Bio-generation of gangliosides in recombinant host cells

Abstract

In various embodiments, host cells, methods, and pharmaceutical compositions comprising complex sphingolipids such as monosialoganglioside 1 (GM1), monosialoganglioside 2 (GM2), and monosialoganglioside 3 (GM3) are provided herein. In some embodiments, the disclosure provides the bioproduction of sphingoglycolipids. In yet another embodiment, the disclosure provides the bioproduction of gangliosides.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 489,919, filed Mar. 13, 2023. The entire teachings of the above application are incorporated herein by reference.

[0002] Incorporation by Reference of Materials in XML This application incorporates by reference the Sequence Listing contained in the following Extensible Markup Language (XML) file, which is filed concurrently with this specification a) File name: 57671008001_Sequence_Listing.xml; created on Mar. 12, 2024, size: 100,172 bytes.

Background Art

[0003] Gangliosides are an important class of molecules composed of a lipid-based ceramide backbone and a sugar head group. Gangliosides play a variety of important roles in healthcare, but the production and purification of gangliosides remain challenging.

Summary of the Invention

[0004] Transgenic yeasts and methods for producing complex sphingolipids such as monosialoganglioside 1 (GM1), monosialoganglioside 2 (GM2), and monosialoganglioside 3 (GM3) are described herein. In some embodiments, the production of the complex sphingolipids occurs within the transgenic yeast.

[0005] In one aspect, the disclosure provides a transgenic yeast comprising a transgene encoding ceramide synthase and a transgene encoding sphingolipid delta-4 desaturase, wherein the yeast does not express the endogenous yeast genes SUR2, SCS7, LAG1, and LAC1. In another aspect, the disclosure provides a transgenic yeast cell comprising ceramide synthase having the sequence of SEQ ID NO: 4, SEQ ID NO: 46, or SEQ ID NO: 48. In another aspect, the disclosure provides a transgenic yeast comprising sphingolipid delta-4 desaturase having the sequence of SEQ ID NO: 2, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In another aspect, the disclosure provides a transgenic yeast comprising a transgene encoding UDP-glucose ceramide glucosyltransferase. In another aspect, the disclosure provides a transgenic yeast comprising UDP-glucose ceramide glucosyltransferase having the sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In another aspect, the Disclosure provides a transgenic yeast comprising beta-1,4-galactosyltransferase. In another aspect, the Disclosure provides that the transgenic yeast comprising beta-1,4-galactosyltransferase is HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), or HsB4GALT6 (SEQ ID NO: 16). In another aspect, the Disclosure provides a transgene yeast comprising a transgene encoding bacterial Neu5Ac synthase, a transgene encoding bacterial UDP-N-acetylglucosamine 2-epimerase, a transgene encoding mammalian CMP N-acetylneuraminate synthetase, and a transgene encoding beta-galactoside alpha-2,3-sialyltransferase. In another aspect, the Disclosure provides a transgenic yeast comprising Neu5Ac having the sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32. In another embodiment, the present disclosure provides a transgenic yeast comprising UDP-N-acetylglucosamine 2-epimerase having the sequence of SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO: 34.In another aspect, the Disclosure provides a transgenic yeast comprising CMP N-acetylneuraminate synthetase having the sequence of SEQ ID NO: 24 or SEQ ID NO: 30. In another aspect, the Disclosure provides a transgenic yeast comprising beta-galactoside alpha-2-3-sialytransferase having the sequence of SEQ ID NO: 18. In another aspect, the Disclosure provides a transgenic yeast comprising a transgene encoding N-acetylglucosamine epimerase enzyme and a transgene encoding beta-1,4N-acetylgalactosaminyltransferase. In another aspect, the Disclosure provides a transgenic yeast comprising a transgene encoding beta-1,3-galactosyltransferase.

[0006] In another aspect, the disclosure provides a method for producing sphingolipids in yeast cells. In some aspects, the method comprises yeast cells in a culture medium, the yeast cells expressing a transgene encoding GhLAG1-1 and a transgene encoding DEGS1, and the yeast cells do not express the endogenous yeast genes SUR2, SCS7, LAG1, and LAC1. In another aspect, the culture medium comprises stearic acid. In another aspect, the yeast cells produce C18 ceramide. In another aspect, the yeast cells include a transgene encoding UDP-glucose ceramide glucosyltransferase. In another aspect, the disclosure provides yeast cells including UDP-glucose ceramide glucosyltransferase having the sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In another aspect, the culture medium comprises glucose. In another aspect, the yeast cells produce glucosylceramide (GlcCer). In another aspect, the yeast cells include a transgene encoding beta-1,4-galactosyltransferase. In another aspect, the disclosure provides that yeast cells containing beta-1,4-galactosyltransferase are HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), or HsB4GALT6 (SEQ ID NO: 16). In another aspect, the culture medium contains UDP-galactose. In another aspect, the yeast cells produce lactosylceramide (LacCer). In another aspect, the yeast cells contain a transgene encoding bacterial Neu5Ac synthase, a transgene encoding bacterial UDP-N-acetylglucosamine 2-epimerase, a transgene encoding mammalian CMP N-acetylneuraminate synthetase, and a transgene encoding beta-galactoside alpha-2,3-sialyltransferase. In another aspect, the disclosure provides a transgenic yeast containing Neu5Ac having the sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32. In another embodiment, the present disclosure provides a transgenic yeast comprising UDP-N-acetylglucosamine 2-epimerase having the sequence of SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO: 34.In another aspect, the disclosure provides a transgenic yeast comprising CMP N-acetylneuraminate synthetase having the sequence of SEQ ID NO: 24 or SEQ ID NO: 30. In another aspect, the disclosure provides a transgenic yeast comprising beta-galactoside alpha-2-3-sialitransferase having the sequence of SEQ ID NO: 18. In another aspect, the culture medium comprises CMP-N-acetylneuraminate (CMP-Neu5NAc). In another aspect, the yeast cells produce monosialoganglioside 3 (GM3). In another aspect, the yeast cells comprise a transgene encoding N-acetylglucosamine epimerase enzyme and a transgene encoding beta-1,4N-acetylgalactosaminyltransferase. In another aspect, the culture medium comprises UDP-N-acetylgalactosamine. In another aspect, the yeast cells produce monosialoganglioside 2 (GM2). In another aspect, the yeast cells comprise a transgene encoding beta-1,3-galactosyltransferase. In another embodiment, the culture medium contains galactose. In another embodiment, yeast cells produce monosialoganglioside 1 (GM1).

[0007] The foregoing will become clear from the following more detailed description of exemplary embodiments. Similar reference numerals refer to the same parts in different drawings, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, but rather are intended to illustrate embodiments. [Brief explanation of the drawing]

[0008] [Figure 1] Structure of ganglioside GM1. GM1 consists of a ceramide (C18-ceramide) lipid backbone and pentasaccharide head groups. These sugars are sequentially added to the ceramide backbone. Different glycosylation patterns on the ceramide backbone result in different types of gangliosides. [Figure 2]Flowchart of ganglioside formation in a heterologous host. To produce gangliosides, C18-ceramide biosynthesis is achieved first. Next, a series of five sugar transferases and two sugar biosynthetic pathways must be introduced to enable ganglioside biosynthesis up to the major ganglioside GM1. Cer: Ceramide (Structure 1). GlcCer: Glucosylceramide (Structure 2). LacCer: Lactosylceramide (Structure 3). GM3: Monosialoganglioside 3 (Structure 4). GM2: Monosialoganglioside 2 (Structure 5). GM1: Monosialoganglioside 1 (Structure 6). UDP-Glc: UDP-glucose. UDP-Gal: UDP-galactose. CMP-Neu5Ac: N-acetylneuraminic acid (CMP-sialic acid). UDP-GalNAc: UDP-N-acetylgalactosamine. [Figure 3] An overview of sphingolipids in yeast. Based on current knowledge of yeast ceramide biosynthesis, specific methods for inhibiting the metabolism of S. cerevisiae to produce and accumulate C18 ceramide can be understood. [Figure 4] Modifications necessary to transform yeast ceramide biosynthesis into a process capable of producing C18 ceramide. Based on previous research, a method has been realized to produce C18-ceramide, the primary lipid backbone of all gangliosides, within yeast. To enable the production and accumulation of C18-ceramide, it is necessary to remove four native yeast genes and express two genes. [Figure 5] LC-MS / MS chromatogram of human ceramide in yeast. After 5 days of fermentation, lipids from the S. cerevisiae strain were extracted in methanol and analyzed. Extracts from the manipulated strain yielded a peak at 23.4 min, which is absent in the wild-type strain. A standard of C18-ceramide was used for identification. This figure shows a chromatogram filtered specifically for the MS-MS transition of C18-ceramide. [Figure 6]LC-MS / MS chromatogram showing glucosylceramide production after heterologous expression of UDP-glucoseceramide glucosyltransferase. After 5 days of fermentation, lipids from the S. cerevisiae strain were extracted in methanol and analyzed. Two candidate enzymes (UGCG, SEQ ID NO: 6, and GCS1, SEQ ID NO: 8) yielded the expected peak at 5.6 minutes, which is consistent with the retention time observed for the glucosylceramide standard. This figure shows a chromatogram filtered specifically for the MS-MS transfer of glucosylceramide. [Figure 7] LC-MS / MS chromatogram showing lactosylceramide production after heterologous expression of glucosylceramide beta-1,4-galactosyltransferase. After 5 days of fermentation, lipids from the S. cerevisiae strain were extracted in methanol and analyzed. Four candidate enzymes, HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), and HsB4GALT6 (SEQ ID NO: 16), yielded the expected peak at 5.4 min. Lactosylceramide standards were used to identify the strains tested. This figure shows a chromatogram filtered specifically for the MS-MS transition of lactosylceramide. [Figure 8] This is a schematic diagram of the manipulated GM3 biosynthesis pathway in yeast. Heterogenetics were supplied from eukaryotes, with the exception of NeuC and NeuB, which have bacterial origins. Arrows with stop symbols indicate knockout. [Figure 9]LC-MS / MS chromatograms showing GM3 production after heterologous expression of the hybrid CMP-Sia pathway and lactosylceramide alpha-2-3-sialyltransferase. After 7 days of fermentation, lipids from S. cerevisiae strains containing two episomal plasmids with the GM3 biosynthesis pathway were extracted in methanol and analyzed. Six different gene combinations yielded expected peaks at 4.5–4.6, which were absent in the lactosylceramide-producing strain used as background. GM3 was added to wild-type S. cerevisiae extracts and used to identify the tested strains. This figure shows chromatograms filtered specifically for MS-MS transitions of GM3. [Modes for carrying out the invention]

[0009] The following describes an exemplary embodiment.

[0010] structure Structure 1: Ceramide-18 (C18 ceramide NS (N-acyl sphingosine), human ceramide) is a compound having the following structure. [ka]

[0011] Structure 2: Glucosylceramide is a compound having the following structure. [ka]

[0012] Structure 3: Lactosylceramide is a compound having the following structure. [ka]

[0013] Structure 4: GM3 is a compound having the following structure. [ka]

[0014] Structure 5: GM2 is a compound having the following structure.

Chem.

[0015] Structure 6: GM1 is a compound having the following structure.

Chem.

[0016] Sequence Table 1 is a list of sequences used in the experiments described in this specification.

Table 1-1

Table 1-2

Table 1-3

[0017] Homologs of GhLag1-1: Human CerS1, and Pichia pastoris bar1

[0018] Homologs of DEGS1: A.thaliana Δ4-desaturase At4g04930, C albicans Des1p, D.melanogaster DES, S.pombe dsd1, Pichia pastoris DES1

[0019] As used herein, the term “sequence identity” refers to the percentage of the degree to which two sequences have the same residues at the same positions when aligned to achieve the highest level of identity. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, and a test sequence is compared to it. Sequence identity between a reference sequence and a test sequence is expressed as the percentage of positions across the entire length of the reference sequence where the reference sequence and the test sequence share the same nucleotide or amino acid when aligned to achieve the highest level of identity. For example, if, when aligned to achieve the highest level of identity, the test sequence has the same nucleotide residues at 70% of the same positions across the entire length of the reference sequence, the two sequences are considered to have 70% sequence identity.

[0020] Aligning of comparison sequences to achieve the maximum level of identity can be easily carried out by those skilled in the art using appropriate alignment methods or algorithms. In some examples, the alignment may include the introduction of gaps to provide the maximum level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity methods of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology). In some embodiments, codon-optimized sequences for efficient expression in different cells, tissues, and / or organisms reflect patterns of codon use in such cells, tissues, and / or organisms, including conserved (or non-conserved) amino acid substitutions that do not adversely affect normal activity.

[0021] In some embodiments, the disclosure provides a ceramide synthase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 46, or SEQ ID NO: 48. In some embodiments, the ceramide synthase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 45, or SEQ ID NO: 47.

[0022] In some embodiments, the Disclosure provides a sphingolipid delta-4 desaturase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44. In some embodiments, the sphingolipid delta-4 desaturase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0023] In some embodiments, the disclosure provides a UDP-glucose ceramide glucosyltransferase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments, the UDP-glucose ceramide glucosyltransferase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

[0024] In some embodiments, the Disclosure provides a beta-1,4-galactosyltransferase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16. In some embodiments, the beta-1,4-galactosyltransferase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15.

[0025] In some embodiments, the Disclosure provides a Neu5AC synthase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32. In some embodiments, the Neu5AC synthase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 25, or SEQ ID NO: 31.

[0026] In some embodiments, the Disclosure provides UDP-N-acetylglucosamine 2-epimerase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO: 34. In some embodiments, UDP-N-acetylglucosamine 2-epimerase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 21, SEQ ID NO: 27, or SEQ ID NO: 33.

[0027] In some embodiments, the Disclosure provides CMP N-acetylneuraminate synthase having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 30. In some embodiments, the CMP N-acetylneuraminate synthase is encoded by a nucleotide sequence having at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity with the nucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 29.

[0028] Overview Gangliosides are an important class of molecules found throughout the human body, particularly in the brain. Gangliosides consist of a lipid-based ceramide backbone (also known as human ceramide or ceramide NS) and a glycosphagnum group (Sipione et al. 2020). These molecules play a wide variety of important roles in human health. GM1 (monosialoganglioside 1) is one of the most abundant gangliosides in brain tissue, and GM1 depletion is associated with neurological disorders such as Huntington's disease and Parkinson's disease (Magistretti et al. 2019) (Figure 1, Structure 6). GM1 administration has been shown to alleviate the symptoms and progression of these diseases (Shneider et al. 2010, Alpaugh et al. 2017). Other gangliosides, such as GM3, GD3, and GD2, are highly abundant on the surface of cancer cell membranes (Dobrenkov et al. 2016) and are essential components of emerging immunotherapy-based cancer treatments (Grabowska et al. 2021, Zang et al. 2022) (Structure 4).

[0029] Gangliosides have important applications across many aspects of healthcare, but their production and purification remain challenges. Currently, GM1 is used as a therapeutic agent in several countries, but this GM1 is sourced from animal brain tissue. Furthermore, gangliosides, which are not very abundant, are difficult to accumulate and purify. Therefore, there is a need for a solution that can provide gangliosides on a scale suitable for commercialization and use them as pharmaceuticals.

[0030] To address the challenges surrounding the production of GM1 and other gangliosides, this disclosure provides a synthetic biological platform for generating gangliosides in a heterologous yeast host (Figure 2). Genes necessary to enable host cells to denominate gangliosides from simple carbon sources, as well as key host genes that must be removed to allow for high levels of biosynthesis, are identified. Currently, these manipulation efforts have yielded strains capable of producing the ganglioside GM3.

[0031] This synthetic biological strategy for generating gangliosides not only enables the production of the proven therapeutic GM1, but also allows for the production of ganglioside intermediates and substitutes. These include GM2, GM3, lactosylceramide, and glucosylceramide. These gangliosides have never been produced in heterologous organisms before.

[0032] Under specified fermentation conditions, the designed strain accumulates gangliosides for final purification and can be used in a variety of forms, including direct administration as a therapy, as well as integration into emerging drug delivery methods to induce specific immune responses.

[0033] A yeast strain capable of producing glucosylceramide, the first glycosylated molecule in the pathway toward GM1 biosynthesis, is described herein. Furthermore, the subsequent addition of UGT enzyme to this strain to enable the formation of the pentasaccharide head group of GM1 is described herein.

[0034] General method Culture medium and formulations Yeast peptone dextrose (YPD) broth consisted of 10 g / L of yeast extract, 20 g / L of peptone, and 20 g / L of dextrose. 20 g / L of agar was added to the YPD plate.

[0035] Selective media were prepared using 6.7 g / L of yeast nitrogen base without amino acids, 1.92 g / L of synthetic complete medium without uracil, tryptophan, or histidine, or 1.6 g / L of leucine if selection by leucine was required. 20 g / L of agar was added to the plates. After sterilization, dextrose was added to a final concentration of 20 g / L.

[0036] Nitrogen-restricted synthetic medium (NLS) consisted of 1.7 g / L yeast nitrogen base without amino acids and ammonium sulfate, 1 g / L ammonium sulfate, an amino acid dropout mixture excluding 1.92 g / L uracil, and 76 mg / L uracil. After autoclaving, 50 ml of 40% dextrose or galactose was added to obtain dextrose-containing nitrogen-restricted synthetic medium (NLSD) or galactose-containing nitrogen-restricted synthetic medium (NLSGal). The pH of the medium was then adjusted to 7.0. A 95% ethanol solution of 25 mg / ml stearic acid (SA) was prepared, and 20 ml was slowly added to the medium while vigorously agitating.

[0037] Genome deletion procedure Deletion of the native S. cerevisiae gene was performed according to Akada et al. 2006. Briefly, a 1kb fragment directly upstream of the desired gene to be knocked out was used to direct the URA3 expression cassette to the knockout locus. Gene removal was indicated by growth of the strain in uracil-deficient medium. Subsequently, the URA3 cassette was removed by growing the strain on a medium containing 5-fluoroorotic acid (5-FOA), which allowed for removal of the URA3 cassette by homologous recombination facilitated by repeats designed in the genome insert. Knockout was confirmed by genomic DNA extraction and PCR. For CRISPR-Cas9-based deletions, the method was modified from Lee et al. 2015. Briefly, the guide RNA was designed to have a 65bp facile region homologous to the genome directly upstream and downstream of the gene to be removed. A 20bp barcode was added for clonal confirmation. Recipient strains were transformed with a plasmid containing guide RNA and the Cas9 mechanism, and streaked out on selective medium. Knockout was confirmed by PCR. Re-streaking of positive clones was performed to remove the Cas9 plasmid, successfully knocking out the target gene.

[0038] Cloning procedure Integrated plasmids and episomal plasmids were constructed using golden gate assembly as previously described (Lee et al. 2015). For the embedded plasmids, intermediate plasmids containing expression cassettes of test genes at different promoter intensities, as well as plasmids containing expression cassettes adjacent to homologous arms and malnutrition markers (e.g., URA3, HIS3, LEU2), were constructed.

[0039] To construct episomal plasmids with pathway modules, three types of plasmids were constructed: a plasmid containing an expression cassette of a test gene under the control of a relatively strong promoter; a plasmid backbone containing elements necessary for yeast growth, namely antibiotic resistance and a 2-micron size; and a plasmid containing multiple expression cassettes for episomal expression.

[0040] In short, 5 μL of Golden Gate reaction solution was transformed into 20–50 μL of E. coli DH-alpha, and after harvesting, the cells were seeded onto appropriate selections. The plates were incubated overnight at 37C, and typically two clones were screened for proper assembly using restriction enzymes. Clones exhibiting the expected pattern were further confirmed by DNA sequencing.

[0041] Transformation procedure Cells were transformed using chemical transformation according to Schietz & Gietz (1989) or using electroporation as described by Shao & Zhao (2014). In both methods, seed cultures grown overnight were inoculated into 50 mL of YPD medium in a 250 mL baffled flask. Cells were grown until at least two replications had occurred (i.e., 4–5 hours). For chemical transformation, cells were centrifuged at 2500 rpm for 5 minutes. Cells were washed once with water, followed by washing with 100 mM lithium acetate. Cells were then concentrated approximately 100-fold in 100 mM lithium acetate. A transformation mixture consisting of 50 mL of cell suspension, 240 mL of 50% PEG, 36 mL of 1 M lithium acetate, and 50 mL of salmon sperm (2 mg / ml) was combined with 50 mL of digested reaction product to produce linear DNA fragments containing expression cassettes and selection markers. The cells were subjected to heat shock by incubation at 30C for 30 minutes, followed by incubation at 42C for 30 minutes. The transformation mixture was centrifuged, and the pellet was gently washed with 1 mL of sterile water. Finally, the cells were resuspended in 150 mL of sterile water and seeded onto appropriate selective plates.

[0042] Cells to be electroporated were collected and treated at 4C. Two washes with water were performed at 4000 rpm for 10 minutes, followed by a sorbitol wash to help concentrate the cells approximately 150-fold. The cells were resuspended in sorbitol and combined with 50 mL aliquots of less than 4 mL of DNA transformation mixture, i.e., pairs of episomal plasmids. The cells were electroporated at 1.5 kV using a 2 mm gap cuvette. Immediately after electroporation, 1 mL of YPD was added, and the cells were collected at 30C for 1 hour, then washed twice with sorbitol and seeded on appropriate selective plates. Validation was performed by PCR of the incorporated strain or by seeding on selective medium to confirm plasmid retention.

[0043] Analysis of sphingolipids (ceramide, glucosylceramide, lactosylceramide) by LC-MS Generally, 1-2 colonies were inoculated into liquid medium and cultured for 5-7 days, and the generation of the target molecule was evaluated using a modified version of a previously described protocol (Bielawski et al. 2009).

[0044] To detect lactosylceramide, each strain was inoculated into culture tubes containing 5 ml of NLSD+0.05%SA and NLSGal+0.05%SA. Liquid culture was performed at 30C and 250 rpm for 5 days. Samples were collected for analysis after 3 and 5 days of fermentation.

[0045] To detect GM3, each strain was inoculated into a 50 ml vented tube containing 10 ml of either YPD, YPGal, or NLSGal + 0.05% SA medium. Liquid culture was performed at 30C and 250 rpm for 7 days. Samples were collected for analysis after 2.5, 4.5, and 7.5 days of fermentation.

[0046] Lipid extraction was performed according to Bielawski et al. 2009. 3 The procedure was carried out as previously described. Briefly, 1 mL of cell culture was centrifuged at maximum speed for 5 minutes. The cell pellet was then resuspended in a yeast lipid extract mixture. The sample was then sonicated for 3 minutes and vortexed briefly. The sample was centrifuged at maximum speed for 5 minutes and the extract (approximately 1 mL) was collected. The process was repeated by adding 500 mL of yeast lipid extract mixture to the remaining pellet, except that the sonication time was reduced to 1 minute. After centrifugation, both extracts were combined and placed in a centrifuge concentrator overnight at medium temperature. For analysis, the lipid extract was reconstituted in 0.15–1 mL of mobile phase B.

[0047] Starting with previously described LC-MS parameters (Bielawski et al. 2009), MS / MS fragmentation was used to verify the identity of each ganglioside, and each putative product was compared to a true standard.

[0048] Results and Discussion Operation for C18 ceramide productionTo access gangliosides, we manipulated the production of human ceramide (C18 ceramide) (structure 1). Yeast already produces various ceramides, and its pathway has been previously characterized (Figure 3). By comparing major yeast ceramides with ceramides that are part of GM1, the key modifications that needed to be made were identified (Figure 4). First, the genes sur2 and scs7, which attach hydroxyl groups to yeast ceramide, had to be removed, which Murakami et al. have shown to be a non-toxic modification resulting in hydroxyl-free ceramide (Murakami et al. 2015). Next, desaturation needed to be introduced into the sphingosine portion of the ceramide, which has also been shown by Murakami and collaborators by adding human sphingolipid delta-4 desaturase (DEGS1, SEQ ID NO: 2) with an endoplasmic reticulum target sequence added to the C-terminus. See U.S. Patent No. 8,367,375 (Kodama et al.). However, Kodama et al. did not disclose any modification of the lipid length on ceramide. Finally, the lipid that binds to sphingosine must be reduced from C26 (typical yeast lipid length) to C18. It has been previously shown that the introduction of the plant ceramide synthase GhLag1-1 (SEQ ID NO: 4) and the knockout of the yeast native ceramide synthases Lag1 and LAC1 enabled the production of C18-chain-length ceramide (Epstein et al. 2012). With this information, we first constructed a strain using homologous recombination by knocking out sur2 and scs7, two hydroxylase enzymes that attach hydroxyl groups to yeast ceramide. Next, we incorporated DEGS1 (SEQ ID NO: 2) and GhLAG1-1 (SEQ ID NO: 4) into the locus. Finally, we removed Lag1 and LAC1 using CRISPR-Cas9 technology to obtain a strain capable of producing C18 ceramide. When this strain was cultured in a medium containing stearic acid, the production of C18 ceramide was observed via LC-MS. This was verified by comparing it with a standard (Figure 5).While ceramides with longer chain lengths have been previously produced in yeast (Murakami et al. 2015), this represents the first demonstration of heterogeneity of C18 ceramide, which functions as the ceramide skeleton of gangliosides and is identical to ceramides found in mammals.

[0049] Natural ceramides play many important roles in normal yeast cellular processes, including membrane stability and the formation of more complex (e.g., modified) lipids, most notably inositol phosphorylceramide, which plays an essential role in cell growth and division processes. By knocking out four endogenous genes (SUR2, SCS7, LAG1, and LAC1) and expressing two exogenous genes (GhLAG1-1 and DEGS1), yeast cells no longer produce natural ceramides. Compared to natural ceramides, the resulting sphingolipids have double bonds, which introduce rigidity to the molecular chains that can disrupt sphingolipid membrane formation. In addition, two hydroxyl groups (one on each molecular chain) are no longer added. These hydroxyl groups are known to be important for maintaining membrane fluidity and proper circulation of membrane components in yeast cells. Previously, it has been shown that the introduction of double bonds and the removal of hydroxyl groups do not cause cell death, but significantly increase sensitivity to inhibitors of the enzyme that produces inositol phosphorylceramide. Removal of both hydroxyl groups by knockout of sur2 and scs7 was observed to reduce the growth rate and indicate some form of growth defect or deficiency caused by the alternative ceramide (Murakami et al., 2015). Furthermore, in our strain, the length of the ceramide fat-acyl chain was shortened from 26 carbons to 18 carbons, which would affect membrane fluidity and membrane structure. This has been independently shown to be non-toxic to yeast. However, it was noted that ceramide production was significantly enhanced when the fat-acyl chain was shortened from 26 carbons to 18 carbons (Epstein et al., 2012). Therefore, we predicted that introducing a structural change to ceramide and shortening the length of the acyl chain would lead to further growth of the non-natural ceramide species causing the growth defect reported by Murakami et al. However, no other growth defects were observed because our manipulated strain accumulated more non-natural ceramide species.Therefore, the ceramide modification combinations described herein enable the production of C18 ceramide and provide the first evidence that modification of the ceramide sphingosine base and the lipid-acyl chain of endogenous yeast ceramide does not cause cell death and does not have any additional adverse effects on cell viability.

[0050] Operation for glucosylceramide production In a strain producing C18 ceramide, the next step in ganglioside biosynthesis involves transferring glucose to ceramide to form glucosylceramide (structure 2). This requires the action of UDP-glucose ceramide glucosyltransferase (UGCG). Two different enzymes (UGCG, SEQ ID NO: 6; GCS1, SEQ ID NO: 8) were screened for activity by cloning them into entry vectors and incorporated into the genome of the C18 ceramide-producing strain under the strong constitutive promoter pTDH3. During culture of the newly constructed strain, the ceramide profile was analyzed by LC-MS, and the presence of glucosylceramide was detected. This activity was validated by comparison with a true glucosylceramide standard (Figure 6). This is the first explanation for the heterologous production of glucosylceramide in the same single-cell host as glucosylceramide found in mammalian gangliosides.

[0051] Procedure for lactosylceramide production The next glycosyltransferase reaction for ganglioside formation is the addition of galactose units to glucosylceramide by beta-1,4-galactosyltransferase (B4GalT) to form lactosylceramide (structure 3). The B4GalT enzyme was cloned into an entry vector and incorporated into the genome of a glucosylceramide-producing strain under the strong constitutive promoter pTDH3. To test its activity, the resulting strain was grown in a medium supplemented with stearic acid and galactose. After sample extraction and measurement on LC-MS, these four enzymes (HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), and HsB4GAL6 (SEQ ID NO: 16)) were found to be able to convert glucosylceramide to lactosylceramide (Figure 7). This is the first explanation for the heterologous production of lactosylceramide in the same single-celled host as lactosylceramide found in mammalian gangliosides.

[0052] GM3 generation operation To achieve GM3 biosynthesis, two different pathways were manipulated. GM3 contains a sugar moiety, Neu5Ac (also known as sialic acid or N-acetylneuraminate), which is not a naturally occurring sugar in the Saccharomyces cerevisiae host (Structure 4). Therefore, the biosynthesis of the sugar donor CMP-Neu5Ac was first manipulated in the yeast host. US2008 / 0085540A1 (Hamilton) discloses the manipulation of CMP-Neu5Ac biosynthesis into the yeast host Pichia pastoris to enable protein sialylation, and a similar approach is used herein. To achieve this biosynthesis, combinations of bacterial and mammalian CMP-Neu5Ac biosynthesis genes were heterologously expressed on plasmids within yeast strains. These include bacterial Neu5Ac synthase (NeuB), bacterial UDP-N-acetylglucosamine 2-epimerase (NeuC), and mammalian CMP N-acetylneuraminate synthetase (CMAS). Simultaneously, beta-galactoside alpha-2,3-sialyltransferase, which transfers the Neu5Ac sugar from CMP-Neu5Ac onto lactosylceramide to produce GM3, was expressed on a separate plasmid. Strains producing lactosylceramide were transformed with both the CMP-Neu5Ac biosynthesis pathway and beta-galactoside alpha-2,3-sialyltransferase, then cultured and GM3 production was evaluated (Figure 8). After growth and sample preparation, six gene combinations were found to result in GM3 production. For each combination, Neu5Ac biosynthesis was enabled by using bacterial NeuB and NeuC supplied from N. meningitis (T1 and T4 in Figure 9), S. agalactiae (T2 and T5 in Figure 9), or E. coli (T3 and T6 in Figure 9). Activation of Neu5Ac to CMP-Neu5Ac was facilitated by mammalian CMAS supplied from either mouse or human (T1-T3 and T4-T6 in Figure 9, respectively), and human beta-galactoside alpha-2,3-sialyltransferase was used for GM3 biosynthesis in all combinations. GM3 production was confirmed by comparison with genuine purchased specifications (Figure 9).This is the first explanation for the heterogeneity of GM3 in a single-celled host.

[0053] Table 2 lists the six combinations of genes used. [Table 2]

[0054] GM2 biosynthesis manipulation (hypothetical example) To achieve GM2 biosynthesis, two genes need to be manipulated in the GM3-producing strain (structure 5). GM2 biosynthesis requires UDP-N-acetylgalactosamine (UDP-GalNAc), a sugar precursor not naturally present in S. cerevisiae. Therefore, UDP-galactose / UDP-N-acetylglucosamine epimerase (GALE) enzymes such as BsGALE (SEQ ID NO: 50), MmGALE (SEQ ID NO: 52), and HsGALE (SEQ ID NO: 54) are introduced into the host. Secondly, beta-1,4N-acetylgalactosaminyltransferases such as HsB4GalNT1 (SEQ ID NO: 56) or CjCgtA (SEQ ID NO: 62) are subsequently manipulated to promote the transfer of GalNAc to GM3. Combining these two genes enables the biosynthesis of GM2, the second-to-last precursor of GM1.

[0055] GM1 biosynthesis manipulation (hypothetical example) To manipulate strains capable of producing the therapeutic molecule GM1, a final gene addition must be performed (Figure 1, Structure 6). Beta-1,3-galactosyltransferases such as HsB3GalT4 (SEQ ID NO: 60) or CjGgtB (SEQ ID NO: 62) are manipulated into strains that produce GM2, and GM2 is converted to GM1 by utilizing UDP-galactose. This result represents the first description of GM1 biosynthesis in heterologous hosts for pharmaceutical applications aimed at treating neurological disorders and conditions.

[0056] Enhancement of UDP-galactose and manipulation of UDP-N-acetylgalactosamine biosynthesis (hypothetical example) UDP-galactose and UDP-N-acetylgalactosamine must be present in host cells to provide the sugar donor molecules essential for ganglioside biosynthesis (Figure 2). UDP-galactose is produced in yeast but does not accumulate significantly. However, UDP-N-acetylgalactosamine is not produced at all in S. cerevisiae. Therefore, three dual-function UDP-glucose / UDP-N-acetylglucosamine epimerase (GALE) enzymes, BsGALE (SEQ ID NO: 50), MmGALE (SEQ ID NO: 52), and HsGALE (SEQ ID NO: 54), have been identified that can convert UDP-glucose and UDP-N-acetylglucosamine to UDP-galactose and UDP-N-acetylgalactosamine, respectively. This represents an essential gene addition when pursuing the biosynthesis of gangliosides GM2 and GM1.

[0057] References Zang,H.;Siddiqui,M.;Gummuluru,S.;Wong,WW;Reinhard,BMGanglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse.ACS Nano 2022,16(11),18408-18420.doi.org / 10.1021 / acsnano.2c06516. Galleguillos,D.;Wang,Q.;Steinberg,N.;Zaidi,A.;Shrivastava,G.;Dhami,K.;Daskhan,G.C.;Schmidt,E.N.;Dworsky-Fried,Z.;Giuliani,F.;Churchward,M.;Power,C.;Todd,K.;Taylor,A.;Macauley,M.S.;Sipione,S.Anti-Inflammatory Role of GM1 and Other Gangliosides on Microglia.Journal of Neuroinflammation 2022,19(1),9.doi.org / 10.1186 / s12974-021-02374-x. Grabowska,J.;Stolk,D.A.;Nijen Twilhaar,M.K.;Ambrosini,M.;Storm,G.;van der Vliet,H.J.;de Gruijl,T.D.;van Kooyk,Y.;den Haan,J.M.M.Liposomal Nanovaccine Containing α-Galactosylceramide and Ganglioside GM3 Stimulates Robust CD8+T Cell Responses via CD169+Macrophages and CDC1.Vaccines 2021,9(1),56.doi.org / 10.3390 / vaccines9010056. Sipione,S.;Monyror,J.;Galleguillos,D.;Steinberg,N.;Kadam,V.Gangliosides in the Brain:Physiology,Pathophysiology and Therapeutic Applications.Front Neurosci 2020,14,572965.doi.org / 10.3389 / fnins.2020.572965. Patry,R.T.;Stahl,M.;Perez-Munoz,M.E.;Nothaft,H.;Wenzel,C.Q.;Sacher,J.C.;Coros,C.;Walter,J.;Vallance,B.A.;Szymanski,C.M.Bacterial AB5 Toxins Inhibit the Growth of Gut Bacteria by Targeting Ganglioside-like Glycoconjugates.Nat Commun 2019,10(1),1390.doi.org / 10.1038 / s41467-019-09362-z. Magistretti,P.J.;Geisler,F.H.;Schneider,J.S.;Li,P.A.;Fiumelli,H.;Sipione,S.Gangliosides:Treatment Avenues in Neurodegenerative Disease.Frontiers in Neurology 2019,10. Alpaugh,M.;Galleguillos,D.;Forero,J.;Morales,L.C.;Lackey,S.W.;Kar,P.;Di Pardo,A.;Holt,A.;Kerr,B.J.;Todd,K.G.;Baker,G.B.;Fouad,K.;Sipione,S.Disease-Modifying Effects of Ganglioside GM1 in Huntington’s Disease Models.EMBO Molecular Medicine 2017,9(11),1537-1557.doi.org / 10.15252 / emmm.201707763. Dobrenkov,K.;Ostrovnaya,I.;Gu,J.;Cheung,I.Y.;Cheung,N.-K.V.Oncotargets GD2 and GD3 Are Highly Expressed in Sarcomas of Children,Adolescents,and Young Adults.Pediatric Blood & Cancer 2016,63(10),1780-1785.doi.org / 10.1002 / pbc.26097. Murakami,S.;Shimamoto,T.;Nagano,H.;Tsuruno,M.;Okuhara,H.;Hatanaka,H.;Tojo,H.;Kodama,Y.;Funato,K.Producing Human Ceramide-NS by Metabolic Engineering Using Yeast Saccharomyces Cerevisiae.Sci Rep 2015,5(1),16319.doi.org / 10.1038 / srep16319. Lee,M.E.;DeLoache,W.C.;Cervantes,B.;Dueber,J.E.A Highly Characterized Yeast Toolkit for Modular,Multipart Assembly.ACS Synth.Biol.2015,4(9),975-986.doi.org / 10.1021 / sb500366v. Shao,Z.;Zhao,H.Manipulating Natural Product Biosynthetic Pathways via DNA Assembler.Current Protocols in Chemical Biology 2014,6(2),65-100.doi.org / 10.1002 / 9780470559277.ch130191. Kodama,Y.;Okuhara,H.;Funato,K.Methods for Producing Human Ceramide Using Yeast Transformants.US8367375B2,February 5,2013.patents.google.com / patent / US8367375 / en Epstein,S.;Castillon,G.A.;Qin,Y.;Riezman,H.An Essential Function of Sphingolipids in Yeast Cell Division.Molecular Microbiology 2012,84(6),1018-1032.doi.org / 10.1111 / j.1365-2958.2012.08087.x. Schneider,J.S.;Sendek,S.;Daskalakis,C.;Cambi,F.GM1 Ganglioside in Parkinson’s Disease:Results of a Five Year Open Study.Journal of the Neurological Sciences 2010,292(1),45-51.doi.org / 10.1016 / j.jns.2010.02.009. Bielawski,J.;Pierce,J.S.;Snider,J.;Rembiesa,B.;Szulc,Z.M.;Bielawska,A.Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography-Tandem Mass Spectrometry.In Lipidomics;Armstrong,D.,Ed.;Methods in Molecular Biology;Humana Press:Totowa,NJ,2009;Vol.579,pp 443-467.doi.org / 10.1007 / 978-1-60761-322-0_22. Hamilton,S.Method of Engineering a Cytidine Monophosphate-Sialic Acid Synthetic Pathway in Fungi and Yeast.US20080085540A1,April 10,2008.patents.google.com / patent / US20080085540A1 / en Akada, R.; Kitagawa, T.; Kaneko, S.; Toyonaga, D.; Ito, S.; Kakihara, Y.; Hoshida, H.; Morimura, S.; Kondo, A.; Kida, K. PCR-Mediated Seamless Gene Deletion and Marker Recycling In Saccharomyces Cerevisiae.Yeast 2006,23(5),399-405.doi.org / 10.1002 / yea.1365. Schiestl,RH;Gietz,RDHigh Efficiency Transformation of Intact Yeast Cells Using Single Stranded Nucleic Acids as a Carrier.Curr Genet 1989,16(5-6),339-346.doi.org / 10.1007 / BF00340712.

[0058] Incorporation by reference, equivalent All patents, published applications, and references cited herein are incorporated in their entirety by reference.

[0059] While exemplary embodiments have been specifically shown and described, it will be understood by those skilled in the art that various modifications can be made to the form and details within the scope of embodiments covered by the appended claims without departing from that scope.

Claims

1. A transgenic yeast cell comprising a transgene encoding ceramide synthase and a transgene encoding sphingolipid delta-4 desaturase, wherein the yeast cell does not express the endogenous yeast genes SUR2, SCS7, LAG1, and LAC1.

2. The transgenic yeast cell according to claim 1, wherein the ceramide synthase is Sequence ID No. 4, Sequence ID No. 46, or Sequence ID No.

48.

3. The transgenic yeast cell according to claim 1 or 2, wherein the sphingolipid delta-4 desaturase is SEQ ID NO: 2, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO:

44.

4. A transgenic yeast cell according to any one of claims 1 to 3, further comprising a transgene encoding UDP-glucose ceramide glucosyltransferase.

5. The transgenic yeast cell according to claim 4, wherein the UDP-glucose ceramide glucosyltransferase is SEQ ID NO: 6 or SEQ ID NO:

8.

6. The transgenic yeast cell according to claim 4, further comprising a transgene encoding beta-1,4-galactosyltransferase.

7. The transgenic yeast cell according to claim 6, wherein the beta-1,4-galactosyltransferase is HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), or HsB4GALT6 (SEQ ID NO: 16).

8. The transgenic yeast cell according to claim 6, further comprising a transgene encoding Neu5Ac synthase, a transgene encoding UDP-N-acetylglucosamine 2-epimerase, a transgene encoding CMP N-acetylneuraminate synthetase, and a transgene encoding beta-galactoside alpha-2,3-sialyltransferase.

9. The transgenic yeast cell according to claim 8, wherein the Neu5Ac synthase is Sequence ID No. 20, Sequence ID No. 26, or Sequence ID No.

32.

10. The transgenic yeast cell according to claim 8, wherein the bacterial UDP-N-acetylglucosamine 2-epimerase is SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO:

34.

11. The transgenic yeast cell according to claim 8, wherein the mammalian CMP N-acetylneuraminate synthetase is SEQ ID NO: 24 or SEQ ID NO:

30.

12. The transgenic yeast cell according to claim 8, wherein beta-galactoside alpha-2-3-sialytransferase is sequence number 18.

13. The transgenic yeast cell according to claim 8, further comprising a transgene encoding the N-acetylglucosamine epimerase enzyme and a transgene encoding beta-1,4N-acetylgalactosaminyltransferase.

14. The transgenic yeast cell according to claim 13, further comprising a transgene encoding beta-1,3-galactosyltransferase.

15. A method for producing sphingolipids in yeast cells, comprising culturing the yeast cells in a culture medium, The yeast cells express the transgene encoding GhLAG1-1 and the transgene encoding DEGS1, A method wherein the yeast cells do not express the endogenous yeast genes SUR2, SCS7, LAG1, and LAC1.

16. The method according to claim 15, wherein the culture medium contains stearic acid.

17. The method according to claim 16, wherein the yeast cells produce C18 ceramide.

18. The method according to any one of claims 15 to 17, wherein the yeast cells further comprise a transgene encoding UDP-glucose ceramide glucosyltransferase.

19. The method according to claim 18, wherein the UDP-glucose ceramide glucosyltransferase is SEQ ID NO: 6 or SEQ ID NO:

8.

20. The method according to claim 18, wherein the culture medium contains glucose.

21. The method according to claim 20, wherein the yeast cells produce glucosylceramide (GlcCer).

22. The method according to claim 18, wherein the yeast cells further comprise a transgene encoding beta-1,4-galactosyltransferase.

23. The method according to claim 22, wherein the beta-1,4-galactosyltransferase is HsB4GALT5 (SEQ ID NO: 10), MmB4GALT5 (SEQ ID NO: 12), PrB4GALT5 (SEQ ID NO: 14), or HsB4GALT6 (SEQ ID NO: 16).

24. The method according to claim 22, wherein the culture medium comprises galactose (Gal).

25. The method according to claim 24, wherein the yeast cells produce lactosylceramide (LacCer).

26. The method according to claim 22, wherein the yeast cells further comprise a transgene encoding bacterial Neu5Ac synthase, a transgene encoding bacterial UDP-N-acetylglucosamine 2-epimerase, a transgene encoding mammalian CMP N-acetylneuraminate synthetase, and a transgene encoding beta-galactoside alpha-2,3-sialyltransferase.

27. The method according to claim 26, wherein the Neu5Ac synthase is sequence number 20, sequence number 26, or sequence number 32.

28. The method according to claim 26, wherein the bacterial UDP-N-acetylglucosamine 2-epimerase is SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO:

34.

29. The method according to claim 26, wherein the mammalian CMP N-acetylneuraminate synthetase is SEQ ID NO: 24 or SEQ ID NO:

30.

30. The method according to claim 26, wherein beta-galactoside alpha-2-3-sialytransferase is sequence number 18.

31. The method according to claim 26, wherein the culture medium comprises CMP-N-acetylneuraminate (CMP-Neu5NAc).

32. The method according to claim 31, wherein the yeast cells produce monosialoganglioside 3 (GM3).

33. The method according to claim 26, wherein the yeast cells further comprise a transgene encoding an N-acetylglucosamine epimerase enzyme and a transgene encoding beta-1,4N-acetylgalactosaminyltransferase.

34. The method according to claim 33, wherein the culture medium contains N-acetylgalactosamine (GalNAc).

35. The method according to claim 34, wherein the yeast cells produce monosialoganglioside 2 (GM2).

36. The method according to claim 33, wherein the yeast cells further comprise a transgene encoding beta-1,3-galactosyltransferase.

37. The method according to claim 36, wherein the culture medium contains galactose.

38. The method according to claim 37, wherein the yeast cells produce monosialoganglioside 1 (GM1).

39. Sphingoglycolipids produced by transgenic yeast cells according to any one of claims 1 to 14, or according to the method according to any one of claims 15 to 38.

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