprises a LPAAT gene called OambLPAAT2. In some embodiments, the amino acid sequence of OambLPAAT2 is SEQ ID NO: 100. In some embodiments, OambLPAAT2 is expressed in cells by transformation with a plasmid comprising a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 82.

[0140] The green alga Edaphochlamys debaryana_SRR13719274 comprises a LPAAT gene called EdLPAAT2. In some embodiments, the amino acid sequence of EdLPAAT2 (Edaphochlamys debaryana_SRR13719274) is SEQ ID NO:101.

[0141] The colony green alga Volvulina steinii comprises a LPAAT gene called VsteLPAAT2. In some embodiments, the amino acid sequence of VsteLPAAT2 is SEQ ID NO:102.

[0142] The colony green algae Gonium octonarium comprises a LPAAT gene called GoctLPAAT2. In some embodiments, the amino acid sequence of GoctLPAAT2 is SEQ ID NO: 103.

[0143] The unicellular green algae Chlamydomonas nivalis contains a LPAAT gene called CnivLPAAT2. In some embodiments, the amino acid sequence of CnivLPAAT2 is SEQ ID NO: 104.

[0144] The green alga Pycnococcus provasolii comprises a LPAAT gene called PproLPAAT2. In some embodiments, the amino acid sequence of PproLPAAT2 is SEQ ID NO:105.

[0145] The green alga Bathycoccus prasinos contains a LPAAT gene called BpraLPAAT2. In some embodiments, the amino acid sequence of GoctLPAAT2 is SEQ ID NO:106.

[0146] CrLPAAT1-ΔCST * It is a Chlamydomonas reinhardtii LPAAT with a total of 30 amino acids (aa 12-41; CST signal) removed from CrLPAAT1. BnBAT2-ΔCST * is a Brassica napus LPAAT with a total of 86 amino acids (aa 2-87) removed from BnBAT2. In some embodiments, CrLPAAT1-ΔCST * The amino acid sequence of BnBAT2-ΔCST is SEQ ID NO: 155. In some embodiments, BnBAT2-ΔCST * The amino acid sequence is SEQ ID NO:156.

[0147] TAG oil of the present disclosure

[0148] Provided herein are TAG oil compositions having TAG species with saturated fatty acids at the sn-2 position. In some embodiments, the TAG species is OPO.

[0149] The complexity of oil and physical property can be assessed by fatty acid profile and the TAG profile of the TAG component of oil.Fatty acid profile is the measurement of fatty acid composition, and can be determined by making oil experience transesterification to generate fatty acid methyl ester (FAME) and subsequently by the gas chromatography (GC / FID) quantitative fatty acid type that is equipped with flame ionization detector.Therefore, the fatty acid content of TAG oil provided herein can be determined by GC / FID.Because TAG comprises three kinds of fatty acids arranged along the glycerol backbone in the triglyceride molecule, the number of the possible different region isomers of TAG can be defined by the cube of the number of fatty acid species in oil.

[0150] The genetic expression of the enzyme described herein can be regulated to increase OPO production. The expression of the gene described herein can be regulated separately or in combination with the expression of one or more enzymes described herein to increase OPO production. The genetic expression of the enzyme described herein can be regulated to enhance LPAAT activity. The expression of the gene described herein can be regulated separately or in combination with the expression of one or more enzymes described herein to enhance LPAAT activity. In some embodiments, the cell described herein is modified to express an exogenous gene to enhance LPAAT activity. In some embodiments, the cell described herein is modified to delete an endogenous gene to enhance LPAAT activity.

[0151] Provided herein are genetic modifications and cultured cells to increase the method for producing the oil provided herein.Genetic modifications are also provided herein to increase the cells producing the oil described herein.The cell provided herein can contain recombinant nucleic acids operable to increase the expression of the gene described herein to enhance the activity of LPAAT.In addition, the cell provided herein can exclude endogenous nucleic acids operable to reduce the expression of the gene described herein to enhance the activity of LPAAT.In some embodiments, modify microalgae cells to increase the oil provided herein.

[0152] Oil composition provided herein is produced by microorganism such as microalgae cell.Therefore, the oil composition produced therefrom can include one or more sterol components with algae oil characteristics.The amount of sterol components in oil can be determined based on weight / weight, for example mg sterol in every 100g oil.Sterol composition (i.e. sterol spectrum) can be determined by mass spectrometry, for example gas chromatography-mass spectrometry (GC-MS); Liquid chromatography-mass spectrometry (LC-MS); Tandem mass spectrometry (MS / MS), and coupled liquid phase and gas chromatography with subsequent flame ionization detection (LC-GC-FID).The exemplary sterol spectrum determined by the GC / MS analysis of the TMS ester of the non-saponifiable fraction of the RBD oil produced by CHK22 is presented in Table 26.Cholestane-3-ol (3a, 5b) is used as internal standard (ISTD).

[0153] Table 26: CHK22 RBD sterol profile

[0154]

[0155]

[0156] In some embodiments, the oil contains a positive amount of ergosta-7,22-dien-3-ol, (3β, 22E)-. In some embodiments, the amount of ergosta-7,22-dien-3-ol, (3β, 22E)- in the oil provided herein is at least 2 mg, at least 5 mg, or at least 8 mg or at least 9 mg per 100 g of oil. In some embodiments, the amount of ergosta-7,22-dien-3-ol, (3β, 22E)- in the oil is 1-100 mg, 2-30 mg, 3-20 mg or 5-15 mg per 100 g of oil.

[0157] In some embodiments, oil contains positive amount of ergosterol.In some embodiments, the amount of ergosterol in oil provided herein is every 100g oil at least 50mg, at least 100mg or at least 125mg.In some embodiments, the amount of ergosterol in oil provided herein is every 100g oil at least 10mg, at least 20mg, at least 30mg, at least 40mg, at least 50mg, at least 60mg, at least 70mg, at least 80mg, at least 90mg, at least 100mg, at least 110mg, at least 120mg, at least 130mg, at least 140mg, at least 150mg, at least 160mg, at least 170mg, at least 180mg, at least 190mg or at least 200mg. In some embodiments, the amount of ergosterol in the oil is 10-50 mg, 50-100 mg, 100-150 mg, 150-200 mg, 10-100 mg, 100-200 mg, 100-2000 mg, or 10-200 mg per 100 g of oil.

[0158] In some embodiments, oil contains a positive amount of campesterol, a positive amount of β-sitosterol or a positive amount of stigmasterol. In some embodiments, the amount of campesterol, β-sitosterol or stigmasterol is no more than 5mg per 100g oil. In some embodiments, oil does not contain campesterol. In some embodiments, the amount of campesterol in the oil provided herein is no more than 5mg campesterol per 100g oil. In some embodiments, oil does not contain stigmasterol. In some embodiments, the amount of stigmasterol in the oil is no more than 5mg per 100g oil. In some embodiments, oil does not contain β-sitosterol. In some embodiments, the amount of β-sitosterol in the oil is no more than 5mg per 100g oil. In some embodiments, oil does not contain β-sitosterol. In some embodiments, the amount of β-sitosterol in the oil is no more than 5mg per 100g oil.

[0159] In some embodiments, the oil contains a positive amount of ergosta-5,8-dien-3-ol, (3β)-. In some embodiments, the amount of ergosta-5,8-dien-3-ol, (3β)- is at least 1 mg per 100 g of oil. In some embodiments, the amount of ergosta-5,8-dien-3-ol, (3β)- is at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 6 mg, at least 7 mg, at least 8 mg, at least 9 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg per 100 g of oil. In some embodiments, the amount of ergosta-5,8-dien-3-ol,(3β)- is 1-5 mg, 10-20 mg, 50-100 mg, 100-150 mg, 150-200 mg, 1-50 mg, 20-100 mg, 100-200 mg or 20-200 mg per 100 g of oil.

[0160] In some embodiments, the oil contains a positive amount of 5.xi.-ergost-7-ene-3β-ol. In some embodiments, the amount of 5.xi.-ergost-7-ene-3β-ol is at least 15 mg per 100 g of oil. In some embodiments, the amount of 5.xi.-ergost-7-ene-3β-ol is at least 20 mg per 100 g of oil. In some embodiments, the amount of 5.xi.-ergost-7-en-3β-ol is at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, at least 100 mg, at least 110 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, or at least 200 mg per 100 g of oil. In some embodiments, the amount of 5.xi.-ergost-7-en-3β-ol is 1-50 mg, 20-50 mg, 50-100 mg, 100-150 mg, 150-200 mg, 20-100 mg, 100-2000 mg, or 20-200 mg per 100 g of oil.

[0161] In some embodiments, the oil contains a positive amount of 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)-. In some embodiments, the amount of 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)- is at least 5 mg per 100 g of oil. In some embodiments, the amount of 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)- is at least 1 mg, at least 2 mg, at least 3 mg, at least 4 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, or at least 50 mg per 100 g of oil. In some embodiments, the amount of 9,19-cyclolanostan-3-ol,24-methylene-,(3β)- is 5-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 5-25 mg, 25-50 mg or 5-50 mg per 100 g of oil.

[0162] The TAG spectral analysis of oil composition provided herein can be carried out using liquid chromatography-mass spectrometry (LC-MS). The TAG oil provided herein can be extracted into a solution of 3:1 toluene / 2-propyl alcohol (v / v) from cells by using the mechanical destruction and vigorous stirring of ceramic beads. The filtered extract can then be injected into the LC mass spectrometer for spectral analysis.

[0163] Chromatographic separation of TAG regioisomers cannot be achieved by the described LCMS method. Instead, a qualitative assessment of the regiospecificity of the major TAG species can be made based on the abundance of diacylglycerol (DAG) ions generated by fragmentation of TAG species ( Figure 1 ). In the LC-APCI-MS analysis of TAG, the loss of fatty acids at the sn-1 and sn-3 positions is energetically more favorable than that at the sn-2 position, which results in a greater abundance of DAG ions formed from those fragments. In the case of the OOP regioisomer, an approximately 1:1 ratio of OP DAG ions (m / z 577) and OO DAG ions (m / z 603) can be observed, as shown in Figure 1 In the case of OPO regioisomers, a more abundant OP DAG ion (m / z 577) can be observed, as shown in Figure A. Figure 1 As shown in Figure B.

[0164] Figure 1TAG species with the same molecular weight are shown to show different daughter ion ratios. Two kinds of TAG species OOP and OPO in Figure A and Figure B have the same mass / charge (m / z) ratio with parent ion (859) respectively, but as shown, due to the influence of different collision energies, molecular fragments are very different. Due to the relative inaccessibility of the acyl group at the sn-2 position, low-energy collisions eliminate fatty acyl groups from sn-1 or sn-3 positions with greater frequency. In the case of OOP, as shown in Figure A, low-energy collisions produce daughter ions of m / z of 577 (OP) and 603 (OO), and the less frequent high-energy collisions that cause O to be eliminated from the sn-2 position also produce daughter ions OP of m / z of 577. Therefore, the mass spectrum of the resulting daughter ions is the summation of all these fragments. The peak at 577 (OP) is produced by eliminating O more frequently at the sn-1 place and eliminating O less frequently at the sn-2 place. Thus, the 577 fragment is approximately 1.5-2× the height of the 603 fragment (OO) produced by the more frequent elimination of P at the sn-3 position. Alternatively, and as shown in Figure B, the OPO TAG species displays a very different daughter ion ratio because the products of low energy collisions, PO and OP, have the same m / z of 577, while the daughter ion of the less frequently generated high energy collisions representing OO has an m / z of 603. For this TAG species, the 577 peak height produced by the sum of the low energy derived PO and OP daughter ions is significantly greater than the peak height of the 603 fragment, which requires much higher energy to generate.

[0165] In some embodiments, the oil may have an OP:OO m / z ratio as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. In some embodiments, the oil may have an OP:OO m / z ratio of at least 1.6 as determined by the abundance of DAG ions produced by mass spectrometry fragmentation of the TAG component in the oil. In some embodiments, the oil can have an OP:OO m / z ratio of at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4.0, at least 4.1, at least 4.2, at least 4.3, at least 4.4, at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, or at least 5.0, as determined by the abundance of DAG ions produced by mass spectrometric fragmentation of the TAG component in the oil.

[0166] TAG regiospecificity can also be determined by a porcine pancreatic lipase assay. This method can be used to determine the identity of the sn-2 fatty acids of TAGs by incubating the TAGs with porcine pancreatic lipase. The lipase reaction results in deacylation of the fatty acids at the sn-1 and sn-3 positions. The remaining sn-2-monoacylglycerol (MAG) can then be separated and identified by solid phase extraction and analysis of the fat composition by gas chromatography using a direct transesterification reaction method.

[0167] The oil composition provided herein can comprise a TAG component, wherein in the TAG component, at least 40% of the TAG is a TAG species with a saturated fatty acid at the sn-2 position. In some embodiments, in the TAG component, at least about 50% of the TAG is a TAG species with a saturated fatty acid at the sn-2 position. In some embodiments, in the TAG component, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59% or about 60% of the TAG is a TAG species with a saturated fatty acid at the sn-2 position. In some embodiments, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 40%-50%, 50%-60% or 40%-60% of the TAGs in the TAG component are TAG species with saturated fatty acids at the sn-2 position. In some embodiments, the saturated fatty acid at the sn-2 position is palmitate.

[0168] The oil compositions provided herein can include a TAG component, wherein at least some of the acyl chains of the TAG component are C18: 1. In some embodiments, at least about 50% of the acyl chains in the TAG component are C18: 1. In some embodiments, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, or at least about 70% of the acyl chains in the TAG component are C18: 1. In some embodiments, at least about 50%-55%, at least about 55%-60%, at least about 60%-65%, at least about 60%-67%, at least about 65%-70%, at least about 50%-60%, at least about 50%-67%, at least about 60%-70%, or at least about 50%-70% of the acyl chains in the TAG component are C18:1.

[0169] Microbial Oil

[0170] In some embodiments, oil provided herein is from the microorganism of genetic modification, for example microalgae, oleaginous yeast or oleaginous bacteria obtain.In some embodiments, the microorganism of genetic modification is the Protocystis species algae strain of genetic modification.In some embodiments, the microorganism of genetic modification comprises foreign gene or exogenous nucleotide.Alternatively or additionally, the microorganism of genetic modification does not comprise endogenous gene or endogenous nucleotide.Genetic modification method described herein can be used for giving the oleic acid and / or OPO TAG production of increase in microorganism.

[0171] Oil provided herein can be obtained from microalgae, oleaginous yeast or oleaginous bacteria.In some embodiments, microorganism is the Protocystis algae strain of modification.The Protocystis algae strain of non-genetically modified can be produced by classical algae strain improvement strategy.The Protocystis algae strain of genetic modification can be produced by expressing foreign genes and / or lacking endogenous genes in microorganism.In some embodiments, microorganism provided herein is the Protocystis algae strain of genetic modification, which produces (for example, at least 50% oleic acid) and the OPO production (for example, at least 70% OPO) of the oleic acid that optimizes increase by classical algae strain improvement strategy.In some embodiments, microorganism provided herein is the Protocystis algae strain of genetic modification, which produces (for example, at least 30% palmitic acid) and the OPO production (for example, at least 70% OPO) of the palmitic acid that optimizes increase by classical algae strain improvement strategy.

[0172] The oil provided herein can be produced by Protocystis cells. The produced oil can be from UTEX 1533 Protocystis strain cells. The produced oil can be from cells of strains that do not express exogenous thioesterases.

[0173] In some embodiments, the oils provided herein are produced from microalgae. In some embodiments, the microalgae is a species of a genus selected from the group consisting of Chlorella sp., Pseudochlorella sp., Heterochlorella sp., Prototheca sp., Arthrospira sp., Euglena sp., Nannochloropsis sp., Phaeodactylum sp., Chlamydomonas sp., Scenedesmus sp., Ostreococcus sp., Selenastrum sp., Haematococcus sp., Nitzschia, Dunaliella, Navicula sp., Trebouxia sp. sp.), Pseudotrebouxia sp., Vavicula sp., Bracteococcus sp., Gomphonema sp., Watanabea sp., Botryococcus sp., Tetraselmis sp., and Isochrysis sp. In some embodiments, the microalgae is a Protocystis sp. In some embodiments, the microalgae is P. moriformis. In some embodiments, the microalgae is P. wickerhamii.

[0174] Genetic engineering and improvement of classic algae strains

[0175] The generation of TAG oil with specific phenotype can be realized by the genetic technology and non-genetic modification technology of microorganism.Although genetic engineering technology can be used for the phenotype that causes in the target host oil-producing microorganism, can adopt classical algae strain to improve and other non-genetic engineering technology to further strengthen these phenotypes.Similarly, can adopt classical algae strain to improve or other non-genetic engineering technology to strengthen some phenotype selected by genetic engineering technology.Phenotype can comprise elaborating specific fatty acid profile, specific TAG profile, carbon yield, growth productivity, volume oil accumulation (for example, g oil / L culture), oil productivity (for example, g oil / L culture) and oil as the dry cell weight percentage (DCW) of algae strain performance measure.

[0176] In some embodiments, the microalgae cells provided herein produce at least 50% lipid by dry cell weight. In some embodiments, the microalgae cells provided herein produce at least 60% lipid by dry cell weight.

[0177] For example, a microorganism that has been non-genetically modified to confer a first phenotype (e.g., increased oleic acid production) can be genetically modified to confer a second phenotype (e.g., increased OPO production). Then, the resulting microorganism can confer both the first phenotype and the second phenotype (e.g., increased oleic acid and OPO production).

[0178] Microorganisms provided herein can be produced by classical algae strain improvement strategies to select microorganisms with desired phenotypes (e.g., high oleic acid production). Classical algae strain improvement (also referred to as "mutation breeding") involves exposing organisms to chemicals or radiation to generate mutants with desirable traits. Ultraviolet (UV) light can be used to introduce random mutations in the nuclear genome of microorganisms. Chemical mutagens include compounds that inhibit or destroy microbial biosynthesis processes, such as antibiotics, antifungals, or carcinogens. Non-limiting examples of chemical mutagens include ICR-191, ethyl methanesulfonate (EMS), and 4-nitroquinoline-1-oxide (4-NQO). Non-limiting examples of chemical mutagens also include acridine mutagens, amino acid analogs, fatty acid biosynthesis inhibitors, cholesterol biosynthesis inhibitors, mTOR inhibitors, and membrane solubilizers. Mutagenesis can also be induced using a combination of chemical mutagens simultaneously. After mutagenesis, a selection agent or enrichment agent can be used to select or enrich the algae strain of interest. Non-limiting examples of enrichment agents include L-canavanine, cerulenin, triparaol, clomiphene, clomiphene citrate, clotrimazole, terfenadine, fluphenazine, AZD8055, BASF 13-338, cafenstrole, clomiphene, PF-042110, and phenylethanol.

[0179] The microorganism with enhanced or changed lysophosphatidic acid acyltransferase activity provided herein can be produced by genetic engineering techniques such as gene recombination. The enhancement of lysophosphatidic acid acyltransferase activity can be achieved by enhancing the expression of exogenous genes in the microorganism and / or reducing the expression of endogenous genes. The enhancement of gene expression can be achieved by overexpression of endogenous genes in the microorganism or the expression of exogenous genes. The reduction of gene expression can be achieved by lacking or removing endogenous genes in the microorganism.

[0180] For example, the non-naturally occurring microorganisms provided herein can be genetically modified to enhance or alter the lysophosphatidic acid acyltransferase activity in the microorganism. The enhancement or alteration of the lysophosphatidic acid acyltransferase activity can be achieved by enhancing the expression of exogenous LPAAT genes in the microorganism and / or reducing the expression of endogenous LPAAT genes.

[0181] The methods provided herein include classical algae strain improvements and / or genetic engineering methods to improve algae strain productivity, carbon yield, oleic acid content, and OPO content. Glucose consumption rate can be a highly predictive indicator of lipid titer. Therefore, glucose consumption rate can be used as an enrichment tool in mutant selection processes.

[0182] Polyol Applications and End Products

[0183] Oil described herein can be used as a substrate in a chemical reaction for producing polyols, such as hydroformylation / reduction or epoxidation and ring opening. The oleic acid moiety at sn-1 and sn-3 positions, when experiencing the chemical reaction mentioned above, will produce diols, that is, when formulated with one or more excipients for various applications, including but not limited to process oils (for example, for tires), waxes, lubricants, other polyols, macromolecular diols, other polyester diols, can be used to produce polyurethane products, such as hard foam, soft foam, cast polyurethane, thermoplastic polyurethane (TPU), elastomers, adhesives, coatings, laminates, films and dispersions. Polyurethane products can be used to build aerospace, automobiles, medical treatment, electronics, construction and building products; sporting goods or recreational equipment, such as skis, snowboards, sidewalls, boating equipment, kayaks; and other consumer products, such as industrial containers, coolers, mattresses, leather products, clothing, footwear, mannequins and mobile phone cases. These polyurethane applications can be used as sustainable replacements for petroleum-based non-renewable materials such as acrylonitrile butadiene styrene (ABS), ultra-high molecular weight polyethylene (UHMWPE), or high-density polyethylene (HDPE).

[0184] Oil provided herein can have improved production efficiency and enhanced TAG composition, to improve the control to urethane chemistry.With respect to the oil (for example, oilseed or plant-derived oil) with larger TAG heterogeneity (therefore, lower purity) and / or diversity, these characteristics of microbial oil can cause greater hydroxyl group (-OH) consistency.The polyol derived from the oil of the height that is rich in single hydroxylated TAG species provided herein may be preferred when generating polymer, be included in the physical property of polymer and may be present in the case of molecular impurities (such as randomness of the regioselectivity of non-hydroxylated fatty acids or fatty acid moiety insertion on glycerol backbone) damage in the oil with more varied or heterogeneous TAG spectrum.

[0185] Polyvalent alcohol described herein can be used for producing polyurethane material particularly.For example, oil provided herein can have relatively low TAG diversity, low fatty acid diversity, and the fatty acid that is present in the oil can be hydroxylated fatty acid.The higher ratio of hydroxylated fatty acid and non-hydroxylated fatty acid can allow to increase chemical reactivity.The oil with low TAG diversity and high ratio hydroxylated fatty acid may be desirable especially in the generation of polyurethane, because hydroxylated fatty acid can participate in the cross-linking reaction with isocyanate.Therefore, the polyvalent alcohol produced by the highly hydroxylated fatty acid that comprises oil can produce the polyurethane material with excellent properties.

[0186] Polyol production

[0187] The hydroxyl group functionality of the polyol can be introduced via chemical transformation of the triglyceride oil. This transformation generally involves the presence of a double bond on the acyl portion of the fatty acid, which can then be transformed to incorporate one or more hydroxyl groups using several different chemical reactions including, for example, epoxidation / ring opening, ozonolysis, and hydroformylation and reduction.

[0188] Epoxidation and subsequent ring opening across the carbon-carbon double bond of the acyl chain can be carried out using a variety of reagents, including, for example, water, hydrogen, methanol, ethanol, propanol, isopropanol or a polyol. Ring opening can be promoted by reacting with an alcohol (including, for example, a β-substituted alcohol). Ring opening of epoxidized oils can also be achieved by using a hydrogenation catalyst such as nickel to produce a hydroxyl moiety.

[0189] Hydroformylation with synthesis gas (syngas) can be carried out using rhodium or cobalt catalysts to form aldehydes at the olefin groups. The aldehydes can then undergo reduction to alcohols in the presence of hydrogen and a nickel catalyst to form polyols.

[0190] The hydroformylation chemical reaction results in the preservation of fatty acid length and the formation of primary hydroxyl moieties. Primary hydroxyl functional groups may be desirable in some PU applications due to increased reactivity compared to secondary hydroxyl moieties. The hydroxyl groups introduced into the olefin groups in the acyl chain can participate in subsequent downstream chemical reactions, i.e., react with isocyanate moieties to form urethane bonds or react with methyl esters to form polyesters. Saturated fatty acids that do not contain double bonds cannot participate in crosslinking reactions with isocyanates. Therefore, saturated fatty acids can compromise structural integrity and reduce the properties of the polymers produced therefrom.

[0191] In some embodiments, the polyols provided herein contain a considerable proportion of primary hydroxyl groups. In some embodiments, the polyols provided herein contain secondary hydroxyl groups. In some embodiments, the polyols provided herein can be modified to increase the proportion of primary hydroxyl groups.

[0192] The derivative of TAG oil provided herein can be the parent material for the production of polyvalent alcohol.The limiting examples of these TAG derivatives comprise fatty acid, fatty acid methyl ester, fatty acid ethyl ester, hydroxylated fatty acid, hydroxylated fatty methyl ester and hydroxylated fatty ethyl ester.The limiting examples of polyvalent alcohol comprises polyester glycol, polyether glycol, hydrogenated polyol, hydroformylation polyol and epoxidation ring-opening polyol.Alternatively, TAG oil provided herein, without the need for further chemical modification, can directly be used as the parent material for generating polyvalent alcohol.

[0193] Fatty acid methyl esters (FAME) can be generated by ester chemistry. For example, TAG can be cleaved into FAME and glycerol by transesterification. Then, FAME can undergo epoxidation and ring opening, for example, to produce FAME of alcohol. Alternatively, polyols can first be generated from TAG by epoxidation and ring opening, for example, followed by transesterification to produce FAME of alcohol and glycerol. Glycerol and potassium methoxide catalysts can be removed by washing with water.

[0194] Catalysts, including potassium methoxide (KOCH3), 1,5,7-triazabicyclo[4.4.0]dodec-5-ene (TBD), titanium(IV)isopropoxide (TIP), dibutyltin dilaurate (DBTDL), tris(pentafluorophenyl)borane (BCF), and potassium tert-butoxide, among others, can be used to re-esterify the ester groups to the alcohol moiety. The dual functionality of alcohol FAME can be used to generate polymer networks using only the methyl ester of the alcohol. Due to the polarity of the molecule (ester on one end and alcohol at the other end), the resulting polymer network can be extended in a linear, unidirectional manner and terminated at a single hydroxyl group.

[0195] The polymer network can also be extended in a bidirectional manner by incorporating diols such as low molecular weight diols or polyols provided herein. Non-limiting examples of diols include propylene glycol, alkyl diols, 1,4-butanediol, 1,3-propylene glycol, and 1,6-hexanediol. In some embodiments, diols can be produced using a microbial host.

[0196] Hyperbranched polyols can be prepared to achieve a range of properties, such as molecular weight, viscosity, branching, and reactivity. For example, hyperbranched polyols can be combined with isocyanates, ionizable molecules, or hydrophobic compounds to produce higher order polyols.

[0197] In some embodiments, the TAG oils described herein can be used to produce materials for polymer applications. Figure 7As shown in , OPO-rich TAG oils can undergo chemical transformations to produce oils containing functional groups such as epoxy, ethoxy, methoxy, or hydroxyl groups. Possible modifications of OPO include (from left to right): hydroformylation and reduction to produce diols; epoxidation to produce diepoxides; and epoxidation followed by ring opening (e.g., with ethanol) to produce diols. OPO-rich TAGs can allow a variety of chemicals to be deployed around the carbon-carbon double bond of the oleic acid moiety, which can produce a wide variety of monomers for use as chain extenders in polymer chemistry applications.

[0198] In some embodiments, strains producing OPO-rich TAGs, such as those described herein, can be useful substrates for additional genetic modification with a delta-12 fatty acid desaturase (FAH12). Upregulating FAH12 expression in such OPO-accumulating strains can increase the production of ricinoleic acid, thereby increasing the level of ricinoleic acid incorporated into TAGs containing oleic acid. Thus, such strains can thereby produce diols, Figure 7 , but in vivo without the need for additional chemical modifications. Non-limiting examples of oleate 12-hydroxylases include Lesquerella fendleri FAH12, Ricinus communis FAH12, or Claviceps purpurea FAH12. Examples of such diols are shown in Figure 8 middle. Example

[0199] Example 1: Expression of Chlamydomonas reinhardtii (CrLPAAT2) in Prospora moriformis strain CHK22.

[0200] The microalgae Protocystis moriformis strain CHK22 (UTEX1533) obtained from the University of Texas at Austin Culture Collection of Algae produces a fatty acid ratio of about 2:1 oleate:palmitate, which can be suitable for OPO production. However, the Protocystis moriformis lysophosphatidic acid acyltransferase (LPAAT) exhibits a strong preference for inserting unsaturated fatty acids rather than saturated fatty acids at the sn-2 position. In this embodiment, heterologous LPAAT genes were tested to identify genes that can effectively guide palmitic acid (saturated fatty acid) to the sn-2 position of TAGs produced by microalgae.

[0201] C. reinhardtii CrLPAAT2 was introduced into P. moriformis strain CHK22. The expression construct pCHK385 contained 5' and 3' homology arms to allow targeted integration of CrLPAAT2 into the genome and is shown in Table 1.

[0202] Table 1: Integration sequences used for transformation of Prospora moriformis with pCHK385 encoding CrLPAAT2.

[0203]

[0204]

[0205]

[0206] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:CrLPAAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination. The Chlamydomonas reinhardtii β-tubulin promoter that drives the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated in uppercase bold italics, while the coding region is indicated in lowercase italics. The PmGH 3'-UTR of Prosporangium moriformis is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of CrLPAAT2. The initiator ATG and terminator TGA codons of CrLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0207] Algal growth and transformation

[0208] Unless otherwise indicated, the strains described herein were grown in 50-mL conical tube format at 28°C with shaking at 200 rpm for 96 hours for lipid production. The culture supernatant was harvested via centrifugation and washed once with MilliQ water. The resulting cell pellet was lyophilized to dryness and subjected to direct transesterification to generate fatty acid methyl esters (FAMEs) for subsequent quantification and characterization by gas chromatography-flame ionization detection (GC / FID).

[0209] The strains to be transformed were grown in nutrient growth medium for 24 hours before transformation. The cells were pelleted by centrifugation, resuspended in medium, and then centrifuged again. The resulting cell pellet was resuspended in medium and 5×10 7The cell is plated to appropriate selection medium, and allowed to dry in a sterile biosafety cabinet. The transformant of CHK22 is generated via particle bombardment of the cells previously plated using gold nanoparticles. Primary transformant (primarytransformant) is selected for the ability to grow on a plate containing sucrose as the sole carbon source. Transformant is grown in lipid production medium, and its resulting biomass is processed as previously described to carry out fatty acid analysis.

[0210] LCMS analysis method

[0211] TAG spectrum analysis was performed using LCMS. TAGs were extracted from cells into a solution of 3:1 toluene / 2-propanol (v / v) using mechanical disruption and vigorous stirring of ceramic beads. The filtered extract was then injected into an Agilent 1290 Infinity II UHPLC system coupled to a 6470B triple quadrupole mass spectrometer and an APCI ionization source.

[0212] LCMS determination of regioisomers

[0213] Based on the abundance of DAG ions generated by the fragmentation of TAG species, a qualitative assessment of the regiospecificity of the major TAG species of the resulting oil was performed ( Figure 1 ).

[0214] Example 2: Determination of sn-2 fatty acid profile after digestion with porcine pancreatic lipase.

[0215] The fatty acid composition at the sn-2 position will be determined after incubation of TAG with porcine pancreatic lipase. The lipase reaction causes TAG to deacylate at the sn-1 and sn-3 positions, leaving sn-2-MAG. The sn-2-MAG is then separated using an Agilent BondElut NH2 propyl solid phase extraction (SPE) cartridge and subjected to fatty acid composition analysis via gas chromatography using a direct transesterification reaction.

[0216] The mass spectra and DAG ion ratios of the non-transgenic algae strain CHK22 and CHK22 transformed with pCHK385 (D552-1, D552-3, and D552-4) are shown in Figure 2 and Table 2. Figure 2 The mass spectrum of OOP / OPO triglycerides is shown. Although the regioisomers were not chromatographically separated by this analytical method, the higher ratio of m / z 577 (OP) to m / z 603 (OO) indicates that the OPO regioisomer is more abundant compared to CHK22.

[0217] Table 2: Screening of primary transformants of CHK22 containing pCHK385.

[0218]

[0219] Table 2 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK385. The moriformis basic strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization GC / FID. TAG analysis was performed using the LCMS method as described above.

[0220] The fatty acid composition at the sn-2 position of transgenic lines CHK22, D552;1, D552;3 and D552;4 was further determined after incubation of TAG with porcine pancreatic lipase. The results are shown in Table 3.

[0221] Table 3: Determination of the percentage of palmitic acid at the sn-2 position after digestion with porcine pancreatic lipase for primary transformants of CHK22 containing pCHK385.

[0222]

[0223] In Table 3, the moriformis base strain CHK22 is shown as a non-transgenic control. The strains were grown in 1-L flasks in lipid production medium at 28°C at 200 rpm for 120 hours as 200-mL cultures. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. The fatty acid composition at the sn-2 position was determined using a porcine pancreatic lipase assay as described above. Figure 5 A corresponding bar graph showing the percentage of palmitic acid at the sn-2 position for various strains that have been transformed with pCHK385 is shown in . Figure 6 A graph showing LC-MS results of oils from CHK22 and strain D552-3 is shown in , which shows that D552-3 has similar molecular characteristics to CHK22, but the amount of palmitic acid in the sn-2 position is significantly increased.

[0224] Example 3: Expression of Chlamydomonas reinhardtii LPAAT1 (CrLPAAT1) in Prosporangium moriformis strain CHK22.

[0225] C. reinhardtii CrLPAAT1 was introduced into P. moriformis strain CHK22. The expression construct pCHK391 contained 5' and 3' homology arms to allow targeted integration of CrLPAAT1 into the genome and is shown in Table 4.

[0226] Table 4: Integration sequences used for transformation of Prospora moriformis with pCHK391 encoding CrLPAAT1.

[0227]

[0228]

[0229]

[0230] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:CrLPAAT1:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of CrLPAAT1. The initiator ATG and terminator TGA codons of CrLPAAT1 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0231] Table 5 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK391. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0232] Table 5: Screening of primary transformants of CHK22 containing pCHK391.

[0233]

[0234] Example 4: Expression of Brassica napus BnBAT2 in Prospora moriformis strain CHK22.

[0235] B. napus BnBAT2 was introduced into P. moriformis strain CHK22. The expression construct pCHK384 contained 5' and 3' homology arms to allow targeted integration of BnBAT2 into the genome and is shown in Table 6.

[0236] Table 6: Integration sequences used for transformation of Prospora moriformis with pCHK384 encoding BnBAT2.

[0237]

[0238]

[0239]

[0240] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:BnBAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of BnBAT2. The initiator ATG and terminator TGA codons of BnBAT2 are indicated by uppercase bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0241] Table 7 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK384. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0242] Table 7: Screening of primary transformants of CHK22 containing pCHK384.

[0243]

[0244] Example 5: Expression of Nannochloropsis sp. NoLPAT4 in Prospora moriformis strain CHK22.

[0245] Nannochloropsis sp. NoLPAT4 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK386 contained 5' and 3' homology arms to allow targeted integration of NoLPAT4 into the genome and is shown in Table 8.

[0246] Table 8: Integration sequences used for transformation of Prospora moriformis with pCHK386 encoding NoLPAT4.

[0247]

[0248]

[0249]

[0250] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:NoLPAT4:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriform prosporangium PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The moriform prosporangium PmAMT03 promoter indicated by boxed italic text drives the expression of NoLPAT4. The initiator ATG and terminator TGA codons of NoLPAT4 are indicated by uppercase bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0251] Table 9 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK386. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0252] Table 9: Screening of primary transformants of CHK22 containing pCHK386.

[0253]

[0254] Example 6: Expression of Synechocystis sp. SII1848 in Prospora moriformis strain CHK22.

[0255] Synechocystis sp. Sll1848 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK387 contained 5' and 3' homology arms to allow targeted integration of Sll1848 into the genome and is shown in Table 10.

[0256] Table 10: Integration sequences used for transformation of Prospora moriformis with pCHK387 encoding Synechocystis sp. SII1848.

[0257]

[0258]

[0259]

[0260] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:Sll1848:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of Sll1848. The initiator ATG and terminator TGA codons of Sll1848 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0261] Table 11 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK387. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0262] Table 11: Screening of primary transformants of CHK22 containing pCHK387.

[0263]

[0264]

[0265] Example 7: Expression of Escherichia coli EcPlsC in Prospora moriformis strain CHK22.

[0266] E. coli EcPlsC was introduced into Prospora moriformis strain CHK22. The expression construct pCHK388 contained 5' and 3' homology arms to allow targeted integration of EcPlsC into the genome and is shown in Table 12.

[0267] Table 12: Integration sequences used for transformation of Prospora moriformis with pCHK388 encoding EcPlsC from E. coli.

[0268]

[0269]

[0270]

[0271] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:EcPlsC:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of EcPlsC. The initiator ATG and terminator TGA codons of EcPlsC are indicated by uppercase bold italics, while the remainder of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0272] Table 13 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK388. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm for 96 hours in 50-mL conical tubes. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0273] Table 13: Screening of primary transformants of CHK22 containing pCHK388.

[0274]

[0275] Example 8: Expression of Nannochloropsis sp. NoLPAT3 in Prospora moriformis strain CHK22.

[0276] Nannochloropsis sp. NoLPAT3 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK453 contained 5' and 3' homology arms to allow targeted integration of NoLPAT3 into the genome and is shown in Table 14.

[0277] Table 14: Integration sequences used for transformation of Prospora moriformis with pCHK453 encoding Nannochloropsis sp. NoLPAT3.

[0278]

[0279]

[0280]

[0281] The construct can be written as 5'Thi4::PmHXT1v2:ScMEL1:PmPGK:CvNR:PmSAD2-2:NoLPAT3:CvNR::3'Thi4. Going in the 5' to 3' direction, bold lowercase sequences represent genomic DNA from strain CHK22, which allows targeted integration at the Thi4 locus via homologous recombination. The moriformis hexose transporter 1 (HXT1 v2) promoter driving expression of S. carlbergensis melibiase (ScMEL1) is represented by boxed uppercase text. The initiator ATG and terminator TAA of ScMEL1 are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis phosphoglucokinase (PGK) 3'-UTR is indicated by capital underlined text, followed by the moriformis stearoyl ACP desaturase-2 (SAD2-2) promoter indicated by boxed italic text, driving the expression of NoLPAT3. The initiator ATG and terminator TGA codons of NoLPAT3 are indicated by capital bold italics, while the rest of the gene is indicated by bold italics. The vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the moriformis nitrate reductase Thi4 3' flanking region indicated by bold lowercase text.

[0282] Table 15 shows the fatty acid / TAG profiles of four primary transformants of CHK22 transformed with pCHK453. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0283] Table 15: Screening of primary transformants of CHK22 containing pCHK453.

[0284]

[0285] Example 9: Expression of Volvox carterii LPAAT2 (VcLPAAT2) in Prospora moriformis strain CHK22.

[0286] Volvox carterii VcLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK785 contained 5' and 3' homology arms to allow targeted integration of VcLPAAT2 into the genome and is shown in Table 16.

[0287] Table 16: Integration sequences used for transformation of Prospora moriformis with pCHK785 encoding VcLPAAT2.

[0288]

[0289]

[0290]

[0291] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:VcLPAAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmGH 3'-UTR is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of VcLPAAT2. The initiator ATG and terminator TGA codons of VcLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0292] Table 17 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK785. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0293] Table 17: Screening of primary transformants of CHK22 containing pCHK785.

[0294]

[0295] Example 10: Expression of Chlamdomonase schloesseriLPAAT2 (ChsLPAAT2) in Prospora moriformis strain CHK22.

[0296] C. schloesseri ChsLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK786 contained 5' and 3' homology arms to allow targeted integration of ChsLPAAT2 into the genome and is shown in Table 18.

[0297] Table 18: Integration sequences used for transformation of Prospora moriformis with pCHK786 encoding ChsLPAAT2.

[0298]

[0299]

[0300]

[0301] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ChsLPAAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis pmGH 3'-UTR is indicated by uppercase underlined text. This is followed by a linker. The moriformis pmGH promoter, indicated by boxed italic text, drives the expression of ChsLPAAT2. The initiator ATG and terminator TGA codons of ChsLPAAT2 are indicated by uppercase bold italics, while the remainder of the gene is indicated by bold italics. The Chlorella vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0302] Table 19 shows the fatty acid / TAG profiles of six primary transformants of CHK22 transformed with pCHK786. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0303] Table 19: Screening of primary transformants of CHK22 containing pCHK786.

[0304]

[0305] Example 11: Expression of Chlamydomonas incerta LPAAT2 (CiLPAAT2) in Prospora moriformis strain CHK22.

[0306] C. incerta CiLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK787 contained 5' and 3' homology arms to allow targeted integration of CiLPAAT2 into the genome and is shown in Table 20.

[0307] Table 20: Integration sequences used for transformation of Prospora moriformis with pCHK787 encoding CiLPAAT2.

[0308]

[0309]

[0310]

[0311] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:CiLPAAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmGH 3'-UTR of Prosporangium moriformis is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of CiLPAAT2. The initiator ATG and terminator TGA codons of CiLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0312] Table 21 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK787. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0313] Table 21: Screening of primary transformants of CHK22 containing pCHK787.

[0314]

[0315] Example 12: Expression of Volvox africanus LPAAT2 (VaLPAAT2) in Prospora moriformis strain CHK22.

[0316] Volvox africanus VaLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK788 contained 5' and 3' homology arms to allow targeted integration of VaLPAAT2 into the genome and is shown in Table 22.

[0317] Table 22: Integration sequences used for transformation of Prospora moriformis with pCHK788 encoding VaLPAAT2.

[0318]

[0319]

[0320]

[0321] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:VaLPAAT2:CvNR::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmGH 3'-UTR of Prosporangium moriformis is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of VaLPAAT2. The initiator ATG and terminator TGA codons of VaLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The C. vulgaris nitrate reductase 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0322] Table 23 shows the fatty acid / TAG profiles of six primary transformants of CHK22 transformed with pCHK788. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0323] Table 23: Screening of primary transformants of CHK22 containing pCHK788.

[0324]

[0325]

[0326] Since CHK22 transformants expressing CrLPAAT2 produced higher OP:OO ratios, the percent identity (%) across all amino acid sequences of the complete LPAAT protein was compared to that of CrLPAAT2. The results are shown in Table 24. The resulting phenotypes of expressing these LPAATs in CHK22 are indicated by the m / z (577.5 / 603.5) OP:OO ratios; the OP:OO ratios shown in the rows for PmLPAAT1 or PmLPAAT2 represent the results of the analysis of the non-transgenic line CHK22.

[0327] In some embodiments, the microalgae cells provided herein comprise an exogenous gene encoding an LPAAT in Table 24. In some embodiments, the exogenous gene can comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an LPAAT in Table 24.

[0328] Table 24: OP:OO ratio

[0329]

[0330]

[0331] Table 25 shows the amino acid sequences of LPAATs described in the Examples herein. Figure 3 , Figures A-C show an amino acid sequence alignment of the amino acid sequences of the LPAATs described herein, including EcPlsC (SEQ ID NO:22), OlLPAAT2 (SEQ ID NO:63), PmLPAAT2 (SEQ ID NO:24), CosLPAAT2 (SEQ ID NO:64), CrLPAAT2 (SEQ ID NO:13), ChsLPAAT2 (SEQ ID NO:14), CiLPAAT2 (SEQ ID NO:15), VaLPAAT2 (SEQ ID NO:21), VcLPAAT2 (SEQ ID NO:20), NoLPAT4 (SEQ ID NO:17), NoLPAT3 (SEQ ID NO:16), PmLPAAT1 (SEQ ID NO:23), Sll1848 (SEQ ID NO:19), BnBAT2 (SEQ ID NO:18), and CrLPAAT1 (SEQ ID NO:12).

[0332] In some embodiments, the microalgae cells provided herein comprise an exogenous gene encoding an LPAAT in Table 25. In some embodiments, the exogenous gene may comprise a sequence encoding an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an LPAAT in Table 25. In some embodiments, the exogenous gene may comprise a sequence encoding an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 12-24, 63, and 64.

[0333] Table 25: LPAAT amino acid sequence

[0334]

[0335]

[0336]

[0337]

[0338] Example 13: Expression of Volvox nagariensis LPAAT2 (VcLPAAT2V2) in Prospora moriformis strain CHK22.

[0339] VcLPAAT2 V2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK967 contained 5' and 3' homology arms to allow targeted integration of VcLPAAT2 V2 into the genome and is shown in Table 27.

[0340] Table 27: Integration sequences used for transformation of Prospora moriformis with pCHK967 encoding VcLPAAT2 V2.

[0341]

[0342]

[0343]

[0344] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:VcLPAAT2V2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the VcLPAAT2 V2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of VcLPAAT2 V2. The initiator ATG and terminator TGA codons of CrLPAAT2 V2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0345] Table 28 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK967. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0346] Table 28: Screening of primary transformants of CHK22 containing pCHK967.

[0347]

[0348] Example 14: Expression of Astrephomene gubernaculifera LPAAT2 (AgLPAAT2) in Prospora moriformis strain CHK22.

[0349] AgLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK968 contained 5' and 3' homology arms to allow targeted integration of AgLPAAT2 into the genome and is shown in Table 29.

[0350] Table 29: Integration sequences used for transformation of Prospora moriformis with pCHK968 encoding AgLPAAT2.

[0351]

[0352]

[0353]

[0354] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:AgLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the AgLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of AgLPAAT2. The initiator ATG and terminator TAG codons of AgLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0355] Table 30 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK968. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0356] Table 30: Screening of primary transformants of CHK22 containing pCHK968.

[0357]

[0358] Example 15: Expression of Edaphochlamys debaryana LPAAT2 (EdLPAAT2) in strain CHK22.

[0359] EdLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK969 contained 5' and 3' homology arms to allow targeted integration of EdLPAAT2 into the genome and is shown in Table 31.

[0360] Table 31: Integration sequences used for transformation of Prospora moriformis with pCHK969 encoding EdLPAAT2.

[0361]

[0362]

[0363]

[0364] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:EdLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the EdLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of EdLPAAT2. The initiator ATG and terminator TAG codons of EdLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0365] Table 32 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK969. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0366] Table 32: Screening of primary transformants of CHK22 containing pCHK969.

[0367]

[0368]

[0369] Example 16: Expression of Dunaliella tertiolecta LPAAT2 (DsLPAAT2) in Prospora moriformis strain CHK22.

[0370] DsLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK971 contained 5' and 3' homology arms to allow targeted integration of DsLPAAT2 into the genome and is shown in Table 33.

[0371] Table 33: Integration sequences used for transformation of Prospora moriformis with pCHK971 encoding DsLPAAT2.

[0372]

[0373]

[0374]

[0375] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:DsLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the DsLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of DsLPAAT2. The initiator ATG and terminator TGA codons of DsLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0376] Table 34 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK971. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0377] Table 34: Screening of primary transformants of CHK22 containing pCHK971.

[0378]

[0379] Example 17: Expression of Nannochloropsis sp. LPAAT2 (SceLPAAT2) in Prospora moriformis strain CHK22.

[0380] SceLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK973 contained 5' and 3' homology arms to allow targeted integration of SceLPAAT2 into the genome and is shown in Table 35.

[0381] Table 35: Integration sequences used for transformation of Prospora moriformis with pCHK973 encoding SceLPAAT2.

[0382]

[0383]

[0384] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:SceLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the SceLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of SceLPAAT2. The initiator ATG and terminator TGA codons of SceLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0385] Table 36 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK973. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0386] Table 36: Screening of primary transformants of CHK22 containing pCHK973.

[0387]

[0388] Example 18: Expression of Micractinum conductrix LPAAT2 (McLPAAT2) in Prospora moriformis strain CHK22.

[0389] McLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK974 contained 5' and 3' homology arms to allow targeted integration of McLPAAT2 into the genome and is shown in Table 37.

[0390] Table 37: Integration sequences used for transformation of Prospora moriformis with pCHK974 encoding McLPAAT2.

[0391]

[0392]

[0393]

[0394] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:McLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the McLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of McLPAAT2. The initiator ATG and terminator TGA codons of McLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0395] Table 38 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK974. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0396] Table 38: Screening of primary transformants of CHK22 containing pCHK974.

[0397]

[0398] Example 19: Expression of Chlorella sorokinensis LPAAT2 (ChsoLPAAT2) in Prospora moriformis strain CHK22.

[0399] ChsoLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK975 contained 5' and 3' homology arms to allow targeted integration of ChsoLPAAT2 into the genome and is shown in Table 39.

[0400] Table 39: Integration sequences used for transformation of Prospora moriformis with pCHK975 encoding ChsoLPAAT2.

[0401]

[0402]

[0403]

[0404] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ChsoLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ChsoLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of ChsoLPAAT2. The initiator ATG and terminator TGA codons of ChsoLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0405] Table 40 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK975. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0406] Table 40: Screening of primary transformants of CHK22 containing pCHK975.

[0407]

[0408] Example 20: Expression of Chlorella variabilis LPAAT2 (ChvaLPAAT2) in Prospora moriformis strain CHK22.

[0409] ChvaLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK976 contained 5' and 3' homology arms to allow targeted integration of ChvaLPAAT2 into the genome and is shown in Table 41.

[0410] Table 41: Integration sequences used for transformation of Prospora moriformis with pCHK976 encoding ChvaLPAAT2.

[0411]

[0412]

[0413]

[0414] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ChvaLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ChvaLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of ChvaLPAAT2. The initiator ATG and terminator TAG codons of ChvaLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0415] Table 42 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK976. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0416] Table 42: Screening of primary transformants of CHK22 containing pCHK976.

[0417]

[0418] Example 21: Expression of R. subcapitata LPAAT2 (RsLPAAT2) in Prospora moriformis strain CHK22.

[0419] RsLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK977 contained 5' and 3' homology arms to allow targeted integration of RsLPAAT2 into the genome and is shown in Table 43.

[0420] Table 43: Integration sequences used for transformation of Prospora moriformis with pCHK977 encoding RsLPAAT2.

[0421]

[0422]

[0423]

[0424] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:RsLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the RsLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of RsLPAAT2. The initiator ATG and terminator TAG codons of RsLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0425] Table 44 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK977. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0426] Table 44: Screening of primary transformants of CHK22 containing pCHK977.

[0427]

[0428]

[0429] Example 22: Expression of dried Chlorella LPAAT2 (ChdeLPAAT2) in Prospora moriformis strain CHK22.

[0430] ChdeLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK978 contained 5' and 3' homology arms to allow targeted integration of ChdeLPAAT2 into the genome and is shown in Table 45.

[0431] Table 45: Integration sequences used for transformation of Prospora moriformis with pCHK978 encoding ChdeLPAAT2.

[0432]

[0433]

[0434]

[0435] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ChdeLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ChdeLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of ChdeLPAAT2. The initiator ATG and terminator TAG codons of ChdeLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0436] Table 46 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK978. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0437] Table 46: Screening of primary transformants of CHK22 containing pCHK978.

[0438]

[0439]

[0440] Example 23: Expression of Auxenochlorella proteothecoides LPAAT2 (ApLPAAT2) in Prospora moriformis strain CHK22.

[0441] ApLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK979 contained 5' and 3' homology arms to allow targeted integration of ApLPAAT2 into the genome and is shown in Table 47.

[0442] Table 47: Integration sequences used for transformation of Prospora moriformis with pCHK979 encoding ApLPAAT2.

[0443]

[0444]

[0445]

[0446] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ApLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ApLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of ApLPAAT2. The initiator ATG and terminator TAG codons of ApLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0447] Table 48 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK979. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0448] Table 48: Screening of primary transformants of CHK22 containing pCHK979.

[0449]

[0450] Example 24: Expression of Chloropicon primus LPAAT2 (ChprLPAAT2) in Prospora moriformis strain CHK22.

[0451] ChprLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK980 contained 5'' and 3' homology arms to allow targeted integration of ChprLPAAT2 into the genome and is shown in Table 49.

[0452] Table 49: Integration sequences used for transformation of Prospora moriformis with pCHK980 encoding ChprLPAAT2.

[0453]

[0454]

[0455] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:ChprLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ChprLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives the expression of ChprLPAAT2. The initiator ATG and terminator TAG codons of ChprLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0456] Table 50 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK980. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0457] Table 50: Screening of primary transformants of CHK22 containing pCHK980.

[0458]

[0459] Example 25: Expression of Homo sapiens AGPAT1 in Prospora moriformis strain CHK22.

[0460] AGPAT1 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK966 contained 5' and 3' homology arms to allow targeted integration of AGPAT1 into the genome and is shown in Table 51.

[0461] Table 51: Integration sequences used for transformation of Prospora moriformis with pCHK966 encoding AGPAT1.

[0462]

[0463]

[0464]

[0465] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:AGPAT1:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which is capable of amplifying the AGPAT1 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of AGPAT1. The initiator ATG and terminator TAG codons of AGPAT1 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0466] Table 52 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK966. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0467] Table 52: Screening of primary transformants of CHK22 containing pCHK966.

[0468]

[0469] Example 26: Expression of Chlamydomonas eustigma LPAAT2 (CeLPAAT2) in Prospora moriformis strain CHK22.

[0470] CeLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK970 contained 5' and 3' homology arms to allow targeted integration of CeLPAAT2 into the genome and is shown in Table 53.

[0471] Table 53: Integration sequences used for transformation of Prospora moriformis with pCHK970 encoding CeLPAAT2.

[0472]

[0473]

[0474]

[0475] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:CeLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the CeLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of CeLPAAT2. The initiator ATG and terminator TAG codons of CeLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0476] Table 54 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK970. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0477] Table 54: Screening of primary transformants of CHK22 containing pCHK970.

[0478]

[0479] Example 27: Expression of PedLPAAT2 (PedLPAAT2) in Prospora moriformis strain CHK22 using the PmAMT03 promoter.

[0480] PedLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK972 contained 5' and 3' homology arms to allow targeted integration of PedLPAAT2 into the genome and is shown in Table 55.

[0481] Table 55: Integration sequences used for transformation of Prospora moriformis with pCHK972 encoding PedLPAAT2.

[0482]

[0483]

[0484]

[0485] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmAMT03:PedLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the PedLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmAMT03 promoter, indicated by boxed italic text, drives expression of PedLPAAT2. The initiator ATG and terminator TAG codons of PedLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0486] Table 56 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK972. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0487] Table 56: Screening of primary transformants of CHK22 containing pCHK972.

[0488]

[0489] Since CHK22 transformants expressing CrLPAAT2 produced higher OP:OO ratios, the percent identity (%) of all amino acid sequences across the complete LPAAT protein was compared to that of CrLPAAT2. The results are shown in Table 57. Pairwise identity (%) between all sequences and CrLPAAT2. The resulting phenotypes expressing these LPAATs in CHK22 are indicated by the m / z (577.5 / 603.5) OP:OO ratios; the OP:OO ratios shown in the rows for PmLPAAT1 or PmLPAAT2 represent the results of the analysis of the non-transgenic line CHK22. The amino acid sequences of the LPAATs described herein are listed in Table 58.

[0490] Table 57: OP:OO ratio

[0491]

[0492]

[0493] Table 58: LPAAT amino acid sequence

[0494]

[0495]

[0496]

[0497] Example 28: Expression of Volvox dictyosporon LPAAT2 (VrLPAAT2) in Prospora moriformis strain CHK22.

[0498] VrLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1133 contained 5' and 3' homology arms to allow targeted integration of VrLPAAT2 into the genome and is shown in Table 59.

[0499] Table 59: Integration sequences used for transformation of Prospora moriformis with pCHK1133 encoding VrLPAAT2.

[0500]

[0501]

[0502] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmACP:VrLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the VrLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The acyl carrier protein promoter (PmACP) from Prospora moriformis, indicated by boxed italic text, drives the expression of VrLPAAT2. The initiator ATG and terminator TAG codons of VrLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The Prospora moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0503] Table 60 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK1133. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0504] Table 60: Screening of primary transformants of CHK22 containing pCHK1133.

[0505]

[0506] Example 29: Expression of Chlorella vulgaris LPAAT2 (ChvuLPAAT2) in Prospora moriformis strain CHK22.

[0507] ChvuLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1134 contained 5' and 3' homology arms to allow targeted integration of ChvuLPAAT2 into the genome and is shown in Table 61.

[0508] Table 61: Integration sequences used for transformation of Prospora moriformis with pCHK1134 encoding ChvuLPAAT2.

[0509]

[0510]

[0511]

[0512] The construct can be written as 5'DAO1b::CrTUB2:ScSUC2:PmPGH:CvNR:PmACP:ChvuLPAAT2:PmPGH::3'DAO1b. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the DAO1b locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The invertase initiator ATG and terminator TAA are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the ChvuLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmACP promoter, indicated by boxed italic text, drives expression of ChvuLPAAT2. The initiator ATG and terminator TAG codons of ChvuLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 DAO1b genomic region indicated by bold lowercase text.

[0513] Table 62 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK1134. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0514] Table 62: Screening of primary transformants of CHK22 containing pCHK1134.

[0515]

[0516]

[0517] Example 30: Generation of a classically improved microalgae strain producing palmitate-rich triglyceride oil.

[0518] The microalgae Prospora moriformis strain CHK22 (UTEX 1533) was subjected to classical strain improvement to improve productivity, carbon yield, and increase palmitic acid content. The mutagenesis, trait selection, and high-throughput automated screening steps of the improvement process are outlined in Fig. 9 In the flow chart in . In brief, algae cells in the logarithmic growth phase are mutagenized by treatment with chemicals or UV light (1). The cells are then subcultured into a lipid production medium and undergo a selection / enrichment strategy (2). The algae strains are then plated onto solid media to obtain clonal isolates (3), which are then interrogated in a 96-well format lipid production medium (4). Using glucose consumption as a proxy for oil production, high glucose consumption algae strains are verified in a lipid production medium in a tube or shake flask format (5). Successfully verified isolates are subcultured for multiple generations to stabilize mutations (6), followed by purification of clonal isolates (7), and then re-interrogated in a lipid production medium (8). Phenotypically stable clones are then verified in fermentation (9). Clones showing variability in (8) are re-verified and subcultured via (6) to generate stable strains.

[0519] CHK22 was chemically mutagenized with 44 μM 4-nitroquinoline 1-oxide (4-NQO) for 30 min at 32 ° C or pseudo-mutagenized by adding only the mutagen solvent DMSO. The mutagen was inactivated by adding sodium thiosulfate and then removed by repeated washing with water. The cells were then allowed to recover for 3 days in limited sugar growth medium. The mutagenized and simulated mutagenized populations were then cultured independently at 38 ° C for 5 days in lipid production medium. The optimal lipid production temperature of CHK22 is usually between 28 ° C and 32 ° C. Application of a temperature higher than the optimal temperature as a stressor provides a growth advantage to mutants with a tendency to produce higher palmitate levels. At the end of 5 days of 38 ° C lipid culture, cells were collected and then incubated at 65 ° C for 4 min. Based on a range discovery experiment performed in the same format, exposure to temperatures within this range during the duration of this time usually kills >99% of the cell population. After heat exposure, the cells were recovered in limited sugar growth medium for three days, diluted, and then plated. Mutant clones were then selected and assessed for glucose consumption rate and fatty acid profile in a 72 h, 96-well block-based lipid production assay ( Fig. 9Table 63 summarizes the glucose consumption rate and fatty acid profile of TAG oil produced by mutant strains. One mutant produced about 8% more palmitate than the CHK22 parent strain. This strain showed a significant reduction in glucose consumption rate, which is a sign of lower lipid titers.

[0520] Table 63: Fatty acid profiles of mutant strains selected for displaying higher glucose consumption (Glc rate) and increased palmitate (C16:0) levels in the 96-well block lipid assay.

[0521]

[0522] In order to further adapt the CHK22 mutant-3 algae strain to the fatty acid composition of the change and improve lipid productivity, the algae strain was continuously passaged in the absence of any selection for more than thirty population doublings, and then plated to solid culture medium to produce single colonies. These subclones were screened in lipid production assays to assess the phenotypic stability of the glucose consumption rate and fatty acid profile of the pedigree. After a cycle of continuous passage, the mutant was still phenotypically unstable. Several subclones that retained higher palmitate level phenotypes and increased glucose consumption rates were passed through other rounds of continuous passage and stability assessment. After a total of five rounds of continuous passage and stability assessment, a stable algae strain was identified and named as CHK100. CHK100 produces about 5% more palmitate than the CHK22 parent algae strain. The oil content (% based on weight / weight; %w / w), dry cell weight (DCW) and lipid titer (g / L) of CHK100 are also greater than those of CHK22 (Table 64 and Table 65). Fig.10 The CHK22 strain improvement strategy to produce CHK100 is shown.

[0523] Table 64: Tube-based assay for CHK100 shows improved lipid titers.

[0524]

[0525]

[0526] NLB, non-lipid biomass; PCP, per cell yield.

[0527] Table 65: Tube-based assay for CHK100 shows increased C16:0 content.

[0528]

[0529] All strains were subjected to standard lipid production conditions, where strains were grown in duplicate in 10 mL lipid production medium. Cultures were grown at 28°C with shaking (200 rpm) for 121 hours, at which time approximately 1 mL of biomass was removed, applied to a polycarbonate filter, washed with an equal volume of Milli Q water, and placed in a tared glass vial at -80°C for 30 minutes. The vials containing the filter and frozen biomass were lyophilized to dryness overnight, the weights recorded, and the filters with dried biomass were subjected to direct transesterification, followed by GC / FID to quantify FAME.

[0530] Example 31: Expression of PedLPAAT2 (PedLPAAT2) in Prospora moriformis strain CHK100 using the PmACP promoter.

[0531] PedLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1126 contained 5' and 3' homology arms to allow targeted integration of PedLPAAT2 into the genome and is shown in Table 66.

[0532] Table 66: Integration sequences used for transformation of Prospora moriformis with pCHK1126 encoding PedLPAAT2.

[0533]

[0534]

[0535]

[0536] The construct can be written as 5'Thi4::CrTUB2:ScSUC2:PmPGH:CvNR:PmACP:PedLPAAT2:PmPGH::3'Thi4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK100, which allows targeted integration at the Thi4 locus via homologous recombination, and the C. reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK100) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the PedLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmACP promoter, indicated by boxed italic text, drives expression of PedLPAAT2. The initiator ATG and terminator TAG codons of PedLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK100 Thi4 genomic region indicated by bold lowercase text.

[0537] Table 67 shows the fatty acid / TAG profiles of the primary transformants of CHK100 transformed with pCHK1126. The moriformis base strain CHK100 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0538] Table 67: Screening of primary transformants of CHK100 containing pCHK1126.

[0539]

[0540] Example 32: Expression of PedLPAAT2 (PedLPAAT2) in Prospora moriformis strain CHK100 using the PmG3PDH promoter.

[0541] PedLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1127 contained 5' and 3' homology arms to allow targeted integration of PedLPAAT2 into the genome and is shown in Table 68.

[0542] Table 68: Integration sequences used for transformation of Prospora moriformis with pCHK1127 encoding PedLPAAT2.

[0543]

[0544]

[0545]

[0546] The construct can be written as 5'Thi4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:PedLPAAT2:PmPGH::3'Thi4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK100, which allows targeted integration at the Thi4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK100) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the PedLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmG3PDH promoter, indicated by boxed italic text, drives expression of PedLPAAT2. The initiator ATG and terminator TAG codons of PedLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK100 Thi4 genomic region indicated by bold lowercase text.

[0547] Table 69 shows the fatty acid / TAG profiles of primary transformants of CHK100 transformed with pCHK1127. Prosporangium moriformis base strain CHK100 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0548] Table 69: Screening of primary transformants of CHK100 containing pCHK1127.

[0549]

[0550]

[0551] Example 33: Expression of PedLPAAT2 (PedLPAAT2) in Prosporangiophora moriformis strain CHK100 using the PmL40-2 promoter.

[0552] PedLPAAT2 was introduced into Prospora moriformis strain CHK100. The expression construct pCHK1128 contained 5' and 3' homology arms to allow targeted integration of PedLPAAT2 into the genome and is shown in Table 70.

[0553] Table 70: Integration sequences used for transformation of Prospora moriformis with pCHK1128 encoding PedLPAAT2.

[0554]

[0555]

[0556]

[0557] The construct can be written as 5'Thi4::CrTUB2:ScSUC2:PmPGH:CvNR:PmL40-2:PedLPAAT2:PmPGH::3'Thi4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK100, which allows targeted integration at the Thi4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK100) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The Prosporangium moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the PedLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmL40-2 promoter, indicated by boxed italic text, drives expression of PedLPAAT2. The initiator ATG and terminator TAG codons of PedLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK100 Thi4 genomic region indicated by bold lowercase text.

[0558] Table 71 shows the fatty acid / TAG profiles of the primary transformants of CHK100 transformed with pCHK1128. The moriformis basic strain CHK100 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0559] Table 71: Screening of primary transformants of CHK100 containing pCHK1128.

[0560]

[0561] Example 34: Expression of PedLPAAT2 from Pedapycetes using the PmMPGp promoter in Prospora moriformis strain CHK100.

[0562] PedLPAAT2 was introduced into Prospora moriformis strain CHK100. The expression construct pCHK1129 contained 5' and 3' homology arms to allow targeted integration of PedLPAAT2 into the genome and is shown in Table 72.

[0563] Table 72: Integration sequences used for transformation of Prospora moriformis with pCHK1129 encoding PedLPAAT2.

[0564]

[0565]

[0566]

[0567] The construct can be written as 5'Thi4::CrTUB2:ScSUC2:PmPGH:CvNR:PmMPGp:PedLPAAT2:PmPGH::3'Thi4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK100, which allows targeted integration at the Thi4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK100) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis pmGH 3'-UTR is indicated by uppercase underlined text, which enables the amplification of the PedLPAAT2 gene, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmMPGp promoter, indicated by boxed italic text, drives expression of PedLPAAT2. The initiator ATG and terminator TAG codons of PedLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The P. moriformis PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK100 Thi4 genomic region indicated by bold lowercase text.

[0568] Table 73 shows the fatty acid / TAG profiles of primary transformants of CHK100 transformed with pCHK1129. The moriformis base strain CHK100 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0569] Table 73: Screening of primary transformants of CHK100 containing pCHK1129.

[0570]

[0571] Example 35: Expression of Volvulina compacta (VcomLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0572] VcomLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1210 contained 5' and 3' homology arms to allow targeted integration of VcomLPAAT2 into the genome and is shown in Table 74.

[0573] Table 74: Integration sequences used for transformation of Prospora moriformis with pCHK1210 encoding VcomLPAAT2.

[0574]

[0575]

[0576]

[0577] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VcomLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of Protocystis moriformis capable of amplifying the VcomLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives expression of VcomLPAAT2. The initiator ATG and terminator TAG codons of VcomLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0578] Table 75 shows the fatty acid / TAG profile of the primary transformants of CHK22 transformed with pCHK1210. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0579] Table 75: Screening of primary transformants of CHK22 containing pCHK1210.

[0580]

[0581] Example 36: Expression of Vitreochlamys sp. CL-2021 (VitrLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0582] VitrLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1211 contained 5' and 3' homology arms to allow targeted integration of VitrLPAAT2 into the genome and is shown in Table 76.

[0583] Table 76: Integration sequences used for transformation of Prospora moriformis with pCHK1211 encoding VitrLPAAT2.

[0584]

[0585]

[0586]

[0587] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VitrLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of Prosporangium moriformis capable of amplifying the VitrLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of VitrLPAAT2. The initiator ATG and terminator TGA codons of VitrLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0588] Table 77 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1211. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0589] Table 77: Screening of primary transformants of CHK22 containing pCHK1211.

[0590]

[0591] Example 37: Expression of Colemanosphaera charkowiensis (CchaLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0592] CchaLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1212 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 78.

[0593] Table 78: Integration sequences used for transformation of Prospora moriformis with pCHK1212 encoding CchaLPAAT2.

[0594]

[0595]

[0596]

[0597] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:CchaLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of Prosporangium moriformis capable of amplifying the CchaLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of CchaLPAAT2. The initiator ATG and terminator TGA codons of CchaLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0598] Table 79 shows the fatty acid / TAG profiles of eight primary transformants of CHK22 transformed with pCHK1212. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0599] Table 79: Screening of primary transformants of CHK22 containing pCHK1212.

[0600]

[0601] Example 38: Expression of Heterozoan japanensis (PjapLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0602] PjapLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1213 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 80.

[0603] Table 80: Integration sequences used for transformation of Prospora moriformis with pCHK1213 encoding PjapLPAAT2.

[0604]

[0605]

[0606]

[0607] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:PjapLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriform protocystis algae capable of amplifying the PjapLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter from Prospora moriformis (indicated by boxed italic text) drives the expression of PjapLPAAT2. The initiator ATG and terminator TGA codons of PjapLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 Thia4 genomic region indicated by bold lowercase text.

[0608] Table 81 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1213. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0609] Table 81: Screening of primary transformants of CHK22 containing pCHK1213.

[0610]

[0611] Example 39: Expression of Volvulinaboldii (VbolLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0612] VbolLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1214 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 82.

[0613] Table 82: Integration sequences used for transformation of Prospora moriformis with pCHK1214 encoding VbolLPAAT2.

[0614]

[0615]

[0616]

[0617] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VbolLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents the genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter that drives the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose) to express is indicated by the boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriform protocystis PmPGH 3'-UTR that can amplify the VbolLPAAT2 gene is indicated by uppercase underlined text, followed by a joint indicated by lowercase bold italics, which contains the common Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of VbolLPAAT2. The initiator ATG and terminator TGA codons of VbolLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0618] Table 83 shows the fatty acid / TAG profiles of eight primary transformants of CHK22 transformed with pCHK1214. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0619] Table 83: Screening of primary transformants of CHK22 containing pCHK1214.

[0620]

[0621]

[0622] Example 40: Expression of PmorLPAAT2 in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0623] PmorLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1215 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 84.

[0624] Table 84: Integration sequences used for transformation of Prospora moriformis with pCHK1215 encoding PmorLPAAT2.

[0625]

[0626]

[0627]

[0628] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:PmorLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter that drives the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriform prosporangium PmPGH 3'-UTR capable of amplifying the PmorLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the common Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmG3PDH promoter, indicated by boxed italic text, drives the expression of PmorLPAAT2. The initiator ATG and terminator TGA codons of PmorLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0629] Table 85 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1215. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0630] Table 85: Screening of primary transformants of CHK22 containing pCHK1215.

[0631]

[0632] Example 41: Expression of Volvox weismannia (VcarfLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0633] VcarfLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1216 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 86.

[0634] Table 86: Integration sequences used for transformation of Prospora moriformis with pCHK1216 encoding VcarfLPAAT2.

[0635]

[0636]

[0637]

[0638] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VcarfLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH that is capable of amplifying the VcarfLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives expression of VcarfLPAAT2. The initiator ATG and terminator TGA codons of VcarfLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0639] Table 87 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1216. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0640] Table 87: Screening of primary transformants of CHK22 containing pCHK1216.

[0641]

[0642] Example 42: Expression of Eudorinacylindrica (EcylLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0643] EcylLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1217 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 88.

[0644] Table 88: Integration sequences used for transformation of Prospora moriformis with pCHK1217 encoding EcylLPAAT2.

[0645]

[0646]

[0647] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:EcylLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH that can amplify the EcylLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives expression of EcylLPAAT2. The initiator ATG and terminator TGA codons of EcylLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0648] Table 89 shows the fatty acid / TAG profiles of eight primary transformants of CHK22 transformed with pCHK1217. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0649] Table 89: Screening of primary transformants of CHK22 containing pCHK1217.

[0650]

[0651] Example 43: Expression of Gonium multicoccum (GmulLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0652] GmulLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1218 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 90.

[0653] Table 90: Integration sequences used for transformation of Prospora moriformis with pCHK1218 encoding GmulLPAAT2.

[0654]

[0655]

[0656]

[0657] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:GmulLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH that can amplify the GmulLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The P. moriformis PmG3PDH promoter, indicated by boxed italic text, drives expression of GmulLPAAT2. The initiator ATG and terminator TGA codons of GmulLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0658] Table 91 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1218. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 96 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0659] Table 91: Screening of primary transformants of CHK22 containing pCHK1218.

[0660]

[0661] Example 44: Expression of Gonium viridistellatum (GvirLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0662] GvirLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1219 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 92.

[0663] Table 92: Integration sequences used for transformation of Prospora moriformis with pCHK1219 encoding GvirLPAAT2.

[0664]

[0665]

[0666]

[0667] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:GvirLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriform protocystis algae capable of amplifying the GvirLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of GvirLPAAT2. The initiator ATG and terminator TGA codons of GvirLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0668] Table 93 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1219. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0669] Table 93: Screening of primary transformants of CHK22 containing pCHK1219.

[0670]

[0671] Example 45: Expression of Volvox ferrisii (VferLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0672] VferLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1220 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 94.

[0673] Table 94: Integration sequences used for transformation of Prospora moriformis with pCHK1220 encoding VferLPAAT2.

[0674]

[0675]

[0676]

[0677] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VferLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriform protocystis algae capable of amplifying the VferLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the 3'-UTR of Chlorella vulgaris nitrate reductase. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives expression of VferLPAAT2. The initiator ATG and terminator TGA codons of VferLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0678] Table 95 shows the fatty acid / TAG profiles of eight primary transformants of CHK22 transformed with pCHK1220. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0679] Table 95: Screening of primary transformants of CHK22 containing pCHK1220.

[0680]

[0681] Example 46: Expression of Vitreochlamys aulata (VaulLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0682] VaulLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1222 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 96.

[0683] Table 96: Integration sequences used for transformation of Prospora moriformis with pCHK1222 encoding VaulLPAAT2.

[0684]

[0685]

[0686]

[0687] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:VaulLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH capable of amplifying the VaulLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of VaulLPAAT2. The initiator ATG and terminator TGA codons of VaulLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0688] Table 97 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1222. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0689] Table 97: Screening of primary transformants of CHK22 containing pCHK1222.

[0690]

[0691] Example 47: Expression of Chlamydomonas sp. CCAC2762_B (Ch_CCAC2762_LPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0692] Ch_CCAC2762_LPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1224 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 98.

[0693] Table 98: Integration sequences used for transformation of Prospora moriformis with pCHK1224 encoding Ch_CCAC2762_LPAAT2.

[0694]

[0695]

[0696]

[0697] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:Ch_CCAC2762_LPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriformis PmPGH 3'-UTR capable of amplifying the Ch_CCAC2762_LPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter from moriformis (indicated by boxed italic text) drives the expression of Ch_CCAC2762_LPAAT2. The initiator ATG and terminator TGA codons of Ch_CCAC2762_LPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 Thia4 genomic region indicated by bold lowercase text.

[0698] Table 99 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1224. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0699] Table 99: Screening of primary transformants of CHK22 containing pCHK1224.

[0700]

[0701]

[0702] Example 48: Expression of Dunaliella tertiolecta (DsalLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDG promoter.

[0703] DsalLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1225 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 100.

[0704] Table 100: Integration sequences used to transform Prospora moriformis with pCHK1225 encoding DsalLPAAT2.

[0705]

[0706]

[0707]

[0708] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:DsalLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter that drives the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The moriform prosporangium PmPGH 3'-UTR capable of amplifying the DsalLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the common Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDG promoter from Protocystis moriformis (indicated by boxed italic text) drives the expression of DsalLPAAT2. The initiator ATG and terminator TAG codons of DsalLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 Thia4 genomic region indicated by bold lowercase text.

[0709] Table 101 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1225. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0710] Table 101: Screening of primary transformants of CHK22 containing pCHK1225.

[0711]

[0712]

[0713] Example 49: Expression of Myarc sp. YARC (MyarcLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0714] MyarcLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1227 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 102.

[0715] Table 102: Integration sequences used for transformation of Prospora moriformis with pCHK1227 encoding MyarcLPAAT2.

[0716]

[0717]

[0718]

[0719] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:MyarcLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability of CHK22 to metabolize sucrose) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH that can amplify the MyarcLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter from Prospora moriformis (indicated by boxed italic text) drives the expression of MyarcLPAAT2. The initiator ATG and terminator TGA codons of MyarcLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22 Thia4 genomic region indicated by bold lowercase text.

[0720] Table 103 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1227. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0721] Table 103: Screening of primary transformants of CHK22 containing pCHK1227.

[0722]

[0723] Example 50: Expression of Chlamydomonas sp. UWO_241 (CuwoLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0724] CuwoLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1228 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 104.

[0725] Table 104: Integration sequences used for transformation of Prospora moriformis with pCHK1228 encoding CuwoLPAAT2.

[0726]

[0727]

[0728]

[0729] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:CuwoLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH capable of amplifying the CuwoLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of CuwoLPAAT2. The initiator ATG and terminator TGA codons of CuwoLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0730] Table 105 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1228. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0731] Table 105: Screening of primary transformants of CHK22 containing pCHK1228.

[0732]

[0733] Example 51: Expression of Chlamydomonas moewusii (CmoeLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0734] CmoeLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1231 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 106.

[0735] Table 106: Integration sequences used for transformation of Prospora moriformis with pCHK1231 encoding CmoeLPAAT2.

[0736]

[0737]

[0738] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:CmoeLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH that can amplify the CmoeLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives expression of CmoeLPAAT2. The initiator ATG and terminator TGA codons of CmoeLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0739] Table 107 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1231. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0740] Table 107: Screening of primary transformants of CHK22 containing pCHK1231.

[0741]

[0742] Example 52: Expression of Oophila amblystomatis (OambLPAAT2) in Prospora moriformis strain CHK22 using the PmG3PDH promoter.

[0743] OambLPAAT2 was introduced into Prospora moriformis strain CHK22. The expression construct pCHK1232 contains 5' and 3' homology arms to allow its targeted integration into the genome and is shown in Table 108.

[0744] Table 108: Integration sequences used for transformation of Prospora moriformis with pCHK1232 encoding OambLPAAT2.

[0745]

[0746]

[0747]

[0748] The construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:OambLPAAT2:PmPGH::3'Thia4. Going in the 5' to 3' direction, the bold lowercase sequence represents genomic DNA from CHK22, which allows targeted integration at the Thia4 locus via homologous recombination, and the Chlamydomonas reinhardtii β-tubulin promoter driving the expression of the yeast sucrose invertase gene (which confers the ability to metabolize sucrose to CHK22) is indicated by boxed uppercase text. The initiator ATG and terminator TAA of the invertase are indicated by uppercase bold italics, while the coding region is indicated in lowercase italics. The PmPGH 3'-UTR of the moriformis PmPGH capable of amplifying the OambLPAAT2 gene is indicated by uppercase underlined text, followed by a linker indicated by lowercase bold italics, which contains the Chlorella vulgaris nitrate reductase 3'-UTR. The PmG3PDH promoter of Prospora moriformis, indicated by boxed italic text, drives the expression of OambLPAAT2. The initiator ATG and terminator TAG codons of OambLPAAT2 are indicated by uppercase bold italics, while the rest of the gene is indicated by bold italics. The PmGH 3'-UTR is indicated by lowercase underlined text, followed by the CHK22Thia4 genomic region indicated by bold lowercase text.

[0749] Table 109 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 transformed with pCHK1232. The moriformis protocystis base strain CHK22 is shown as a non-transgenic control. The strains were grown in lipid production medium at 28°C at 200 rpm in 50-mL conical tubes for 120 hours. The biomass was harvested, lyophilized to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method as described above.

[0750] Table 109: Screening of primary transformants of CHK22 containing pCHK1232.

[0751]

[0752] The amino acid sequences of the various LPAATs disclosed herein were compared to the amino acid sequence of CrLPAAT2 to generate a percent (%) sequence identity. The results are shown in Table 110. The resulting phenotype of expressing these LPAATs in the non-transgenic cell line CHK22 is indicated by the OP:OO ratio.

[0753] In some embodiments, the microalgae cells provided herein comprise an exogenous gene encoding an LPAAT in Table 110. In some embodiments, the exogenous gene can comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an LPAAT in Table 110.

[0754] Table 110: Percent Identity and OP:OO Ratios for CrLPAAT2

[0755]

[0756]

[0757]

[0758] The amino acid sequences were compared to that of PedLPAAT2 to generate percent sequence identity (%). The results are shown in Table 111. The resulting phenotypes of expressing these LPAATs in CHK22 are indicated by the OP:OO ratio.

[0759] In some embodiments, the microalgae cells provided herein comprise an exogenous gene encoding an LPAAT in Table 111. In some embodiments, the exogenous gene can comprise a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an LPAAT in Table 111.

[0760] Table 111: Percent Identity and OP:OO Ratios for PedLPAAT2

[0761]

[0762]

[0763]

[0764] Table 112 shows the amino acid sequences of LPAATs described herein.

[0765] In some embodiments, the microalgae cells provided herein comprise an exogenous gene encoding an LPAAT in Table 112. In some embodiments, the exogenous gene may comprise a sequence encoding an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an LPAAT in Table 112. In some embodiments, the exogenous gene may comprise a sequence encoding an enzyme having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 83-106, 155, and 156.

[0766] Table 112: LPAAT amino acid sequence

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773] Table 113 shows the conserved LPAAT motifs and the percent identity (%) of the most active LPAATs across the complete LPAAT protein relative to PedLPAAT2. The transmembrane domains of these LPAATs are compared, and a schematic diagram of the LPAAT is shown in Fig.11 The LPAAT catalytic domain NHX4D is shown as a shaded bar, the binding motif EGTR or EGHR is shown as a black bar, and the transmembrane domain is shown as a grey bar.

[0774] Table 113: Percent identity to PedLPAAT2 and LPAAT motifs

[0775]

[0776]

[0777] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now expect multiple variations, changes and substitutions without departing from the present invention. It should be understood that the various alternatives of the embodiments of the present invention described herein can be used to implement the present invention. The following claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. An oil comprising: at least 10 mg of ergosterol per 100 g of said oil; and triacylglycerol (TAG) component, wherein at least 40% of the TAGs in the TAG component are TAG species having a saturated fatty acid at the sn-2 position, Wherein at least 50% of the acyl chains in the TAG component are C18:

1.

2. The oil of claim 1, wherein the saturated fatty acid is C16:

0.

3. The oil according to any one of the preceding claims, wherein at least 50% of the TAGs in the TAG component are TAG species having a saturated fatty acid at the sn-2 position.

4. The oil according to any one of the preceding claims, wherein the TAG species comprises C18: 1 at the sn-1 and sn-3 positions.

5. The oil according to any one of the preceding claims, wherein the TAG species comprises C16:0 at the sn-2 position.

6. The oil according to any one of the preceding claims, wherein the TAG species comprises or consists of: 1,3-Dioleoyl-2-palmitoylglycerol (OPO).

7. The oil according to any one of the preceding claims, wherein at least 20% of the acyl chains in the TAG component are C16:

0.

8. The oil according to any one of the preceding claims, wherein at least 60% of the acyl chains in the TAG component are C18:1 and at least 30% of the acyl chains in the TAG component are C16:

0.

9. The oil according to any one of the preceding claims, wherein 50%-67% or 60%-67% of the acyl chains in the TAG component are C18:1, and 20%-33% of the acyl chains in the TAG component are C16:

0.

10. The oil of any one of the preceding claims, wherein the oil has an OP:OO m / z ratio of at least 1.6, as determined by the abundance of diacylglycerol (DAG) ions produced by mass spectrometric fragmentation of the TAG component in the oil.

11. The oil according to any one of the preceding claims, wherein the oil has an OP:OO m / z ratio of at least 2, the OP:OO m / z ratio being determined by the abundance of DAG ions produced by mass spectrometric fragmentation of the TAG component in the oil.

12. The oil of any one of the preceding claims, wherein the oil has an OP:OO m / z ratio of at least 3, the OP:OO m / z ratio being determined by the abundance of DAG ions produced by mass spectrometric fragmentation of the TAG component in the oil.

13. The oil of any of the preceding claims, wherein the oil has an OP:OO m / z ratio of at least 4, the OP:OO m / z ratio being determined by the abundance of DAG ions produced by mass spectrometric fragmentation of the TAG component in the oil.

14. The oil according to any one of the preceding claims, wherein the oil comprises at least 50 mg ergosterol per 100 g of the oil.

15. The oil according to any one of the preceding claims, wherein the oil comprises at least 100 mg ergosterol per 100 g of the oil.

16. The oil according to any one of the preceding claims, wherein the oil comprises 100-200 mg ergosterol per 100 g of the oil.

17. The oil according to any one of the preceding claims, wherein the oil comprises, per 100 g of the oil, no more than 5 mg campesterol, no more than 5 mg β-sitosterol or no more than 5 mg stigmasterol.

18. The oil according to any one of the preceding claims, wherein the oil comprises no more than 5 mg campesterol per 100 g of the oil.

19. The oil according to any one of the preceding claims, wherein the oil does not comprise campesterol.

20. The oil according to any one of the preceding claims, wherein the oil comprises no more than 5 mg stigmasterol per 100 g of the oil.

21. The oil according to any one of the preceding claims, wherein the oil does not contain stigmasterol.

22. The oil according to any one of the preceding claims, wherein the oil comprises no more than 5 mg β-sitosterol per 100 g of the oil.

23. The oil according to any one of the preceding claims, wherein the oil does not comprise β-sitosterol.

24. The oil according to any one of the preceding claims, wherein the oil further comprises one or more of ergosta-5,8-dien-3-ol, (3β)-; 5.xi.-ergosta-7-en-3β-ol; 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)-; and ergosta-7,22-dien-3-ol, (3β).

25. The oil according to claim 24, wherein the oil comprises at least 1 mg of ergosta-5,8-dien-3-ol, (3β)-per 100 g of the oil.

26. The oil according to claim 24, wherein the oil comprises 1-50 mg of ergosta-5,8-dien-3-ol, (3β)-per 100 g of the oil.

27. The oil of claim 24, wherein the oil comprises at least 15 mg 5.xi.-ergost-7-en-3β-ol per 100 g of the oil.

28. The oil of claim 24, wherein the oil comprises 1-50 mg 5.xi.-ergost-7-en-3β-ol per 100 g of the oil.

29. The oil of claim 24, wherein the oil comprises at least 20 mg 5.xi.-ergost-7-en-3β-ol per 100 g of the oil.

30. The oil of claim 24, wherein the oil comprises at least 5 mg of 9,19-cyclolanostan-3-ol, 24-methylene-, (3β)- per 100 g of the oil.

31. The oil of any one of the preceding claims, wherein the oil is algae oil.

32. according to the oil in any one of the preceding claims, wherein said oil is a genetically modified algae oil.

33. The oil of any one of the preceding claims, wherein the oil is produced by algal cells.

34. according to the oil in any one of the preceding claims, wherein said oil is produced by genetically modified algae cells.

35. The oil of any one of the preceding claims, wherein the oil is produced by Prototheca cells.

36. The oil of any one of the preceding claims, wherein the oil is non-naturally occurring.

37. A composition comprising the oil of any one of claims 1-36; and one or more excipients.

38. The composition of claim 37, wherein the composition is a nutritional supplement.

39. The composition of claim 37, wherein the composition is an infant formula.

40. The composition of claim 39, wherein the infant formula comprises one or more of whey, casein, lactose, vitamin D, human milk oligosaccharides (HMOs), vegetable oils, and antibodies.

41. The composition of claim 40, wherein the vegetable oil comprises soybean oil, rapeseed oil, sunflower oil, coconut oil, palm oil or palm kernel oil.

42. The composition of claim 39, wherein the infant formula comprises soy protein.

43. A TAG polyol produced from the oil of any one of claims 1-36.

44. A method for producing a TAG polyol, the method comprising: a) subjecting the oil according to any one of claims 1 to 36 to epoxidation, thereby producing an epoxidized oil; and b) ring opening the epoxidized oil in the presence of an alcohol, an acid or hydrogen and a suitable catalyst to produce the TAG polyol.

45. The method of claim 44, wherein the epoxidized oil is ring-opened in the presence of an alcohol.

46. ​​The method of claim 44, wherein the epoxidized oil is ring-opened in the presence of hydrogen and a suitable catalyst.

47. The method of claim 44, wherein the epoxidized oil is ring-opened in the presence of an acid.

48. A method for producing a TAG polyol, the method comprising: a) subjecting the oil according to any one of claims 1 to 36 to hydroformylation, thereby producing a hydroformylated oil; and b) reducing the hydroformylated oil in the presence of hydrogen and a suitable catalyst to produce the TAG polyol.

49. A TAG polyol produced by the method of any one of claims 44-48.

50. A microalgae cell comprising an exogenous gene encoding an enzyme having lysophosphatidic acid acyltransferase activity, wherein the cell produces an oil comprising a TAG component, wherein at least 40% of the TAGs in the TAG component are TAG species having a saturated fatty acid at the sn-2 position, Wherein at least 50% of the acyl chains in the TAG component are C18:

1.

51. The microalgae cell of claim 50, wherein the enzyme is lysophosphatidic acid acyltransferase (LPAAT).

52. The microalgae cell of claim 50 or 51, wherein the enzyme is algal lysophosphatidic acid acyltransferase (LPAAT).

53. The microalgae cell of any one of claims 50-52, wherein the enzyme comprises a sequence motif selected from any one of (I) NHXXXXD (or NHX4D); (II) (F / Y) XXR; (III) EGXR; and (IV) proline, wherein X is any amino acid.

54. The microalgae cell of any one of claims 50-53, wherein the enzyme comprises NHXXXXD (or NHX4D), wherein X is any amino acid.

55. The microalgae cell of any one of claims 50-54, wherein the enzyme comprises a sequence having at least 70%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 107-109.

56. The microalgae cell of any one of claims 50-55, wherein the enzyme comprises (F / Y)XXR, wherein X is any amino acid.

57. The microalgae cell of any one of claims 50-56, wherein the enzyme comprises a sequence having at least 60%, at least 75%, at least 85%, or 100% sequence identity to any one of SEQ ID NOs: 110-119.

58. The microalgae cell of any one of claims 50-57, wherein the enzyme comprises EGXR, wherein X is any amino acid.

59. The microalgae cell of any one of claims 50-58, wherein the enzyme comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 120-134.

60. The microalgae cell of any one of claims 50-59, wherein the enzyme comprises a sequence having a conserved proline.

61. The microalgae cell of any one of claims 50-60, wherein the enzyme comprises a sequence having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or 100% sequence identity to any one of SEQ ID NOs: 135-154.

62. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas reinhardtii lysophosphatidic acid acyltransferase (CrLPAAT1).

63. The microalgae cell of any one of claims 50-52 and 62, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

12.

64. The microalgae cell of any one of claims 50-52, 62, and 63, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

2.

65. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas incerta lysophosphatidic acid acyltransferase (CiLPAAT2).

66. The microalgae cell of any one of claims 50-52 and 65, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

15.

67. The microalgae cell of any one of claims 50-52, 65, and 66, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

10.

68. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonasschloesseri lysophosphatidic acid acyltransferase (ChsLPAAT2).

69. The microalgae cell of any one of claims 50-52 and 68, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

14.

70. The microalgae cell of any one of claims 50-52, 68, and 69, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

9.

71. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvoxafricanus lysophosphatidic acid acyltransferase (VaLPAAT2).

72. The microalgae cell of any one of claims 50-52 and 71, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

21.

73. The microalgae cell of any one of claims 50-52, 71 and 72, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

11.

74. The microalgae cell of any one of claims 50-52, wherein the enzyme is Nannochloropsis oceanica lysophosphatidic acid acyltransferase (NoLPAT3).

75. The microalgae cell of any one of claims 50-52 and 74, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

16.

76. The microalgae cell of any one of claims 50-52, 74, and 75, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

7.

77. The microalgae cell of any one of claims 50-52, wherein the enzyme is Synechocystis sp. 1-acyl-sn-glycerol-3-phosphate acyltransferase (Sll1848).

78. The microalgae cell of any one of claims 50-52 and 77, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

19.

79. The microalgae cell of any one of claims 50-52, 77, and 78, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

5.

80. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas reinhardtii lysophosphatidic acid acyltransferase (CrLPAAT2).

81. The microalgae cell of any one of claims 50-52 and 80, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

13.

82. The microalgae cell of any one of claims 50-52, 80, and 81, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

1.

83. The microalgae cell of any one of claims 50-52, wherein the enzyme is Escherichia coli 1-acyl-sn-glycerol-3-phosphate acyltransferase (EcPlsC).

84. The microalgae cell of any one of claims 50-52 and 83, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

22.

85. The microalgae cell of any one of claims 50-52, 83, and 84, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

6.

86. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvox carteri lysophosphatidic acid acyltransferase (VcLPAAT2).

87. The microalgae cell of any one of claims 50-52 and 86, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

20.

88. The microalgae cell of any one of claims 50-52, 86, and 87, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

8.

89. The microalgae cell of any one of claims 50-52, wherein the enzyme is Brassicanapus acyltransferase (BnBAT2).

90. The microalgae cell of any one of claims 50-52 and 89, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

18.

91. The microalgae cell of any one of claims 50-52, 89, and 90, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

3.

92. The microalgae cell of any one of claims 50-52, wherein the enzyme is Nannochloropsis marineus lysophosphatidic acid acyltransferase (NoLPAT4).

93. The microalgae cell of any one of claims 50-52 and 92, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

17.

94. The microalgae cell of any one of claims 50-52, 92, and 93, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

4.

95. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvox nagariensis lysophosphatidic acid acyltransferase (VcLPAAT2).

96. The microalgae cell of any one of claims 50-52 and 95, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

40.

97. The microalgae cell of any one of claims 50-52, 95, and 96, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

25.

98. The microalgae cell of any one of claims 50-52, wherein the enzyme is Astrephomenegubernaculifera lysophosphatidic acid acyltransferase (AgLPAAT2).

99. The microalgae cell of any one of claims 50-52 and 98, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

41.

100. The microalgae cell of any one of claims 50-52, 98, and 99, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

26.

101. The microalgae cell of any one of claims 50-52, wherein the enzyme is Edaphochlamysdebaryana lysophosphatidic acid acyltransferase (EdLPAAT2).

102. The microalgae cell of any one of claims 50-52 and 101, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

42.

103. The microalgae cell of any one of claims 50-52, 101 and 102, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

27.

104. The microalgae cell of any one of claims 50-52, wherein the enzyme is Dunaliella salina lysophosphatidic acid acyltransferase (DsLPAAT2).

105. The microalgae cell of any one of claims 50-52 and 104, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

43.

106. The microalgae cell of any one of claims 50-52, 104, and 105, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

28.

107. The microalgae cell of any one of claims 50-52, wherein the enzyme is Scenedesmus sp. lysophosphatidic acid acyltransferase (SceLPAAT2).

108. The microalgae cell of any one of claims 50-52 and 107, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

44.

109. The microalgae cell of any one of claims 50-52, 107, and 108, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

29.

110. The microalgae cell of any one of claims 50-52, wherein the enzyme is Micractiniumconductrix lysophosphatidic acid acyltransferase (McLPAAT2).

111. The microalgae cell of any one of claims 50-52 and 110, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

45.

112. The microalgae cell of any one of claims 50-52, 110, and 111, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

30.

113. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlorella sorokiniana lysophosphatidic acid acyltransferase (ChsoLPAAT2).

114. The microalgae cell of any one of claims 50-52 and 113, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

46.

115. The microalgae cell of any one of claims 50-52, 113, and 114, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

31.

116. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlorella variabilis lysophosphatidic acid acyltransferase (ChvaLPAAT2).

117. The microalgae cell of any one of claims 50-52 and 116, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

47.

118. The microalgae cell of any one of claims 50-52, 116, and 117, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

32.

119. The microalgae cell of any one of claims 50-52, wherein the enzyme is Raphidocelis subcapitata lysophosphatidic acid acyltransferase (RsLPAAT2).

120. The microalgae cell of any one of claims 50-52 and 119, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

48.

121. The microalgae cell of any one of claims 50-52, 119, and 120, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

33.

122. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlorella desiccata lysophosphatidic acid acyltransferase (ChdeLPAAT2).

123. The microalgae cell of any one of claims 50-52 and 122, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

49.

124. The microalgae cell of any one of claims 50-52, 122, and 123, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

34.

125. The microalgae cell of any one of claims 50-52, wherein the enzyme is Auxenochlorella protothecoides lysophosphatidic acid acyltransferase (ApLPAAT2).

126. The microalgae cell of any one of claims 50-52 and 125, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

50.

127. The microalgae cell of any one of claims 50-52, 125, and 126, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

35.

128. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chloropiconprimus lysophosphatidic acid acyltransferase (ChprLPAAT2).

129. The microalgae cell of any one of claims 50-52 and 128, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

51.

130. The microalgae cell of any one of claims 50-52, 128, and 129, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

36.

131. The microalgae cell of any one of claims 50-52, wherein the enzyme is Homo sapiens lysophosphatidic acid acyltransferase (AGPAT1).

132. The microalgae cell of any one of claims 50-52 and 131, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

52.

133. The microalgae cell of any one of claims 50-52, 131, and 132, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

37.

134. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonaseustigma lysophosphatidic acid acyltransferase (CeLPAAT2).

135. The microalgae cell of any one of claims 50-52 and 134, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

53.

136. The microalgae cell of any one of claims 50-52, 134, and 135, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

38.

137. The microalgae cell of any one of claims 50-52, wherein the enzyme is Pedinophyceae lysophosphatidic acid acyltransferase (PedLPAAT2).

138. The microalgae cell of any one of claims 50-52 and 137, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

54.

139. The microalgae cell of any one of claims 50-52, 137, and 138, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

39.

140. The microalgae cell of any one of claims 50-52, 137, and 138, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

59.

141. The microalgae cell of any one of claims 50-52, 137, and 138, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

60.

142. The microalgae cell of any one of claims 50-52, 137, and 138, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

61.

143. The microalgae cell of any one of claims 50-52, 137, and 138, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

62.

144. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvox reticuliferus lysophosphatidic acid acyltransferase (VrLPAAT2).

145. The microalgae cell of any one of claims 50-52 and 144, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

55.

146. The microalgae cell of any one of claims 50-52, 144, and 145, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

57.

147. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlorella vulgaris lysophosphatidic acid acyltransferase (ChvuLPAAT2).

148. The microalgae cell of any one of claims 50-52 and 147, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

56.

149. The microalgae cell of any one of claims 50-52, 147, and 148, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

58.

150. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvulinacompacta lysophosphatidic acid acyltransferase (VcomLPAAT2).

151. The microalgae cell of any one of claims 50-52 and 150, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

83.

152. The microalgae cell of any one of claims 50-52, 150, and 151, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

65.

153. The microalgae cell of any one of claims 50-52, wherein the enzyme is Vitreochlamys sp. CL-2021 lysophosphatidic acid acyltransferase (VitrLPAAT2).

154. The microalgae cell of any one of claims 50-52 and 153, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

84.

155. The microalgae cell of any one of claims 50-52, 153, and 154, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

66.

156. The microalgae cell of any one of claims 50-52, wherein the enzyme is Colemanosphaera charkowiensis lysophosphatidic acid acyltransferase (CchaLPAAT2).

157. The microalgae cell of any one of claims 50-52 and 156, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

85.

158. The microalgae cell of any one of claims 50-52, 156, and 157, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

67.

159. The microalgae cell of any one of claims 50-52, wherein the enzyme is Pleodorina japonica lysophosphatidic acid acyltransferase (PjapLPAAT2).

160. The microalgae cell of any one of claims 50-52 and 159, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

86.

161. The microalgae cell of any one of claims 50-52, 159, and 160, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

68.

162. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvulina boldii lysophosphatidic acid acyltransferase (VbolLPAAT2).

163. The microalgae cell of any one of claims 50-52 and 162, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

87.

164. The microalgae cell of any one of claims 50-52, 162, and 163, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

69.

165. The microalgae cell of any one of claims 50-52, wherein the enzyme is Pandorinamorum lysophosphatidic acid acyltransferase (PmorLPAAT2).

166. The microalgae cell of any one of claims 50-52 and 165, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

88.

167. The microalgae cell of any one of claims 50-52, 165, and 166, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

70.

168. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvox weismannia lysophosphatidic acid acyltransferase (VcarfLPAAT2).

169. The microalgae cell of any one of claims 50-52 and 168, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

89.

170. The microalgae cell of any one of claims 50-52, 168, and 169, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

71.

171. The microalgae cell of any one of claims 50-52, wherein the enzyme is Eudorinacylindrica lysophosphatidic acid acyltransferase (EcylLPAAT2).

172. The microalgae cell of any one of claims 50-52 and 171, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

90.

173. The microalgae cell of any one of claims 50-52, 171, and 172, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

72.

174. The microalgae cell of any one of claims 50-52, wherein the enzyme is Goniummulticoccum lysophosphatidic acid acyltransferase (GmulLPAAT2).

175. The microalgae cell of any one of claims 50-52 and 174, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

91.

176. The microalgae cell of any one of claims 50-52, 174, and 175, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

73.

177. The microalgae cell of any one of claims 50-52, wherein the enzyme is Gonium viridistellatum lysophosphatidic acid acyltransferase (GvirLPAAT2).

178. The microalgae cell of any one of claims 50-52 and 177, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

92.

179. The microalgae cell of any one of claims 50-52, 177, and 178, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

74.

180. The microalgae cell of any one of claims 50-52, wherein the enzyme is Volvox ferrisii lysophosphatidic acid acyltransferase (VferLPAAT2).

181. The microalgae cell of any one of claims 50-52 and 180, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

93.

182. The microalgae cell of any one of claims 50-52, 180, and 181, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

75.

183. The microalgae cell of any one of claims 50-52, wherein the enzyme is Vitreochlamysaulata lysophosphatidic acid acyltransferase (VaulLPAAT2).

184. The microalgae cell of any one of claims 50-52 and 183, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

94.

185. The microalgae cell of any one of claims 50-52, 183, and 184, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

76.

186. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas sp. CCAC2762_B lysophosphatidic acid acyltransferase (Ch_CCAC2762_LPAAT2).

187. The microalgae cell of any one of claims 50-52 and 186, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

95.

188. The microalgae cell of any one of claims 50-52, 186, and 187, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

77.

189. The microalgae cell of any one of claims 50-52, wherein the enzyme is Dunaliella tertiolecta lysophosphatidic acid acyltransferase (DsalLPAAT2).

190. The microalgae cell of any one of claims 50-52 and 189, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

96.

191. The microalgae cell of any one of claims 50-52, 189, and 190, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

78.

192. The microalgae cell of any one of claims 50-52, wherein the enzyme is Microglena sp. YARC lysophosphatidic acid acyltransferase (MyarcLPAAT2).

193. The microalgae cell of any one of claims 50-52 and 192, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

97.

194. The microalgae cell of any one of claims 50-52, 192, and 193, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

79.

195. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas sp. UWO_241 lysophosphatidic acid acyltransferase (CuwoLPAAT2).

196. The microalgae cell of any one of claims 50-52 and 195, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

98.

197. The microalgae cell of any one of claims 50-52, 195, and 196, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

80.

198. The microalgae cell of any one of claims 50-52, wherein the enzyme is Chlamydomonas moewusii lysophosphatidic acid acyltransferase (CmoeLPAAT2).

199. The microalgae cell of any one of claims 50-52 and 198, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

99.

200. The microalgae cell of any one of claims 50-52, 198, and 199, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

81.

201. The microalgae cell of any one of claims 50-52, wherein the enzyme is Oophilaamblystomatis lysophosphatidic acid acyltransferase (OambLPAAT2).

202. The microalgae cell of any one of claims 50-52 and 201, wherein the enzyme comprises a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

100.

203. The microalgae cell of any one of claims 50-52, 201 and 202, wherein the cell comprises a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

82.

204. The microalgae cell of any one of claims 50-203, wherein the exogenous gene is codon-optimized for expression in a Protocystis strain.

205. The microalgal cell of any one of claims 50-204, wherein the cell does not comprise exogenous glycerol-3-phosphate acyltransferase (GPAT1).

206. The microalgae cell of any one of claims 50-205, wherein the cell is from a basal strain of the genus Protocystis.

207. The microalgal cell of any one of claims 50-206, wherein the cell is from Prototheca moriformis base strain UTEX 1533.

208. The microalgae cell of any one of claims 50-207, wherein the cell is derived from a classically improved strain from the Prospora moriformis base strain UTEX 1533.

209. The microalgae cell of any one of claims 50-208, wherein the cell is from a non-genetically modified base strain of Protocystis that produces an oil having a fatty acid profile of at least 50% oleic acid.

210. The microalgae cell of any one of claims 50-209, wherein the cell is from a non-genetically modified base strain of Protocystis that produces an oil having a fatty acid profile of at least 30% palmitic acid.

211. The microalgae cell of any one of claims 50-210, wherein the Prototheca basal strain is Prototheca wickerhamii.

212. The microalgae cell of any one of claims 50-210, wherein the basal strain of the genus Prosporaminaceae is Prospora moriformis.

213. The microalgal cell of any one of claims 50-212, wherein the microalgal cell produces at least 50% lipid by dry cell weight.

214. The microalgae cell of any one of claims 50-213, wherein the oil is the oil of any one of claims 1-36.

215. A method of producing a non-naturally occurring oil, the method comprising culturing microalgae cells in a culture medium, wherein the oil comprises a TAG component, wherein at least 40% of the TAG species in the TAG component have a saturated fatty acid at the sn-2 position, Wherein at least 50% of the acyl chains in the TAG component are C18:

1.

216. The method of claim 215, further comprising isolating the oil composition from the culture medium.

217. The method of claim 215, further comprising expressing in the microalgae cells an exogenous enzyme having lysophosphatidic acid acyltransferase activity.

218. The method of any one of claims 215-217, wherein the microalgae cell is a microalgae cell according to any one of claims 50-214.

219. The method of any one of claims 215-218, wherein the oil is the oil of any one of claims 1-36.

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