Bioproduction of gangliosides in recombinant host cells

Abstract

Provided herein are, in various embodiments, host cells, methods, and pharmaceutical compositions including complex sphingolipids, such as monosialoganglioside 1 (GM1), monosialoganglioside 2 (GM2), and monosialoganglioside 3 (GM3). In some embodiments, the disclosure provides for the bioproduction of glycosphingolipids. In still further embodiments, the disclosure provides for the bioproduction of gangliosides.

Description

BIOPRODUCTION OF GANGLIOSIDES IN RECOMBINANT HOST CELLSRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 489,919, filed on March 13, 2023. The entire teachings of the above application are incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith: a) File name: 5767100800 l_Sequence_Listing.xml; created March 12, 2024, 100,172 Bytes in size.BACKGROUND

[0003] Gangliosides are an important class of molecules, composed of a lipid-based ceramide backbone and a sugar head-group. Although gangliosides play a wide variety of important roles in healthcare, production and purification of gangliosides remains a challenge.SUMMARY

[0004] Described herein are transgenic yeast and methods for producing complex sphingolipids, such as monosial oganglioside 1 (GM1), monosial oganglioside 2 (GM2), and monosialoganglioside 3 (GM3). In several aspects, the production of complex sphingolipids occurs in a transgenic yeast.

[0005] In an aspect, the present disclosure provides for a transgenic yeast including a transgene encoding a ceramide synthase and a transgene encoding a sphingolipid delta-4 desaturase, wherein the yeast does not express endogenous yeast genes SUR2, SCS7, LAG1, and LAC1. In another aspect, the disclosure provides for a transgenic yeast cell including a ceramide synthase having a sequence of SEQ ID NO: 4, SEQ ID NO: 46, or SEQ ID NO: 48. In another aspect, the disclosure provides for a transgenic yeast including a sphingolipid delt-4 desaturase having a 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 for a transgenic yeast including a transgene encoding a UDP-glucose ceramide glucosyltransferase. In another aspect, the disclosure provides for a transgenic yeast including a UDP-glucose ceramide glucosyltransferase having a sequenceof SEQ ID NO: 6 or SEQ ID NO: 8. In another aspect, the disclosure provides for a transgenic yeast including a beta- 1,4 galactosyltransferase. In another aspect, the disclosure provides for a transgenic yeast including a 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 for a transgenic yeast including a transgene encoding bacterial Neu5Ac synthase, a transgene encoding bacterial UDP-N-acetylglucosamine 2-epimerase, a transgene encoding mammalian CMP N-acetylneuraminic acid synthetase, and a transgene encoding beta-galactoside alpha-2, 3-sialyltransferase. In another aspect, the disclosure provides for a transgenic yeast including a Neu5Ac having a sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO:32. In another aspect, the disclosure provides for a transgenic yeast including a UDP-N-acetylglucosamine 2-epimerase having a sequence of SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO: 34. In another aspect, the disclosure provides for a transgenic yeast including a CMP N-acetylneuraminic acid synthetase having a sequence of SEQ ID NO: 24 or SEQ ID NO: 30. In another aspect, the disclosure provides for a transgenic yeast including a beta-galactoside alpha-2-3 -sialytransf erase having a sequence of SEQ ID NO: 18. In another aspect, the disclosure provides for a transgenic yeast including a transgene encoding an N- acetylglucosamine epimerase enzyme and a transgene encoding a beta-1,4 N- acetylgalactosaminyltransferase. In another aspect, the disclosure provides for a transgenic yeast including a transgene encoding a beta-1, 3 -galactosyltransferase.

[0006] In another aspect, the disclosure provides for a method of producing a sphingolipid in a yeast cell. In several aspects, the method includes the yeast cell in a culture medium, wherein the yeast cell expresses a transgene encoding GhLAGl-1 and a transgene encoding DEGS1 and wherein the yeast cell does not express endogenous yeast genes SUR2, SCS7, LAG1, and LAC1. In another aspect, the culture medium comprises stearic acid. In another aspect, the yeast cell produces Cl 8 ceramide. In another aspect, the yeast cell includes a transgene encoding a UDP- glucose ceramide glucosyltransferase. In another aspect, the disclosure provides for a yeast cell including a UDP-glucose ceramide glucosyltransferase having a sequence of SEQ ID NO: 6 or SEQ ID NO: 8. In another aspect, the culture medium includes glucose. In another aspect, the yeast cell produces glucosylceramide (GlcCer). In another aspect, the yeast cell includes a transgene encoding a beta-1,4 galactosyltransferase. In another aspect, the disclosure provides for a yeast cell including a 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 culture medium includes UDP-galactose. In another aspect, theyeast cell produces lactosylceramide (LacCer). In another aspect, the yeast cell includes a transgene encoding bacterial Neu5Ac synthase, a transgene encoding bacterial UDP-N- acetylglucosamin 2-epimerase, a transgene encoding mammalian CMP N-acetylneuraminic acid synthetase, and a transgene encoding a beta-galactoside alpha-2, 3-sialyltransferase. In another aspect, the disclosure provides for a transgenic yeast including a Neu5Ac having a sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO:32. In another aspect, the disclosure provides for a transgenic yeast including a UDP-N-acetylglucosamine 2-epimerase having a sequence of SEQ ID NO: 22, SEQ ID NO: 28, or SEQ ID NO: 34. In another aspect, the disclosure provides for a transgenic yeast including a CMP N-acetylneuraminic acid synthetase having a sequence of SEQ ID NO: 24 or SEQ ID NO: 30. In another aspect, the disclosure provides for a transgenic yeast including a beta-galactoside alpha-2-3 -sialytransf erase having a sequence of SEQ ID NO: 18. In another aspect, the culture medium includes CMP-N-acetylneuraminate (CMP-Neu5NAc). In another aspect, the yeast cell produces monosialoganglioside 3 (GM3). In another aspect, the yeast cell includes a transgene encoding an N-acetylglucosamine epimerase enzyme and a transgene encoding a beta-1,4 N-acetylgalactosaminyltransferase. In another aspect, the culture medium includes UDP-N-acetylgalactosamine. In another aspect, the yeast cell produces monosialoganglioside 2 (GM2). In another aspect, the yeast cell includes a transgene encoding a beta- 1,3 -galactosyltransferase. In another aspect, the culture medium includes galactose. In another aspect, the yeast cell produces monosialoganglioside 1 (GM1).BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0008] FIG. 1. Structure of the ganglioside GM1. GM1 consists of a ceramide (Cl 8- ceramide) lipid backbone and a pentasaccharide head group. These sugars are added sequentially to the ceramide backbone. Different glycosylation patterns on the ceramide backbone yield different types of gangliosides.

[0009] FIG. 2. Flowchart for the production of gangliosides in a heterologous host. In order to produce gangliosides, the biosynthesis of C18-ceramide is first achieved. Then, a series of 5 sugar transferases and two sugar biosynthetic pathways must be introduced to enable biosynthesis of gangliosides, up to the key 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 : monosial oganglioside 1 (Structure 6). UDP-Glc: UDP-glucose. UDP-Gal: UDP -galactose. CMP- Neu5Ac: N-acetylneuraminic acid (CMP-Sialic acid). UDP-GalNAc: UDP-N- acetylgalactosamine.

[0010] FIG. 3. Overview of sphingolipids in yeast. Based on current knowledge of yeast ceramide biosynthesis, specific ways in which to disrupt the metabolism of S. cerevisiae in order to produce and accumulate C18-ceramide can be understood.

[0011] FIG. 4. Modifications required to convert yeast ceramide biosynthesis into a process that can produce C18 ceramide. Based on previous work, a way to produce Cl 8- ceramide, the primary lipid backbone for all gangliosides, in yeast was realized. Four native yeast genes must be removed and two genes must be expressed to enable the production and accumulation of C18-ceramide.

[0012] FIG. 5. LC-MS / MS chromatogram of human ceramide in yeast. After 5 days of fermentation, lipids from S. cerevisiae strains were extracted in methanol and analyzed. The extract from an engineered strain resulted in a peak at 23.4 min which is absent from the wildtype strain. A standard of C18-ceramide was used for identification. This figure shows chromatograms that have been filtered specifically for the MS-MS transition of C18-ceramide.

[0013] FIG. 6. LC-MS / MS chromatogram depicting the production of glucosylceramide after the heterologous expression of a UDP-glucose ceramide glucosyltransferase. After 5 days of fermentation, lipids from S. cerevisiae strains were extracted in methanol and analyzed. Two candidate enzymes (UGCG, SEQ ID NO: 6, and GCS1, SEQ ID NO: 8) resulted in an expected peak at 5.6 min which coincides with the retention time observed for the glucosylceramide standard. This figure shows chromatograms that have been filtered specifically for the MS-MS transition of glucosylceramide.

[0014] FIG. 7. LC-MS / MS chromatogram depicting the production of lactosylceramide after the heterologous expression of a glucosylceramide beia- -galaciosyltransferase.After 5 days of fermentation, lipids from S. cerevisiae strains 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) resulted in an expected peak at 5.4 min. A standard of lactosylceramide was used for identification in the tested strains. This figure shows chromatograms that have been filtered specifically for the MS-MS transition of lactosylceramide.

[0015] FIG. 8. Schematic representation of the engineered GM3 biosynthetic pathway in yeast. Heterologous genes were sourced from eukaryotic systems, except for NeuC and NeuB which have bacterial origin. Arrows with stop sign indicate knockouts.

[0016] FIG. 9. LC-MS / MS chromatogram depicting GM3 production after the heterologous expression of a hybrid CMP-Sia pathway and a lactosylceramide alpha-2-3- sialyltransferase. After 7 days of fermentation, lipids from S. cerevisiae strains containing two episomal plasmids bearing GM3 biosynthetic pathways were extracted in methanol and analyzed. Six distinct combinations of genes resulted in an expected peak at 4.5-4.6 which is absent from the lactosylceramide producer strain that was used as background. The extract of wildtype S. cerevisiae was spiked with GM3 and was used for identification in the tested strains. This figure shows chromatograms that have been filtered specifically for the MS-MS transition of GM3.DETAILED DESCRIPTION

[0017] A description of example embodiments follows.Structures

[0018] Structure 1 : Ceramide-18 (C18 ceramide NS (N-acyl Sphingosine), human ceramide) is a compound having the following structure:

[0019] Structure 2: glucosylceramide is a compound having the following structure:

[0020] Structure 3: lactosylceramide is a compound having the following structure:

[0021] Structure 4: GM3 is a compound having the following structure:

[0022] Structure 5: GM2 is a compound having the following structure:

[0023] Structure 6: GM1 is a compound having the following structure:Sequences

[0024] Table 1 is a listing of the sequences utilized in experiments described herein.

[0025] Homologs of GhLagl-1 : Human CerSl; and Pichia pastoris barl

[0026] Homologs of DEGS1 : A. thaliana A4-desaturase At4g04930; C albicans Deslp; D. melanogaster DES; S. pombe dsdl; Pichia pastoris DES1

[0027] As used herein, the term “sequence identity,” refers to the extent to which two sequences have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which a test sequences are compared. Sequence identity between reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide residue at 70% of the same positions over the entire length of the reference sequence.

[0028] Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, alignment can include introduced gaps to provide for the maximal 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 method 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, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally 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 the pattern of codon usage in such cells, tissues, and / or organisms containing conservative (or nonconservative) amino acid substitutions that do not adversely affect normal activity.

[0029] In some aspects, the disclosure provides for 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 aspects, 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.

[0030] In some aspects, the disclosure provides for 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 aspects, 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.

[0031] In some aspects, the disclosure provides for 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 aspects, 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.

[0032] In some aspects, the disclosure provides for 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 aspects, 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.

[0033] In some aspects, the disclosure provides for 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 aspects, 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.

[0034] In some aspects, the disclosure provides for a 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 aspects, the 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.

[0035] In some aspects, the disclosure provides for a CMP N-acetylneuraminic acid 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 I DNO: 24 or SEQ ID NO: 30. In some aspects, the CMP N-acetylneuraminic acid 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.Introduction

[0036] Gangliosides are an important class of molecules that are found throughout the human body, and particularly in the brain. Gangliosides are composed of a lipid-based ceramide backbone (also known as human ceramide or ceramide NS) and a sugar head-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 the depletion of GM1 is correlated with neurological diseases such as Huntington’s and Parkinson’s disease (Magistretti et al. 2019) (FIG. 1, Structure 6). It has been shown that administration of GM1 can 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 cancer cell membrane surfaces (Dobrenkov et al. 2016) and are an essential component for emerging immunotherapy -based cancer treatments (Grabowska et al. 2021, Zang et al. 2022) (Structure 4).

[0037] While gangliosides have important applications throughout many aspects of healthcare, production and purification of gangliosides remains a challenge. Currently, GM1 is utilized in several countries as a therapeutic, but this GM1 is sourced from animal brain tissue. Furthermore, less abundant gangliosides are difficult to accumulate and purify. Therefore, a solution to providing gangliosides at a scale amenable to commercialization and use as a pharmaceutical is necessary.

[0038] To address the issues surrounding production of GM1 and other gangliosides, the present disclosure provides a synthetic biology platform to produce gangliosides in a heterologous yeast host (FIG. 2). The genes required to enable a host cell to produce gangliosides de novo from simple carbon sources are identified, as well as key host genes that must be eliminated to enable high-level biosynthesis. Currently, these engineering efforts have resulted in a strain that can produce the ganglioside GM3.

[0039] This synthetic biology strategy for producing gangliosides will enable not only the production of the proven therapeutic GM1, but also the production of ganglioside intermediates and alternatives. This includes GM2, GM3, lactosylceramide, and glucosylceramide. These gangliosides have never been produced in a heterologous organism to date.

[0040] Under designated fermentation conditions, the designed strain can accumulate gangliosides for the eventual purification and use in various formats, including direct administration as a treatment, as well as incorporation into emerging drug delivery methods to elicit specific immune responses.

[0041] Described herein is a yeast strain that is capable of producing glucosylceramide, the first glycosylated molecule on the pathway towards GM1 biosynthesis. Also described herein is the subsequent addition of UGT enzymes into this strain to enable production of the pentasaccharide head-group of GM1.General MethodsMedia and formulations

[0042] Yeast peptone dextrose (YPD) broth was composed of 10 g / L of yeast extract, 20 g / L of peptone and 20 g / L dextrose. For YPD plates, 20 g / L of agar was added.

[0043] Selective media was prepared using 6.7 g / L of yeast nitrogen base without amino acids, 1.92 g / L of synthetic complete media lacking uracil, tryptophan or histidine or 1.6 g / L if selection with leucine was needed. For plates, 20 g / L of agar was added. Following sterilization, dextrose was added at a final concentration of 20 g / L.

[0044] Nitrogen-limited synthetic media (NLS) was composed of 1.7 g / L of yeast nitrogen base without amino acids and ammonium sulfate, 1 g / L of ammonium sulfate, 1.92 g / L of amino acid dropout mix minus uracil and 76 mg / L of uracil. Following autoclave sterilization, 50 ml of 40% dextrose or galactose was added to obtain either nitrogen-limited synthetic media with dextrose (NLSD) or nitrogen-limited synthetic media with galactose (NLSGal_. The pH of the media was then adjusted to 7.0. A solution of 25 mg / ml of stearic acid (SA) in 95% ethanol was prepared and 20 ml was added slowly while medium was stirred vigorously.Genomic deletion procedures

[0045] Deletion of native S. cerevisiae genes was carried out according to Akada et al. 2006. Briefly, a Ikb fragment directly upstream of the desired gene to be knocked out was used to direct a URA3 expression cassette to the knockout locus. Removal of the gene was indicated by growth of the strain in media lacking uracil. The URA3 cassette was subsequently removed by growing the strain on media containing 5-fluoroorotic acid (5-FOA), which allowed the URA3 cassette to be removed through homologous recombination that was facilitated by repeats designed into the genomic insert. Knockouts were confirmed via genomic DNA extraction and PCR. For CRISPR-Cas9-based deletions, methods were modified from Lee et al. 2015. Briefly, guide RNA was designed with 65 bp flanking regions with homology to the genome directly up- and downstream of the gene to be removed. A 20 bp barcode was added for clone confirmation. A plasmid containing the guide RNA and the Cas9 machinery was transformed into the recipient strain and streaked out on selective media. Knockouts were confirmed by PCR. Re-streaking of positive clones was performed to remove the Cas9 plasmid, resulting in the successful knockout of the target gene.Cloning procedures

[0046] Integration and episomal plasmids were constructed using Golden gate assembly as described before (Lee et al. 2015). For integration plasmids, an intermediate plasmid containing an expression cassette for the tested gene under different promoter strengths and the plasmid containing the expression cassette flanked with homologous arms and an auxotrophic marker (e.g., URA3, HIS3, LEU2) were constructed.

[0047] For construction of episomal plasmids bearing pathway modules, three types of plasmids were constructed: a plasmid containing the expression cassette for the tested gene under the control of a relatively strong promoter, a plasmid backbone with the required elements foryeast propagation, i.e. antibiotic resistance and 2 micron and plasmid containing multiple expression cassettes for episomal expression.

[0048] Briefly, 5 uL of Golden gate reactions were transformed to 20-50 uL of E. coli DH- alpha and plated on appropriate selection after recovery. Plates were incubated overnight at 37 C and usually 2 clones were screened for proper assembly using restriction enzymes. A clone that showed the expected pattern was further confirmed by DNA sequencing.Transformation procedures

[0049] Cells were transformed either using chemical transformation according to Schiestl & Gietz (1989) or electroporation as described by Shao & Zhao (2014). In both methods, a seed culture that had been grown overnight was used to inoculate 50 ml of YPD media in a 250-ml baffled flask. Cells were allowed to grow for at least two duplications (i.e., 4-5 h). For chemical transformation, cells were centrifuged at 2500 rpm for 5 min. Cells were washed with water once followed by a wash with 100 mM lithium acetate. Then, cells were concentrated approximately 100-fold in 100 mM lithium acetate. A transformation mix consisting in 50 mL of cell suspension, 240 mL of 50% PEG, 36 mL of IM lithium acetate and 50 mL of salmon sperm (2 mg / ml) was combined with 50 mL of a digestion reaction that generated a linear DNA fragment comprising an expression cassette and a selection marker. Heat shock was performed by incubation of cells at 30 C for 30 min followed by 42 C for 30 min. Transformation mixes were centrifuged and pellets were washed gently with 1 mL of sterile water. Finally, cells were resuspended in 150 mL of sterile water and plated on appropriate selection plates.

[0050] Cells being electroporated were collected and processed at 4 C. Two washes with water were performed at 4000 rpm for 10 min followed by a sorbitol wash that helped to concentrate cells by approximately 150-fold. Cells were resuspended in sorbitol and 50-mL aliquots were combined with less than 4 mL of DNA transformation mix, i.e., pair of episomal plasmids. Cells were electroporated at 1.5 kV using 2 mm-gap cuvettes. Immediately after electroporation, 1 mL of YPD was added and cells were recovered for 1 h at 30 C before being washed with sorbitol twice and plated on appropriate selection plates. Verification was conducted via PCR for integration strains or by plating on selective media to confirm plasmid maintenance.Analysis of sphingolipids (ceramide, glucosylceramide and lactosylceramide) by LC-MS

[0051] Generally, 1-2 colonies were inoculated to liquid media and cultured over 5-7 days to assess the production of the target molecule using a previously described protocol (Bielawski et al. 2009) with adaptations.

[0052] For detection of lactosylceramide, each strain was inoculated to culture tubes containing 5-ml of NLSD+0.05%SA and NLSGal+0.05%SA. Liquid culture was carried out at 30 C and 250 rpm for 5 days. Samples were collected after 3 and 5 days of fermentation for analysis.

[0053] For detection of GM3, each strain was inoculated to 50-ml tubes with vent caps containing 10 ml of either YPD, YPGal or NLSGal+0.05%SA media. Liquid culture was carried out at 30 C and 250 rpm for 7 days. Samples were collected after 2.5, 4.5 and 7.5 days of fermentation for analysis.

[0054] Lipid extraction was conducted as described before in Bielawski et al. 20093. Briefly, 1 mL of cell culture was centrifuged at maximum speed for 5 min. Then, cell pellets were resuspended in yeast lipid extraction mixture. Samples were then sonicated for 3 min and vortexed briefly. Samples were centrifuged at maximum speed for 5 min and extract (~1 mL) was collected. 500 mL of yeast lipid extraction mixture was added to the remaining pellet and the process is repeated, except that sonication time was reduced to 1 min. After centrifugation, both extracts were combined and placed in a centrifuge concentrator overnight at medium temperature. Lipid extracts were reconstituted in 0.15-1 mL of mobile phase B for analysis.

[0055] Beginning with LC-MS parameters described previously (Bielawski et al. 2009), MS / MS fragmentation was used to verify the identity of each ganglioside and compare each putative product to that of an authentic standard.Results and DiscussionEngineering production of C18 ceramide

[0056] In order to access gangliosides, the production of human ceramide (Cl 8 ceramide) (Structure 1) was engineered. Yeast already produce a variety of ceramides, and the pathways have been characterized previously (FIG. 3). In comparing the major yeast ceramide with the ceramide that is part of GM1, the key changes that must be made were identified (FIG. 4). First, the genes that install hydroxyl groups on yeast ceramides, the genes sur2 and scs7, must be removed, and this was shown by Murakami et al. to be a non-toxic modification that resulted in hydroxyl-free ceramides (Murakami et al. 2015). Second, a desaturation must be introduced intothe sphingosine portion of the ceramide, which was also shown by Murakami and coworkers by addition of the human sphingolipid delta-4 desaturase (DEGS1; SEQ ID NO: 2) with an endoplasmic reticulum targeting sequence added to the C-terminus. See US Patent No. 8,367,375 (Kodama et al.). However, Kodama et al. does not disclose modifying the length of the lipid on the ceramide. Finally, the lipid that is attached to sphingosine must be reduced from C26 (typical yeast lipid length) to Cl 8. It was previously shown that introduction of a plant ceramide synthase GhLagl-1 (SEQ ID NO: 4) and knockout of the yeast native ceramide synthases Lagl and LAC1 enabled production of ceramides with a C18 chain length (Epstein et al. 2012). With this information in hand, a strain was constructed by first knocking out sur2 and scs7, the two hydroxylase enzymes that install hydroxyl groups on yeast ceramides, using homologous recombination. Next, DEGS1 (SEQ ID NO: 2) and GhLAGl-1 (SEQ ID NO: 4) were integrated into a genetic locus. Finally, Lagl and LAC1 were removed via CRISPR-Cas9 technology to arrive at a strain that had the capability to produce C18 ceramide. Upon culturing of this strain in media that contained stearic acid, the production of Cl 8 ceramide via LC-MS was observed. This was verified by comparison to a standard (FIG. 5). While ceramides with longer chain lengths have been produced in yeast previously (Murakami et al. 2015), this is the first demonstration of heterologous production of a C18 ceramide that serves as a ceramide backbone for gangliosides and that is identical to ceramides found in mammals.

[0057] Native ceramide has many important roles in normal cellular processes for yeast, such as membrane stability and forming more complex (e.g., modified) lipids, most notably inositol phosphorylceramides, which play essential roles in cell growth and division processes. Knocking out four endogenous genes (SUR2, SCS7, LAG1, and LAC1) and expressing two exogenous genes (GhLAGl-1 and DEGS1) causes the yeast cell to no longer produce native ceramide. Relative to the native ceramide, the sphingolipid produced has a double bond, which introduces rigidity into the molecular chain, which may disrupt membrane formation by the sphingolipid. In addition, two hydroxyl groups, one on each of the molecular chains, are no longer added. These hydroxyl groups are known to be important for maintaining membrane fluidity and proper cycling of membrane components of the yeast cell. Previously, it has been shown that introduction of a double bond and removal of hydroxyl groups does not cause cell death, but significantly increases sensitivity to inhibitors of the enzyme that produces inositol phopshorylceramides. It was observed that removal of both hydroxyl groups, through knockout of sur2 and scs7, reduced growth speed, indicating some form of growth defect or deficiency caused by the alternative ceramides (Murakami et al., 2015). Furthermore, in our strain, thelength of the fatty-acyl chain of the ceramide has been reduced from 26 carbons to 18 carbons, which will affect membrane fluidity and membrane structure. This has independently been shown to not be toxic to yeast. However, it was noted that ceramide production was greatly enhanced when the fatty-acyl chain was reduced from 26 to 18 carbons (Epstein et al., 2012). Therefore, we would predict that introducing structural changes on the ceramide, as well as shortening the acy-chain length, would lead to further increase of the non-natural ceramide species that is causing the growth defect reported by Murakami et al. However, we did not observe any additional growth defect as more non-native ceramide species were accumulated in our engineered strain. Thus the combination of ceramide modifications described herein enable production of C18 ceramide and provide the first evidence that modification of the ceramide sphingosine base and of the fatty-acyl chain of endogenous yeast ceramides does not cause cell death or have an additive negative impact on cell viability.Engineering of glucosylceramide production

[0058] With a strain that produces Cl 8 ceramide in hand, the next step of ganglioside biosynthesis involves transfer of a glucose onto ceramide to form glucosylceramide (Structure 2). This requires the action of a UDP -glucose ceramide glucosyltransferase (UGCG). Two different enzymes (UGCG, SEQ ID NO: 6; GCS1, SEQ ID NO: 8) were screened for activity by cloning into an entry vector and integrated into the genome of a C18-ceramide-producing strain under the strong constitutive promoter pTDH3. Upon culturing of the newly constructed strains, the ceramide profiles were analyzed via LCMS, and the presence of glucosylceramide was detected. This activity was verified by comparison to an authentic glucosylceramide standard (FIG. 6). This is the first description of heterologous production of glucosylceramide in a unicellular host that is identical to glucosylceramide found in mammalian gangliosides.Engineering of lactosylceramide production

[0059] The next sugar transfer reaction towards the production of gangliosides is to add a galactose unit onto glucosylceramide, which is performed by a beta- 1,4 galactosyltransferase (B4GalT) to form lactosylceramide (Structure 3). B4GalT enzymes were cloned into an entry vector and integrated into the genome of a glucosylceramide-producing strain under the strong constitutive promoter, pTDH3. To test for activity, the resulting strains were grown in media supplemented with stearic acid and galactose. After sample extraction and measurement on LCMS, it was discovered that 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 capable of converting glucosylceramide to lactosylceramide (FIG. 7). This is the first description of heterologous production of lactosylceramide in a unicellular host that is identical to the lactosylceramide found in mammalian gangliosides.Engineering of GM3 production

[0060] In order to achieve the biosynthesis of GM3, two distinct pathways were engineered. GM3 contains a Neu5 Ac (also known as sialic acid or N-acetylneuraminate) sugar moiety, which is not a sugar that is natively found in Saccharomyces cerevisiae host (Structure 4). Therefore, biosynthesis of the sugar donor CMP-Neu5Ac was first engineered into the yeast host. US 2008 / 0085540 Al (Hamilton) discloses engineering CMP-Neu5Ac biosynthesis into the yeast host Pichia pastoris to enable sialylation of proteins, and a similar approach is employed herein. To achieve this biosynthesis, a combination of bacterial and mammalian CMP-Neu5Ac biosynthetic genes were heterologously expressed on a plasmid in the yeast strain. This includes a bacterial Neu5 Ac synthase (NeuB), a bacterial UDP-N-acetylglucosamine 2-epimerase (NeuC), and a mammalian CMP N-acetylneuraminic acid synthetase (CMAS). Simultaneously, a 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. A strain that produces lactosylceramide was transformed both with the CMP-Neu5 Ac biosynthetic pathway and a beta-galactoside alpha-2, 3-sialyltransferase and then cultured to assess GM3 production (FIG. 8). After growth and sample preparation, we found that six combinations of genes resulted in GM3 production. For each combination, biosynthesis of Neu5Ac was enabled by using bacterial NeuB and NeuC sourced from N meningitis (T1 and T4 in FIG. 9), S. agalactiae (T2 and T5 in FIG. 9) or E. coli (T3 and T6 in FIG. 9). Activation of Neu5Ac to CMP-Neu5Ac was facilitated by a mammalian CMAS sourced from either mouse or human (TITS and T4-T6, respectively in FIG. 9), and in all combinations the human beta-galactoside alpha- 2,3 -sialyltransferase was employed for GM3 biosynthesis. GM3 production was confirmed with comparison to an authentic purchased standard (FIG. 9). This is the first description of heterologous production of GM3 in a unicellular host.

[0061] Table 2 is a listing of the six combinations of genes usedEngineering GM2 biosynthesis (Prophetic example)

[0062] In order to achieve the biosynthesis of GM2, two genes are required to be engineered into the GM3-producing strain (structure 5). GM2 biosynthesis requires the saccharide precursor UDP-N-acetylgalactosamine (UDP-GalNAc), which is not native to S. cerevisiae. Therefore, an UDP-Galactose / UDP-N-acetylglucosamine epimerase (GALE) enzyme, such as BsGALE (SEQ ID NO: 50), MmGALE (SEQ ID NO: 52), and HsGALE (SEQ ID NO: 54), will be introduced into the host. Second, a beta- 1,4 N-acetylgalactosaminyltransferase, such as HsB4GalNTl (SEQ ID NO: 56) or CjCgtA (SEQ ID NO: 62), will subsequently be engineered to facilitate the transfer of GalNAc onto GM3. These two genes together will enable the biosynthesis of GM2, the penultimate precursor to GM1.Engineering GM1 biosynthesis (Prophetic example)

[0063] To engineer a strain that is capable of producing the therapeutic molecule GM1, a final gene addition must be performed (FIG. 1, Structure 6). A beta- 1,3 -galactosyltransferase, such as HsB3GalT4 (SEQ ID NO: 60) or CjGgtB (SEQ ID NO: 62), will be engineered into astrain that produces GM2, and by utilizing UDP-galactose, will convert GM2 into GM1. This result will mark the first description of GM1 biosynthesis in a heterologous host for the pharmaceutical application towards treating neurological disorders and conditions.Enhance UDP-galactose and engineering UDP-N-acetylgalactosamine biosynthesis (Prophetic example)

[0064] To provide the essential sugar donor molecules for ganglioside biosynthesis, UDP- galactose and UDP-N-acetylgalactosamine must be present in the host cell (FIG. 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-functional UDP- glucose / UDP-N-acetylglucosamine epimerase (GALE) enzymes have been identified, BsGALE (SEQ ID NO: 50), MmGALE (SEQ ID NO: 52), and HsGALE (SEQ ID NO: 54), which can convert UDP-glucose and UDP-N-acetylglucosamine into UDP-galactose and UDP-N- acetylgalactosamine, respectively. This will be an essential gene addition when pursuing the biosynthesis of gangliosides GM2 and GM1.REFERENCES

[0065] Zang, H.; Siddiqui, M.; Gummuluru, S.; Wong, W. W.; Reinhard, B. M. Ganglioside- Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse. ACS Nano 2022, 76 (11), 18408-18420. doi.org / 10.1021 / acsnano.2c06516.

[0066] 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 / s 12974-021 -02374-x.

[0067] 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 a-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.

[0068] 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.

[0069] Patry, R. T.; Stahl, M.; Perez-Munoz, M. E.; Nothaft, EL; 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.

[0070] Magistretti, P. J.; Geisler, F. EL; Schneider, J. S.; Li, P. A.; Fiumelli, EL; Sipione, S. Gangliosides: Treatment Avenues in Neurodegenerative Disease. Frontiers in Neurology 2019, 10.

[0071] 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.

[0072] 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.

[0073] 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 / srepl6319.

[0074] 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.

[0075] 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. chl30191.

[0076] Kodama, Y.; Okuhara, H.; Funato, K. Methods for Producing Human Ceramide Using Yeast Transformants. US8367375B2, February 5, 2013. patents.google.com / patent / US8367375 / en

[0077] 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.

[0078] 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.

[0079] 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 I.ipidomics: Armstrong, D., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2009; Vol. 579, pp 443-467. doi.org / 10.1007 / 978-l-60761-322-0_22.

[0080] Hamilton, S. Method of Engineering a Cytidine Monophosphate-Sialic Acid Synthetic Pathway in Fungi and Yeast. US20080085540A1, April 10, 2008. patents. google. com / patent / US20080085540Al / en

[0081] 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. FeaV 2006, 23 (5), 399-405. doi.org / 10.1002 / yea.1365.

[0082] Schiestl, R. H.; Gietz, R. D. High 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.INCORPORATION BY REFERENCE; EQUIVALENTS

[0083] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0084] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

CLAIMSWhat is claimed is:

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

2. The transgenic yeast cell of claim 1, wherein the ceramide synthase is SEQ ID NO: 4, SEQ ID NO: 46, or SEQ ID NO: 48.

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

5. The transgenic yeast cell of claim 4, wherein the UDP-glucose ceramide glucosyltransferase is SEQ ID NO: 6 or SEQ ID NO: 8.

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

7. The transgenic yeast cell of 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 of claim 6, further comprising a transgene encoding a Neu5Ac synthase, a transgene encoding a UDP-N-acetylglucosamine 2-epimerase, a transgene encoding a CMP N-acetylneuraminic acid synthetase, and a transgene encoding a betagalactoside alpha-2, 3-sialyltransferase.

9. The transgenic yeast cell of claim 8, wherein the Neu5Ac synthase is SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32.

10. The transgenic yeast cell of 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 of claim 8, wherein the mammalian CMP N-acetylneuraminic acid synthetase is SEQ ID NO: 24 or SEQ ID NO: 30.

12. The transgenic yeast cell of claim 8, wherein the beta-galactoside alpha-2-3- sialytransferase is SEQ ID NO: 18.

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

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

15. A method of producing a sphingolipid in a yeast cell, the method comprising culturing the yeast cell in a culture medium, wherein the yeast cell expresses a transgene encoding GhLAGl-1 and a transgene encoding DEGS1 and wherein the yeast cell does not express endogenous yeast genes SUR2, SCS7, LAG1, and LAC 1.

16. The method of claim 15, wherein the culture medium comprises stearic acid.

17. The method of claim 16, wherein the yeast cell produces C18 ceramide.

18. The method of any one of claims 15-17, wherein the yeast cell further comprises a transgene encoding a UDP-glucose ceramide glucosyltransferase.

19. The method of claim 18, wherein the UDP-glucose ceramide glucosyltransferase is SEQ ID NO: 6 or SEQ ID NO: 8.

20. The method of claim 18, wherein the culture medium comprises glucose.

21. The method of claim 20, wherein the yeast cell produces glucosylceramide (GlcCer).

22. The method of claim 18, wherein the yeast cell further comprises a transgene encoding a beta- 1,4 galactosyltransferase.

23. The method of 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 of claim 22, wherein the culture medium comprises galactose (Gal).

25. The method of claim 24, wherein the yeast cell produces lactosylceramide (LacCer).

26. The method of claim 22, wherein the yeast cell further comprises a transgene encoding bacterial Neu5 Ac synthase, a transgene encoding bacterial UDP-N-acetylglucosamin 2- epimerase, a transgene encoding mammalian CMP N-acetylneuraminic acid synthetase, and a transgene encoding a beta-galactoside alpha-2, 3-sialyltransferase.

27. The method of claim 26, wherein the Neu5Ac synthase is SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32.

28. The method of 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 of claim 26, wherein the mammalian CMP N-acetylneuraminic acid synthetase is SEQ ID NO: 24 or SEQ ID NO: 30.

30. The method of claim 26, wherein the beta-galactoside alpha-2-3 -sialytransferase is SEQ ID NO: 18.

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

32. The method of claim 31, wherein the yeast cell produces monosial oganglioside 3 (GM3).

33. The method of claim 26, wherein the yeast cell further comprises a transgene encoding an N-acetylglucosamine epimerase enzyme and a transgene encoding a beta- 1,4 N- acetylgalactosaminyltransferase.

34. The method of claim 33, wherein the culture medium comprises N-acetylgalactosamine (GalNAc).

35. The method of claim 34, wherein the yeast cell produces monosial oganglioside 2 (GM2).

36. The method of claim 33, wherein the yeast cell further comprises a transgene encoding a beta- 1 ,3 -galactosyltransferase.

37. The method of claim 36, wherein the culture medium comprises galactose.

38. The method of claim 37, wherein the yeast cell produces monosialoganglioside 1 (GM1).

39. A glycosphingolipid produced by the transgenic yeast cell of any one of claims 1-14 or according to the method of any one of claims 15-38.

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