Region-specific triglyceride oils and mixtures thereof and related food and nutritional applications

Abstract

This article provides triglyceride oil compositions rich in 1,3-dioleoyl-2-palmitoylglycerol (OPO) for food applications and infant nutritional formulations. This article also provides methods for producing OPO-rich, non-naturally occurring triglyceride oil compositions from non-naturally occurring microorganisms, and their applications in human and animal foods, infant formulations, and nutritional supplements.

Description

Background Technology

[0001] Oil-producing microorganisms can convert carbon substrates into oils (including triglycerides (TAGs) and lipids) and accumulate these oils intracellularly. TAGs are important components in foods and nutrients. Human milk TAGs play a crucial role in infant nutrition and development. These TAGs present challenges in formulating infant foods and formula milk powders that mimic the composition and functional properties of human milk. In particular, TAG structure affects the digestion, absorption, and bioavailability of lipophilic micronutrients and essential fatty acids, which influence infant feeding and nutrition. This invention aims to provide an oil with a high oleic-palmitic-oleic acid content for use in the food and human nutrition fields.

[0002] Cross-references to related applications

[0003] This international application claims the benefit of U.S. Provisional Application No. 63 / 595,229, filed November 1, 2023.

[0004] sequence list

[0005] This application includes a sequence list XML submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The sequence list XML was created on October 30, 2024, named 009919_00093_WO_SL.xml, and has a size of 535,010 bytes.

[0006] Reference merging

[0007] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0008] The features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of the invention, and in the accompanying drawings: Figure 1 shows two types of TAGs with the same molecular weight, displaying different daughter ion ratios. Figure A shows OOP, and Figure B shows OPO.

[0009] Figure 2 shows mass spectra of OOP / OPO triglycerides in oils produced by various microalgal strains provided in this paper. Figure A shows CHK22, D552-1; Figure B shows CHK22, D552-3; Figure C shows CHK22, D552-4; and Figure D shows the non-transgenic strain CHK22.

[0010] Figure 3, section A shows the amino acid sequence alignment of the LPAAT described herein. The sequences are: 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), CrLPAAT1 (SEQ ID NO: 12), and a common sequence (SEQ ID NO: 157).

[0011] Figure 4 A schematic diagram showing the structural domains of the LPAAT described herein is shown.

[0012] Figure 5 The bar graph shows the lipid content of various strains (including those transformed with pCHK385) in... sn Fatty acid composition at the -2 position.

[0013] Figure 6 The LC-MS results of oils from CHK22 and strain D552-3 are shown.

[0014] Figure 7 The diagram shows TAG oils rich in OPO and downstream TAG intermediates generated through a chemical conversion process.

[0015] Figure 8 An example of a diol produced from a TAG oil rich in OPO is shown.

[0016] Figure 9 An overview of an exemplary conventional strain improvement strategy for generating mutants derived from CHK22 (UTEX 1533) is shown.

[0017] Figure 10 The strategy for obtaining CHK100 from CHK22 through strain modification is shown.

[0018] Figure 11 The transmembrane domain of LPAAT disclosed herein is shown.

[0019] Figure 12 The health benefits of sn-2 palmitate for infants are shown in the figure, which is cited from Havlicekova et al., 2015.

[0020] Figure 13 The LC-MS triglyceride (TAG) spectra of algal OPO oil and a first-generation human milk fat substitute obtained by transesterification catalyzed by plant olease were compared according to one aspect of this disclosure. Summary of the Invention

[0021] In some embodiments, this document provides a naturally occurring oil comprising: at least 10 mg of ergosterol per 100 g of oil; and a triglyceride (TAG) component, wherein at least 40% of the TAG component is a TAG type having a saturated fatty acid at the sn-2 position, and at least 50% of the acyl chain in the TAG component is C18:1. The saturated fatty acid may be C16:0. At least 50% of the TAG component may be a TAG type having a saturated fatty acid at the sn-2 position. At least 50% of the acyl chain in the TAG component may be C18:1. The TAG type may contain C18:1 at the sn-1 and sn-3 positions. The TAG type may contain C16:0 at the sn-2 position. The TAG type may contain or consist of 1,3-dioleoyl-2-palmitoylglycerol (OPO). At least 50% of the TAG component may be C18:1, and at least 20% of the acyl chain 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 OP:OO m / z ratio of the oil may be at least 1.6, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 2, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 3, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 4, determined by the abundance of DAG ions generated by mass spectrometric 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 contain at least 100-200 mg of ergosterol per 100 g of oil. The oil may also contain no more than 5 mg of campesterol, no more than 5 mg of β-sitosterol, or no more than 5 mg of stigmasterol per 100 g of oil. For example, the oil may contain no more than 5 mg of campesterol per 100 g of oil. In some embodiments, the oil does not contain campesterol. For example, the oil may contain no more than 5 mg of stigmasterol per 100 g of oil. In some embodiments, the oil does not contain stigmasterol. For example, the oil may contain no more than 5 mg of β-sitosterol per 100 g of oil. In some embodiments, the oil does not contain β-sitosterol.The oil may also contain one or more of (3β)-ergoster-5,9-dien-3-ol, 5ξ-ergoster-7-en-3β-ol, (3β)-24-methylene-9,19-cyclolanostane-3-ol, and (3β)-ergoster-7,22-dien-3-ol. The oil may contain at least 1 mg of (3β)-ergoster-5,9-dien-3-ol per 100 g of oil. The oil may contain 1-50 mg of (3β)-ergoster-5,9-dien-3-ol per 100 g of oil. The oil may contain at least 15 mg of 5ξ-ergoster-7-en-3β-ol per 100 g of oil. The oil may contain 1-50 mg of 5ξ-ergoster-7-en-3β-ol per 100 g of oil. The oil may contain at least 20 mg of 5ξ-ergoster-7-en-3β-ol per 100 g of oil. The oil may contain at least 5 mg of (3β)-24-methylene-9,19-cyclolanostane-3-ol per 100 g of oil. The oil may be algal oil. The oil may be genetically modified algal oil. The oil can be produced from algal cells. The oil can be produced from genetically modified algal cells. The oil may be from the genus *Protocol*. Prototheca ) produced by cells.

[0022] This document discloses a composition comprising oils and one or more excipients. The composition can be a nutritional supplement. The composition can be infant formula. Infant formula may contain one or more of whey, casein, lactose, vitamin D, human milk oligosaccharides (HMOs), vegetable oils, and antibodies. Non-limiting examples of vegetable oils include soybean oil, low-erucic acid rapeseed oil, sunflower oil, coconut oil, palm oil, and palm kernel oil. Infant formula may contain soy protein. This invention discloses a TAG polyol derived from oils.

[0023] This document discloses a method for producing TAG polyols, comprising: epoxidizing the oils described herein to produce epoxidized oils; and ring-opening the epoxidized oils in the presence of an alcohol, an acid, or hydrogen and a suitable catalyst to produce TAG polyols. The epoxidized oils can be ring-opened in the presence of an alcohol. The epoxidized oils can be ring-opened in the presence of hydrogen and a suitable catalyst. The epoxidized oils can be ring-opened in the presence of an acid. This document discloses a TAG polyol prepared by the method described herein.

[0024] This document discloses a method for producing TAG polyols, comprising: hydroformylating the oil described herein to produce hydroformylated oil; and reducing the hydroformylated oil in the presence of hydrogen and a suitable catalyst to produce TAG polyols. This document also discloses a TAG polyol prepared by the method described herein.

[0025] This paper discloses a microalgal cell containing an exogenous gene encoding an enzyme with lysophosphatidylacyltransferase activity, wherein the cell produces an oil containing a TAG component, wherein at least 40% of the TAG component can be a TAG species having a saturated fatty acid at the sn-2 position, and wherein at least 50% of the acyl chain of the TAG component can be C18:1. The enzyme can be a lysophosphatidylacyltransferase (LPAAT). The enzyme can be an algal, higher plant, or mammalian lysophosphatidylacyltransferase (LPAAT).

[0026] In some embodiments, the enzyme disclosed herein may comprise any sequence motif selected from the following: (I) NHXXXXD (or NHX4D); (II) (F / Y) XXR; (III) EGXR; and (IV) proline, wherein X is any amino acid.

[0027] In some embodiments, the enzyme may comprise NHXXXXD (or NHX4D), where X is any amino acid. In some embodiments, the enzyme may comprise a sequence having at least 70% or at least 100% identity with any one of SEQ ID NO: 107-109.

[0028] In some embodiments, the enzyme may comprise (F / Y)XXR, where X is any amino acid. In some embodiments, the HNH domain comprises a sequence having at least 60%, at least 75%, at least 85%, or at least 100% identity with any one of SEQ ID NO: 110-119.

[0029] In some embodiments, the enzyme may comprise EGXR, where 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 at least 100% sequence identity with any one of SEQ ID NO: 120-134.

[0030] In some embodiments, the enzyme may comprise sequence A 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 at least 100% sequence identity with any one of SEQ ID NO: 135-154.

[0031] The enzyme can be Chlamydomonas reinhardtii ( Chlamydomonas reinhardtiiLysophosphatidyl acyltransferase (CrLPAAT1). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 12. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 2.

[0032] The enzyme can be Chlamydomonas aeruginosa ( Chlamydomonas incerta Lysophosphatidyl acyltransferase (CiLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 15. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 10.

[0033] The enzyme can be Chlamydomonas stearothermiae ( Chlamydomonas schloesseri Lysophosphatidyl acyltransferase (ChsLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 14. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 9.

[0034] The enzyme can be *Volvox africanus* (…). Volvox africanus Lysophosphatidyl acyltransferase (VaLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 21. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 11.

[0035] The enzyme can be marine microbiococcus ( Nannochloropsis oceanicaLysophosphatidyl acyltransferase (NoLPAT3). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 16. Cells may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7.

[0036] The enzyme can be from the genus Synechocystis (… Synechocystis sp. The enzyme may contain a 1-acyl-sn-glycerol-3-phosphoacyltransferase (Sll1848) 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 at least 100% sequence identity with SEQ ID NO: 19. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 5.

[0037] The enzyme can be Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii Lysophosphatidyl acyltransferase (CrLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 13. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 1.

[0038] The enzyme may be *E. coli* 1-acyl-sn-glycerol-3-phosphoacyltransferase (EcPlsC). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 22. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 6.

[0039] The enzyme can be Volvox catatini ( Volvox carteriLysophosphatidyl acyltransferase (VcLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 20. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 8. The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 40. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 25.

[0040] The enzyme can be European rapeseed ( Brassica napus Acyltransferase (BnBAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 18. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 3. The enzyme may be a marine microcystis (BnBAT2). Nannochloropsis oceanica Lysophosphatidyl acyltransferase (NoLPAT4). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 17. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 4.

[0041] The enzyme may be a lysophosphatidyl acyltransferase (AgLPAAT2) from the stalked star algae (Astrephomene gubernaculifera). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 41. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 26.

[0042] The enzyme may be EdLPAAT2, a lysophosphatidyl acyltransferase from *Edaphochlamys debaryana*. The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 42. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27.

[0043] This enzyme can be used for Dunaliella salina ( Dunaliella salina Lysophosphatidyl acyltransferase (DsLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 43. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 28.

[0044] Enzymes can be found in the genus *Scenedesmus* ( Scenedesmus sp. Lysophosphatidyl acyltransferase (SceLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 44. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 29.

[0045] Enzymes can guide microalgae ( Micractinium conductrix Lysophosphatidyl acyltransferase (McLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 45. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 30.

[0046] Enzymes can be Chlorella sorokinensis ( Chlorella sorokiniana Lysophosphatidyl acyltransferase (ChsoLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 46. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 31.

[0047] Enzymes can be derived from Chlorella vulgaris ( Chlorella variabilis Lysophosphatidyl acyltransferase (ChvaLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 47. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 32.

[0048] The enzyme can be *Neptunus cephalopoda* (…). Raphidocelis subcapitata Lysophosphatidyl acyltransferase (RsLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 48. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 33.

[0049] Enzymes can be dehydrated Chlorella (Chlorella desiccata Lysophosphatidyl acyltransferase (ChdeLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 49. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 34.

[0050] The enzyme can be *Chlorella vulgaris* (a type of algae). Auxenochlorella protothecoides Lysophosphatidyl acyltransferase (ApLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 50. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 35.

[0051] This enzyme can help Chlorophytum ( Chloropicon primus Lysophosphatidyl acyltransferase (ChprPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 51. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 36.

[0052] Enzymes can be used by Homo sapiens ( Homo sapiens Lysophosphatidyl acyltransferase (AGPAT1). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 52. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 37.

[0053] This enzyme can be used for *Variegata* ( Chlamydomonas eustigmaLysophosphatidyl acyltransferase (CeLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 53. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 38.

[0054] The enzyme may be a Pedinophyceae lysophosphatidyl acyltransferase (PedLPAAT2). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 54. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 39. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 59. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 50. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 61. The cell may contain 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 at least 100% sequence identity with SEQ ID NO: 62.

[0055] The enzyme can be *Volvariella reticulata*. Volvox reticuliferusLysophosphatidyl acyltransferase (VrLPAAT2). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 55. Cells may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 57. The enzyme may be from Chlorella vulgaris (VrLPAAT2). Chlorella vulgaris Lysophosphatidyl acyltransferase (ChvuLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 56. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 58.

[0056] The enzyme can be a dense, volcanic algae-like organism (Volvox). Volvulina compacta) Lysophosphatidyl acyltransferase (VcomLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 83. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 65.

[0057] This enzyme can be found in the genus *Chlamydomonas* (…). Vitreochlamys sp.)CL-2021 Lysophosphatidyl acyltransferase (VitrLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 84. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 66.

[0058] Enzymes can be used for *Coccus chalcogenoides* ( Colemanosphaera charkowiensisLysophosphatidyl acyltransferase (CchaLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 85. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 67.

[0059] Enzymes can be found in Japanese polycystic algae ( Pleodorina japonica) Lysophosphatidyl acyltransferase (PjapLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 86. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 68.

[0060] The enzyme may be a Volvulina boldii lysophosphatidyl acyltransferase (VbolLPAAT2). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 87. The cell may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 69.

[0061] Enzymes can be derived from *Chlorella vulgaris* ( Pandorina morum Lysophosphatidyl acyltransferase (PmorLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 88. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 70.

[0062] The enzyme can be of the Weismann type from Volcanobacter catechu ( ). Volvox carteri f. weismannia Lysophosphatidyl acyltransferase (VcarfLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 89. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 71.

[0063] Enzymes can be found in Cylindrical Hollow Globe ( Eudorina cylindrica Lysophosphatidyl acyltransferase (EcylLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 90. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 72.

[0064] The enzyme can be *Polycholenoptera* (…). Gonium multicoccum Lysophosphatidyl acyltransferase (GmulLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 91. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 73.

[0065] Enzymes can be derived from *Plasmodium chloroticum* (a type of algae). Gonium viridistellatum Lysophosphatidyl acyltransferase (GvirLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 92. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 74.

[0066] The enzyme can be *Volvariella ferri* (… Volvox ferrisiiLysophosphatidyl acyltransferase (VferLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 93. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 75.

[0067] The enzyme can be Chlamydomonas cystis ( ) Vitreochlamys aulata Lysophosphatidyl acyltransferase (VaulLPAAT2). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 94. Cells may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 76.

[0068] Enzymes can be Chlamydomonas ( Chlamydomonas sp. ) CCAC2762_B Lysophosphatidyl acyltransferase (Ch_CCAC2762_LPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 95. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 77.

[0069] Enzymes can be Dunaliella salina (Dunaliella salina) Dunaliella salina Lysophosphatidyl acyltransferase (DsalLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 96. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 78.

[0070] Enzymes can be found in the genus *Pleurotus* ( Microglena sp.)YARCLysophosphatidyl acyltransferase (MyarcLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 97. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 79.

[0071] Enzymes can be Chlamydomonas ( Chlamydomonas sp. UWO_241 lysophosphatidyl acyltransferase (CuwoLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 98. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 80.

[0072] Enzymes can be Chlamydomonas mossii ( Chlamydomonas moewusii Lysophosphatidyl acyltransferase (CmoeLPAAT2). The enzyme may contain 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 at least 100% sequence identity with SEQ ID NO: 99. Cells may contain 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 at least 100% sequence identity with SEQ ID NO: 81.

[0073] The enzyme can be a salamander symbiotic algae ( Oophila amblystomatis Lysophosphatidyl acyltransferase (OambLPAAT2). The enzyme may contain a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 100. Cells may contain a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 82.

[0074] The exogenous gene can be codon-optimized for expression in *Protozoa* strains. In some embodiments, the cells do not contain the exogenous glycerol-3-phosphoacyltransferase (GPAT1). The cells can be derived from *Protozoa* strains. Prototheca The basic strain. Cells can be derived from *Procambarus mulberryii* (…). Prototheca moriformis The basic strain is UTEX 1533. Cells can be derived from *Procambarus mulberryii* (…). Prototheca moriformis The basic strain UTEX 1533 is a strain improved using traditional methods. Cells can be derived from non-genetically modified *Protozoa* species. Prototheca The basic strain produces oils with a fatty acid profile of at least 50% palmitic acid. Cells can originate from non-genetically modified *Protozoa* species. Prototheca The basic strain produces oils with a fatty acid profile containing at least 30% palmitic acid. The *Protozoa* genus (… Prototheca The basic strain can be *Aeromonas viride* (a type of algae). Prototheca wickerhamii The genus *Protocolospora* ( Prototheca The basic strain can be *Procambarus mulberryii* (…). Prototheca moriformisMicroalgal cells can produce at least 60% lipids by weight of stem cells. Microalgal cells can produce at least 50% lipids by weight of stem cells. The oil may contain a triglyceride (TAG) component, wherein at least 40% of the TAG component may be a TAG type having a saturated fatty acid at the sn-2 position, and wherein at least 50% of the acyl chain in the TAG component may be C18:1. The oil may contain at least 10 mg of ergosterol per 100 g of oil. The saturated fatty acid may be C16:0. At least 50% of the TAG component may be a TAG type having a saturated fatty acid at the sn-2 position. At least 50% of the acyl chain in the TAG component may be C18:1. The TAG type may contain C18:1 at the sn-1 and sn-3 positions. The TAG type may contain C16:0 at the sn-2 position. The TAG type may contain or be composed 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. Alternatively, 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 OP:OO m / z ratio of the oil may be at least 1.6, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. Alternatively, the OP:OO m / z ratio of the oil may be at least 2, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 3, determined by the abundance of DAG ions generated by mass spectrometric 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 5ξ-ergoster-7-en-3β-ol, (3β)-24-methylene-9,19-cyclolanostane-3-ol, and (3β)-ergoster-7,22-dien-3-ol. The oil may contain at least 20 mg of 5ξ-ergoster-7-en-3β-ol per 100 g of oil. The oil may contain at least 5 mg of (3β)-24-methylene-9,19-cyclolanostane-3-ol per 100 g of oil. The oil may be algal oil. The oil may be genetically modified algal oil. The oil may be produced from algal cells. Lipids can be produced from genetically modified algal cells. Lipids can be produced by algae of the genus *Protocellus* (…). Prototheca ) produced by cells.

[0075] This document discloses a method for producing non-naturally occurring lipids, the method comprising culturing microalgal cells in a culture medium, wherein the lipids comprise 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, and wherein at least 50% of the acyl chains in the TAG component may be C18:1. The method may further comprise isolating the lipid composition from the culture medium. The method may further comprise expressing an exogenous enzyme with lysophosphatidyl acyltransferase activity in the microalgal cells. The microalgal cells may be the microalgal cells described herein. The lipids may comprise a triglyceride (TAG) component, wherein at least 40% of the TAG component may be a TAG species having a saturated fatty acid at the sn-2 position, and wherein at least 50% of the acyl chains in the TAG component may be C18:1. The lipids may contain at least 10 mg of ergosterol per 100 g of lipid. The saturated fatty acid may be C16:0. At least 50% of the TAG component may be a TAG species having a saturated fatty acid at the sn-2 position. At least 50% of the acyl chains in the TAG component may be C18:1. The TAG may contain C18:1 at the sn-1 and sn-3 positions. The TAG may contain C16:0 at the sn-2 position. The TAG may contain 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 OP:OO m / z ratio of the oil may be at least 1.6, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 2, determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil. The OP:OO m / z ratio of the oil may be at least 3, determined by the abundance of DAG ions generated 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 5ξ-ergoster-7-en-3β-ol, (3β)-24-methylene-9,19-cyclolanostane-3-ol, and (3β)-ergoster-7,22-dien-3-ol. The oil may contain at least 20 mg of 5ξ-ergoster-7-en-3β-ol per 100 g of oil. The oil may contain at least 5 mg of (3β)-24-methylene-9,19-cyclolanostane-3-ol per 100 g of oil. The oil may be algal oil.Lipids can be genetically modified algal lipids. Lipids can be produced from algal cells. Lipids can be produced from genetically modified algal cells. Lipids can be produced from Algae of the genus *Protozoa*. Prototheca ) produced by cells.

[0076] Traditionally, microalgae are known to be used to produce long-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), which are commonly used in human and animal infant formulas, supplements, and foods. Industrially, there is a need to optimize the fatty acid composition and triglyceride structure of oils produced from microalgae to expand the application potential of microalgae oils beyond long-chain LC-PUFAs, making them suitable for specific nutritional or physicochemical properties. Specifically, aspects of this application contain oils derived from microalgae that are rich in saturated fatty acids, particularly palmitate esters, at the sn-2 position.

[0077] In some embodiments, this document provides a naturally occurring oil comprising: at least 10 mg of ergosterol per 100 g of oil; and a triglyceride (TAG) component, wherein at least 40% of the TAG component comprises a TAG species having a saturated fatty acid at the sn-2 position, and wherein at least 50% of the TAG component comprises an acyl chain of C18:1. The oil can be an algal oil. The oil can be a genetically modified algal oil. The oil can be produced from algal cells. The oil can be produced from genetically modified algal cells. The oil can be produced from *Protozoa* (…). Prototheca ) produced by cells.

[0078] This document discloses a composition comprising oils and one or more excipients. The composition can be a food product. It can be a food or beverage. Furthermore, the composition can be a nutritional supplement. More specifically, the composition can be infant formula.

[0079] This document discloses a composition comprising oils and one or more excipients. The composition can be a nutritional supplement. The composition can be infant formula. Infant formula may contain one or more of whey, casein, lactose, vitamin D, human milk oligosaccharides (HMOs), vegetable oils, and antibodies. Non-limiting examples of vegetable oils include soybean oil, low-erucic acid rapeseed oil, sunflower oil, coconut oil, palm oil, and palm kernel oil. Infant formula may contain soy protein.

[0080] Lipids can be algal lipids. Lipids can be genetically modified algal lipids. Lipids can be produced from algal cells. Lipids can be produced from genetically modified algal cells. Lipids can be produced from Algae of the genus *Protozoa* (…). Prototheca ) produced by cells. Detailed Implementation

[0081] This document provides oil and fat compositions comprising a TAG rich in 1,3-dioleoyl-2-palmitoylglycerol (OPO), methods for their preparation, formulations, and applications. The oil and fat compositions provided herein can be produced by genetically modified microorganisms, such as the microalgae cells described herein. Methods for producing and culturing microorganisms that produce the oil and fat compositions provided herein are also provided. A bioreactor comprising non-naturally occurring microorganisms described herein, which have been modified to produce the oil and fat compositions provided herein, is also provided.

[0082] definition

[0083] As used herein, the term “microbial oil” refers to oil produced or extracted from microorganisms (microbes), such as oil-producing, single-celled, eukaryotic or prokaryotic microorganisms, including but not limited to microalgae, yeasts, bacteria and fungi.

[0084] As used in this article, the terms “triacylglycerol,” “triglyceride,” or “TAG” refer to esters between glycerol and three fatty acids.

[0085] As used herein, the terms "OPO" or "1,3-dioleoyl-2-palmitoylglycerol" refer to those found in TAG. sn -2 position has palmitate (C16:0) and in TAG sn -1 and sn -3 TAG types with oleic acid ester (C18:1) at position -3.

[0086] As used herein, the term "OOP" or "1,2-dioleoyl-3-palmitoylglycerol" refers to the TAG. sn -3 position has palmitate (C16:0) and in TAG sn -1 and sn -2 positions contain oleic acid esters (C18:1) TAG types.

[0087] 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 oils" generally refers to a polyol obtained by the chemical transformation of TAG oils (e.g., through epoxidation and ring-opening reactions, ozone decomposition and reduction reactions, or hydroformylation and reduction reactions).

[0088] As used herein, the terms “polyurethane,” “PU,” or “urethane” refer to a class of polymers consisting of urethane bonds formed between the polyol and the isocyanate moiety.

[0089] As used in this article, the term "high oleic" can refer to oleic acid with a content of more than 60%, more than 70%, more than 80%, or more than 90%.

[0090] As used in this article, the term “about” means and can be ±10% of the provided value.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this teaching, some exemplary methods and materials are described herein.

[0092] Human breast milk

[0093] Human breast milk is the primary source of nutrition for newborns. It consists of fats, proteins, carbohydrates, and a variety of vitamins and minerals essential for healthy growth and development. Breast milk also contains important factors that protect infants from infection and inflammation, and contribute to the healthy development of the immune system and gut microbiota.

[0094] Breast milk contains approximately 3-55% fat, of which 98% is in the form of TAG (tartary acid-tannin derivative), containing over 200 different fatty acids. Breast milk also contains approximately 17-25% palmitic acid, of which 70-75% is uniquely distributed in the stereospecific TAG region, numbered 2(tartary acid-tannin derivative). sn -2) position, and unsaturated fatty acids, such as oleic acid and linoleic acid, are mainly distributed in sn -1 and sn -3 digits. sn High levels of palmitic acid at the -2 position can help enhance the absorption of calcium and essential fats in the infant's gut and benefit overall digestive health. A key TAG in human breast milk is 1,3-dioleoyl-2-palmitoylglycerol, also known as... sn -2 palmitate, oleic-palmitic-oleic acid, or OPO. OPO is found in human breast milk but is not normally present in cow's milk.

[0095] Given the complexity of human breast milk, infant formula manufacturers have struggled to replicate even a fraction of the characteristics of this extraordinary natural food. Efforts have been made to achieve the desired relative proportions of fatty acids in infant formula to replicate the composition of breast milk. Several products have also been developed that can replicate OPO-rich triglycerides and can be combined with other fat components to mimic human breast milk. Human milk fat substitutes (HMFS) are a class of structured lipids widely used as ingredients in infant formula. Like human milk fat, HMFS are characterized by the specific enrichment of palmitoyl (C16:0) groups in the middle of the glycerol backbone. sn-2(or β). However, no single-source fat substitute can fully replicate all the benefits of natural products.

[0096] Although most infant formula uses plant-based fats, many plants tend to esterify saturated fatty acids. sn-1 and sn-3 Position, not in sn-2 Palmitic acid is bound at this position. In contrast, engineered oil crops and / or oil-producing microorganisms can accumulate high levels of palmitic acid. sn-2 TAGs rich in saturated fatty acids such as OPO. These alternative TAG sources may better mimic the stereoisomer structure of human milk fat, thus providing a higher quality HMFS source for infant nutrition.

[0097] Infant formula made from vegetable oils or dairy products has a similar fat content to the fatty acids in human milk, but exhibits a different lipid structure. For example, approximately 70% of the palmitate in human milk is esterified in TAGs. sn-2 In contrast, vegetable oils and the milk of other mammals (e.g., cows, sheep, and goats) are esterified in... sn-2 Palmitic acid at positions 1 and 2 account for only about 5-20% and 30-40% of total palmitic acid, respectively. Therefore, in most infant formulas derived from plant or dairy products, the majority of the lipid components are palmitic acid esterified at positions 1 and 2. sn-1 / 3 Bit.

[0098] Human milk fat substitutes are a class of structured lipids developed to mimic the TAG distribution of human breast milk and obtained by altering the composition, structure, and distribution of fatty acids linked to the glycerol backbone. The inventors of this application have discovered that the oils disclosed in this application, including microalgae-derived TAG oils rich in OPO, can be used as a substitute for... sn-2 Human milk fat substitutes composed of structured TAGs rich in palmitic acid. The strains can be derived from modified traditional strains or genetically modified.

[0099] On the one hand, OPO-rich TAG lipids from algae are produced by microalgae obtained through modification of traditional strains. On the other hand, OPO-rich TAG lipids from algae are produced by genetically modified microalgae. Microalgae strains can be selected from, but are not limited to, *Procambarus mulberryii* (…). P. moriformi OPO oils can be incorporated as a partial substitute for vegetable oils in regular infant nutrition products, such as infant formula, follow-up formula, and pediatric supplements for full-term or premature infants. For example, Figure 13 LC-MS triglyceride (TAG) spectra of algal OPO oil and a first-generation human milk fat alternative obtained through enzymatic transesterification of vegetable oil were compared, representing one aspect of this disclosure. Figure 13It can be seen that the amount of OPO in the OPO oil of the present invention significantly exceeds that of commercially available plant-derived alternatives.

[0100] Triglycerides sn-2 Naturally occurring palmitic acid is associated with key biological functions in infants, such as... [[ID= As shown (adapted from Havlicekova et al., 2016, Beta-palmitate - a natural component of human milk in supplemental milk formulas. Nutr J. 15:28. doi: 10.1186 / s12937-016-0145-1). The structural properties of human milk triglycerides are designed to optimize fatty acid absorption, primarily due to the position of palmitic acid in... ​ Achieving adequate absorption of essential nutrients from breast milk is crucial for healthy infant development, as lipids are a primary energy source in infancy and play a key role in various developmental processes. Furthermore, there is a close correlation between the absorption of key micronutrients such as calcium and the absorption of palmitic acid.

[0101] When the triglyceride molecules provided to infants contain palmitate esters located on the outer side, the palmitic acid released by lipases often binds with calcium to form an insoluble salt.

[0102] The accumulation of these insoluble salts in the infant's gastrointestinal tract (GI tract) can significantly affect microbiome development and lead to discomfort in infants, specifically hardened stools. Disruption of microbiome development caused by excessive insoluble palmitate in the GI tract may potentially trigger inflammatory and immune-related problems. Using products containing... ​ Using palmitic acid-rich oils (such as algal-derived OPO oil) to formulate infant nutritional products can replicate the natural composition of palmitic acid found in human breast milk, compared to traditional alternatives. ​ Nutritional benefits related to palmitic acid.

[0103] TAG biosynthesis

[0104] Lysophosphatidyl acyltransferase (LPAAT), a membrane-bound O-acyltransferase (MBOAT) encoded by a multi-gene family, is the rate-limiting enzyme in the Kennedy pathway in higher plants. The Kennedy pathway converts glycerol-3-phosphate to TAG using glycerol-3-phosphate acyltransferase (GPAT), LPAAT, phosphatidyl phosphatase (PAP), and diacylglycerol acyltransferase (DGAT). In this pathway, LPAAT controls the production of phosphatidic acid (PA), a key intermediate.

[0105] LPAAT genes are present in both prokaryotes and eukaryotes. In mammals, LPAAT has been characterized as a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. For example, Homo sapiens (… ​ The AGPAT gene is ​ 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 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 with SEQ ID NO:37. Plants also frequently possess several LPAAT genes; for example, Arabidopsis thaliana (… ​ It possesses 5 LPAAT genes. In most microalgae and higher plants, TAG biosynthesis occurs simultaneously in chloroplasts and the endoplasmic reticulum (ER), and the substrate preference of LPAAT enzymes is typically oleoyl-CoA. However, in other eukaryotes, including animals and fungi, the primary organelle for TAG biosynthesis is the endoplasmic reticulum (ER), and again, the substrate preference of LPAAT enzymes is typically oleoyl-CoA.

[0106] Enzymes with lysophosphatidyl acyltransferase activity may contain four catalytic motifs: (I) NHXXXXD (or NHX4D); (II) (F / Y)XXR; (III) EGXR; and (IV) proline, where X is any amino acid. Motif I may contain a conserved NHXXXXD (or NHX4D) sequence, where the residues following histidine (H) are generally hydrophilic, while the residues preceding aspartic acid (D) are almost always hydrophobic. Motif II may contain a (F / Y)XXR sequence, where the first residue is either phenylalanine (F) or tyrosine (Y), followed by two residues and a conserved arginine (R). The residues preceding the R may be hydrophilic, while the residues preceding the hydrophilic residues may be hydrophobic. Motif III may contain a conserved EGXR sequence. Motif IV may contain a conserved proline (P). Enzymes described herein may contain sequences having one, two, three, or four of these catalytic motifs. The cells described herein may contain exogenous genes, which contain sequences encoding enzymes having one, two, three, or four of these catalytic motifs.

[0107] In some embodiments, the enzyme may comprise a sequence having at least 70%, at least 85%, or at least 100% identity with any one of SEQ ID NO: 107-109. In some embodiments, the cell may comprise a foreign gene comprising a sequence encoding an enzyme having at least 70%, at least 85%, or at least 100% sequence identity with any one of SEQ ID NO: 107-109.

[0108] In some embodiments, the enzyme may comprise a sequence having at least 60%, at least 75%, at least 85%, or at least 100% identity with any of SEQ ID NO: 110-119. In some embodiments, the cell may comprise a foreign gene comprising a sequence encoding an enzyme having at least 60%, at least 75%, at least 85%, or at least 100% sequence identity with any of SEQ ID NO: 110-119.

[0109] 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 at least 100% sequence identity with any of SEQ ID NO: 120-134. In some embodiments, the cell may comprise a foreign 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 at least 100% sequence identity with any of SEQ ID NO: 120-134.

[0110] In some embodiments, the enzyme described herein may comprise catalytic motif IV: conserved proline. In some embodiments, the enzyme described herein may comprise a third domain having a conserved proline sequence. 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 at least 100% sequence identity with any of SEQ ID NO: 135-154. In some embodiments, the cell may comprise a foreign 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 at least 100% sequence identity with any of SEQ ID NO: 135-154.

[0111] Chlamydomonas reinhardtii ( ​CrLPAAT1, a single-celled green alga, comprises two LPAAT genes: CrLPAAT1 and CrLPAAT2. The chlorophyll-specific CrLPAAT2 enzyme is located on the ER membrane. However, unlike typical LPAAT located on the ER, CrLPAAT2 prefers palmitoyl-CoA rather than oleoyl-CoA as its 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 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 with SEQ ID NO: 2. In some embodiments, the amino acid sequence of CrLPAAT2 is SEQ ID NO: 13. In some embodiments, expression in CrLPAAT2 cells is achieved by transforming 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 with SEQ ID NO: 10. Related species: Chlamydomonas schlegelii ( ​ ), Chlamydomonas indeterminate ​ Chlamydomonas spp. ( ​ They each have similar genes— ​ , ​ and ​ In some embodiments, the amino acid sequence of ChsLPAAT2 is SEQ ID NO: 14. In some embodiments, ChsLPAAT1 is expressed in cells by transformation of 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 with 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 cells by transformation of 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 with 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 cells by transforming 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 with SEQ ID NO: 38.

[0112] Marine microalgae—*Micrococcus* genus ​ It has 4 LPAAT genes. Two of these genes, ​ and ​ These genes are crucial for TAG biosynthesis. NoLPAT3 and NoLPAT4 are typically found around lipid droplets, although these two genes are not associated with other lipid droplet-related proteins. The location of NoLPAT3 and NoLPAT4 around lipid droplets can enhance TAG accumulation, especially during periods of nutrient deficiency. In some embodiments, the amino acid sequence of NoLPAT3 is SEQ ID NO: 16. In some embodiments, NoLPAT3 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 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 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 with SEQ ID NO: 4.

[0113] European rapeseed ( ​ In TAG biosynthesis, one of the LPAAT genes is LPAAT. ​ . ​ It 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 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 with SEQ ID NO: 3.

[0114] Synechocystis ( ​ The LPAAT gene is called Sll1848 Sll 1848 in ​ The positions introduced C18:0 and C18:1, and are in the genus Synechocystis ( ​ The major LPAAT specific to C16:0 in .) is present. In some embodiments, the amino acid sequence of Sll1848 is SEQ ID NO: 19. In some embodiments, Sll1848 is expressed in cells by transformation of 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 with SEQ ID NO: 5.

[0115] Green algae species such as Volvox catatini ( ​ ), African Volvox ( ​ ) and Volvox reticulata ( ​ They also have similar LPAAT genes. ​ , ​ and ​ 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 of 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 with 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 cells by transformation of 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 with 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 cells by transforming 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 with SEQ ID NO: 57.

[0116] The LPAAT gene in E. coli is called ​ Gene. In *E. coli*, the EcPlsC mutant strain is characterized by a deficiency in LPAAT activity. This 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 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 with SEQ ID NO: 6.

[0117] Microalgae mulberry-shaped protozoa ( ​ LPAAT showed that it was effective against... ​A strong preference is shown for inserting unsaturated fatty acids, with very little insertion of 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.

[0118] Green algae species such as Chlorella sorokinensis ( ​ ), Mutant Chlorella ( ​ ​ ), dehydrated Chlorella ( ​ ) and common Chlorella ( ​ They also have similar LPAAT genes. ​ In some embodiments, the amino acid sequence of ChsoLPAAT2 is SEQ ID NO: 46. In some embodiments, ChsoLPAAT2 is expressed in cells by transformation of 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 with 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 cells by transformation of 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 with 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 cells by transformation of a plasmid containing a sequence that is 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: 56. 34. In some embodiments, the amino acid sequence of ChvuLPAAT2 is SEQ ID NO: 56. In some embodiments, ChvuLPAAT2 is expressed in cells by transformation of a plasmid containing a sequence that is 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: 58.

[0119] Multicellular green algae, stalked star algae ( ​ ) contains a called ​The LPAAT gene. 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 containing a sequence that is 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: 26.

[0120] Green algae, *Tetraphyta debarei* ( ​ ) contains a called ​ The LPAAT gene. 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 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 with SEQ ID NO: 27.

[0121] Dunaliella salina, a single-celled green algae ​ ) contains a called ​ The LPAAT gene. 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 with SEQ ID NO: 28.

[0122] Community green algae genus Scenedesmus ( ​ ) contains a called ​ The LPAAT gene. In some embodiments, the amino acid sequence of SceLPAAT2 is SEQ ID NO: 44. In some embodiments, SceLPAAT2 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 with SEQ ID NO: 29.

[0123] Dense, communal green algae resembling Volvox ( ​ Includes a called ​The LPAAT gene. 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 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 with SEQ ID NO: 65.

[0124] Colonial green algae, Japanese multicellular algae ( ​ ) contains a term ​ The LPAAT gene. 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 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 with SEQ ID NO: 68.

[0125] Colonial green algae, *Bauerella salina* ( ​ ) contains a called ​ The LPAAT gene. 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 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 with SEQ ID NO: 69.

[0126] Community green algae, Polycholenaceae ( ​ ) contains a called ​ The LPAAT gene. 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 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 with SEQ ID NO: 73.

[0127] Community green algae, *Discocephalum glomeratum* ( ​ ) contains a called ​The LPAAT gene. 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 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 with SEQ ID NO: 74.

[0128] Single-celled green algae guide microalgae ( ​ ) contains a called ​ The LPAAT gene. In some embodiments, the amino acid sequence of McLPAAT2 is SEQ ID NO: 45. In some embodiments, McLPAAT2 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 with SEQ ID NO: 30.

[0129] Dunaliella salina, a single-celled green algae ​ ) contains a called ​ The LPAAT gene. 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 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 with SEQ ID NO: 78.

[0130] Microalgae Near-capitalized Sharp-celled Algae ( ​ ) contains a called ​ The LPAAT gene. 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 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 with SEQ ID NO: 33.

[0131] Heterotrophic green algae, Chlorella vulgaris ( ​ ) contains a called ​In some embodiments, the amino acid sequence of ApLPAAT2 is SEQ ID NO: 50. In some embodiments, ApLPAAT2 is expressed in cells by transformation of 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 with SEQ ID NO: 35.

[0132] Green algae and chlorophyll ( Chloropicon primus ) contains a called ChprLPAAT2 The LPAAT gene. In some embodiments, the amino acid sequence of ChprLPAAT2 is SEQ ID NO: 51. In some embodiments, ChprLPAAT2 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 with SEQ ID NO: 36.

[0133] The class Pedinophyceae, a group of green algae, includes a group known as PedLPAAT2The LPAAT gene. 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 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 with SEQ ID NO: 39. In some embodiments, PedLPAAT2 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 with SEQ ID NO: 60. In some embodiments, PedLPAAT2 is expressed in cells by transforming 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 with SEQ ID NO: 61. In some embodiments, PedLPAAT2 is expressed in cells by transforming 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 with SEQ ID NO: 62.

[0134] Chlamydomonas (a type of green algae) Vitreochlamys sp.). Includes a called VitrLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 66.

[0135] Chlorella vulgaris (C. chalcogenide) Colemanosphaera charkowiensis ) contains a called CchaLPAAT2The LPAAT gene. 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 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 with SEQ ID NO: 67.

[0136] Green algae Bauerella ( Volvulina boldii ) contains a called VbolLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 69.

[0137] Chlorella vulgaris ( Pandorina morum ) contains a term PmorLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 70.

[0138] Green algae Caterpillarella Weismann type ( Volvox carteri f . weismannia ) contains a term VcarfLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO:71.

[0139] Green algae, Cylindrical hollow sphere algae ( Eudorina cylindrica ) contains a term EcylLPAAT2The LPAAT gene. In some embodiments, the amino acid sequence of EcylLPAAT2 is SEQ ID NO: 90. In some embodiments, EcylLPAAT2 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 with SEQ ID NO: 72.

[0140] Green algae Ferris Volvox ( Volvox ferrisii ) contains a called VferLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 75.

[0141] Chlorella vulgaris (a green algae) Vitreochlamys aulata ) contains a called VaulLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 76.

[0142] Chlamydomonas (a type of green algae) Chlamydomonas sp.)CCAC2762_B Includes a called Ch_CCAC2762_ LPAAT2 The LPAAT gene. 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 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 with SEQ ID NO:77.

[0143] Green algae, genus *Cyperus* ( Microglena sp.)YARC Includes a called MyarcLPAAT2The LPAAT gene. In some embodiments, the amino acid sequence of MyarcLPAAT2 is SEQ ID NO: 97. In some embodiments, by transforming 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 with SEQ ID NO: 79, the LPAAT gene is transformed. MyarcLPAAT2 It is expressed in cells.

[0144] Chlamydomonas (a type of green algae) Chlamydomonas sp. ) UWO_241 Includes a called CuwoLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 80.

[0145] Chlamydomonas mossii (a green algae) Chlamydomonas moewusii ) contains a called CmoeLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 81.

[0146] Green algae and salamander symbiotic algae ( Oophila amblystomatis ) contains a called OambLPAAT2 The LPAAT gene. 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 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 with SEQ ID NO: 82.

[0147] Green algae, *Tetraphyta debarei* ( Edaphochlamys debaryana)_SRR13719274 Includes a called EdLPAAT2 The LPAAT gene. In some implementations, EdLPAAT2 (debaranthellae) Edaphochlamys debaryana)_SRR13719274 The amino acid sequence of ) is SEQ ID NO: 101.

[0148] Colonial green algae, *Schönleinii* ( Volvulina steinii ) contains a called VsteLPAAT2 The LPAAT gene. In some embodiments, the amino acid sequence of VsteLPAAT2 is SEQ ID NO: 102.

[0149] Colonial green algae, octocyte discus ( Gonium octonarium ) contains a called GoctLPAAT2 The LPAAT gene. In some embodiments, the amino acid sequence of GoctLPAAT2 is SEQ ID NO: 103.

[0150] Single-celled green algae Chlamydomonas aeruginosa ( Chlamydomonas nivalis ) contains a called CnivLPAAT2 The LPAAT gene. In some embodiments, the amino acid sequence of CnivLPAAT2 is SEQ ID NO: 104.

[0151] Green algae, Rhodococcus prolifera ( Pycnococcus provasolii ) contains a called PproLPAAT2 The LPAAT gene. In some implementations, PproLPAAT2 The amino acid sequence is SEQ ID NO: 105.

[0152] Green algae grass deep globulus ( Bathycoccus prasinos ) contains a called BpraLPAAT2 The LPAAT gene. In some embodiments, the amino acid sequence of GoctLPAAT2 is SEQ ID NO: 106.

[0153] CrLPAAT1-ΔCST It is a type of Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii )LPAAT, in which a total of 30 amino acids (aa 12-41; CST signal sequence) were removed from CrLPAAT1. BnBAT2-ΔCST A type of European rapeseed ( Brassica napus )LPAAT, wherein a total of 86 amino acids (aa 2-87) have been removed from BnBAT2. In some embodiments, CrLPAAT1-ΔCST The amino acid sequence is SEQ ID NO: 155. In some embodiments, BnBAT2-ΔCST The amino acid sequence is SEQ ID NO: 156.

[0154] This disclosure of TAG grease

[0155] This invention provides a TAG oil composition having TAG types, wherein the TAG types are in sn-2 The TAG contains saturated fatty acids. In some implementations, the TAG type is OPO.

[0156] The complexity and physical properties of oils and fats can be evaluated using fatty acid profiles and TAG profiles of their TAG components. A fatty acid profile is a measure of fatty acid composition, which can be determined by transesterifying the oil to generate fatty acid methyl esters (FAMEs), followed by quantification of fatty acid types using gas chromatography / FID equipped with a flame ionization detector. Therefore, the fatty acid content of the TAG oils presented in this paper can be determined by GC / FID. Since TAGs consist of three fatty acids arranged along the glycerol backbone in the triglyceride molecule, the number of different regional isomers that may exist in TAGs can be defined as the cube of the number of fatty acid types in the oil.

[0157] Gene expression of the enzymes described herein can be modulated to produce high levels of OPO. The expression of the genes described herein can be modulated individually or in combination with the expression of one or more enzymes described herein to produce high levels of OPO. Gene expression of the enzymes described herein can be modulated to enhance LPAAT activity. The expression of the genes described herein can be modulated individually or in combination with the expression of one or more enzymes described herein to enhance LPAAT activity. In some embodiments, the cells described herein are modified to express exogenous genes, thereby enhancing LPAAT activity. In some embodiments, the cells described herein are modified to delete endogenous genes, thereby enhancing LPAAT activity.

[0158] This article provides methods for genetically modifying and culturing cells to produce high yields of the lipids described herein. This article also provides genetically modified cells to produce high yields of the lipids described herein. The cells provided herein may contain recombinant nucleic acids operable to increase the expression of the genes described herein to enhance LPAAT activity. Furthermore, the cells provided herein may exclude endogenous nucleic acids operable to decrease the expression of the genes described herein, thereby enhancing LPAAT activity. In some embodiments, microalgal cells are modified to produce high yields of the lipids described herein.

[0159] The oil compositions described herein are produced by microorganisms such as microalgae cells. Therefore, the oil compositions produced therefrom may contain one or more sterol components characteristic of algal oils. The content of a particular sterol component in the oil can be determined by weight ratio, for example, milligrams of sterol per 100 grams of oil. The sterol composition (i.e., sterol profile) can be obtained by mass spectrometry, such as gas chromatography-mass spectrometry (GC-MS); liquid chromatography-mass spectrometry (LC-MS); tandem mass spectrometry (MS / MS); and liquid chromatography coupled with gas chromatography followed by flame ionization detection (LC-GC-FID). Exemplary sterol profiles of the TMS esters of the unsaponifiable fraction of RBD oil produced from CHK22, determined by GC / MS analysis, are shown in Table 26. Cholesterol-3-ol (3a, 5b) were used as internal standards (ISTD).

[0160] Table 26: CHK22 RBD lipid sterol spectrum

[0161] In some embodiments, the oil contains a detectable amount of (3β,22E)-ergoster-7,22-dien-3-ol. In some embodiments, the amount of (3β,22E)-ergoster-7,22-dien-3-ol in the oil provided herein is at least 2 mg, at least 5 mg, at least 8 mg, or at least 9 mg per 100 g of oil. In some embodiments, the amount of ergoster(3β,22E)-ergoster-7,22-dien-3-ol in the oil is 1-100 mg, 2-30 mg, 3-20 mg, or 5-15 mg per 100 g of oil.

[0162] In some embodiments, the oil contains a detectable amount of ergosterol. In some embodiments, the amount of ergosterol in the oil provided herein is at least 50 mg, at least 100 mg, or at least 125 mg per 100 g of oil. In some embodiments, the amount of ergosterol in the oil provided herein is at least 10 mg, at least 20 mg, at least 30 mg, at least 40 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 implementations, 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 100g of oil.

[0163] In some embodiments, the oil contains detectable amounts of campesterol, β-sitosterol, or stigmasterol. In some embodiments, the amount of campesterol, β-sitosterol, or stigmasterol is no more than 5 mg per 100 g of oil. In some embodiments, the oil does not contain campesterol. In some embodiments, the amount of campesterol in the oil provided herein is no more than 5 mg per 100 g of oil. In some embodiments, the oil does not contain stigmasterol. In some embodiments, the amount of stigmasterol in the oil is no more than 5 mg per 100 g of oil. In some embodiments, the oil does not contain β-sitosterol. In some embodiments, the amount of β-sitosterol in the oil is no more than 5 mg per 100 g of oil.

[0164] In some embodiments, the oil contains a detectable amount of (3β)-ergoster-5,8-dien-3-ol. In some embodiments, the amount of (3β)-ergoster-5,8-dien-3-ol is at least 1 mg per 100 g of oil. In some embodiments, the amount of (3β)-ergoster-5,8-dien-3-ol 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 mg of oil. In some embodiments, the amount of (3β)-ergoster-5,8-dien-3-ol 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.

[0165] In some embodiments, the oil contains a detectable amount of 5.xyl-ergoster-7-en-3β-ol. In some embodiments, the amount of 5.xyl-ergoster-7-en-3β-ol is at least 15 mg per 100 g of oil. In some embodiments, the amount of 5.xyl-ergoster-7-en-3β-ol is at least 20 mg per 100 g of oil. In some embodiments, the amount of 5.xi.-ergoster-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 implementations, the amount of 5.xi.-ergoster-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.

[0166] In some embodiments, the oil contains a detectable amount of (3β)-24-methylene-9,19-cyclolanostane-3-ol. In some embodiments, the amount of (3β)-24-methylene-9,19-cyclolanostane-3-ol is at least 5 mg per 100 g of oil. In some embodiments, the amount of (3β)-24-methylene-9,19-cyclolanostane-3-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, or at least 50 mg per 100 g of oil. In some embodiments, the amount of (3β)-24-methylene-9,19-cyclolanostane-3-ol 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.

[0167] The TAG spectral analysis of the oil and fat compositions presented in this article can be performed using liquid chromatography-mass spectrometry (LC-MS). The TAG oil presented in this article was extracted from cells into a 3:1 toluene / 2-propanol (v / v) solution by mechanical disruption and vigorous agitation using ceramic beads. The filtered extract was then injected into the LC-MS instrument for profiling.

[0168] Chromatographic separation of TAG regioisomers cannot be achieved using the aforementioned LCMS method. Instead, qualitative assessment of the regiospecificity of major TAG species can be performed based on the abundance of diacylglycerol (DAG) ions generated from TAG fragmentation (Figure 1). In LC-APCI-MS analysis of TAGs, compared with... sn-2 Compared to the previous position, sn-1 and sn-3 Fatty acids at the OPO regiomerex are more prone to energy loss, leading to a higher abundance of DAG ions formed from these fragments. In the case of the OOP regiomerex, the ratio of OPDAG ions (m / z 577) to OODAG ions (m / z 603) is approximately 1:1, as shown in Figure 1, A. In the case of the OPO regiomerex, a greater amount of OP DAG ions (m / z 577) is observed, as shown in Figure 1, B.

[0169] Figure 1 demonstrates that TAG species with the same molecular weight exhibit different daughter ion ratios. The two TAG species in Figures A and B—OOP and OPO—have parent ions with the same mass-to-charge ratio. m / z (859), but as shown in the figure, due to the different effects of collision energy, the fragmentation modes of these two molecules are significantly different. Because in sn-2 The acyl group at the position is relatively inaccessible, and low-energy collisions occur at a higher frequency from... sn-1 or sn-3 The fatty acyl group is eliminated at the site. As shown in Figure A, in the case of OOP, low-energy collisions produce daughter ions with m / z of 577 (OP) and 603 (OO), while high-energy collisions at lower frequencies lead to the elimination of fatty acyl groups. sn-2 The elimination of O at the position also produces the fragment ion OP with m / z 577. Therefore, the mass spectrum of the resulting fragment ion is the sum of all these fragments. The generation of the 577(OP) peak is due to, on the one hand, the... sn-1 The more frequent elimination of bit 0 stems from, on the other hand, the fact that bit 0 is eliminated more often. sn-2 The elimination of low-frequency 0 bits is therefore necessary. Consequently, the peak height of fragment 577 is approximately 1.5–2 times that of fragment 603 (OO), the latter originating from… sn-3More frequent elimination of P. Alternatively, as shown in Figure B, the OPO TAG species exhibits a very different daughter ion ratio because the low-energy collision products PO and OP have the same m / z 577, while the less frequently generated OO daughter ions via high-energy collisions have an m / z of 603. For this TAG species, the 577 peak height resulting from the sum of PO and OP daughter ions generated by low-energy collisions is significantly higher than the 603 fragment peak height, which requires higher energies to form.

[0170] In some embodiments, the oil may have an OP:OO m / z ratio, which is determined by the abundance of DAG ions generated by mass spectrometric fragmentation of the TAG component in the oil. In some embodiments, the OP:OO m / z ratio of the oil may be at least 1.6, which is determined by the abundance of DAG ions generated by mass spectrometric fragmentation of the TAG component in the oil. In some embodiments, the OP:OO m / z ratio of the oil may be 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, and this ratio is determined by the abundance of DAG ions generated by mass spectrometry fragmentation of the TAG component in the oil.

[0171] The specificity of TAG regions can be further determined by a porcine pancreatic lipase assay. This method can be used to determine the specificity of TAG by incubating TAG with porcine pancreatic lipase. sn-2 Identification of fatty acids. Lipase reaction leads to... sn-1 and sn-3 The fatty acids at position 1 are deacylated. Then, by solid-phase extraction, followed by direct transesterification and analysis of fatty acid composition by GC / FID (gas chromatography / flame ionization detection), the remaining fatty acids can be separated and identified. sn-2 - Monoacylglycerol (MAG).

[0172] The oil and fat compositions provided herein may include a TAG component, wherein at least 40% of the TAG component comprises a TAG type having a saturated fatty acid at the sn-2 position. In some embodiments, at least about 50% of the TAG component comprises a TAG type having a saturated fatty acid at the sn-2 position. sn-2The TAGs are types containing saturated fatty acids. In some embodiments, approximately 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of the TAG component is... sn-2 The TAG type contains saturated fatty acids. In some embodiments, 40-45%, 45-50%, 50-55%, 55-60%, 40-50%, 50-60%, or 40-60% of the TAG component is... sn-2 The TAG species contain saturated fatty acids. In some implementations, in sn-2 The saturated fatty acid at position 1 is palmitate.

[0173] The oil and fat compositions provided herein may 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 chain in the TAG component is C18:1.

[0174] Microbial oils

[0175] In some embodiments, the oils provided herein are obtained from genetically modified microorganisms, such as microalgae, oleogenetic yeasts, or oleogenetic bacteria. In some embodiments, the genetically modified microorganisms are genetically modified *Protozoa* species (…). Prototheca sp. (Strain). In some embodiments, the genetically modified microorganism contains exogenous genes or exogenous nucleotides. Alternatively or additionally, the genetically modified microorganism does not contain endogenous genes or endogenous nucleotides. The genetic modification methods described herein can be used to confer the ability of microorganisms to produce high levels of oleic acid and / or OPO TAG.

[0176] The oils provided herein can be obtained from microalgae, oleogenetic yeasts, or oleogenetic bacteria. In some embodiments, the microorganisms are modified Protocellae (…). Prototheca sp. ) strain. A non-genetically modified protocellar alga ( Prototheca sp. Strains can be produced through traditional strain modification strategies. A genetically modified *Protocellus* species (…) Prototheca sp. The strain can be produced by overexpressing endogenous / exogenous genes and / or deleting endogenous genes in a microorganism. In some embodiments, the microorganism provided herein is a genetically modified *Protocolospora* species produced through conventional strain modification strategies. Prototheca sp. The strain is designed to optimize high oleic acid production (e.g., at least 50% oleic acid) and high OPO production (e.g., at least 70% OPO). In some embodiments, the microorganism provided herein is a genetically modified Protocellella genus produced through conventional strain improvement strategies. Prototheca sp. The strain is designed to optimize the ability to produce high levels of palmitic acid (e.g., at least 30% palmitic acid) and high levels of OPO (e.g., at least 70% OPO).

[0177] The oils provided in this article can be derived from the genus *Protozoa* (… Prototheca The lipids produced can originate from the genus *UTEX* 1533 (*Protocolospora*). Prototheca The lipids produced can come from strains that do not express exogenous thioesterases.

[0178] In some embodiments, the lipids provided herein are produced via microalgae. In some embodiments, the microalgae are species selected from one of the following genera: Chlorella ( Chlorella sp. ), *Chlorella* genus ( Pseudochlorella sp. ), *Heterocarya* genus ( Heterochlorella sp. ), Protocellus ( Prototheca sp. ), genus Arthrospira ( Arthrospira sp. Euglena ( ) Euglena sp.), genus Microcystis ( Nannochloropsis sp. ), genus *Phaeodactylum* ( Phaeodactylum sp. Chlamydomonas ( ) Chlamydomonas sp .), *Scenedesmus* ( Scenedesmus sp. ), Microgreen algae ( Ostreococcus sp. ), genus Creepingula ( Selenastrum sp.) genus Haematococcus ( Haematococcus sp. ), Rhomboidella ( Nitzschia) genus Dunaliella ( Dunaliella ), Navicula ( Navicula sp. ), genus *Cyclophora* Trebouxia sp. ), *Pseudococcus* ( Pseudotrebouxia sp. ), Vavicula sp. genus *Bombyx* Bracteococcus sp. ), Heterophyta ( Gomphonema sp .), Watanabe nori ( Watanabea sp. ), genus *Botrytis* Botryococcus sp. ), genus Tetraphyta ( Tetraselmis sp. ) and Isochrysis ( Isochrysis sp. ) 。 In some implementation schemes, the microalgae are those of the genus *Protocolospora* (…). Prototheca sp. ) 。 In some implementation schemes, the microalgae are mulberry-shaped protozoa (… P. moriformis ) 。 In some implementations, the microalgae are *Vibrio vulgaris* (a type of green algae). P. wickerhamii ).

[0179] Genetic engineering and improvement of traditional strains

[0180] The production of TAG-specific lipids with specific phenotypes can be achieved through both genetic and non-genetic modification techniques in microorganisms. While genetic engineering techniques can be used to target phenotypes induced in host oleogenetic microorganisms, conventional strain modification and other non-genetic engineering techniques can be used to further enhance these phenotypes. Similarly, conventional strain modification or other non-genetic engineering techniques can be used to enhance certain phenotypes selected through genetic engineering. Phenotypic characteristics may include specific fatty acid profiles, specific TAG profiles, carbon yield, growth productivity, volumetric oil production (e.g., g oil / L culture), oil production rate (e.g., g oil / L culture·day), and the percentage of oil to dry cell weight (DCW) as an indicator of strain performance.

[0181] In some embodiments, the microalgae cells provided herein produce at least 50% lipids on a dry weight basis. In some embodiments, the microalgae cells provided herein produce at least 60% lipids on a dry weight basis.

[0182] For example, a microorganism that has not been genetically modified to confer a first phenotype (e.g., the ability to produce high levels of oleic acid) can be genetically modified to confer a second phenotype (e.g., the ability to produce high levels of OPO). The resulting microorganism can then be simultaneously conferred the first and second phenotypes (e.g., the ability to produce both high levels of oleic acid and OPO).

[0183] The microorganisms described herein can be generated using conventional strain improvement strategies to select microorganisms with desired phenotypes, such as high oleic acid production. Conventional strain improvement (also known as "mutant breeding") involves exposing organisms to chemicals or radiation to produce mutants with the desired traits. Ultraviolet (UV) light can be used to introduce random mutations within the nuclear genome of microorganisms. Chemical mutagens include compounds that inhibit or disrupt microbial synthetic 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. Combinations of chemical mutagens can also be used simultaneously to induce mutagenesis. After mutagenesis treatment, selectors or enrichment agents can be used to screen or enrich the target strain. Non-limiting examples of enriching agents include L-canavanine, cyanobacterium, tripalaprol, clomiphene, clomiphene citrate, clotrimazole, terfenadine, fluphenazine, AZD8055, BASF 13-338, cyclophosphamide, clomiphene, PF-042110 and phenylethanol.

[0184] The microorganisms with enhanced or altered lysophosphatidyl acyltransferase activity described in this article can be generated through genetic engineering techniques such as gene recombination. Enhancement of lysophosphatidyl acyltransferase activity can be achieved by increasing the expression of exogenous genes and / or decreasing the expression of endogenous genes in the microorganisms. Increased gene expression can be achieved through overexpression of endogenous genes or expression of exogenous genes in the microorganisms. Decreased gene expression can be achieved by deleting or removing endogenous genes in the microorganisms.

[0185] For example, the non-naturally occurring microorganisms described in this article can be genetically modified to enhance or alter the activity of lysophosphatidyl acyltransferases (LPAATs). Enhancement or alteration of LPAAT activity can be achieved by increasing the expression of exogenous LPAAT genes and / or decreasing the expression of endogenous LPAAT genes in the microorganisms.

[0186] The methods presented in this article include traditional strain improvement and / or genetic engineering methods to improve strain productivity, carbon production, oleic acid content, and OPO content. Glucose consumption rate can be a highly predictive indicator of lipid titers. Therefore, glucose consumption rate can be used as an enrichment tool in mutant screening processes.

[0187] Polyol Applications and End Products

[0188] The greases described herein can be used as substrates in chemical processes for the preparation of polyols, such as hydroformylation / reduction reactions, epoxidation reactions, and ring-opening reactions. The oleic acid moiety at the sn-1 and sn-3 positions, when subjected to the above chemical processes, will generate diols; these diols, when formulated with one or more excipients, can be used in a variety of applications, including but not limited to processed greases (e.g., for tires), waxes, lubricants, other polyols, macromolecular diols, other polyester diols, and can be used to prepare polyurethane products, such as rigid foams, flexible foams, cast polyurethane, thermoplastic polyurethane (TPU), elastomers, adhesives, coatings, laminates, films, and dispersions. Polyurethane products can be used to manufacture aerospace, automotive, medical, electronic, construction and building supplies; sporting or leisure equipment, such as skis, snowboards, sidewalls, marine equipment, kayaks; and other consumer products, such as industrial containers, refrigerators, mattresses, leather goods, clothing, footwear, mannequins, and mobile phone cases. These polyurethane applications can serve as sustainable alternatives to petroleum-based, non-renewable materials such as acrylonitrile-butadiene-styrene (ABS), ultra-high molecular weight polyethylene (UHMWPE), or high-density polyethylene (HDPE).

[0189] The oils and fats provided herein can have improved production efficiency and enhanced TAG composition for improved urethane chemical control. These characteristics of microbial oils can result in oils with a higher degree of hydroxyl (-OH) homogeneity relative to higher TAG heterogeneity (and therefore lower purity) and / or greater diversity (e.g., oils from oilseeds or plants). Polyols derived from oils and fats provided herein that are highly rich in hydroxylated TAG species may be preferred in the formation of polymers, including in cases where the physical properties of the polymer may be impaired by molecular impurities (e.g., non-hydroxylated fatty acids) or by the randomness of selective insertion of fatty acid portions on the glycerol backbone, which may be present in oils and fats with a more diverse or heterogeneous TAG composition.

[0190] The polyols described herein are particularly useful for preparing polyurethane materials. For example, the oils provided herein may have relatively low TAG diversity, low fatty acid diversity, and the fatty acids present in the oils may be hydroxylated fatty acids. A higher ratio of hydroxylated to non-hydroxylated fatty acids allows for increased chemical reactivity. Oils with low TAG diversity and a high proportion of hydroxylated fatty acids are particularly desirable in polyurethane production because hydroxylated fatty acids can participate in crosslinking reactions with isocyanates. Therefore, polyols derived from highly hydroxylated fatty acids contained in oils can produce polyurethane materials with excellent properties.

[0191] Preparation of polyols

[0192] The hydroxyl functional groups of polyols can be introduced through the chemical conversion of triglyceride oils. This conversion typically involves the presence of a double bond on the acyl moiety of a fatty acid, which can then be converted to contain one or more hydroxyl groups using several different chemical treatments, including, for example, epoxidation / ring-opening, ozone decomposition, and hydroformylation and reduction.

[0193] Epoxidation of the carbon-carbon double bond in the acyl chain and subsequent ring-opening can be carried out using a variety of reagents, including, for example, water, hydrogen, methanol, ethanol, propanol, isopropanol, or polyols. Ring-opening can be promoted by reaction with alcohols, including, for example, β-substituted alcohols. Ring-opening of epoxidized fats can also be achieved by using hydrogenation catalysts such as nickel to generate the hydroxyl moiety.

[0194] Hydroformylation using syngas can be carried out in the presence of a rhodium or cobalt catalyst to form an aldehyde on an olefinic group. This aldehyde can then be reduced in the presence of hydrogen and a nickel catalyst to produce an alcohol, thereby yielding a polyol.

[0195] Hydroformylation preserves the fatty acid chain length and forms a primary hydroxyl moiety. Primary hydroxyl functional groups are desirable in some polyurethane (PU) applications because they offer increased reactivity compared to secondary hydroxyl moieties. The hydroxyl groups introduced into the olefinic group of the acyl chain can participate in subsequent downstream chemical processing, i.e., reacting with the isocyanate moiety to form a urethane bond or reacting with methyl esters to form a polyester. Saturated fatty acids without double bonds cannot participate in crosslinking reactions with isocyanates. Therefore, saturated fatty acids compromise structural integrity and reduce the properties of the resulting polymers.

[0196] In some embodiments, the polyols provided herein contain a significant 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.

[0197] The TAG oil derivatives provided herein can be used as starting materials for the generation of polyols. Non-limiting examples of these TAG derivatives include fatty acids, fatty acid methyl esters, fatty acid ethyl esters, hydroxylated fatty acids, hydroxylated fatty acid methyl esters, and hydroxylated fatty acid ethyl esters. Non-limiting examples of polyols include polyester glycols, polyether glycols, hydrogenated polyols, hydroformylated polyols, and epoxidized ring-opening polyols. Alternatively, the TAG oils provided herein can be used directly as starting materials for the generation of polyols without further chemical modification.

[0198] Fatty acid methyl esters (FAMEs) can be produced through esterification. For example, TAG can be cleaved into FAMEs and glycerol via transesterification. Furthermore, FAMEs can undergo epoxidation and ring-opening reactions, for example, to generate alcoholic FAMEs. Alternatively, polyols can be first generated from TAGs via epoxidation and ring-opening, for example, followed by transesterification to generate alcoholic FAMEs and glycerol. Glycerol and potassium methoxide catalyst can be removed by washing with water.

[0199] Catalysts, including potassium methoxide (KOCH3), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), titanium isopropoxide (IV), dibutyltin dilaurate (DBTDL), tris(pentafluorophenyl)borane (BCF), and potassium tert-butoxide, can be used to re-esterify ester groups to alcohol moieties. The bifunctional groups of alcoholic FAMEs can be used to create polymer networks using only the methyl esters of the alcohols. Due to the polarity of the molecules (one end ester, the other alcohol), the resulting polymer networks can extend in a linear, unidirectional manner and are capped with a single hydroxyl group.

[0200] The polymer network can also be extended bidirectionally by introducing 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-propanediol, and 1,6-hexanediol. In some embodiments, the diols can be generated using a microbial host.

[0201] 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, ionic molecules, or hydrophobic compounds to produce higher-order polyols.

[0202] In some implementations, the TAG oil described herein can be used to generate materials for polymer applications. For example... Figure 7 As shown, OPO-rich TAG oils can undergo chemical transformation to produce oils containing functional groups, such as epoxy, ethoxy, methoxy, or hydroxyl groups. Possible modifications to OPO include (from left to right): hydroformylation and reduction reactions to produce diols; epoxidation reactions to produce diepoxide products; and ring-opening reactions following epoxidation (e.g., ring-opening with ethanol) to produce diols. OPO-rich TAGs allow for various chemical treatments around the carbon-carbon double bonds in the oleic acid moiety, thereby enabling the production of a variety of monomers that can be used as chain extenders in polymer chemistry applications.

[0203] In some implementations, strains such as those described herein capable of producing OPO-rich TAGs can serve as useful substrates for further genetic modification using the Δ-12 fatty acid desaturase (FAH12). Upregulating FAH12 expression in such OPO-accumulating strains increases ricinoleic acid production, thereby increasing the level of ricinoleic acid incorporated into oleic acid-containing TAGs. Therefore, these strains can thus produce diols, similar to... Figure 7 Those described herein, but which do not require additional chemical modification in vivo. Non-restrictive examples of oleic acid 12-hydroxylases include Rescler (…). Lesquerella fendleri FAH12, castor bean ( Ricinus communis FAH12 or ergot ( Claviceps purpurea )FAH12. Examples of this diol are shown in Figure 8 middle.

[0204] Example

[0205] Example 1: In mulberry-shaped algae ( P. moriformis Chlamydomonas reinhardtii was expressed in strain CHK22. Chlamydomonas reinhardtii ()( CrLPAAT2 ).

[0206] Microalgae *Mulberry-shaped Protozoa* obtained from the Algal Culture Collection at the University of Texas at Austin ( Prototheca moriformis The strain CHK22 (UTEX 1533) produces a fatty acid with an oleic acid to palmitic acid ratio of approximately 2:1, making it suitable for OPO production. However, P. moriformis Lysophosphatidyl acyltransferase (LPAAT) in sn The -2 position exhibits a strong preference for incorporating unsaturated fatty acids rather than saturated fatty acids. In this embodiment, heterologous LPAAT genes were tested to identify those capable of efficiently directing palmitic acid (a saturated fatty acid) to TAGs produced by microalgae. sn -2 genes.

[0207] Chlamydomonas reinhardtii ( C. reinhardtii ) CrLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK385 contains 5' and 3' homologous arms to allow CrLPAAT2 Targeted integration into the genome, as shown in Table 1.

[0208] Table 1: Codes Used CrLPAAT2 pCHK385 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0209]

[0210] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: CrLPAAT2 :CvNR::3'DAO1b. The sequence is from 5' to 3', and the bold lowercase letter sequence represents the CHK22 genomic DNA, which allows for homologous recombination in... DAO1b Locus-targeted integration. Chlamydomonas reinhardtii (a yeast strain that drives the expression of the sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose) C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver CrLPAAT2 The expression. CrLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris Nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region in bold lowercase text.

[0211] Strain growth and transformation

[0212] Unless otherwise stated, the strains described herein were cultured in 50 mL conical tubes at 28°C. The cells were cultured at 200 rpm for 96 hours at C to produce lipids. The culture supernatant was collected by centrifugation and washed once with MilliQ water. The resulting cell pellet was lyophilized and subjected to direct transesterification to generate fatty acid methyl esters (FAME), which were used for subsequent quantification and characterization by gas chromatography-flame ionization detection (GC / FID).

[0213] The strains used for transformation were grown in nutrient growth medium for 24 hours before transformation. Cells were precipitated by centrifugation, resuspended in medium, and then centrifuged again. The resulting cell pellet was resuspended in medium and then centrifuged at 5 × 10⁻⁶. 7Cells were plated in a suitable selective medium and dried in a sterile biosafety cabinet. Transformants of CHK22 were generated by bombarding the previously plated cells with gold nanoparticles. Primary transformants were selected based on their ability to grow on plates containing sucrose as the sole carbon source. The transformants were grown in a lipid-producing medium, and the resulting biomass was used for fatty acid analysis as described previously.

[0214] LCMS Analysis Method

[0215] TAG spectroscopy analysis was performed using LCMS. TAGs were extracted from cells into a 3:1 toluene / 2-propanol (v / v) solution by mechanical disruption and vigorous agitation using ceramic beads. The filtered extract was then injected into an Agilent 1290 Infinity II UHPLC system connected to a 6470B triple quadrupole mass spectrometer and an APCI ionization source.

[0216] LCMS determination of regioisomers

[0217] Based on the abundance of DAG ions generated by the fragmentation of TAG species, a qualitative assessment of the region-specificity of the main TAG species in the resulting oils was conducted (Figure 1).

[0218] Example 2: Digestion with pancreatic porcine lipase sn-2 Determination of fatty acid profiles.

[0219] Measurements were performed after incubating TAG with porcine pancreatic lipase. sn-2 The fatty acid composition at position 1. Lipase reactions lead to TAG at position 2. sn-1 and sn-3 Deacylation at position, thus leaving sn-2 -MAG. Then, separation was performed using an Agilent Bond Elut NH2propyl solid-phase extraction (SPE) column. sn-2 -MAG, and analyzed the fatty acid composition by gas chromatography using direct transesterification.

[0220] Mass spectra and DAG ion ratios of non-GMO strain CHK22 and CHK22 (D552-1, D552-3, and D552-4) transformed with pCHK385 are shown in Figure 2 and Table 2. Figure 2 shows the mass spectrum of OOP / OPO triglycerides. Although the regiomeric isomers were not separated chromatographically by this analytical method, m / z 577 (OP) and m / z The higher ratio of 603 (OO) indicates that the abundance of the OPO regioisomer is higher than that of CHK22.

[0221] Table 2: Screening of primary transformants of CHK22 obtained by pCHK385 transformation.

[0222]

[0223] Table 2 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK385. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0224] After incubating TAG with porcine pancreatic lipase, further assays were performed on the transgenic lines CHK22, D552-1, D552-3, and D552-4. sn-2 Fatty acid composition. The results are shown in Table 3.

[0225] Table 3: Measurement of the content of pCHK385-transformed CHK22 primary transformant after digestion with porcine pancreatic lipase sn-2 Percentage of palmitate.

[0226]

[0227] In Table 3, the mulberry-shaped algae ( P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium, with 200 mL of culture placed in a 1 L shake flask and incubated for 28 hours. C. Incubate at 200 rpm for 120 hours.

[0228] Biomass was harvested, freeze-dried to dryness, and subjected to direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. The assay was performed using porcine pancreatic lipase as described above. sn-2 Fatty acid composition at position 1. Figure 5 The corresponding bar charts show the various strains transformed by pCHK385. sn-2 Percentage of palmitic acid at position. Figure 6 The LC-MS results of the oils from strains CHK22 and D552-3 are shown, indicating that D552-3 has similar molecular characteristics to CHK22, but... sn The content of palmitic acid at the -2 position increased significantly.

[0229] Example 3: In mulberry-shaped algae ( P. moriformis Chlamydomonas reinhardtii was expressed in strain CHK22.C. reinhardtii ) LPAAT1 ( CrLPAAT1 ).

[0230] Chlamydomonas reinhardtii ( C. reinhardtii ) CrLPAAT1 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK391 contains 5' and 3' homologous arms to allow CrLPAAT1 Targeted integration into the genome, as shown in Table 4.

[0231] Table 4: Codes Used CrLPAAT1 pCHK391 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0232]

[0233] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: CrLPAAT1 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver CrLPAAT1 The expression. CrLPAAT1 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0234] Table 5 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK391. (Mulberry-shaped algae) P. moriformisThe basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0235] Table 5: Screening of primary transformants of CHK22 obtained by pCHK391 transformation.

[0236]

[0237] Example 4: In mulberry-shaped algae ( P. moriformis European rapeseed () was expressed in strain CHK22 Brassica napus ) BnBAT2 .

[0238] European rapeseed ( B. napus ) BnBAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK384 contains 5' and 3' homologous arms to allow BnBAT2 Targeted integration into the genome, as shown in Table 6.

[0239] Table 6: Table of materials used for the transformation of *Mulberry-shaped Algae* using pCHK384 encoding BnBAT2 (… P. moriformis The integrated sequence of ).

[0240]

[0241] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: BnBAT2 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented by boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* (… P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver BnBAT2 The expression. BnBAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0242] Table 7 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK384. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. In the lipid production medium of this strain, it was cultured in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0243] Table 7: Screening of primary transformants of CHK22 obtained by pCHK384 transformation.

[0244]

[0245] Example 5: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses the genus *Microcystis*. Nannochloropsis ) NoLPAT4 .

[0246] The genus *Microcystis* ( Nannochloropsis ) NoLPAT4 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK386 contains 5' and 3' homologous arms to allow NoLPAT4 Targeted integration into the genome, as shown in Table 8.

[0247] Table 8: Used for encoding NoLPAT4 pCHK386 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0248]

[0249] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: NoLPAT4 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented by boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* (… P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver NoLPAT4 The expression. NoLPAT4 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0250] Table 9 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK386. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0251] Table 9: Screening of primary transformants of CHK22 obtained by pCHK386 transformation.

[0252]

[0253] Example 6: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses the genus Syncytium ( Synechocystis sp ) SII1848 .

[0254] Synechocystis ( Synechocystis sp ) Sll1848 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK387 contains 5' and 3' homologous arms to allow Sll1848 Targeted integration into the genome, as shown in Table 10.

[0255] Table 10: Codes for the genus *Syntropha* (… Synechocystis sp ) SII1848 pCHK387 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0256]

[0257] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: Sll1848 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented by boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* (… P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver Sll1848 The expression. Sll1848 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0258] Table 11 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK387. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0259] Table 11: Screening of primary transformants of CHK22 converted by pCHK387.

[0260]

[0261] Example 7: In mulberry-shaped algae ( P. moriformis Escherichia coli expressed in strain CHK22 Escherichia coli ) EcPlsC .

[0262] E. coli EcPlsC Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK388 contains 5' and 3' homologous arms to allow EcPlsC Targeted integration into the genome, as shown in Table 12.

[0263] Table 12: Used for encoding Escherichia coli (E. coli) Escherichia coli ) EcPlsC pCHK388 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0264]

[0265] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: EcPlsC :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae ( ) is represented in boxed italics. P. moriformis PmAMT03 Startup Driver EcPlsC The expression. EcPlsC The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented by lowercase underlined text, followed by the CHK22DAO1b genomic region, represented by bold lowercase text.

[0266] Table 13 shows the fatty acid / TAG profiles of the four primary transformants of CHK22 converted from pCHK388. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0267] Table 13: Screening of primary transformants of CHK22 obtained by pCHK388 transformation.

[0268]

[0269] Example 8: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses the genus *Microcystis*. Nannochloropsis ) NoLPAT3 .

[0270] The genus *Microcystis* ( Nannochloropsis ) NoLPAT3 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK453 contains 5' and 3' homologous arms to allow NoLPAT3 Targeted integration into the genome, as shown in Table 14.

[0271] Table 14: Codes for the genus *Microcystis* ( Nannochloropsis ) NoLPAT3 pCHK453 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0272]

[0273] This constructor can be written as 5’Thi4::PmHXT1v2:ScMEL1:PmPGK:CvNR:PmSAD2-2:NoLPAT3: CvNR::3’Thi4 Along the 5' to 3' direction, the bold lowercase letter sequence represents the genomic DNA of strain CHK22, which allows for homologous recombination in... Thi4 Targeted integration at gene loci. Driven S. Carlbergensis melibiose enzyme ( ScMEL1 The expression of mulberry-shaped protozoa ( P. moriformis Hexotransfer protein 1 ( HXT1 v2 The promoter is indicated by boxed uppercase text. ScMEL1 The start codon ATG and stop codon TAA are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Mulberry-shaped algae* ( P. moriformis 3′-UTR of phosphoglucokinase (PGK) is represented in uppercase and underscore text, followed by the driving force. NoLPAT3 The expressed mulberry-like algae stearoyl ACP desaturase-2 ( SAD2-2 The starter is indicated by boxed italic text. NoLPAT3 The start codon ATG and stop codon TGA are represented in uppercase, bold italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented by lowercase underlined text, followed by *Procambarus mulberryii* (…). P. moriformis ) Thi4 3′ Flanking area, indicated by bold lowercase text.

[0274] Table 15 shows the fatty acid / TAG profiles of four primary transformants of CHK22 converted from pCHK453. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0275] Table 15: Screening of primary transformants of CHK22 obtained by pCHK453 transformation.

[0276]

[0277] Example 9: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses Volvox catechu ( Volvox carteri) LPAAT2 ( VcLPAAT2 ).

[0278] Volvox catechu ( V.carteri ()( VcLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK785 contains 5' and 3' homologous arms to allow VcLPAAT2 Targeted integration into the genome, as shown in Table 16.

[0279] Table 16: Used for coding VcLPAAT2 pCHK785 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0280]

[0281] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: VcLPAAT2 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriform The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver VcLPAAT2 The expression. VcLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0282] Table 17 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK785. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0283] Table 17: Screening of primary transformants of CHK22 obtained by pCHK785 transformation.

[0284]

[0285] Example 10: In mulberry-shaped algae ( P. moriformis strain CHK22 Chlamydomonas schloesser LPAAT2 (ChsLPAAT2) .

[0286] Will C. schloesseri ChsLPAAT2 Introducing mulberry-shaped algae ( P. moriformisIn strain CHK22. The expression construct pCHK786 contains 5' and 3' homologous arms to allow ChsLPAAT2 Targeted integration into the genome, as shown in Table 18.

[0287] Table 18: Used for coding ChsLPAAT2 pCHK786 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0288]

[0289] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ChsLPAAT2 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriform )PmPGH 3′-UTR is represented in uppercase underscore text, followed by a header. The mulberry-shaped algae is represented in boxed italic text. P. moriformis PmAMT03 Startup Driver ChsLPAAT2 The expression. ChsLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0290] Table 19 shows the fatty acid / TAG profiles of the six primary transformants of CHK22 converted from pCHK786. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0291] Table 19: Screening of primary transformants of CHK22 obtained by pCHK786 transformation.

[0292]

[0293] Example 11: In mulberry-shaped algae ( P. moriformis Chlamydomonas indeterminate was expressed in strain CHK22. Chlamydomonas incerta ) LPAAT2 (CiLPAAT2) .

[0294] The undetermined Chlamydomonas ( C. uncertain) CiLPAAT2 Import of mulberry-shaped algae ( P. moriformis strain CHK22. The expression construct pCHK787 contains 5' and 3' homologous arms to allow CiLPAAT2 Targeted integration into the genome, as shown in Table 20.

[0295] Table 20: Used for coding CiLPAAT2 pCHK787 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0296]

[0297] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: CiLPAAT2 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriform The PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver CiLPAAT2 The expression. CiLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented by lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented by bold lowercase text.

[0298] Table 21 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK787. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0299] Table 21: Screening of primary transformants of CHK22 obtained by pCHK787 transformation.

[0300]

[0301] Example 12: In mulberry-shaped algae ( P. moriformis African Volvox (Expression of Volvox africanus) in strain CHK22 Volvo africanus) LPAAT2 (VaLPAAT2) .

[0302] African Volvox ( V. africanus)VaLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK788 contains 5' and 3' homologous arms to allow ValPAAT2 Targeted integration into the genome, as shown in Table 22.

[0303] Table 22: Used for coding ValPAAT2 pCHK788 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0304]

[0305] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ValPAAT2 :CvNR::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformThe PmPGH 3′-UTR is represented in uppercase underlined text, followed by a header in lowercase bold italics. The mulberry-shaped algae (represented in boxed italics) is represented in... P. moriformis PmAMT03 Startup Driver ValPAAT2 The expression. ValPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. Common Chlorella ( C. vulgaris The nitrate reductase 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genomic region, represented in bold lowercase text.

[0306] Table 23 shows the fatty acid / TAG profiles of the six primary transformants of CHK22 converted by pCHK788. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMES for subsequent quantification and characterization by GC / FID. Perform TAG analysis using the LCMS method described above.

[0307] Table 23: Screening of primary transformants of CHK22 obtained by pCHK788 transformation.

[0308]

[0309] Because the CHK22 transformant expressing CrLPAAT2 produced a higher OP:OO ratio, the amino acid sequences of all full-length LPAAT proteins were compared for percentage identity (%) with CrLPAAT2. The results are shown in Table 24. The phenotypes produced by expressing these LPAATs in CHK22 are expressed as m / z (577.5 / 603.5) OP:OO ratios; the OP:OO ratios shown in the rows of PmLPAAT1 or PmLPAAT2 represent the analysis results of the non-transgenic line CHK22.

[0310] In some embodiments, the microalgal cells provided herein contain a foreign gene encoding LPAAT as shown in Table 24. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with LPAAT as shown in Table 24.

[0311] Table 24: OP:OO ratio

[0312] Table 25 shows the amino acid sequence of LPAAT described in the embodiments of this article. Figure 3, Figures A and C show the amino acid sequence alignment of the LPAATs described herein, which include 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). The common sequence is reflected in SEQ ID NO: 157 below.

[0313] In some embodiments, the microalgal cells provided herein contain a foreign gene encoding LPAAT as listed in Table 25. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with LPAAT as listed in Table 25. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with any of SEQ ID NO: 12-24, 63, and 64.

[0314] Table 25: LPAAT amino acid sequence

[0315] Example 13: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses *Volvariella nagari* ( Volvo Carteri f. nagariensis )LPAAT2 (VcLPAAT2 V2).

[0316] Will VcLPAAT2 V2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK967 contains 5' and 3' homologous arms to allow VcLPAAT2 V2 Targeted integration into the genome, as shown in Table 27.

[0317] Table 27: Used for coding VcLPAAT2 V2 pCHK967 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0318]

[0319] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: VcLPAAT2 V2 :PmPGH::3'DAO1b. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of the yeast invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriform )PmPGH 3′-UTR is represented by uppercase and underscore text, which makes VcLPAAT2 V2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver VcLPAAT2 V2 The expression. CrLPAAT2 V2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0320] Table 28 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted from pCHK967. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMES for subsequent quantification and characterization by GC / FID. Perform TAG analysis using the LCMS method described above.

[0321] Table 28: Screening of CHK22 primary transformants converted by pCHK967.

[0322]

[0323] Example 14: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses the stalked dark star algae ( ) Astrephomene gubernaculifera) LPAAT2 (AgLPAAT2) .

[0324] Will AgLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK968 contains 5' and 3' homologous arms to allow AgLPAAT2 Targeted integration into the genome, as shown in Table 29.

[0325] Table 29: Used for coding AgLPAAT2 pCHK968 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0326]

[0327] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: AgLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented by uppercase and underscore text, which makes AgLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver AgLPAAT2 The expression. AgLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0328] Table 30 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK968. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0329] Table 30: Screening of primary transformants of CHK22 converted by pCHK968.

[0330]

[0331] Example 15: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses *Tetracentron debarei* (a type of algae). Edaphochlamys debaryana) LPAAT2 (EdLPAAT2) .

[0332] Will EdLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK969 contains 5' and 3' homologous arms to allow EdLPAAT2 Targeted integration into the genome, as shown in Table 31.

[0333] Table 31: Used for encoding EdLPAAT2 pCHK969 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0334]

[0335] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: EdLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes EdLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver EdLPAAT2 The expression. EdLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0336] Table 32 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK969. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0337] Table 32: Screening of primary transformants of CHK22 obtained by pCHK969 transformation.

[0338]

[0339] Example 16: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses Dunaliella salina (Dunaliella salina). Dunaliella salina) LPAAT2 (DsLPAAT2) .

[0340] Will DsLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK971 contains 5' and 3' homologous arms to allow DsLPAAT2 Targeted integration into the genome, as shown in Table 33.

[0341] Table 33: Used for encoding DsLPAAT2 pCHK971 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0342]

[0343] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: DsLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes DsLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver DsLPAAT2 The expression. DsLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0344] Table 34 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK971. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0345] Table 34: Screening of primary transformants of CHK22 obtained by pCHK971 transformation.

[0346]

[0347] Example 17: In mulberry-shaped algae ( P. moriformis Strain CHK22 expresses Scenedesmus (genus) Scenedesmus sp.)LPAAT2 (SceLPAAT2) .

[0348] Will SceLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK973 contains 5' and 3' homologous arms to allow SceLPAAT2 Targeted integration into the genome, as shown in Table 35.

[0349] Table 35: Used for encoding Sce LPAAT2 pCHK973 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0350]

[0351] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: SceLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes SceLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver SceLPAAT2 The expression. SceLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0352] Table 36 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK973. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0353] Table 36: Screening of primary transformants of CHK22 obtained by pCHK973 transformation.

[0354]

[0355] Example 18: In mulberry-shaped algae ( P. moriformis Expression in strain CHK22 Micractinum conductrix LPAAT2 (McLPAAT2) .

[0356] Will McLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK974 contains 5' and 3' homologous arms to allow McLPAAT2 Targeted integration into the genome, as shown in Table 37.

[0357] Table 37: Used for coding McLPAAT2 pCHK974 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0358]

[0359] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: McLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes McLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver McLPAAT2 The expression. McLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0360] Table 38 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK974. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0361] Table 38: Screening of primary transformants of CHK22 converted by pCHK974.

[0362]

[0363] Example 19: In mulberry-shaped algae ( P. moriformis Chlorella sorokinesi was expressed in strain CHK22. Chlorella sorokiniana) LPAAT2 (ChsoLPAAT2) .

[0364] Will ChsoLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK975 contains 5' and 3' homologous arms to allowChsoLPAAT2 Targeted integration into the genome, as shown in Table 39.

[0365] Table 39: Used for coding ChsoLPAAT2 pCHK975 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0366]

[0367] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ChsoLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ChsoLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( Chlorella vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver ChsoLPAAT2 The expression. ChsoLPAAT2 The start codon ATG and stop codon TGA are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0368] Table 40 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK975. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0369] Table 40: Screening of primary transformants of CHK22 obtained by pCHK975 transformation.

[0370]

[0371] Example 20: In mulberry-shaped algae ( P. moriformis The variant Chlorella expressed in strain CHK22 Chlorella variabilis) LPAAT2 (ChvaLPAAT2) .

[0372] Will ChvaLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK976 contains 5' and 3' homologous arms to allow ChvaLPAAT2 Targeted integration into the genome, as shown in Table 41.

[0373] Table 41: Used for encoding ChvaLPAAT2 pCHK976 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0374]

[0375] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ChvaLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ChvaLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris Nitrate reductase 3′-UTR. The PmAMT03 promoter of *Prorocentrum moriformis* (represented in boxed italics) drives this process. ChvaLPAAT2 The expression. ChvaLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformisThe PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0376] Table 42 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK976. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0377] Table 42: Screening of primary transformants of CHK22 obtained by pCHK976 transformation.

[0378]

[0379] Example 21: In mulberry-shaped algae ( P. moriformis Expression in strain CHK22 Raphidocelis subcapitata (Raphidocelis subcapitata) LPAAT2 (RsLPAAT2) .

[0380] Will RsLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK977 contains 5' and 3' homologous arms to allow RsLPAAT2 Targeted integration into the genome, as shown in Table 43.

[0381] Table 44: Used for encoding RsLPAAT2 pCHK977 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0382]

[0383] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03:Rs LPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtiiThe β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes RsLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup Driver RsLPAAT2 The expression. RsLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0384] Table 44 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted from pCHK977. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0385] Table 44: Screening of primary transformants of CHK22 converted by pCHK977.

[0386]

[0387] Example 22: In mulberry-shaped algae ( P. moriformis Dehydrated Chlorella was expressed in strain CHK22. Chlorella desiccata) LPAAT2 (ChdeLPAAT2) .

[0388] Will ChdeLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK978 contains 5' and 3' homologous arms to allow ChdeLPAAT2 Targeted integration into the genome, as shown in Table 45.

[0389] Table 45: Used for coding ChdeLPAAT2 pCHK978 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0390]

[0391] This constructor can be written as

[0392] 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ChdeLPAAT :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. ​ The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( ​ PmPGH3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​ PmAMT03 Startup Driver ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0393] Table 46 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK978. (Mulberry-shaped algae) ​ The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0394] Table 46: Screening of CHK22 primary transformants converted by pCHK978.

[0395]

[0396] Example 23: In mulberry-shaped algae ( ​ The strain CHK22 expresses Chlorella protochaeta ( Auxenochlorella proteothecoides) LPAAT2 (ApLPAAT2) .

[0397] Will ApLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK979 contains 5' and 3' homologous arms to allow ApLPAAT2 Targeted integration into the genome, as shown in Table 47.

[0398] Table 47: Used for coding ApLPAAT2 pCHK979 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0399]

[0400] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmAMT03: ApLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​ )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​ PmAMT03 Startup Driver ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0401] Table 48 shows the fatty acid / TAG profiles of the CHK22 primary transformant obtained from pCHK979. (Mulberry-shaped algae) P. ​ The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0402] Table 48: Screening of CHK22 primary transformants converted by pCHK979.

[0403]

[0404] Example 24: In mulberry-shaped algae ( ​ Expression in strain CHK22 ​ ​ .

[0405] Will ​ Introducing mulberry-shaped algae ( ​ In strain CHK22. The expression construct pCHK980 contains 5' and 3' homologous arms to allow ​ Targeted integration into the genome, as shown in Table 49.

[0406] Table 49: Used for encoding ​ pCHK980 transformed mulberry-shaped algae ( ​ The integrated sequence of ).

[0407]

[0408] This constructor can be written as 5'DAO1b::CrTUB2: ​ :PmPGH:CvNR:PmAMT03: ​ :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). ​ The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​)PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​ PmAMT03 Startup Driver ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0409] Table 50 shows the fatty acid / TAG profiles of the CHK22 primary transformant obtained via pCHK980. (Mulberry-shaped algae) P. ​ The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0410] Table 50: Screening of CHK22 primary transformants converted by pCHK980.

[0411]

[0412] Example 25: In mulberry-shaped algae ( ​ The strain CHK22 expresses Homo sapiens ( ​ ​ .

[0413] Will ​ Introducing mulberry-shaped algae ( ​ In strain CHK22. The expression construct pCHK966 contains 5' and 3' homologous arms to allow ​ Targeted integration into the genome, as shown in Table 51.

[0414] Table 51: Codes Used ​ pCHK966 transformed mulberry-shaped algae ( ​ The integrated sequence of ).

[0415]

[0416] This constructor can be written as 5'DAO1b::CrTUB2: ​ :PmPGH:CvNR:PmAMT03: ​ :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). ​ The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​ )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​ PmAMT03 Startup Driver ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0417] Table 52 shows the fatty acid / TAG profiles of the primary transformant of CHK22 converted by pCHK966. (Mulberry-shaped algae) ​ The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0418] Table 52: Screening of CHK22 primary transformants obtained by pCHK966 transformation.

[0419]

[0420] Example 26: In mulberry-shaped algae ( ​ The strain CHK22 expresses *Variegata* (a type of algae). ​ .

[0421] Will ​ Introducing mulberry-shaped algae ( ​ In strain CHK22. The expression construct pCHK970 contains 5' and 3' homologous arms to allow ​ Targeted integration into the genome, as shown in Table 53.

[0422] Table 53: Used for encoding ​ pCHK970 transformed mulberry-shaped algae ( ​ The integrated sequence of ).

[0423]

[0424] This constructor can be written as 5'DAO1b::CrTUB2: ​ :PmPGH:CvNR:PmAMT03: ​ :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). ​ The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​ )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​ PmAMT03 Startup Driver ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22DAO1b genome region, represented in bold lowercase text.

[0425] Table 54 shows the fatty acid / TAG profiles of the CHK22 primary transformant obtained via pCHK970. (Mulberry-shaped algae) P. ​ The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0426] Table 54: Screening of CHK22 primary transformants converted by pCHK970.

[0427]

[0428] Example 27: Using the PmAMT03 promoter in *Procambarus mulberryii* (… ​ ) strain CHK22 expresses species of the class Alpha phylum ( ​ .

[0429] Will ​ Introducing mulberry-shaped algae ( ​ In strain CHK22. The expression construct pCHK972 contains 5' and 3' homologous arms to allow ​ Targeted integration into the genome, as shown in Table 55.

[0430] Table 55: Used for encoding ​ pCHK972 transformed mulberry-shaped algae ( ​ The integrated sequence of ).

[0431]

[0432] This constructor can be written as 5'DAO1b::CrTUB2: ​ :PmPGH:CvNR:PmAMT03: ​ :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). ​ The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​ )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmAMT03 Startup DriverPedLPAAT2 The expression. PedLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0433] Table 56 shows the fatty acid / TAG profiles of the CHK22 primary transformant obtained by pCHK972 conversion. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0434] Table 56: Screening of CHK22 primary transformants converted by pCHK972.

[0435]

[0436] Because the CHK22 transformant expressing CrLPAAT2 produced a higher OP:OO ratio, the percentage identity (%) of all full-length amino acid sequences of LPAAT proteins with CrLPAAT2 was compared, and the results are shown in Table 57. Pairwise identity (%) between all sequences and CrLPAAT2. The phenotypes produced by expressing these LPAATs in CHK22 are expressed as m / z (577.5 / 603.5) OP:OO ratios; the OP:OO ratios shown in the rows of PmLPAAT1 or PmLPAAT2 represent the analysis results of the non-transgenic line CHK22. The amino acid sequences of the LPAATs described in this paper are listed in Table 58.

[0437] Table 57: OP:OO ratio

[0438] Table 58: LPAAT amino acid sequence

[0439] Example 28: In mulberry-shaped algae ( P. moriformis The strain CHK22 expresses *Volvariella reticulata* (…). Volvoxreticuliferus ) LPAAT2 ( VrLPAAT2 ).

[0440] Will VrLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1133 contains 5' and 3' homologous arms to allow VrLPAAT2 Targeted integration into the genome, as shown in Table 59.

[0441] Table 59: Used for coding VrLPAAT2 pCHK1133 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0442]

[0443] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmACP: VrLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes VrLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris nitrate reductase 3′-UTR. (Indicated by boxed italic text from *Procambarus mulberryii*) P. moriformis The acyl carrier protein promoter (PmACP) drives... VrLPAAT2 The expression. VrLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0444] Table 60 shows the fatty acid / TAG profiles of the CHK22 primary transformant converted by pCHK1133. (Mulberry-shaped algae) P.moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0445] Table 60: Screening of CHK22 primary transformants converted by pCHK1133.

[0446]

[0447] Example 29: In mulberry-shaped algae ( P. moriformis Chlorella vulgaris was expressed in strain CHK22. Chlorella vulgaris)LPAAT2 (ChvuLPAAT2 ).

[0448] Will ChvuLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1134 contains 5' and 3' homologous arms to allow ChvuLPAAT2 Targeted integration into the genome, as shown in Table 61.

[0449] Table 61: Used for encoding ChvuLPAAT2 pCHK1134 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0450]

[0451] This constructor can be written as 5'DAO1b::CrTUB2: ScSUC2 :PmPGH:CvNR:PmACP: ChvuLPAAT2 :PmPGH::3'DAO1b. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the DAO1b locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of the yeast sucrose invertase gene (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ChvuLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmACP bootloader ChvuLPAAT2 The expression. ChvuLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 DAO1b genome region, represented in bold lowercase text.

[0452] Table 62 shows the fatty acid / TAG profiles of the CHK22 primary transformant converted by pCHK1134. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0453] Table 62: Screening of CHK22 primary transformants converted by pCHK1134.

[0454]

[0455] Example 30: Generate a microalgae strain modified by conventional methods that produces triglyceride oil rich in palmitate.

[0456] microalgae ( P. moriformis Strain CHK22 (UTEX 1533) underwent conventional strain improvement to enhance yield, carbon yield, and palmitic acid content. The improvement process included mutagenesis, phenotypic selection, and high-throughput automated screening steps, such as... Figure 9The flowchart is shown. In short, algal cells in the logarithmic growth phase are mutagenized by chemical treatment or UV light treatment (1). The cells are then passaged into lipid production medium and a selection / enrichment strategy is employed (2). The strains are plated on solid medium to obtain clonal isolates (3), which are then interrogated in lipid production medium in 96-well plates (4). High glucose-consuming strains are validated in lipid production medium in tubes or shake flasks using glucose consumption as a surrogate indicator of lipid production (5). Successfully validated isolates are passaged multiple times to stabilize the mutation (6), followed by purification of the clonal isolates (7), and then re-screened in lipid production medium (8). Phenotypic stable clones are then validated in fermentation (9). The clones showing variability in (8) are revalidated by (6) and passaged to produce stable cell lines.

[0457] CHK22 cells were chemically mutagenized at 32°C for 30 min with 44 μM 4-nitroquinoline 1-oxide (4-NQO), or sham-mutated by adding only the mutagenic solvent DMSO. The mutagen was inactivated by adding sodium thiosulfate followed by repeated washing with water. Cells were then allowed to recover in a glucose-limited growth medium for 3 days. Subsequently, the mutagenized and mock-mutaged populations were independently cultured in lipid production medium at 38°C for five days. The optimal lipid production temperature for CHK22 is typically 28–32°C. Applying conditions above the optimum temperature acts as a stress factor, thus providing a growth advantage for mutants that tend to produce higher palmitic acid levels. After 5 days of lipid culture at 38°C, cells were harvested and then incubated at 65°C for 4 minutes. Experiments were determined based on the range of conditions performed in the same manner; exposure for this period typically killed >99% of the cell population. Following heat exposure, cells were recovered in a glucose-limited growth medium for three days, diluted, and then plated. Mutant clones were then selected, and their glucose consumption rate and fatty acid profiles (e.g., 72-hour lipid production assays based on 96-well plates) were evaluated. Figure 9 (As shown). Table 63 summarizes the glucose consumption rate and fatty acid profile of TAG oil produced by the mutant strain. One mutant produced approximately 8% more palmitic acid than the parent CHK22 strain. This strain exhibited a significantly reduced glucose consumption rate, a hallmark of lower lipid titers.

[0458] Table 63: Fatty acid profiles of selected mutant strains that exhibited higher glucose consumption (Glc rate) and increased palmitate (C16:0) levels in 96-well plate lipid assays.

[0459]

[0460] To further adapt the CHK22 mutant-3 strain to the altered fatty acid composition and improve lipid production, the strain was passaged more than thirty times without any selective pressure, and then plated on solid media to obtain single colonies. These subclones were screened in lipid production assays to assess the phenotypic stability of the lineage to glucose consumption and fatty acid profiles. After one consecutive passage cycle, the mutant remained phenotypically unstable. Several subclones retaining higher palmitate levels and increased glucose consumption were obtained through several more consecutive passages and stability assessments. After a total of five consecutive passages and stability assessments, a stable strain, designated CHK100, was identified. CHK100 produced approximately 5% more palmitate than its parental strain CHK22. CHK100 also had higher lipid content (%w / w), dry cell weight (DCW), and lipid titer (g / L) than CHK22 (Tables 64 and 65). Figure 10 The strategy for obtaining CHK100 from CHK22 through strain modification is shown.

[0461] Table 64: Test tube-based CHK100 assays showed increased lipid titers.

[0462]

[0463] NLB, non-lipid biomass; PCP, yield per cell.

[0464] Table 65: Test tube-based CHK100 measurements show an increase in C16:0 content.

[0465]

[0466] All strains were cultured under standard lipid production conditions, with two parallel cultures per 10 mL lipid production medium. Cultures were grown at 28°C with shaking (200 rpm) for 121 hours. At this point, 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 pre-weighed glass vial. The vial containing the filter and frozen biomass was then lyophilized overnight, and the weight was recorded. The filter containing the dried biomass was subjected to direct transesterification, followed by GC / FID to quantify the fat-to-metabolite (FAME).

[0467] Example 31: Using the PmACP promoter in *Procambarus mulberryii* (… P. moriformis ) strain CHK100 expresses species of the class Alpha phylum ( Pedinophyceae sp.) LPAAT2 (PedLPAAT2) .

[0468] Will PedLPAAT2 Introducing mulberry-shaped algae ( P. moriformisIn strain CHK22. The expression construct pCHK1126 contains 5' and 3' homologous arms to allow PedLPAAT2 Targeted integration into the genome, as shown in Table 66.

[0469] Table 66: Used for encoding PedLPAAT2 pCHK1126 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0470]

[0471] This constructor can be written as 5'Thi4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmACP: PedLPAAT2 :PmPGH::3'Thi4. The bold lowercase sequence along the 5' to 3' direction represents the CHK100 genomic DNA, which allows for targeted integration at the Thi4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK100 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes PedLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmACP bootloader PedLPAAT2 The expression. PedLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK100 Thi4 genomic region, represented in bold lowercase text.

[0472] Table 67 shows the fatty acid / TAG profiles of the CHK100 primary transformant converted by pCHK1126. (Mulberry-shaped algae) P. moriformis The basic strain CHK100 is shown as a non-transgenic control. The strain was cultured in lipid production medium, in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0473] Table 67: Screening of CHK100 primary transformants converted by pCHK1126.

[0474]

[0475] Example 32: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis ) strain CHK100 expresses species of the class Alpha phylum ( Pedinophyceae sp.) LPAAT2 (PedLPAAT2) .

[0476] Will PedLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK100. The expression construct pCHK1127 contains 5' and 3' homologous arms to allow PedLPAAT2 Targeted integration into the genome, as shown in Table 68.

[0477] Table 68: Used for encoding PedLPAAT2 pCHK1127 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0478]

[0479] This constructor can be written as 5'Thi4::CrTUB2: ScSUC2 :PmPGH:CvNR: PmG3PDH: PedLPAAT2 :PmPGH::3'Thi4. The bold lowercase sequence along the 5' to 3' direction represents the CHK100 genomic DNA, which allows for targeted integration at the Thi4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK100 to metabolize sucrose). [[ID=7 The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. ​ )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes ​ Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( ​ 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). ​PmG3PDH bootloader ​ The expression. ​ The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( ​ The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK100 Thi4 genomic region, represented in bold lowercase text.

[0480] Table 69 shows the fatty acid / TAG profiles of the CHK100 primary transformant converted by pCHK1127. (Mulberry-shaped algae) ​ The basic strain CHK100 is shown as a non-transgenic control. The strain was cultured in lipid production medium, in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0481] Table 69: Screening of CHK100 primary transformants converted by pCHK1127.

[0482]

[0483] Example 33: Using the PmL40-2 promoter in *Procambarus mulberryii* (… ​ ) strain CHK100 expresses species of the class Alpha phylum ( ​ .

[0484] Will ​ Introducing mulberry-shaped algae ( P. moriformis In strain CHK100. The expression construct pCHK1128 contains 5' and 3' homologous arms to allow PedLPAAT2 Targeted integration into the genome, as shown in Table 70.

[0485] Table 70: Used for coding PedLPAAT2 pCHK1128 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0486]

[0487] This constructor can be written as 5'Thi4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmL40-2: PedLPAAT2 :PmPGH::3'Thi4. The bold lowercase sequence along the 5' to 3' direction represents the CHK100 genomic DNA, which allows for targeted integration at the Thi4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK100 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes PedLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmL40-2 promoter drive PedLPAAT2 The expression. PedLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK100 Thi4 genomic region, represented in bold lowercase text.

[0488] Table 71 shows the fatty acid / TAG profiles of the CHK100 primary transformant converted by pCHK1128. (Mulberry-shaped algae) P. moriformis The basic strain CHK100 is shown as a non-transgenic control. The strain was cultured in lipid production medium, in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0489] Table 71: Screening of CHK100 primary transformants converted by pCHK1128.

[0490]

[0491] Example 34: Using the PmMPGp promoter in *Procambarus mulberryii* (… P. moriformis ) strain CHK100 expresses species of the class Alpha phylum ( Pedinophyceae sp.) LPAAT2 (PedLPAAT2) .

[0492] Will PedLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK100. The expression construct pCHK1129 contains 5' and 3' homologous arms to allow PedLPAAT2 Targeted integration into the genome, as shown in Table 72.

[0493] Table 72: Used for coding PedLPAAT2 pCHK1129 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0494]

[0495] This constructor can be written as 5'Thi4::CrTUB2: ScSUC2 :PmPGH:CvNR: PmMPGp: PedLPAAT2 :PmPGH::3'Thi4. The bold lowercase sequence along the 5' to 3' direction represents the CHK100 genomic DNA, which allows for targeted integration at the Thi4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK100 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. *Procambarus mulberryii* ( P. moriformis )PmPGH 3′-UTR is represented in uppercase and underscore text, which makes PedLPAAT2 Genes can amplify, followed by adapters, represented in lowercase bold italic text, which contains common Chlorella ( C. vulgaris 3′-UTR of nitrate reductase. The text in italics indicates the mulberry-shaped algae (…). P. moriformis PmMPGp bootloader PedLPAAT2 The expression. PedLPAAT2 The start codon ATG and stop codon TAG are represented in bold uppercase italics, while the rest of the gene is represented in bold italics. *Mulberry-shaped algae* ( P. moriformis The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK100 Thi4 genomic region, represented in bold lowercase text.

[0496] Table 73 shows the fatty acid / TAG profiles of the CHK100 primary transformant converted by pCHK1129. (Mulberry-shaped algae) P. moriformis The basic strain CHK100 is shown as a non-transgenic control. The strain was cultured in lipid production medium, in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0497] Table 73: Screening of CHK100 primary transformants converted by pCHK1129.

[0498]

[0499] Example 35: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses densely packed Volvox (a type of algae). Volvulina compacta) (VcomLPAAT2) .

[0500] Will VcomLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1210 contains 5' and 3' homologous arms to allow VcomLPAAT2 Targeted integration into the genome, as shown in Table 74.

[0501] Table 74: Used for coding VcomLPAAT2 pCHK1210 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0502]

[0503] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VcomLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VcomLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader VcomLPAAT2 The expression. VcomLPAAT2 The start codon ATG and stop codon TAG are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0504] Table 75 shows the fatty acid / TAG profiles of the CHK22 primary transformant obtained from pCHK1210. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0505] Table 75: Screening of CHK22 primary transformants converted by pCHK1210.

[0506]

[0507] Example 36: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses the genus Chlamydomonas ( ). Vitreochlamys sp.) CL-2021 (VitrLPAAT2) .

[0508] Will VitrLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1211 contains 5' and 3' homologous arms to allow VitrLPAAT2 Targeted integration into the genome, as shown in Table 76.

[0509] Table 76: Used for coding VitrLPAAT2 pCHK1211 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0510]

[0511] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VitrLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VitrLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader VitrLPAAT2 The expression. VitrLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0512] Table 77 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1211. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0513] Table 77: Screening of CHK22 primary transformants converted by pCHK1211.

[0514]

[0515] Example 37: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Strain CHK22 expresses *Coccus chalcogenoides* ( Colemanosphaera charkowiensis) (CchaLPAAT2) .

[0516] Will CchaLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1212 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 78.

[0517] Table 78: Used for coding CchaLPAAT2 pCHK1212 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0518]

[0519] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: CchaLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... CchaLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader CchaLPAAT2 The expression. CchaLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0520] Table 79 shows the fatty acid / TAG profiles of the CHK22 primary transformant converted by pCHK1212. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0521] Table 79: Screening of CHK22 primary transformants converted by pCHK1212.

[0522]

[0523] Example 38: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Pleodorina japonica (PjapLPAAT2) is expressed in strain CHK22.

[0524] Will PjapLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1213 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 80.

[0525] Table 80: Used for coding PjapLPAAT2 pCHK1213 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0526]

[0527] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: PjapLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... PjapLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is indicated in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR from *C. vulgaris*. The text in boxed italics is from *Procambarus mulberryii* (…). P. moriformis PmG3PDH bootloader PjapLPAAT2 The expression. PjapLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0528] Table 81 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1213. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0529] Table 81: Screening of CHK22 primary transformants converted by pCHK1213.

[0530]

[0531] Example 39: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Strain CHK22 expresses *Cyclocarya baumannii* (…). Volvulin boldii) (VbolLPAAT2) .

[0532] Will VbolLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1214 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 82.

[0533] Table 82: Used for coding VbolLPAAT2 pCHK1214 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0534]

[0535] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VbolLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VbolLPAAT2 Mulberry-shaped protozoan (genes) P. moriformisPmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader VbolLPAAT2 The expression. VbolLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0536] Table 83 shows the fatty acid / TAG profiles of the CHK22 primary transformant converted from pCHK1214. (Mulberry-shaped algae) P. moriform The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0537] Table 83: Screening of CHK22 primary transformants converted by pCHK1214.

[0538]

[0539] Example 40: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses Chlorella vulgaris ( Pandorina morum) (PmorLPAAT2) .

[0540] Will PmorLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1215 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 84.

[0541] Table 84: Used for encoding PmorLPAAT2 pCHK1215 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0542]

[0543] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2:PmPGH:CvNR:PmG3PDH: PmorLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... PmorLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader PmorLPAAT2 The expression. PmorLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0544] Table 85 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1215. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0545] Table 85: Screening of CHK22 primary transformants converted by pCHK1215.

[0546]

[0547] Example 41: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses the Weissmann type of Volvox catterii ( ) Volvox carter f. weismannia) (VcarfLPAAT2) .

[0548] Will VcarfLPAAT2 Introducing mulberry-shaped algae ( P. moriformisIn strain CHK22. The expression construct pCHK1216 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 86.

[0549] Table 86: Used for encoding VcarfLPAAT2 pCHK1216 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0550]

[0551] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VcarfLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VcarfLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader VcarfLPAAT2 The expression. VcarfLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0552] Table 87 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1216. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0553] Table 87: Screening of CHK22 primary transformants converted by pCHK1216.

[0554]

[0555] Example 42: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses Cylindrical Hollow Globulus ( Eudorina cylindrica) (EcylLPAAT2) .

[0556] Will EcylLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1217 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 88.

[0557] Table 88: Used for encoding EcylLPAAT2 pCHK1217 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0558]

[0559] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: EcylLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... EcylLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader EcylLPAAT2 The expression. EcylLPAAT2The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0560] Table 89 shows the fatty acid / TAG profiles of eight primary transformants of CHK22 converted from pCHK1217. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0561] Table 89: Screening of CHK22 primary transformants converted by pCHK1217.

[0562]

[0563] Example 43: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses Polycholenosa ( Gonium multicoccum) (GmulLPAAT2) .

[0564] Will GmulLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1218 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 90.

[0565] Table 90: Used for coding GmulLPAAT2 pCHK1218 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0566]

[0567] This construct can be written as 5'Thia4::CrTUB2:ScSUC2:PmPGH:CvNR:PmG3PDH:GmulLPAAT2:PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is indicated in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are indicated in bold uppercase italics, while the coding region is indicated in lowercase italics. The *Procambarus mulii* species capable of amplifying the GmulLPAAT2 gene... P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis The PmG3PDH promoter drives the expression of GmulLPAAT2. GmulLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0568] Table 91 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1218. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 96 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0569] Table 91: Screening of CHK22 primary transformants converted by pCHK1218.

[0570]

[0571] Example 44: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses *Discocephalum glomeratum* (a type of algae). Gonium viridistellatum) (GvirLPAAT2) .

[0572] Will GvirLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1219 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 92.

[0573] Table 92: Used for coding GvirLPAAT2 pCHK1219 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0574]

[0575] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: GvirLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... GvirLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader GvirLPAAT2 The expression. GvirLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0576] Table 93 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1219. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0577] Table 93: Screening of CHK22 primary transformants converted by pCHK1219.

[0578]

[0579] Example 45: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis strain CHK22 expresses *Volvariella ferri* ( Volvox ferrisii) (VferLPAAT2) .

[0580] Will VferLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1220 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 94.

[0581] Table 94: Used for encoding VferLPAAT2 pCHK1220 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0582]

[0583] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VferLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VferLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader VferLPAAT2 The expression. VferLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0584] Table 95 shows the fatty acid / TAG profiles of the CHK22 primary transformant converted by pCHK1220. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0585] Table 95: Screening of CHK22 primary transformants converted by pCHK1220.

[0586]

[0587] Example 46: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses Chlamydomonas hygroscopicus ( Vitreochlamys aulata) (VaulLPAAT2) .

[0588] Will VaulLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1222 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 96.

[0589] Table 96: Used for encoding VaulLPAAT2 pCHK1222 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0590]

[0591] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: VaulLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... VaulLPAAT2 The PmPGH 3′-UTR of *P. moriformis* is represented in uppercase underlined text, followed by the linker in lowercase bold italics, which contains the nitrate reductase 3′-UTR of *C. vulgaris*. The *P. moriformis* gene is represented in boxed italics. P. moriformis PmG3PDH bootloader VaulLPAAT2 The expression. VaulLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0592] Table 97 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1222. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0593] Table 97: Screening of CHK22 primary transformants converted by pCHK1222.

[0594]

[0595] Example 47: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Chlamydomonas genus was expressed in strain CHK22. Chlamydomonas sp.) CCAC2762_B (Ch_CCAC2762_LPAAT2) .

[0596] Will Ch_CCAC2762_LPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1224 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 98.

[0597] Table 98: Used for encoding Ch_CCAC2762_LPAAT2 pCHK1224 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0598]

[0599] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: Ch_ CCAC2762_LPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... Ch_CCAC2762_LPAAT2 The PmPGH 3′-UTR of *P. moriformis* is represented in uppercase underlined text, followed by the linker in lowercase bold italics, which contains the nitrate reductase 3′-UTR of *C. vulgaris*. The text from *P. moriformis* is represented in boxed italics. P. moriformis PmG3PDH bootloader Ch_CCAC2762_LPAAT2 The expression. Ch_CCAC2762_LPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0600] Table 99 shows the fatty acid / TAG spectra of seven primary transformants of CHK22 converted from pCHK1224. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0601] Table 99: Screening of CHK22 primary transformants converted by pCHK1224.

[0602]

[0603] Example 48: Using the PmG3PDG promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses Dunaliella salina (Dunaliella salina). Dunaliella salina) (DsalLPAAT2) .

[0604] Will DsalLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1225 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 100.

[0605] Table 100: Used for coding DsalLPAAT2 pCHK1225 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0606]

[0607] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: DsalLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... DsalLPAAT2 The PmPGH 3′-UTR of *P. moriformis* is represented in uppercase underlined text, followed by the linker in lowercase bold italics, which contains the nitrate reductase 3′-UTR of *C. vulgaris*. The text from *P. moriformis* is represented in boxed italics. P. moriformis PmG3PDG bootloader driver DsalLPAAT2 The expression. DsalLPAAT2 The start codon ATG and stop codon TAG are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0608] Table 101 shows the fatty acid / TAG profiles of seven primary transformants of CHK22 converted from pCHK1225. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0609] Table 101: Screening of CHK22 primary transformants converted by pCHK1225.

[0610]

[0611] Example 49: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis The strain CHK22 expresses the genus *Softshell Algae*. Microglena sp.)YARC (MyarcLPAAT2) .

[0612] Will MyarcLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1227 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 102.

[0613] Table 102: Used for coding MyarcLPAAT2 pCHK1227 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0614]

[0615] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: MyarcLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... MyarcLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is indicated in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR from *C. vulgaris*. The text in boxed italics is from *Procambarus mulberryii* (…). P. moriformis PmG3PDH bootloader MyarcLPAAT2 The expression. MyarcLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22Thia4 genomic region, represented in bold lowercase text.

[0616] Table 103 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1227. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0617] Table 103: Screening of CHK22 primary transformants converted by pCHK1227.

[0618]

[0619] Example 50: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Chlamydomonas genus was expressed in strain CHK22. Chlamydomonas sp.) UWO_241 (CuwoLPAAT2) .

[0620] Will CuwoLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1228 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 104.

[0621] Table 104: Used for encoding CuwoLPAAT2 pCHK1228 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0622]

[0623] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2 :PmPGH:CvNR:PmG3PDH: CuwoLPAAT2 :PmPGH::3'Thia4. Along the 5' to 3' direction, the bold lowercase sequence represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; *Chlamydomonas reinhardtii*, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... CuwoLPAAT2 Mulberry-shaped protozoan (genes) P. moriformisPmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader CuwoLPAAT2 The expression. CuwoLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0624] Table 105 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1228. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0625] Table 105: Screening of CHK22 primary transformants converted by pCHK1228.

[0626]

[0627] Example 51: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Chlamydomonas mossae was expressed in strain CHK22. Chlamydomonas moewusii) (CmoeLPAAT2) .

[0628] Will CmoeLPAAT2 Introducing mulberry-shaped algae ( P. moriformis In strain CHK22. The expression construct pCHK1231 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 106.

[0629] Table 106: Used for coding CmoeLPAAT2 pCHK1231 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0630]

[0631] This constructor can be written as 5'Thia4::CrTUB2: ScSUC2:PmPGH:CvNR:PmG3PDH: CmoeLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... CmoeLPAAT2 Mulberry-shaped protozoan (genes) P. moriformis PmPGH 3′-UTR is represented in uppercase underlined text, followed by the connector in lowercase bold italics, and contains the nitrate reductase 3′-UTR of *Chlorella vulgaris*. The *Procambarus vulgaris* is represented in boxed italics. P. moriformis PmG3PDH bootloader CmoeLPAAT2 The expression. CmoeLPAAT2 The start codon ATG and stop codon TGA are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0632] Table 107 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1231. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0633] Table 107: Screening of CHK22 primary transformants converted by pCHK1231.

[0634]

[0635] Example 52: Using the PmG3PDH promoter in *Procambarus mulberryii* (… P. moriformis Salamander symbiotic algae expressed in strain CHK22 Oophila amblystomatis)(OambLPAAT2) .

[0636] Will OambLPAAT2 Introducing mulberry-shaped algae ( P. moriformisIn strain CHK22. The expression construct pCHK1232 contains 5' and 3' homologous arms to allow for targeted integration into the genome, as shown in Table 108.

[0637] Table 108: Used for coding OambLPAAT2 pCHK1232 transformed mulberry-shaped algae ( P. moriformis The integrated sequence of ).

[0638]

[0639] This constructor can be written as 5'Thia4::CrTUB2: ScSUCz :PmPGH:CvNR:PmG3PDH: OambLPAAT2 :PmPGH::3'Thia4. The bold lowercase sequence along the 5' to 3' direction represents the CHK22 genomic DNA, which allows for targeted integration at the Thia4 locus via homologous recombination; Chlamydomonas reinhardtii, which drives the expression of yeast invertase genes (conferring the ability of CHK22 to metabolize sucrose). C. reinhardtii The β-tubulin promoter is represented in boxed uppercase text. The start codon ATG and stop codon TAA of the invertase are represented in bold uppercase italics, while the coding region is represented in lowercase italics. Capable of amplifying... OambLPAAT2 The PmPGH 3′-UTR of *P. moriformis* is represented in uppercase underlined text, followed by the linker in lowercase bold italics, which contains the nitrate reductase 3′-UTR of *C. vulgaris*. The *P. moriformis* gene is represented in boxed italics. P. moriformis PmG3PDH bootloader OambLPAAT2 The expression. OambLPAAT2 The start codon ATG and stop codon TAG are represented in uppercase bold italics, while the rest of the gene is represented in bold italics. The PmPGH 3′-UTR is represented in lowercase underlined text, followed by the CHK22 Thia4 genomic region, represented in bold lowercase text.

[0640] Table 109 shows the fatty acid / TAG profiles of the seven primary transformants of CHK22 converted from pCHK1232. (Mulberry-shaped algae) P. moriformis The basic strain CHK22 is shown as a non-transgenic control. The strain was cultured in lipid production medium in 50 mL conical tubes at 28°C. C. Incubate at 200 rpm for 120 hours. Harvest biomass, freeze-dry to dryness, and perform direct transesterification to generate FAMEs for subsequent quantification and characterization by GC / FID. TAG analysis was performed using the LCMS method described above.

[0641] Table 109: Screening of CHK22 primary transformants converted by pCHK1232.

[0642]

[0643] The amino acid sequences of the various LPAATs disclosed in this paper were compared with the amino acid sequence of CrLPAAT2 to generate the percentage of sequence identity (%). The results are shown in Table 110. The phenotypes obtained by expressing these LPAATs in the non-transgenic cell line CHK22 are expressed as OP:OO ratio.

[0644] In some embodiments, the microalgal cells provided herein contain a foreign gene encoding LPAAT as shown in Table 110. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with LPAAT as shown in Table 110.

[0645] Table 110: Percentage similarity with CrLPAAT2 and OP:OO ratio

[0646] The amino acid sequences were compared with those of PedLPAAT2 to generate the percentage of sequence identity (%). The results are shown in Table 111. The phenotypes obtained by expressing these LPAATs in CHK22 are expressed as OP:OO ratios.

[0647] In some embodiments, the microalgal cells provided herein contain a foreign gene encoding LPAAT as shown in Table 111. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with LPAAT as shown in Table 111.

[0648] Table 111: Percentage similarity to PedLPAAT2 and OP:OO ratio

[0649] Table 112 shows the amino acid sequence of LPAAT described in this paper.

[0650] In some embodiments, the microalgal cells provided herein contain a foreign gene encoding LPAAT as listed in Table 112. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with LPAAT as listed in Table 112. In some embodiments, the foreign gene may contain 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 at least 100% sequence identity with any of SEQ ID NOs: 83-106, 155, and 156.

[0651] Table 112: LPAAT amino acid sequence

[0652] Table 113 shows the conserved LPAAT motifs and the percentage (%) of identity of the most active LPAAT in the entire LPAAT protein relative to PedLPAAT2. The transmembrane domains of these LPAATs are compared, and a schematic diagram of the LPAATs is shown below. Figure 11 In the diagram, 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 gray bar.

[0653] Table 113: Percentage Identities with PedLPAAT2 and LPAAT Motifs

[0654] Example 53: Composition of OPO oil

[0655] Table 114 provides eight representative high-yield samples from the strains and methods described in Example 32 above. sn-2 Palmitic mulberry algae ( Prototheca moriformis Average fatty acid content of algal oils.

[0656] Table 114: High sn-2 Palmitic mulberry algae ( Prototheca moriformis Average fatty acid composition of algal oil TAG

[0657] Oleic acid is primarily esterified to the external sites of TAG (Tag). sn 1 / 3 (Table 115), while the middle position ( sn-2 It is mainly composed of palmitic acid (Table 116). Approximately 70% of the total palmitic acid is in sn-2 Esterification at one site. TAG analysis of eight batches of OPO algal oil confirmed that oleate was esterified at one site. sn-1 and sn-3 Palmitate at position, palmitate sn-2 TAGs at position 1 (i.e., 1,3-dioleoyl-2-palmitoylglycerol, or OPO) account for more than 40% of the TAG types in the OPO algal oils disclosed herein.

[0658] Table 115: High sn-2 Palmitic mulberry algae ( Prototheca moriformis In algal oils sn-1 / 3 Fatty acid profile at position 1

[0659] Table 116: High sn-2 Palmitic mulberry algae ( Prototheca moriformis Fatty acid profile at the sn-2 position in algal oil

[0660] Example 54: Comparison of the composition of oil mixtures formulated with OPO oils

[0661] OPO algal oil can be used as a substitute for human milk fat, intended to at least partially replace vegetable oils in regular infant formula for full-term infants, at levels up to, for example, up to 60% of the total lipid composition of the formula. Examples of oil blends for infant formula made from OPO algal oil in combination with other vegetable oils are reported in Table 117.

[0662] Table 117: Examples of oil blends for infant formula made from a combination of OPO algal oil and conventional vegetable oils. All values ​​are expressed in grams of oil per 100 g of oil blend.

[0663]

[0664] The combination of algal oils and vegetable oils, as illustrated in Table 117, provides a fatty acid composition primarily rich in oleic acid, palmitic acid, linoleic acid, lauric acid, myristic acid, capric acid, and α-linolenic acid, as shown in Table 118.

[0665] Table 118: Fatty acid composition of oil mixtures for infant formula made from a combination of OPO algal oil and conventional vegetable oils.

[0666]

[0667] The total palmitic acid content was within the range of palmitic acid observed in human breast milk compared to the fatty acid composition of human breast milk (Table 119). The dataset shown in Table 119 was calculated from a series of original studies and represents a total of 835 mature human breast milk samples collected in the 10 countries described below: Yuhas et al., 2006: Australia (n = 48), Canada (n = 48), Chile (n = 50), Japan (n = 51), Mexico (n = 46), Philippines (n = 54), UK (n = 44) and the United States (n = 49) Thakkar et al., 2013: Singapore (n=50) Giuffrida et al., 2016: China (n=345) Table 119: Fatty acid composition of human milk samples (N=835) from multiple countries (Yuhas et al., 2006; Thakkar et al., 2013 and Giuffrida et al., 2016)

[0668] Example 55: Palmitic acid levels in microalgal oils

[0669] The combinations of algal oils and vegetable oils illustrated in Table 117 above provide the benefits shown in Table 120. sn-2 High levels of palmitic acid, esterified at one position. As reported in the literature, human milk contains... sn-2 Palmitic acid levels ranged from 67.8 ± 4.6% (Giuffrida et al., 2019). Algal OPO oils, used in combination with other vegetable oils, provide... sn-2 Palmitic acid levels were within the range observed in human milk (Table 116).

[0670] Table 120: Oil mixtures for infant formula made from a combination of OPO algal oil and conventional vegetable oils sn-2The level of palmitic acid

[0671] Table 120 shows (a) the parental *Mulberry-shaped Protozoa* ( Prototheca moriformis It should be noted that in the original text, there is a possible error in line where "ScSUCz" is likely a misspelling and should probably be "ScSUC2" as in other similar lines. This has been left as is in the translation to maintain consistency with the original. (a) Total TAG of microalgae strains, (b) high palmitic acid strains produced from parental strains through mutagenesis, and (c) OPO strains engineered by inserting LPAAT genes, (1) Total TAG of microalgae strains, (2) Total TAG of microalgae strains, (b) high palmitic acid strains produced from parental strains through mutagenesis, and (c) OPO strains engineered by inserting LPAAT genes. sn-1(3) Position and (3) sn-2 The fatty acid profiles of the 100g total fatty acids are illustrated in Table 121. Data are expressed as g / 100g total fatty acids.

[0672] Table 121: Comparison of fatty acid profiles of parental strain (CHK22), high palmitic acid strain (CHK100), and OPO-engineered strain (Example 32).

[0673] When expressed as g / 100g total palmitic acid, internal ( sn The level of palmitic acid esterified at position -2 was 73.3%.

[0674] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these preferred embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. The following claims are intended to define the scope of the invention, and the methods and structures within the scope of these claims, and their equivalents, are thereby covered.

Claims

1. An algal oil containing triglycerides (TAG), wherein: a. The total TAG content of the algal oil has a fatty acid profile comprising: about 25 wt.% to about 35 wt.% of C16:0 fatty acids; about 1.5 wt.% to about 3.0 wt.% of C18:0 fatty acids; about 48 wt.% to about 58 wt.% of C18:1 n-9 fatty acids; about 4.5 wt.% to about 7.5 wt.% of C18:2 n-6 fatty acids; and about 2 wt.% to about 6 wt.% of other fatty acids; b. TAG sn-1(3) The site has a fatty acid profile comprising: about 1.5 wt.% to about 4.5 wt.% of C16:0 fatty acids; about 1.5 wt.% to about 4.5 wt.% of C18:0 fatty acids; about 70 wt.% to about 90 wt.% of C18:1 n-9 fatty acids; about 5.5 wt.% to about 9 wt.% of 18:2 n-6 fatty acids; about 1.5 wt.% to about 4 wt.% of other fatty acids; and c.TAG sn-2 The site has a fatty acid profile comprising: about 65 wt.% to about 75 wt.% of C16:0 fatty acids; about 0.1 wt.% to 1 wt.% of C18:0 fatty acids; about 18 wt.% to 25 wt.% of C18:1 n-9 fatty acids; about 1 wt.% to about 5 wt.% of 18:2 n-6 fatty acids; and about 1 wt.% to about 5 wt.% of other fatty acids; The algal oils mentioned therein are derived from modified microalgal strains.

2. The algal oil according to claim 1, wherein the modified microalgae strain is a genetically modified microalgae strain.

3. The algal oil according to claim 1, wherein the modified microalgae strain is derived from a traditional strain modification.

4. The algal oil according to claim 1, wherein the modified microalgal strain is derived from both conventional strain improvement and genetic modification.

5. The algal oil according to any one of the preceding claims, wherein the algal oil is derived from the genus *Protocellus* (…). Prototheca ) produced by cells.

6. The algal oil according to any one of the preceding claims, wherein the algal oil is not naturally occurring.

7. A composition comprising algal oil as described in any one of claims 1-6 and one or more excipients.

8. The composition according to claim 7, wherein the composition is a nutritional supplement for humans or animals.

9. The composition according to claim 7, wherein the composition is infant formula.

10. The composition of claim 9, wherein the infant formula comprises one or more of whey, casein, lactose, vitamin D, human lactose oligosaccharides (HMOs), and antibodies.

11. The composition according to claim 9, wherein the infant formula further comprises plant protein.

12. A mixture of oils and fats, comprising: a. Approximately 50-60 g of algal oil per 100 g of oil mixture, wherein the algal oil contains TAG. Among them, TAG sn-2 At least about 60% of the fatty acids in the product are C16:0 fatty acids; b. Approximately 20-30 g of sunflower seed oil per 100 g of oil mixture; and c. Approximately 15-25g of canola oil per 100g of oil mixture.

13. The oil mixture according to claim 12, further comprising at least about 3-10 g of coconut oil per 100 g of oil mixture.

14. The oil mixture according to claim 12, wherein the algal oil is the algal oil according to any one of claims 1-6.

15. A composition comprising the oil mixture of any one of claims 12-14 and one or more excipients.

16. The composition of claim 15, wherein the composition is a nutritional supplement for human or animal use.

17. The composition according to claim 15, wherein the composition is infant formula.

18. The composition of claim 15, wherein the infant formula comprises one or more of whey, casein, lactose, vitamin D, human lactose oligosaccharides (HMOs), and antibodies.

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