WO2021019104A2

WO2021019104A2 - A production host for producing human milk oligosaccharides

Info

Publication number: WO2021019104A2
Application number: PCT/EP2020/075434
Authority: WO
Inventors: Woncheol Kim
Original assignee: Basf Se
Priority date: 2019-09-18
Filing date: 2020-09-11
Publication date: 2021-02-04
Also published as: WO2021019104A3

Abstract

The present invention relates to a production host comprising transporters and enzymes involved in the metabolic pathway for the production of human milk oligosaccharides (HMO), preferably a fucosylated and/ or sialylated oligosaccharide, such that the HMO is produced in a secretion organelle in the production host. The invention further relates to transporters targeted for a secretion organelle in the production host, nucleic acid constructs for the expression of the transporters and enzymes, an artificial chromosome, a method for the production of the human milk oligosaccharide and use of heterologous localization sequences for targeting the transporters and the enzymes to the secretion organelle.

Description

A PRODUCTION HOST FOR PRODUCING HUMAN MILK OLIGOSACCHARIDES

SEQUENCE LISTING

This application includes a nucleotide and amino acid sequence listing in computer readable form (CRF) as an ASC II text (.txt) file according to“Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in International Patent Applications Under the Patent Cooperation Treaty (PCT)” ST.25. The sequence listing is identified below and is hereby incorporated by reference into the specification of this application in its entirety and for all purposes.

FIELD OF THE INVENTION

The present invention relates to a production host comprising transporters and enzymes involved in the metabolic pathway for the production of human milk oligosaccharides (HMOs), preferably a fucosylated and/ or sialylated oligosaccharide, such that the HMO is produced in a secretion organelle in the production host. The invention further relates to transporters targeted to a secretion organelle membrane in the production host. Furthermore, the invention provides nucleic acid molecule encoding said transporters and enzymes, nucleic acid construct for the expression of the transporters and enzymes, an artificial chromosome comprising said nucleic acid molecule or the nucleic acid construct, a method for the production of the human milk oligosaccharides in the production host and use of the heterologous localization sequences for targeting the transporters and the enzymes to the secretion organelle and its membrane.

BACKGROUND OF THE INVENTION

Human milk contains diverse oligosaccharides which help to develop and maintain the intestinal flora and prevent binding of pathogens and toxins to human gut. Also, human milk oligosaccahrides (HMOs) have unique biological activities such as anti-bacterial, anti-viral; immune system and cognitive development enhancing activities due to which these compounds are attractive components in the nutritional industry for the production of infant formulas or as dietary supplements for children and adults. Generally, HMOs are characterized by a lactose moiety at the reducing end, and fucose and/ or sialic acid at the non-reducing end. Examples of some of the prominent neutral HMOs are 1 , 2- fucosyllactose, 2’-fucosyllactose, 3’-fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N- neotetraose and lacto-N-fucopentose. Besides such neutral HMOs, acidic HMOs can also be found in human milk, such as 3’-sialyllactose, 6’- sialyllactose, 3-fucosyl-3’-sialyllactose, and disial-lacto-N- tetraose.

In humans, HMOs are produced in a secretion organelle, i.e. endoplasmic reticulum and/or Golgi apparatus of the mammary cells, and secreted into milk. In humans, the substrate GDP-fucose is produced in the mammary cell cytoplasm and transported into the secretion organelle. The substrate lactose is produced in the secretion organelle and a secretion organelle localized fucosyltransferase transfers the fucosyl group from the GDP-fucose to lactose for the production of, for example, 2’- fucosyllactose.

HMOs may be extracted from human breast milk or cow milk. However, in both the cases it is difficult to obtain large amounts and/ or to obtain a satisfactory purity.

HMOs can also be chemically synthesized. However, the HMO production through chemical synthesis is limited by stereo-specificity issues, precursor availability, product impurities and high production cost.

HMO production in genetically modified organisms is being attempted by manipulation of genes and pathways involved in the production of HMOs. For example, different strategies have been adopted for the production of HMOs in bacteria such as Escherichia coli (E. coli).

US 9587241 B2 provides a method of producing HMOs in an engineered bacterium obtained by introduction of an exogenous b-galactosidase gene with reduced b-galactosidase activity and an exogenous fucosyltransferase gene; and inactivation of an endogenous b-galactosidase gene and colanic acid synthesis gene.

US 9758803 B2 provides a method of producing sialylated oligosaccharides in a bacterium comprising providing a bacterium that comprises an exogenous sialyltransferase gene, a deficient sialic acid catabolic pathway, a sialic acid synthetic capability, and a functional lactose permease gene, and culturing the bacterium in the presence of lactose. The sialic acid synthetic capability comprised expressing exogenous CMP-Neu5Ac synthetase, exogenous sialic acid synthase, and exogenous UDP-GlcNAc-2-epimerase.

EP 2379708 B1 provides a method of making a recombinant E. coli cell to produce fucosylated compounds comprising the steps of transforming the cell to express enzymes such as fucose kinase, fucose-1 -phosphate guanyltransferase, fucosyltransferase; and inactivating fucose-1 -phosphate aldolase gene, fucose isomerase gene and a fuculose kinase gene.

US 2018/ 0305724 A1 provides a method for the production of an oligosaccharide in a microbial host cell having a recombinant glycosyltransferase, and a nucleic acid sequence coding for a protein enabling the export of the oligosaccharide, wherein the oligosaccharide is obtainable in free form in the medium i.e. without being bound to the protein on the surface of the host cell.

WO 2019/ 025485 relates to a genetically modified E. coli comprising an inducible lysis system for easy production and extraction of HMOs, which comprises a Mg²⁺ regulated promoter and elements, lysis gene from bacteriophage, exogenous gene encoding a mutant of lactose permease, heterologous gene encoding fucosyltransferase, heterologous genes encoding a chaperone, and wherein the genes encoding the Lon proteases and wcaJ genes are inactivated, and zwf gene and gsk gene are overexpressed.

Although attempts to produce HMOs in E. coli are considered to be successful, yet the outer membrane of E. coli, like that of most gram-negative bacteria, contains the potent immunostimulatory lipopolysaccharides (LPS), also known as endotoxins. In mammalian hosts, endotoxins can induce a pyrogenic response and ultimately trigger septic shock. Contaminating endotoxins must therefore be removed from recombinant proteins expressed in E. coli before they can be safely administered to human. To date, no post-expression methodologies have been described that can remove endotoxin entirely. Thus, HMOs manufactured in E. coli may contain residual endotoxin in low but still sufficient quantities to activate human immune cells. Bacteriophage infection in fermentation process is another area of serious concern. In order to address these concerns, attempts have been made to produce the HMOs in yeast, particularly Saccharomyces cerevisiae (S. cerevisiae), which is generally recognized as safe (GRAS).

Yu et. al. (2018, Production of Human milk oligosaccharide 2’-fucosyllactose by metabolically engineered Sacchromyces cerevisiae. Microbial Cell Factories, 17: 101) discloses a method of constructing a genetically engineered S. cerevisiae to produce 2’-fucosyllactose via the salvage pathway using L-fucose and lactose as the substrates for producing 2’-fucosyllactose. However, the author mentioned that one of the reasons for obtaining low titres of 2’-fucosyllactose is associated with the problem in L-fucose uptake in the yeast which may be attributed to the inefficient transport of the substrate fucose into S. cerevisiae. Other reasons that have been described for the low titre of 2’-fucosyllactose are the production of by-product difucosyllactose and inefficient secretion of considerable amounts of 2’- fucosyllactose. Specifically, the authors described that only about 25% of 2’-fucosyllactose was found in the media, while around 75% was found inside the cells.

Lui et. al. (2018, Biosynthesis of a functional human milk oligosaccharide, 2’ - fucosyllactose, and L- fucose, using engineered S. cerevisiae. ACS Synthetic Biology. 2018. 7 (11), 2529- 2536) discloses a method for producing 2’-fucosyllactose in recombinant S. cerevisiae. Heterologous genes coding for lactose transporter (Lac12), genes coding for enzymes involved in de novo GDP-L-fucose pathway consisting of GDP-D-mannose-4, 6-dehydratase (Gmd) and GDP-4-keto-6-deoxymannose- 3,5-epimerase- 4-reductase (WcaG) were introduced into S. cerevisiae, for the production of 2- fucosyllactose. The Gmd gene and WcaG gene used in Lui et. al. are derived from E. coli and carry a risk regarding the heterologous expression. Furthermore, the authors reported limitations through lactose toxicity in the cytosol and reported inefficient 2’-fucosyllactose secretion.

Thus, it was an object of the presenty claimed invention to provide a production host and a corresponding method for facilitating expression of one or more HMOs with reduced or free from potential toxins at a high yield. SUMMARY OF THE INVENTION

Surprisingly, it has been found that targeting the transporters of the substrates and the enzymes involved in the metabolic pathway for the production of HMOs to the secretion organelle in the production host results in the production of HMOs without having recourse ot bacterial toxins and at a high yield.

The inventors of the present invention genetically engineered a production host wherein the transporters of the substrates and the enzymes involved in the metabolic pathway for the production of HMOs are targeted to the secretion organelle of the production host such that the production of the HMOs take places in the secretion organelle of the production host. The transporters targeted to the secretion organelle may comprise a heterologous localization sequence for integration in the secretion organelle membrane, and enzymes targeted to the secretion organelle comprise a heterologous localization sequence for localization in the secretion organelle lumen.

The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links:

1 . A production host comprising a heterologous nucleic acid encoding a

a) a lactose transporter targeted to a secretion organelle membrane, and/or b) a GDP-fucose transporter targeted to a secretion organelle membrane, and/ or c) cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane, and/ or

d) a fucosyltransferase targeted to a secretion organelle, and/or

e) a sialyltransferase targeted to a secretion organelle.

2. The production host of embodiment 1 , wherein the production host comprises a metabolic pathway, of which at least one reaction is performed in the secretion organelle, for production of a human milk oligosaccharide, preferably a fucosylated and/or sialylated oligosaccharide, more preferably,

a fucosylated oligosaccharide, more preferably 1 , 2-fucosyllactose, 2'-fucosyllactose, 3’-fucosyllactose or difucosyllactose, most preferably 2'-fucosyllactose, and/or a sialylated oligosaccharide, more preferably 3'-sialyllactose or 6'-sialyllactose, or 3'- sialyl-3-fucosyllactose. The production host of any of the preceding embodiments, wherein the production host further comprises a metabolic pathway for the production of GDP-fucose, preferably for the production of GDP-fucose from GDP-mannose, and wherein preferably the metabolic pathway is in the cytoplasm. The production host of any of the preceding embodiments, wherein the production host further comprises a metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic acid, preferably for the production of cytidine 5’- monophosphate N- acetylneuraminic acid from UDP- N-acetyl-glucosamine, and wherein preferably the metabolic pathway is in the cytoplasm. The production host of any of the preceding embodiments, wherein the secretion organelle is the endoplasmatic reticulum and/or the Golgi apparatus, preferably the Golgi apparatus. The production host of any of the preceding embodiments, wherein the production host further comprises a lactose transporter targeted to the host cell membrane. The production host of any of the preceding embodiment, wherein the lactose transporter targeted to the secretion organelle membrane comprises a heterologous localization sequence for integration in the secretion organelle membrane. The production host of any of the preceding embodiments 6 or 7, wherein lactose transporter is targeted for integration in the secretion organelle membrane by a heterologous localization sequence, wherein the localization sequence comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 40, 42, 44, 46, 48, 50, 52, or 54. The production host of embodiment 8, wherein the heterologous localization sequence is derived from the production host or an organism of genera Saccharomyces, Rattus or Homo. The production host of any of the preceding embodiments, wherein

the fucosyltransferase targeted to a secretion organelle, and/or

the sialyltransferase targeted to a secretion organelle

comprises a heterologous localization sequence for localization in the secretion organelle lumen. The production host of any of the preceding embodiments, wherein the heterologous localization sequence for localization of the fucosyltransferase in the secretion organelle lumen comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38. The production host of embodiment 1 1 , wherein the heterologous localization sequence is derived from the production host or an organism of genera Rattus or Homo. The production host of any of the preceding embodiments, wherein the heterologous localization sequence for localization of the sialyltransferase in the secretion organelle lumen comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38. The production host of embodiment 12, wherein the heterologous localization sequence is derived from the production host or an organism of genera Rattusor Homo. The production host of any of the preceding embodiments, wherein

a) the lactose transporter targeted to the secretion organelle membrane and/ or to the host cell membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 22, 24, 26, 28, 30, 32, 34, 36, or 106, and/or b) the GDP-fucose transporter targeted to a secretion organelle membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 12, and/ or

c) the cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 82, and/ or

d) the fucosyltransferase targeted to a secretion organelle comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 20, and/or e) the sialyltransferase targeted to a secretion organelle comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 98, 100, 102, or 104. The production host of any of the preceding embodiments, wherein the metabolic pathway for the production of GDP-fucose comprises f) one or more lyases, preferably a hydrolyase, even more preferably GDP mannose 4, 6- dehydratase; and one or more oxidoreductase, preferably GDP L- fucose synthase. The production host of any of the preceding embodiments, wherein the metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic acid comprises g) one or more enzymes preferably selected from GNE, NANS, NANP, and CMAS. The production host of any of the preceding embodiments, wherein the production host is a yeast,

preferably of order Saccharomycetales or Schizosaccharomycetales,

more preferably of family Alloascoideaceae, Ascoideaceae, Cephaloascaceae, Debaryomycetaceae, Dipodascaceae, Endomycetaceae, Lipomycetaceae, Metschnikowiaceae, Phaffomycetaceae, Pichiaceae, Saccharomycetaceae, Saccharomycodaceae, Saccharomycopsidaceae, Trichomonascaceae,

Trigonopsidaceae or Schozosaccharomycetaceae

more preferably of genus Alloascoidea, Ascoidea, Cephaloascus, Babjeviella, Debaryomyces, Enteroramus, Hyphopichia, Kurtzmaniella, Meyerozyma, Millerozyma, Nematodospora, Peterozyma, Priceomyces, Scheffersomyces, Schwanniomyces, Spathaspora, Suhomyces, Teunomyces, Wickerhamia, Yamadazyma, Arxula, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete, Sporopachydermia, Yarrowia, Endomyces, Dipodascopsis, Lipomyces, Zygozyma, Aciculoconidium, Clavispora, Danielozyma, Kodamaea, Metschnikowia, Nectaromyces, Barnettozyma, Cyberlindnera, Komagataella, Phaffomyces, Starmera, Wickerhamomyces, Allodekkera, Brettanomyces, Kregervanrija, Martiniozyma, Nakazawaea, Ogataea, Pichia (synonym: Hansenula), Saturnispora, Arxiozyma, Citeromyces, Cyniclomyces, Eremothecium, Hagleromyces, Issatchenkia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Pachysolen, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Yueomyces, Zygosaccharomyces, Zygotorulaspora, Zygowilliopsis, Hanseniaspora, Saccharomycodes, Arthroascus, Saccharomycopsis, Blastobotrys, Diddensiella, Groenewaldozyma, Spencermartinsiella, Sugiyamaella, Trichomonascus, Wickerhamiella, Zygoascus, Botryozyma, Tortispora, Trigonopsis, Ambrosiozyma, Candida, Deakozyma, Diutina, Kuraishia, Macrorhabdus, Metahyphopichia, Middelhovenomyces, Myxozyma, Nadsonia, Schizoblastosporion, Starmerella, Sympodiomyces or Schizosaccharomyces,

more preferably of species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces hubeiensis, Kluyveromyces lactis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces wickerhamii, Kluyveromyces marxianus, Kluyveromyces nonfermentans, Kluyveromyces siamensis, Kluyveromyces wickerhamii, Pichia barkeri, Pichia bruneiensis, Pichia cactophila, Pichia cecembensis, Pichia cephalocereana, Pichia chibodasensis, Pichia deserticola, Pichia dushanensis, Pichia eremophila, Pichia exigua, Pichia fermentans, Pichia garciniae, Pichia heedii, Pichia insulana, Pichia kluyveri, Pichia kudriavzevii, Pichia manshurica, Pichia membranifaciens, Pichia nakasei, Pichia nongkratonensis, Pichia norvegensis, Pichia occidentalis, Pichia porticicola, Pichia pseudocactophila, Pichia punctispora, Pichia rarassimilans, Pichia scaptomyzae, Pichia scutulata, Pichia sporocuriosa, Pichia terricola, Pichia membranifaciens, Pichia species, Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomyces cf. kudriavzevii, Saccharomyces bayanus, Saccharomyces kudriavzevii, Saccharomyces bayanus, Saccharomyces uvarum, Saccharomyces cariocanus, Saccharomyces cerevisiae (baker's yeast), Saccharomyces bayanus, Saccharomyces eubayanus, Saccharomyces eubayanus, Saccharomyces uvarum, Saccharomyces cf. kudriavzevii, Saccharomyces kudriavzevii, Saccharomyces kudriavzevii, Saccharomyces bayanus, Saccharomyces kudriavzevii, Saccharomyces uvarum, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces chevalieri, Saccharomyces douglasii, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces eubayanus, Saccharomyces uvarum, Saccharomyces jurei, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces pastorianus, Saccharomyces uvarum, Schizosaccharomyces cryophilus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, Schizosaccharomyces kambucha, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, Schizosaccharomyces pombe (fission yeast), Schizosaccharomyces versatilis, Yarrowia alimentaria, Yarrowia bubula, Yarrowia deformans, Yarrowia divulgata, Yarrowia galli, Yarrowia hollandica, Yarrowia keelungensis, Yarrowia lipolytica, Yarrowia osloensis, Yarrowia parophonii, Yarrowia phangngaensis, Yarrowia porcina or Yarrowia yakushimensis,

most preferably of genus Saccharomyces. A transporter targeted to a secretion organelle membrane, wherein the transporter is a) a lactose transporter, preferably the lactose transporter comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 22, 24, 26, 28, 30, 32, 34, 36, or 106, and/or

A nucleic acid molecule

a) comprising a nucleic acid sequence encoding a lactose transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 21 , 23, 25, 27, 29, 31 , 33, 35, or 105,

and at least one nucleic acid sequence encoding any one of b) a GDP-fucose transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 11 , wherein the nucleic acid sequence is codon optimized; and/ or

c) a cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 81 , wherein the nucleic acid sequence is codon optimized; and/ or

d) a fucosyltransferase targeted to the secretion organelle and having at least 70% sequence identity to SEQ ID No. 19, and/or

e) a sialyltransferase targeted to the secretion organelle and having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103.

A nucleic acid construct for the expression of

a) a lactose transporter targeted to the secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 21 , 23, 25, 27, 29, 31 , 33, 35, or 105,

and at least one polypeptide selected of

b) a GDP-fucose transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 1 1 ; and/or c) a cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 81 ; and/or

d) a fucosyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 19, and/or

e) a sialyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103. A nucleic acid construct of embodiment 21 , wherein the nucleic acid construct is an artificial chromosome. An artificial chromosome comprising a nucleic acid sequence for the expression of a) the lactose transporter targeted to the secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 21 , 23, 25, 27, 29, 31 , 33, 35, or 105, and at least one of

b) the GDP-fucose transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 11 , and/ or;

c) the cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 81 ; and/ or

d) the fucosyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 19; and/or

e) the sialyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103, and/or f) one or more lyases, preferably a hydrolyase, even more preferably, GDP mannose 4, 6- dehydratase; and oxidoreductase, preferably GDP L- fucose synthase; and/ or g) one or more enzymes preferably selected from GNE, NANS, NANP, and CMAS. The artificial chromosome of embodiment 21 , comprising the nucleic acid sequences according to a); b); c); d); e); f); g); a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and d); b) and f); b) and d) and f); c) and e); c) and g); c) and e) and g); a) and b) and d); a) and b) and f); a) and b) and d) and f); a) and c) and e); a) and c) and g); a) and c) and e) and g); or all of a), b), c), d), e), f), g), in any combination. A production host according to any of embodiments 1-18, comprising a nucleic acid according to embodiment 20, and/ or a nucleic acid construct according to any of embodiments 21 - 22, and/or preferably an artificial chromosome according to any of embodiments 23- 24. A method for production of a human milk oligosaccharide, comprising the steps of i) providing a production host according to any of embodiments 1-18 or 25, and ii) culturing the production host in the presence of lactose in a fermentation medium comprising an energy source, wherein the energy source is one or more of glycerol, succinate, malate, pyruvate, lactate, ethanol and citrate. The method of production of embodiment 26, further comprising the step of

iii) obtaining the human milk oligosaccharides from the production host and/or preferably from the fermentation medium. The method of production of any of embodiments 26- 27, wherein the human milk oligosaccharide is a fucosylated and/or sialylated oligosaccharide, more preferably selected from

a fucosylated oligosaccharide, more preferably 1 ,2-fucosyllactose, 2'-fucosyllactose, 3’-fucosyllactose or difucosyllactose, most preferably 2'-fucosyllactose, and/or

a sialylated oligosaccharide, more preferably 3'-sialyllactose or 6'-sialyllactose, or 3'- sialyl-3-fucosyllactose. Use of a heterologous localization sequence for targeting a polypeptide to a secretion organelle or a secretion organelle membrane, wherein

the polypeptide is selected from

a) a lactose transporter targeted to a secretion organelle membrane, and/ or b) a GDP- fucose transporter targeted to a secretion organelle membrane, and/ or c) a cytidine 5’- monophosphate N-acetylneuraminicc acid transporter targeted to a secretion organelle membrane, and/ or

d) a fucosyltransferase targeted to a secretion organelle, and/ or

e) a sialyltransferase targeted to a secretion organelle. Use of a heterologous localization sequence of embodiment 29, wherein the localization sequence is selected from,

a’) the sequence for localization of the lactose transporter, comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 40, 42, 44, 46, 48, 50, 52, 54, and/ or d’) the sequence for localization of the fucosyl transferase, comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38, and/ or

e’) the sequence for localization of the sialytransferase, comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38. and the polypeptide is selected from

a) the lactose transporter comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 22, 24, 26, 28, 30, 32, 34, 36 or 106, and/ or

b) the GDP-fucose transporter comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 12, and/ or

c) the cytidine 5’- monophosphate N-acetylneuraminic acid transporter comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 82, and/ or

d) the fucosyl transferase comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 20, and/ or

e) the sialytransferase comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 98, 100, 102 or 104.

31 . The use according to any of embodiments 29- 30, wherein

the secretion organelle is the endoplasmatic reticulum and/or the Golgi apparatus, preferably the Golgi apparatus, preferably of a yeast of

order Saccharomycetales or Schizosaccharomycetales,

Trigonopsidaceae or Schozosaccharomycetaceae

most preferably of genus Saccharomyces.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

Figure 1 ) provides the design of nucleic acid constructs. A) schematic of nucleic acid construct for first generation lactose transport modules, B) schematic of second-generation lactose transport modules, C) schematic of 2’-fucosyllactose production modules, D) schematic of 6’-sialyllactose production modules, E) schematic of 3’- sialyllactose production modules, F) legend showing annotations of parts encoded on nucleic acid constructs.

Figure 2) provides results of galactose assimilation assay in S. cerevisiae gal2A strain.

Figure 3) provides results of growth assay to test localization of lactose permeases.

Figure 4) provides results for lactose toxicity in wild type yeast cell with plasma membrane (PM) lactose permease (LP) expression vs. PM LP and Golgi LP expression.

Figure 5) provides LCMS analysis of standard curve, 2’- fucosyllactose.

Figure 6) provides LCMS detection of 2’- fucosyllactose in production host (yeast) culture supernatant.

Figure 7) provides LCMS analytical detection of 6’-sialyllactose and 3’-sialyllactose using hydrophilic interaction liquid chromatography (HILIC).

Figure 8) provides LCMS analysis of standard curve, (a) 3’-sialyllactose and (b) 6’- sialyllactose.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense.

Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless stated otherwise or apparent from the nature of the definition, the definitions apply to all methods and uses described herein. As used in this specification and in the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise. In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, and even more preferably ±5 %.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" etc. and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. relate to steps of a method or use or assay, there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below.

It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, a number of terms are defined below.

Definitions

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of the ordinary skill in the art to which this invention pertains.

The term "human milk oligosaccharide" or "HMO", unless otherwise specified, refers generally to any number of complex carbohydrates found in the human breast milk that can be in acidic or neutral form, and to precursors thereof. Non-limiting examples of some of the prominent neutral HMOs are 1 , 2-fucosyllactose, 2’-fucosyllactose, 3’- fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, lacto-N-fucopentose I, lacto-N-fucopentose II, lacto-N-hexose, iso-lacto-N-octaose, iso- lacto-N-neooctaose, and para-lacto-N-octaose. Besides such neutral HMOs, acidic HMOs can also be found in human milk, such as 3’-sialyllactose, 6’-sialyllactose, 3-sialyl-3-fucosyllactose, and disial- lacto-N-tetraose.

The term "production host" as used herein means a cell that comprises a metabolic pathway for the production of one or more HMOs, wherein preferably at least one step of the pathway is performed in the secretion organelle, i.e. an endoplasmic reticulum and/ or Golgi apparatus. The production host preferably contains a lactose transporter, and/ or a fucose transporter, and/ or a cytidine 5’- monophosphate N-acetylneuraminic acid transporter, and/ or a fucosyltransferase, and/ or a sialyltransferase, all transporters - where present - targeted to the secretion organelle membrane and enzymes targeted to the secretion organelle of the production host, and a metabolic pathway and heterologous enzymes for the production of GDP- fucose in the cytoplasm, preferably for the production of GDP- fucose from GDP- mannose, and optionally a metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic acid in the cytoplasm, preferably for the production of cytidine 5’- monophosphate N-acetylneuraminic acid from UDP- N-acetyl- glucosamine. The term“secretion organelle” refers to any cell organelle that is involved in secretory functions in the cell. The preferred examples of secretion organelles are endoplasmic reticulum (ER) and Golgi apparatus.

Heterologous localization sequence refers to a peptide sequence which directs a protein having such sequence to be transported to and retained in a cellular compartment or the membrane of the cellular compartment. In the present context, a heterologous localization sequence preferably directs a lactose transporter for integration in the secretion organelle membrane and/or directs enzymes fucosyltransferase and/or sialyltransferase for localization in the secretion organelle lumen. It is understood that the heterologous localization sequence is either heterologous to the production host and/or heterologous to the respective polypeptide.

The term“heterologous genes” refers to a gene which is introduced into the cell from outside, i.e. which is not naturally present in the production host cell. In the context of the present invention, heterologous genes in the production host are genes coding for lactose transporter, GDP-fucose transporter, cytidine 5’- monophosphate N-acetylneuraminic acid transporter, fucosyltransferase, sialyltransferase, and genes coding for heterologous enzymes involved in metabolic pathway for the production of GDP- fucose and cytidine 5’- monophosphate N-acetylneuraminic acid.

The term“heterologous enzymes” refers to enzymes that do not naturally occur in the production host cell. In the present context, heterologous enzymes are fucosyltransferase, sialyltransferase, and the enzymes involved in metabolic pathway for the production of GDP- fucose, preferably GDP mannose 4, 6- dehydratase and GDP L-fucose synthase and the enzymes involved in metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic acid, preferably GNE, NANS, NANP, and CMAS.

The terms“nucleic acid”, "nucleic acid sequence" or“nucleic acid molecule” have their usual meaning and may include a nucleotide or polynucleotide sequence, and fragments or portions thereof, as well as to DNA, cDNA, and RNA of genomic or synthetic origin which may be double-stranded or single- stranded, whether representing the sense or antisense strand. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may encode a given protein. The nucleic acid sequences used in the present invention further encompass codon-optimized sequences. A nucleic acid is codon-optimized by systematically altering codons in recombinant DNA to be expressed in a production host cell other than the cell from which the nucleic acid was isolated so that the codons match the pattern of codon usage in the organism used for expression and thereby to enhance yields of an expressed protein. The codon-optimized sequence nevertheless encodes a protein with the same amino acid sequence as the native protein.

A particular nucleotide sequence, for example a sequence unerlying a particular heterologous gene or a promoter sequence etc. can either be amplified by polymerase chain reaction from the genomic sequences of a particular organism from which they are derived from, or it can be chemically synthesized by method known the art.

Sequence identity usually is provided as“% sequence identity” or“% identity”. To determine the percent-identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program“NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

The following example is meant to illustrate two nucleotide sequences, but the same calculations apply to protein sequences:

Seq A: AAGATACTG length: 9 bases

Seq B: GATCTGA length: 7 bases

Hence, the shorter sequence is sequence B.

Producing a pairwise global alignment which is showing both sequences over their complete lengths results in Seq A: AAGATACTG- Seq B: -GAT-CTG A

The Ί” symbol in the alignment indicates identical residues (which means bases for DNA or amino acids for proteins). The number of identical residues is 6.

The symbol in the alignment indicates gaps. The number of gaps introduced by alignment within the Seq B is 1. The number of gaps introduced by alignment at borders of Seq B is 2, and at borders of Seq A is 1.

The alignment length showing the aligned sequences over their complete length is 10.

Producing a pairwise alignment which is showing the shorter sequence over its complete length according to the invention consequently results in:

Seq A: GATACTG-

Seq B: GAT-CTG A

Producing a pairwise alignment which is showing sequence A over its complete length according to the invention consequently results in:

Seq A: AAGATACTG

Seq B: -GAT-CTG

Producing a pairwise alignment which is showing sequence B over its complete length according to the invention consequently results in:

Seq A: GATACTG- Seq B: GAT-CTGA

The alignment length showing the shorter sequence over its complete length is 8 (one gap is present which is factored in the alignment length of the shorter sequence).

Accordingly, the alignment length showing Seq A over its complete length would be 9 (meaning Seq A is the sequence of the invention).

Accordingly, the alignment length showing Seq B over its complete length would be 8 (meaning Seq B is the sequence of the invention).

After aligning two sequences, in a second step, an identity value is determined from the alignment produced. For purposes of this description, percent identity is calculated by %-identity = (identical residues / length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give“%-identity”. According to the example provided above, %-identity is: for Seq A being the sequence of the invention (6 / 9) * 100 = 66.7 %; for Seq B being the sequence of the invention (6 / 8) * 100 =75%.

To determine the percent-identity between two nucleic acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e. , a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program“NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters for nucleic acid alignments (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).

For calculating the percent identity of two nucleic acid sequences sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For nucleic acid sequences encoding for a protein or a peptide, the pairwise alignment shall be made over the complete length of the coding region of the sequence of this invention. Introns present in the other sequence may be removed for the pairwise alignment to allow comparison with the sequence of this invention. Percent identity is then calculated by:

%-identity = (identical residues / length of the alignment region which is showing the coding region of the sequence of this invention over its complete length) *100.

As used herein the term "gene" means a segment of DNA involved in producing a polypeptide chain that may or may not include regions preceding and following the coding regions (e.g. 5' untranslated (5' UTR) or leader sequences and 3' untranslated (3' UTR) or trailer sequences, as well as intervening sequence (introns) between individual coding segments (exons)).

The term“coding for" as used herein has its usual meaning and may include, but are not limited to, for example, the property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.

As used herein "amino acid sequences" refers to peptide or protein sequences or portions thereof.

As used herein, the term "nucleic acid construct" refers to a DNA molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (for example, at least to a promoter).

As used herein, the term "artificial chromosome" refers to nucleic acid constructs that contain all the structural elements of natural chromosome, preferably linear, which contains one origin of replication, a centromere, and two telomeric sequences. It is also preferable to provide each construct with at least one selection marker, such as gene to impart drug resistance, or to complement a host metabolic lesion. The presence of markers is useful in the selection of the transformants. For example, in yeast, the URA3, HIS3, LYS2, TRP1 , SUC2, G418, BLA, HPH, or SH BLE genes may be used. The artificial chromosome of the present invention also comprises heterologous genes coding for lactose transporter, GDP-fucose transporter, cytidine 5’- monophosphate N- acetylneuraminic acid transporter, fucosyltransferase, sialyltransferase, and the genes coding for heterologous enzymes involved in metabolic pathway for the production of GDP- fucose.

As used herein, the term "vector" refers to a polynucleotide designed to introduce nucleic acids into one or more host cells. In preferred embodiments, vectors autonomously replicate in different host cells. The term is intended to encompass, but is not limited to cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

As used herein, the term "promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream gene. In preferred embodiments, the promoter is appropriate to the production host cell in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

As used herein, the term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3’ processing and polyadenylation of a primary transcript and termination of transcription.

As used herein, the term "selection marker" refers to a protein capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector or an artificial chromosome.

As used herein, the term "transformed" refers to a cell that has a non-native (heterologous) polynucleotide sequence integrated into its genome or located on an artificial chromosome or plasmid. As used herein the term "express/ expression" refers to a process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

Metabolic pathway for the production of GDP- fucose: GDP- fucose can be generated through two distinct metabolic pathways: the de novo or salvage pathway. In the de novo pathway, GDP- fucose is synthesized from mannose-6-phosphate by GDP mannose 4,6-dehydratase and GDP- fucose synthase. The alternative salvage pathway requires L-fucose as the substrate for producing GDP-L fucose. This pathway is preferably catalyzed by a bifunctional enzyme, L-fucokinase/GDPI-fucose phosphorylase (FKP). The present invention preferably utilizes the de novo pathway for the production of GDP-fucose.

Further, the metabolic pathways for the production of 6’-sialyllactose and 3’-sialyllactose differ by a single gene, the sialyltransferase, and hence the two pathways are described in parallel. Cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) serves as a substrate (along with lactose) for both 6’-sialyllactose and 3’-sialyllactose synthesis in the Golgi apparatus lumen. The cytosolic production of CMP-Neu5Ac requires the expression of 4 genes, namely, i) GNE, a bifunctional enzyme that converts UDP-N-acetyl-glucosamine, an endogenous yeast metabolite, into N-acetylmannosamine 6-phosphate (ManNAc-6-P), ii) NANS, which uses ManNAc-6-P as a substrate to generate a phosphorylated form of N-acetylneuraminic acid (Neu5Ac-9-phosphate), iii) NANP, which dephosphorylates Neu5Ac-9-phosphate to produce Neu5Ac, and iv) CMAS, which converts (Neu5Ac) to CMP-Neu5Ac. The pathways are catalyzed by ST3 beta-galactoside alpha- 2, 3-sialyltransferase 4 and ST6 beta-galactoside alpha- 2, 6-sialyltransferase 1 for the production of 3’-sialyllactose and 6’-sialyllactose, respectively, from CMP-Neu5Ac and lactose.

The presently claimed invention provides a production host that comprises a metabolic pathway for the production of HMOs in the secretion organelle i.e. an endoplasmic reticulum and/ or Golgi apparatus, and contains a lactose transporter, and/ or a fucose transporter, and/ or a Cytidine 5'- monophosphate N-acetylneuraminic acid, and/ or a fucosyltransferase, and/ or a sialyltransferase, the transporters targeted to the secretion organelle membrane and the enzymes targeted to the secretion organelle lumen of the production host, and a metabolic pathway and heterologous enzymes for the production of GDP- fucose in the cytoplasm, preferably for the production of GDP- fucose from GDP- mannose, and/or a metabolic pathway and heterologous enzymes for the production of Cytidine 5'-monophosphate N-acetylneuraminic acid from UDP-N-acetyl-glucosamine.

The production host preferably is a yeast, preferably of genus Alloascoidea, Ascoidea, Cephaloascus, Babjeviella, Debaryomyces, Enteroramus, Hyphopichia, Kurtzmaniella, Meyerozyma, Millerozyma, Nematodospora, Peterozyma, Priceomyces, Scheffersomyces, Schwanniomyces, Spathaspora, Suhomyces, Teunomyces, Wickerhamia, Yamadazyma, Arxula, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete, Sporopachydermia, Yarrowia, Endomyces, Dipodascopsis, Lipomyces, Zygozyma, Aciculoconidium, Clavispora, Danielozyma, Kodamaea, Metschnikowia, Nectaromyces, Barnettozyma, Cyberlindnera, Komagataella, Phaffomyces, Starmera, Wickerhamomyces, Allodekkera, Brettanomyces, Kregervanrija, Martiniozyma, Nakazawaea, Ogataea, Pichia (synonym: Hansenula), Saturnispora, Arxiozyma, Citeromyces, Cyniclomyces, Eremothecium, Hagleromyces, Issatchenkia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Pachysolen, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Yueomyces, Zygosaccharomyces, Zygotorulaspora, Zygowilliopsis, Hanseniaspora, Saccharomycodes, Arthroascus, Saccharomycopsis, Blastobotrys, Diddensiella, Groenewaldozyma, Spencermartinsiella, Sugiyamaella, Trichomonascus, Wickerhamiella, Zygoascus, Botryozyma, Tortispora, Trigonopsis, Ambrosiozyma, Candida, Deakozyma, Diutina, Kuraishia, Macrorhabdus, Metahyphopichia, Middelhovenomyces, Myxozyma, Nadsonia, Schizoblastosporion, Starmerella, Sympodiomyces or Schizosaccharomyces. Most preferably the genus is Saccharomyces.

According to the present invention the HMO is preferably a fucosylated and/ or sialylated oligosaccharide. Preferably, the fucosylated oligosaccharide is selected from the group consisting of 2- fucosyllactose, 2’- fucosyllactose, 3’- fucosyllactose, difucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, lacto-N-fucopentose I, lacto-N-fucopentose II, lacto-N-hexose, iso-lacto-N-octaose, iso- lacto-N-neooctaose, and para-lacto-N-octaose, and the sialylated oligosaccharide is selected from the group consisting of 3’- sialyllactose, 6’- sialyllactose, 3-sialyl-3-fucosyllactose, and disial-lacto-N- tetraose. More preferably, the fucosylated oligosaccharide is selected from 1 ,2-fucosyllactose, 2'- fucosyllactose, 3-fucosyllactose or difucosyllactose, and the sialylated oligosaccharide is selected from 3'-sialyllactose or 6'-sialyllactose, or 3'-sialyl-3-fucosyllactose. Most preferably, the fucosylated oligosaccharide is 2'-fucosyllactose; and the sialylated oligosaccharide most preferably is 3'- sialyllactose or 6'-sialyllactose.

The secretion organelle is the endoplasmic reticulum or the Golgi apparatus, preferably the Golgi apparatus, and even more preferably the Golgi apparatus of S. cerevisiae.

Preferably the metabolic pathway for the production of GDP-fucose is the de novo pathway, wherein GDP- fucose is produced from GDP- mannose, a metabolite native to Saccharomyces, preferably in the presence of GDP- mannose 4, 6- dehydratase and GDP L- fucose synthase. Preferably, the conversion of GDP- mannose to GDP- fucose takes place in the cytoplasm.

The genes coding for GDP- mannose 4, 6- dehydratase and GDP L- fucose synthase are preferably derived from a plant, preferably from the genus Arabidopsis.

Also preferably the genes the GDP- mannose 4, 6- dehydratase and GDP L- fucose synthase are derived from E. coli, preferably from the GmD and WcaG genes from E. coli K-12.

In the metabolic pathway for the production of Cytidine 5'-monophosphate N-acetylneuraminic acid, UDP-N-acetyl-glucosamine, a metabolite native to Saccharomyces, is converted to Cytidine 5'- monophosphate N-acetylneuraminic acid, preferably in the presence of enzymes GNE, NANS, NANP and CMAS.

The genes coding for any of the enzymes GNE, NANS, NANP and CMAS are preferably derived from the genus Homo and Danio. Most preferably, the enzymes GNE, NANS, NANP and CMAS are derived from Homo sapiens and Danio rerio, Dre.

The production host preferably further comprises a lactose transporter targeted to the plasma membrane of the host cell.

The lactose transporter is derived from an organism that can import lactose. Typically, such organism is able to metabolize lactose. Thus, by selection for lactose metabolism it is easy to obtain suitable microorganisms comprising a suitable gene coding for a lactose transporter. Preferably the lactose transporter gene is derived from an organism selected from the genus Alloascoidea, Ascoidea, Cephaloascus, Babjeviella, Debaryomyces, Enteroramus, Hyphopichia, Kurtzmaniella,

Meyerozyma, Millerozyma, Nematodospora, Peterozyma, Priceomyces, Scheffersomyces, Schwanniomyces, Spathaspora, Suhomyces, Teunomyces, Wickerhamia, Yamadazyma, Arxula, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete, Sporopachydermia, Yarrowia, Endomyces, Dipodascopsis, Lipomyces, Zygozyma, Aciculoconidium, Clavispora, Danielozyma, Kodamaea, Metschnikowia, Nectaromyces, Barnettozyma, Cyberlindnera, Komagataella, Phaffomyces, Starmera, Wickerhamomyces, Allodekkera, Brettanomyces,

Kregervanrija, Martiniozyma, Nakazawaea, Ogataea, Pichia (synonym: Hansenula), Saturnispora, Arxiozyma, Citeromyces, Cyniclomyces, Eremothecium, Hagleromyces, Issatchenkia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Pachysolen, Saccharomyces,

Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Yueomyces, Zygosaccharomyces, Zygotorulaspora, Zygowilliopsis, Hanseniaspora, Saccharomycodes, Arthroascus, Saccharomycopsis, Blastobotrys, Diddensiella, Groenewaldozyma, Spencermartinsiella, Sugiyamaella, Trichomonascus, Wickerhamiella, Zygoascus, Botryozyma, Tortispora, Trigonopsis, Ambrosiozyma, Candida, Deakozyma, Diutina, Kuraishia, Macrorhabdus, Metahyphopichia, Middelhovenomyces, Myxozyma, Nadsonia, Schizoblastosporion, Starmerella, Sympodiomyces or Schizosaccharomyces. Most preferably the genus is Saccharomyces..

A preferred lactose transporter is the Lactose permease 12 (Lac12) from Kluveromyces lactis and/or cellodextrin transporter CDT-1 from Neurospora crassa.

The lactose transporter targeted for the Golgi apparatus membrane of the production host preferably comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 22, 24, 26, 28, 30, 32, 34, 36 or 106, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to any of the aforementioned sequences.

The lactose transporter preferably comprises a heterologous localization sequence for integration in the Golgi apparatus membrane. The production host preferably comprises a lactose transporter targeted to the plasma membrane of the production host and a lactose transporter targeted to the Golgi apparatus membrane. The heterologous localization sequence for integration of the lactose transporter in the Golgi apparatus membrane of the production host preferably is derived from a production host organism, most preferably from the production host organism.

Preferably, the GDP- fucose transporter is derived from an organism as described herein in view of the lactose transporter gene.

The GDP- fucose transporter targeted to the Golgi apparatus membrane of the production host preferably comprises a heterologous targeting sequence for targeting to the secretion organelle, wherein the targeting sequence comprises or constists of an amino acid sequence having at least 70% sequence identity to a sequence according to Table 3, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to a sequence according to Table 3.

The GDP- fucose transporter either naturally localizes to the Golgi apparatus membrane and/or is targeted to the Golgi apparatus membrane by a heterologous targeting sequence.

Preferably, the cytidine 5'-monophosphate N-acetylneuraminic acid transporter is derived from an organism selected from any mammalian species.

The cytidine 5'-monophosphate N-acetylneuraminic acid transporter targeted to the Golgi apparatus membrane of the production host preferably comprises a heterologous targeting sequence for targeting to the secretion organelle, wherein the targeting sequence comprises or constists of an amino acid sequence having at least 70% sequence identity to a sequence according to Table 3, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to a sequence according to Table 3.

The cytidine 5'-monophosphate N-acetylneuraminic acid transporter naturally localizes to the Golgi apparatus membrane and/or is targeted to the Golgi apparatus membrane by a heterologous targeting sequence. The fucosyltransferase is preferably derived from a mammalian species.

The fucosyltransferase targeted for the Golgi apparatus of the production host preferably comprises a heterologous targeting sequence for targeting to the secretion organelle, wherein the targeting sequence comprises or constists of an amino acid sequence having at least 70% sequence identity to a sequence according to Table 3, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to a sequence according to Table 3.

In an embodiment, the fucosyltransferase comprises a heterologous localization sequence for localization of fucosyltransferase in the lumen of the Golgi apparatus.

In an embodiment, the heterologous localization sequence for localization of fucosyltransferase in the lumen of Golgi apparatus of the production host is derived from an organism as described for the lactose transporter gene above.

Preferably, the heterologous localization sequence for localization of fucosyltransferase in the lumen of the Golgi apparatus of the production host comprises or constists of an amino acid sequence having at least 70% sequence identity to a sequence according to Table 3, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to a sequence according to Table 3.

In an embodiment, the sialyltransferase is derived from an organism selected from the genus Homo, Helicobactor, Bacteroides, and Escherichia. Preferably, the sialyltransferase is derived from Homo sapiens, Helicobactor pyroli, Escherichia coli, or Bacteriodes fragilis. More preferably, the sialyltransferase is derived from Homo sapiens, Bacteriodes fragilis 9343, or Escherichia coli 126. Most preferably, the sialyltransferase is derived from Homo sapiens.

The sialyltransferase targeted for the Golgi apparatus of the production host comprises a heterologous targeting sequence for targeting to the secretion organelle, wherein the targeting sequence comprises or constists of an amino acid sequence having at least 70% sequence identity to a sequence according to Table 3, preferably having at least 75%, more preferably having at least 80%, even more preferably having at least 85%, further more preferably having at least 90%, and most preferably having at least 95% sequence identity to a sequence according to Table 3.

In an embodiment, the sialyltransferase comprises a heterologous localization sequence for localization of sialyltransferase in the lumen of the Golgi apparatus.

In an embodiment, the heterologous localization sequence for localization of sialyltransferase in the lumen of Golgi apparatus of of the production host is derived from an organism selected from the genus Rattus.

The heterologous localization sequence for localization of sialyltransferase preferably is for the lumen of the Golgi apparatus of the production host.

Related heterologous genes coding for lactose transporters, localization sequence for targeting the lactose transporter to the secretion organelle, GDP-fucose transporter, Cytidine 5'-monophosphate N-acetylneuraminic acid transporter, fucosyltransferase, sialyltransferase, localization sequences for targeting fucosyltransferase, sialyltransferase to the secretion organelle, genes encoding enzymes involved in the production of GDP-fucose, and genes encoding enzymes involved in the production of Cytidine 5'-monophosphate N-acetylneuraminic acid, from other genera and species can be easily identified by any method known to the skilled person, e.g. by performing sequence comparisons or functional complementation assays.

The production host comprises a) a lactose transporter targeted to the plasma membrane and/ or Golgi apparatus membrane of the production host and/ or b) a GDP- fucose transporter targeted to the Golgi apparatus membrane of the production host and/ or; c) a Cytidine 5'-monophosphate N- acetylneuraminic acid transporter targeted to the Golgi apparatus membrane of the production host and/ or d) a fucosyltransferase targeted to the Golgi apparatus of the production host and/ or; e) a sialyltransferase targeted to the Golgi apparatus of the production host The production host preferably further comprises enzymes involved in the metabolic pathway for the production of GDP- fucose from GDP- mannose, preferably GDP- mannose 4, 6- dehydratase and GDP L- fucose synthase, preferably from a plant.

The production host preferably further comprises enzymes involved in the metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic from UDP-N-acetyl-glucosamine, preferably GNE, NANS, NANP and CMAS, preferably from Homo sapiens or Danio rerio. he present invention provides an artificial chromosome comprising a nucleic acid construct for the expression of a) a lactose transporter targeted to the Golgi apparatus membrane of the production host and/ or b) a GDP- fucose transporter targeted to the Golgi apparatus membrane of the production host and/ or; c) a cytidine 5’- monophosphate N-acetylneuraminic transporter targeted to the Golgi apparatus membrane of the production host and/ or d) a fucosyltransferase targeted to the Golgi apparatus of the production host and/ or; e) a sialyltransferase targeted to the Golgi apparatus of the production host and/ or f) GDP mannose 4, 6- dehydratase and GDP L- fucose synthase; and/ or g) GNE, NANS, NANP and CMAS.

In an embodiment, the artificial chromosome further comprises h) at least one yeast replication origin and one centromere, preferably, ORI and ARS1/CEN4 and/or i) a selection marker, preferably URA3; and/ or j) at least one E. coli replication origin and 1 antibiotic resistance gene, preferably ampicillin resistance gene, and/or k) at least one lox P sequence.

In an embodiment, the artificial chromosome comprises the nucleic acid sequences according to a); b); c); d); e); f); g); a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and c); b) and d); b) and e); b) and f); b) and g); a) and b) and c); a) and b) and d); a) and b) and e); a) and b) and f); b) and d) and c); b) and d) and e); b) and d) and f); c) and d); c) and e); c) and f); c) and g); c) and e) and g); c) and d) and g); c) and d) and f); a) and b) and d) and f); a) and c) and e); a) and c) and g); a) and c) and e) and g); or all of a), b), c), d), e), f), g), in any combination.

In a preferred embodiment, the artificial chromosome comprises the nucleic acid sequences according to a); b); c); d); e); f); g); a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and d); b) and f); b) and d) and f); c) and e); c) and g); c) and e) and g); a) and b) and d); a) and b) and f); a) and b) and d) and f); a) and c) and e); a) and c) and g); a) and c) and e) and g); or all of a), b), c), d), e), f), g), in any combination.

The present invention provides a method for production of a human milk oligosaccharide, comprising the steps of i) providing a production host, and ii) culturing the production host in the presence of lactose in a fermentation medium comprising an energy source, wherein the energy source is one or more of glycerol, succinate, malate, pyruvate, lactate, ethanol and citrate, and thereby producing the fucosylated and/or sialylated oligosaccharides in the secretion organelle of the production host.

The present invention provides a method for production of a human milk oligosaccharide, comprising the steps of i) providing a yeast production host, and ii) culturing the yeast production host in the presence of lactose in a fermentation medium comprising an energy source, wherein the energy source is one or more of glycerol, succinate, malate, pyruvate, lactate, ethanol and citrate, and thereby producing the fucosylated and/or sialylated oligosaccharides in the secretion organelle of the yeast production host as described herein.

The present invention provides a method for production of a human milk oligosaccharide, comprising the steps of i) providing a yeast production host, and ii) culturing the yeast production host in the presence of lactose in a fermentation medium comprising an energy source, wherein the energy source is one or more of glycerol, succinate, malate, pyruvate, lactate, ethanol and citrate, and thereby producing the fucosylated and/or sialylated oligosaccharides in the secretion organelle of the production host as described herein.

The production method preferably further comprises the step of iii) obtaining the human milk oligosaccharides from the production host and/or preferably from the fermentation medium as during purification of the oligosaccharides some production host cells will be destroyed, thereby releasing further oligosaccharides into the fermentation medium.

The production method according to the invention produces a human milk oligosaccharide which is a fucosylated and/or sialylated oligosaccharide, more preferably selected from a fucosylated oligosaccharide, more preferably 1 ,2-fucosyllactose, 2'-fucosyllactose, 3-fucosyllactose or difucosyllactose, most preferably 2'-fucosyllactose, and/or a sialylated oligosaccharide, more preferably 3'-sialyllactose or 6'-sialyllactose, or 3'-sialyl-3-fucosyllactose.

By means of the above technical solution, the production host of the present invention demonstrates production of HMOs in the secretion organelle of the production host yeast which lowers the risk regarding the heterologous expression in E. coli and provides a more natural mechanism of secretion of HMOs which mimics the human system more closely. The approach used in the present invention for the production of HMOs involves selection of heterologous genes for lactose transporters, localization sequence for targeting the lactose transporter to the secretion organelle, GDP-fucose transporter, cytidine 5’- monophosphate N-acetylneuraminic transporter, fucosyltransferase, sialyltransferase, localization sequences for targeting fucosyltransferase, sialyltransferase to the secretion organelle, and genes encoding enzymes involved in the production of GDP-fucose and cytidine 5’- monophosphate N-acetylneuraminic. The de novo production of GDP- fucose avoids the necessity to feed L-Fucose for providing GDP-Fucose through the salvage pathway and avoids problems with L-Fucose uptake in yeast. Further, the secretion organelle (Golgi apparatus) localized production of HMOs avoid lactose toxicity in the cytosol and facilitates efficient secretion of HMO. The prior art also describes the problems with difucosyllactose as a by-product through additional fucosylation of 2’-fucosyllactose. The approach used in the present invention avoids this through a lower GDP-Fucose concentration through the de novo pathway.

The following examples are provided for illustrative purposes. It is thus understood that the examples are not to be construed as limiting. The skilled person will clearly be able to envisage further modifications of the principles laid out herein.

EXAMPLES

Example 1

Designing the nucleic acid constructs A nucleic acid construct comprising genes coding for lactose transporter was created for transporting lactose to Golgi apparatus membrane of the production host S. cerevisiae. Another nucleic acid construct comprising genes coding for the metabolic pathway for the production of GDP- fucose, GDP-fucose transporter, and fucosyltransferase, was created for the production of 2’- fucosyllactose in the Golgi apparatus of S. cerevisiae.

(i) Lactose transport to Golgi apparatus of S. cerevisiae

To transport lactose from the culture medium to the Golgi apparatus of the production host S. cerevisiae requires a lactose transporter to be expressed at two locations: the plasma membrane (PM) and the Golgi apparatus membrane. Nucleic acid constructs encoding two LAC12 lactose transporter (Lactose permease) derived from Kluyveromyces lactis (K. lactis) were built. The first expressed transporter targeted for the PM of S. cerevisiae. The sequence details for wild type coding sequence (CDS), codon optimized CDS and transporter protein (SEQ ID No.s 1 to 3) are provided below in Table 1. The second nucleic acid construct was designed to encode a lactose transporter which is a chimeric protein with a heterologous localization sequence i.e. a C-terminal signal sequence targeting the chimeric protein for integration in the Golgi apparatus membrane of S. cerevisiae. The sequence details for codon optimized CDS and for chimeric proteins are provided in Table 2 (SEQ IDs NO.s 21 to 36). The sequence details for heterologous localization sequences targeting for Golgi apparatus are provided in Table 3 (SEQ ID No.s 39 to 54). Two independent versions of the PM LAC12 lactose transporter CDS were used. The first (SEQ ID No. 2) demonstrated genetic instability in yeast due to sequence similarity with the Golgi-localized LAC12 CDS. The second LAC12 re-coded gene (SEQ ID No. 105) was designed to be different in DNA sequence to eliminate the genetic instability.

(ii) Production of 2’-fucosyllactose in the Golgi apparatus of S. cerevisiae

For the production of 2’-fucosyllactose in S. cerevisiae, a nucleic acid construct was designed to facilitate cytoplasmic conversion of GDP-mannose, a metabolite native to S. cerevisiae, to GDP- fucose. Two genes from Arabidopsis thaliana (A. thaliana) {MUR1 (SEQ ID NO.s 4 to 6, Table 1), GER1 (SEQ ID NO.s 7 to 9, Table 1)} were selected to produce GDP-fucose. Further, to localize GDP-fucose to the Golgi apparatus of S. cerevisiae, a transporter derived from Homo sapiens (H. sapiens) that naturally localized to Golgi membrane, SLC35C1 was chosen. The sequence details for wild type CDS, codon optimized CDS and transporter protein (SEQ ID No.s 10 to 12) are provided below in Table 1.

To produce 2’- fucosyllactose, a gene encoding fucosyltransferase derived from H. sapiens called FUT2 which produces 2’- fucosyllactose from lactose and GDP-fucose, was selected. The sequence details for wild type CDS, codon optimized CDS and enzyme (SEQ ID No.s 16 to 18) are provided below in Table 1.

FUT2 gene was designed for coding a chimeric protein comprising a heterologous localization sequence for localization in the lumen of Golgi apparatus of S. cerevisiae i.e. an N-terminal Golgi- targeting sequence derived from Rattus norvegicus (R. norvegicus) gene ST6GAL1 (SEQ ID No.s 13 to 15, Table 1). The sequence details for codon optimized CDS and for chimeric proteins are provided in Table 2 (SEQ IDs NO.s 19 to 20). The sequence details for heterologous localization sequences for localization in the lumen of Golgi apparatus are provided in Table 3 (SEQ ID No.s 37 to 38).

(iii) Production of 6’-sialyllactose or 3’-sialyllactose in the Golgi apparatus of S. cerevisiae

Cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) serves as a substrate (along with lactose) for both 6’-sialyllactose and 3’-sialyllactose synthesis in the Golgi apparatus lumen. The cytosolic production of CMP-Neu5Ac requires the expression of 4 genes, namely, i) GNE, ii) NANS, iii) NANP, and iv) CMAS, which converts UDP- N-acetyl-glucosamine to CMP-Neu5Ac.

Human SLC35A1 (SEQ ID No. 81- 82), an integral Golgi membrane protein, was used to localize CMP-Neu5Ac into the Golgi lumen.

Human St3GAL4 (ST3 beta-galactoside alpha- 2, 3-sialyltransferase 4) and St6GAL1 (ST6 beta- galactoside alpha- 2, 6-sialyltransferase 1) were selected for the production of 3’-sialyllactose and 6’-sialyllactose, respectively, from CMP-Neu5Ac and lactose.

All genes were sourced from the human genome. Homologs with <80% sequence identity to the human sequences GNE, NANS, and CMAS were also identified. Thus, the sequences for GNE, NANS, and CMAS were sourced not only from the human genome but also the genome of zebrafish (Danio rerio, Dre). The zebrafish proteins share 80.5, 78.8, and 57.5 % identity with their human homologs, respectively. Further, native and chimeric versions of both St3GAL4 and St6GAL1 were designed to improve the chances of localizing these enzymes to the S. cerevisiae Golgi apparatus membrane. The native versions of the human enzymes localize to the Golgi membrane in human cells. For the chimeric versions (SEQ ID No.s 97- 98 for St6GAL1 , and SEQ ID No.s 99- 100 for St3GAL4, the human N-terminal Golgi targeting sequence was replaced with one from R. norvegicus St6GAL1 gene (SEQ ID No. 37- 38).

Figure 1 provides the design of a nucleic acid construct.

Table 1

Table 2

Table 3

Method for designing nucleic acid construct

For the heterologous genes described above to be expressed in yeast S. cerevisiae, each coding sequence (CDS) was first codon optimized for expression in S. cerevisiae and assigned a suitable constitutive yeast promoter (PRO) and terminator (TER) sequence to drive gene expression. The resulting sets of transcription units (TUs) were then concatenated in silico, with flanking loxPsym sequences to enable inducible evolution of Nucleic acids in the future. Pathway (PTW) sequences were segmented into parts suitable for commercial gene synthesis and subsequent‘in yeasto’ assembly. Transcription units, including yeast promoter and terminator sequences are shown in Table 4. A description for PROs and TERs is provided in Table 5 and Table 6, respectively.

Table 4

8 FIG/1 SEQ

44

45

47

PRO, TER is promoter, terminator sequence from yeast S. cerevisiae genome

*Chimeric protein designed by replacing LAC12 C-terminus with Golgi targeting region from yeast (See) or human (Hsa) protein as indicated (Sce_TMN3, Sce_TP05, Sce_ALG6, Hsa_ALG6)

indicated Golgi targeting sequence appended to C-terminus of LAC12 coding sequence

**Chimeric protein designed by replacing N-terminus of indicated gene with Golgi-targeting region from ST6GAL1 R. norvegicus gene

Table 5

*all promoter sequences are derived from the published S. cerevisiae genome sequence (S288C) in the Saccharomyces Genome Database (www.yeastgenome.org).

8 FIG/ 1 SEQ Table 6

*all terminator sequences derive from the published S. cerevisiae genome sequence (S288C) in the Saccharomyces Genome Database (www.yeastgenome.org). Example 2

Development of the production host S. cerevisiae strains

Nucleic acid constructs are assembled by homologous recombination in yeast using a standard yeast transformation protocol described in Gietz R. D. (2014). Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods in molecular biology 1205:1-12. Nucleic acid construts were subsequently recovered from S. cerevisiae into One Shot® Top10 E. coli cells ((Invitrogen, Carlsbad, CA, USA), and purified for digestion verification and sequence analysis. Yeast strains to test lactose transport and 2’- fucosyllactose production were produced by transforming a wild-type S. cerevisiae strain BY4741 or a strain lacking the plasma membrane galactose transporter Gal2 (gal2A, yNeoArm0001). Nucleic acid constructs and S. cerevisiae strains used for analysis are listed in Tables 7 and 8.

Table 7.

PM = plasma membrane; LP = lactose permease; Kla = K. lactis; Ani = A. nidulans; Sst = S. stipitis; Hsa = H. sapiens; See = S. cerevisiae; Ath = A. thaliana; Rno = R. norvegicus; n/a = not applicable

Table 8

Culture media and growth conditions

E. coli cells were grown in Luria Broth containing 75 pg/ml Carbenicillin at 30°C and 250 rpm agitation. All yeast experiments were carried out at 30 °C using two different kinds of minimal yeast medium lacking the appropriate amino acids to select for maintenance of nucleic acid constructs. Synthetic complete (SC) drop out medium contains 6.8 g/l yeast nitrogen base, 5 g/l ammonium sulfate, 20 g/l glucose, and 2 g/l uracil amino acid (AA) drop out mix, or 2g/l leucine AA drop out mix, or 2 g/l uracil and leucine AA drop out mix. The AA drop out mixes contain all of the remaining amino acids other than the one(s) indicated. Synthetic dextrose (SD) drop in medium contains 6.8 g/l yeast nitrogen base, 5 g/l ammonium sulfate, 20 g/l glucose, 63 mg/I histidine, and 153 mg/I methionine.

Dot assay

For each strain a single colony was inoculated into one well of a 96-well plate containing 200 pi of synthetic complete uracil drop out medium supplemented with 2% raffinose and grown overnight at 30°C. The following day the density of cells was measured with an OD 600 reading. The cell concentration was adjusted to 0.2 OD units or 2x10⁶ cells/ml. 10-fold serial dilutions were carried out in water. 5 mI of cell suspension corresponding to 2x10² - 2x10⁵ cells/ml was spotted on the appropriate drop in or drop out plate and incubated at 30°C.

Batch growth for 2’ fucosyllactose production

A single yeast colony was inoculated into 1 ml of synthetic drop in medium and grown overnight with shaking at 30 °C. 200 mI of overnight culture was inoculated into 5 ml of fresh synthetic drop in medium supplemented with 0.1 g/l lactose and grown for 24 hours with shaking at 30 °C.

Example 3

Evaluation of lactose transport in the production host S. cerevisiae Experiment 1

Growth on galactose requires a functional lactose permease (LP) at the plasma membrane. A production host S. cerevisiae strain lacking the galactose permease gene GAL2 (gal2A) should not be able to survive when grown on galactose as the sole carbon source. However, by expressing a functional lactose permease (LP) at the plasma membrane, the gal2A strain should survive on galactose as the LP can promiscuously transport galactose.

Biological duplicate yeast transformants were tested for each plasma membrane-Golgi LP pair. The sugar source is either 0.5% dextrose (dex) or 0.5% galactose (gal) supplemented in drop-in medium. Ten-fold serial dilutions of yeast cells grown (left-to-right) are shown in Figure 2. 8 lactose permease nucleic acid constructs as provided in Table 7 were transformed into the gal2A strain and growth on galactose was evaluated. The eight Golgi targeting localization sequences used to C-terminally tag the K. lactis LAC12 gene are indicated.

The bottom panel shows controls where in the absence of a lactose transport nucleic acid construct (empty), the gal2A strain cannot grow and in the presence of GAL2 (expressed from its native location in the genome), yeast can grow robustly.

Consistent with functional lactose permease localization to the plasma membrane, growth was detected for all strains tested and the results are shown in Figure 2. The interpretation of this assay is complicated by expression of LPs on both the plasma membrane as well as the Golgi memberane; once galactose is transported into the cytoplasm, it could either be metabolized, enabling growth, or alternatively transported into the Golgi and secreted.

Experiment 2

The plasma membrane (PM) lactose permease (LP) as well as the eight Golgi localized LPs as provided in Table 7 were individually subcloned and evaluated for growth on galactose in a gal2A S. cerevisiae strain.

Growth assay was conducted to test localization of the lactose permeases. Yeast cells lacking the galactose transporter GAL2 were grown on 1 % galactose or 1 % dextrose at 30 °C for 14 days and 4 days, respectively. Strains carry either an empty vector (empty), a plasmid encoding a plasma membrane (PM) localized lactose permease (LP) from K. lactis (shown in left panel of Figure 3), or a plasmid encoding a Kla LP (Lac12) targeted to the Golgi membrane with a C-terminal localization sequence (tag) (as indicated; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae) (right panel). Ten- fold serial dilutions of yeast cells grown (top-to-bottom) are shown in Figure 3. Galactose plates were prepared with“drop-in” synthetic medium, only supplementing the required amino acids (histidine, methionine, leucine). Dextrose plates were prepared with“drop-out” synthetic complete medium lacking uracil.

As expected the PM expression of the K. lactis (Kla) LP enabled growth on galactose (Figure 3, left panel). It was seen that seven of eight Golgi localized LPs derived from Kla did not support growth on galactose (Figure 3, right panel). The one exception, Kla Lac12 appended with the Golgi targeting sequence KKLL, was able to grow. This suggests that at least some of that expressed chimeric protein mis-localizes to the PM (Figure 3, right panel). The fact that the other seven failed to grow whereas their unfused counterpart robustly promoted growth provides strong evidence the seven fusion proteins are efficiently Golgi localized.

Experiment 3

Previous reports suggest that lactose is toxic to yeast cells expressing LPs. It was hypothesized that if lactose is toxic to yeast cells expressing a PM LP, then co-expression of the Golgi-localized LP may serve as a detoxification mechanism, effectively“pumping” lactose out of the cell via natural secretion.

Figure 4 provides the results for lactose detoxification by co-expressing PM (Kla) and Golgi LPs (Kla + C-term tag as indicated). Relative to the PM LP alone (Kla Lac12 (PM)), cells co-expressing the Golgi LP grow more robustly on both 0.01 % and 0.03% lactose. All plates shown in this figure are “drop-in” synthetic medium and were incubated at 30°C.

The results indicated that lactose is extremely toxic to yeast cells expressing the Kla LP (but not to non-expressers), when grown on dextrose as the carbon source. It was found that Golgi LP expression suppressed lactose toxicity at both 0.01 % and 0.03% lactose providing strong presumptive evidence for detoxification, which was hypothesized to occur via lactose transport into the Golgi and subsequent secretion.

Example 4 Evaluation of the production of 2’-fucosyllactose in the production host S. cerevisiae To evaluate the production of 2’- fucosyllactose metabolomics experiments were performed. Preparation of standard

A 2’-fucosyllactose standard was purchased from Santa Cruz Biotechnology (sc-256371 , >95% purity) and 2’-fucosyllactose detection was evaluated using a known method specifying graphitic carbon high-performance liquid chromatography with tandem mass spectrometry {Bao et al. (2013). Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry. Analytical Biochemistry. 433 (1). 28- 35}. Figure 5 shows that 2’ fucosyllactose was easily detected in this experiment.

LCMS analysis of standard curve, 2’- fucosyllactose

For the LCMS analysis, a Thermo Scientific™ HYPERCARB (2.1 x100 mm, 3 pm) column was used with a Dionex Ultimate 3000™ system and the column oven temperature was set to 25 °C for the isocratic elution. A flow rate of 200 pl/min was used with neat solvents for mobile phase A) LCMS grade water, and B) acetonitrile. Solvent B was kept constant at 12 %. Injection volume was set to 3 pi for all analyses (5 minutes total run time per injection). MS analyses were carried out by coupling the LC system to a Thermo Q Exactive HF™ mass spectrometer operating in heated electrospray ionization mode (HESI). Spray voltage for negative mode was 3.5 kV and capillary temperature was set to 320 °C with a sheath gas rate of 35, aux gas of 10, max spray current of 100 pA, S-lens RF level = 55.0, and Aux gas temperature of 50°C. Method duration was 3 minutes with Parallel Reaction Monitoring (PRM) scan in negative mode for 2’- fucosyllactose from 1-4 minutes post injection. MS resolution was 15,000 with an AGC target of 1e⁶ and a maximum IT of 500 ms, with an isolation window of 0.4 m/z for the 487.1668 m/z parent ion [C₁₈H₃₂O₁₅] , isolation offset of 0.1 m/z, scan range from 50-515 m/z, at a single normalized collision energy (nCE) of 25.

The injection order of samples, standard curve points, and analytical blanks was randomized to mitigate batch and order effects, and to assess carry over and reproducibility of the standard curve. Approximately 1 blank injection was included for every four samples and standards. Standard curve points were prepared in water using authentic chemical standards, and serial-diluted in a pseudolog pattern ranging from 100 nM for the lowest calibrant to 10 mM for the high calibrant. Each standard curve point was analyzed in duplicate (random order). Quantification of 2’-fucosyllactose was carried out using the extracted ion chromatogram (XIC) peak height for the characteristic fragment ion [C₈H_I30₆] at 205.0712 m/z. The resulting peak intensities were fit to a linear regression (GraphPad Prizm 7) with a fixed y-intercept of zero. The best-fit slope was used to interpolate the detected concentration of 2’- fucsosy I lactose in samples, and the sample concentration factor was considered to estimate the original concentration of 2’- fucosyllactose in media supernatant and cell pellet.

Figure 5 (A) provides extracted ion chromatogram (XIC) of 2’- fucosyllactose standard by LCMS, parent mass = 487.1669 m/z. The peak is generated from XIC data of a product reaction monitoring (PRM) scan using a characteristic 205.0716 m/z fragment. High resolution accurate mass was used for both MS1 and MS2 values. Figure 5 (B) provides a standard curve used for quantification with authentic 2’- fucosyllactose chemical standard. Peak height of the negative mode molecular ion [M- H] was used for quantification. The lowest calibrant analyzed was 100 nM and the highest was 10 mM, n=2 for each standard, average of both values is plotted. Figure 5 (C) provides a high resolution accurate mass full scan (MS1) of the intact 2’- fucosyllactose parent ion in negative mode electrospray ionization (ESI). The mass is consistent with the theoretical formula of 2’- fucosyllactose in negative mode (C18H32O15), <5 ppm. Figure 5 (D) provides a high resolution accurate mass tandem mass spectrum (MS2) of the 2’- fucosyllactose parent ion in negative mode. The parent ion and characteristic 205 fragment used for the XIC in panel A are annotated. Other characteristic fragments are visible, which can be used to confirm the 2’-fucosyllactose structure.

To conduct the metabolomics analysis, only 6 of the 8 Golgi LP gene localization sequences (tags) were selected, discarding the KKLL and HsALG6 tags due to poor detoxification at higher lactose concentrations. A lactose concentration of 0.01 % was used, as this clearly showed detoxification and decent growth in Figure 4. The cells were grown in drop-in synthetic medium with 1 % dextrose to be consistent with previous examples.

Yeast cells were co-transformed with one of six lactose transport nucleic acid constructs (Table 7) and the 2’- fucosylalactose production nucleic acid constructs, yielding 6 strains. Empty vectors were also co-transformed to provide a negative control strain. Each strain was inoculated into 5ml of medium containing 0.01 % lactose and grown for 24 hours. After 24 hours, the cultures reached mid log phase (OD₆oo ~0.5-1.0). 5ml of supernatant was collected for each sample and stored at -80 °C. Alternatively, a set of samples was prepared where the cells were grown overnight without lactose, sub-cultured 1 :5, and then treated for 2 hours with lactose. Again, the cells were grown to mid-log phase (OD₆oo ~0.5-1.0) and then 5ml of supernatant was collected and stored at -80 °C. The latter experiment was performed to compensate for lactose toxicity and slow growth of strains at 24 hours. The metabolomics core concentrated the supernatant samples 500-fold and then subjected them to LC-MS/MS as previously optimized for the standard and shown in Figure 5. Experimental and standard curve samples were processed in a random order and no sample carry-over was observed. A total of 16 samples were run, of which four were negative controls.

Absolute quantification and compound identification for 2’- fucosyllactose were confirmed with an authentic standard in a subset of samples grown for 24 hours in lactose (Table 9).

Specifically, all strains carrying the 2’-fucosyllactose production module with the Fut2 enzyme targeted to the Golgi membrane as a chimera with the rat St6gal1 sequence (pNeo0049) produced between 1 2E ¹⁰ and 7.3^{E 10} M 2’- fucosyllactose (59 and 358 ng/l 2’- fucosyllactose). Representative LC-MS/MS data from one sample that produced 2’-fucosyllactose is shown in Fig. 6. No 2’- fucosyllactose was detected in any of the negative control samples or any of the 2-hour lactose samples.

For this experiment, the detection limit for 2’-fucosyllactose is defined as 0.3 of the lowest linear calibrant. The lowest linear calibrant was 100 nM (Figure 5), and considering the 500X concentration factor, this is equivalent to 60 picomolar, or 6.0E ¹¹ M. 2’-fucosyllactose detected in the samples is thus well above the detection limit of the experiment. Specifically, since the detection limit in these experiments is approximately 6.0E ¹¹ M, the highest value we detected here (in the pNeo0026+pNeo0049, 24 hours sample) represents a signal more than ten-fold above the detection limit, and the lowest values reported in Table 9 are about 2X the detection limit. Table 9

2‘-FL ist he abbreviation for 2‘- fucosyllactose; NF not found

Values in Table 9 represent MS intensity (arbitrary units) unless otherwise specified. NF = not found, or otherwise below the detection limit of the method (~60 picomolar equivalent in yeast culture supernatant). Identity of 2’-fucosyllactose was confirmed by authentic standards (Figure 4). The standard curve for 2- fucosyllactose was fit to a linear regression and zero Y-intercept with a slope of 486290181818. The final concentration factor from media supernatant to on-column was 500X. The average molecular weight of 2-fucosyllactose is 488.4377. The detection limit in these experiments is approximately 6.0E ¹¹ M. Therefore, the highest value (for (26+49, 24h) represents a signal more than ten-fold above the detection limit and the lowest values reported are about 2X detection limit.

LCMS detection of 2’-fucosyllactose in yeast culture supernatant

Figure 6 (A) provides extracted ion chromatograms (XICs) of 2’- fucosyllactose from sample “pNeo0025+pNeo0049 mid log 24h 0.01 % lac” (top; Table 9, row 2), and“Empty vector mid log 24h 0.01 % lac” (bottom; Table 9, row 15) by LCMS. The transition from parent mass > fragment is shown for each compound. The peak is generated from XIC data of a product reaction monitoring (PRM) scan using the characteristic fragment m/z. Figure 6 (B) provides high resolution accurate mass tandem mass spectrum (MS2) of the 2’- fucosyllactose parent ion in negative mode, each spectrum at 6 minutes (RT of 2’- fucosyllactose). Top and bottom samples same as panel (A). The retention time, peak shape, and MS2 fragments are consistent with the 2’- fucosyllactose standard detection as shown in Figure 5.

Example 5

Evaluation of the production of 2’-fucosyllactose (2’- FL) in the production host S. cerevisiae using optimized lactose transport and 2’-fucosyllactose production modules

“Second generation" lactose transport (pNeo0328) and 2’-fucosyllactose production (pNeo012) modules were designed and built to increase 2’-fucosyllactose production by improving genetic stability and increasing the expression level of enzymes in the system (Figure 1).

S. cerevisiae cells were transformed with the second generation constructs and empty vectors. Cells were grown overnight in synthetic defined selective medium containing dextrose. The following day, cells were sub-cultured into the same medium supplemented with 0.01 % or 0.05% lactose. After 48 hours of growth, supernatant and pellet samples were collected for extraction and graphitic carbon HPLC-MS/MS analysis (as described in Example 4). This experiment was performed with technical duplicate samples. The average total 2‘- fucosyllactose (2‘- FL) detected in the culture supernatant plus pellet as shown in Table 10. No appreciable increase in 2’- fucosyllactose production was observed when cells were grown in 0.05% lactose compared to 0.01 %. This indicates that lactose is not the limiting substrate in 2’- fucosyllactose production.

Table 10

Sup- cell culture medium; DCW- dry cell weight; NF- not found

8 FIG/ 1 SEQ

Example 6

Evaluation of the production of 6’-sialyllactose (6’- SL) or 3’-sialyllactose (3’- SL)in the production host S. cerevisiae

To evaluate the production of 6’-sialyllactose or 3’-sialyllactose metabolomics experiments were performed.

Preparation of standard

Standards comprising 3’-sialyllactose sodium salt and 6’-sialyllactose sodium salt were purchased from Santa Cruz Biotechnology (sc-216626, sc-2211 10). 6’-sialyllactose or 3’-sialyllactose standards were analyzed with hydrophilic interaction liquid chromatography (HILIC) method. Using HILIC, detection and near-baseline separation was achieved, and the elution order was assigned with individual standards (Figure 7). The molecular ion and corresponding retention times were used for quantification of both 3’SL and 6’SL peaks.

6’-sialyllactose or 3’-sialyllactose standards were mixed and run as a cocktail. The resulting standard curves were linear for both compounds (Figure 8).

To conduct metabolomics analysis, 6’-sialyllactose or 3’-sialyllactose production modules (Figure 1 ) were individually co-transformed with the first generation lactose transport module into yeast. Two empty vectors were co-transformed into yeast to serve as the negative control strain. Cells were grown overnight in minimal selective drop-in medium with 1 % dextrose. The following day cells were sub-cultured into the same medium supplemented with 0.01 % lactose. Cultures were grown for 2 days at 30°C and with agitation to a final Oϋ_qoo of 0.46-0.77. The cell culture volumes were normalized to obtain ~1.5mg of dry cell weight (DCW) worth of cell material (using the internally established conversion formula 1 ml. of cells growing at an OD₆oo of 1 is equal to 0.6 mg DCW). Next, the cells were collected by centrifugation, supernatants discarded, and the cell pellets stored at -80°C until further analysis.

The cell pellets were extracted using 80% acetonitrile and a bead blasting technique. Experimental and standard curve samples were processed in a random order and a blank was run after each

8 FIG/ 1 SEQ standard. Some product carryover was detected in blank samples run immediately after high concentration standards (on average ~1.4% after 1 , 3, and 10 mM 3’SL standards, and ~2.7% after 3, and 10pM 6’SL standards). The results of detection and quantification of 3’- SL and 6’- SL are provided in Table 1 1.

Table 1 1

DCW- dry cell weight, #Chimeric protein with Golgi targeting signal from R. norvegicus (Rn)

It was found that yeast strains carrying the human versions of GNE, NANS and CMAS led to production of 8-9 pg of 3’-SL and 79-1 13 pg of 6’-SL per gram of dry cell weight. This demonstrates that the 6 genes in each of the 3’-SL and 6’-SL pathways, including SLC53A1 , NANP, St3GAL4, and St6GAL1 (native and chimeric), are functionally expressed in yeast. On the other hand, when zebrafish orthologs for GNE, NANS and CMAS were encoded on the neochromosomes, a lower yield for 6’-SL (6-15 pg /g DCW), and no 3’-SL was observed above the intensity cut off (E⁺⁰⁴). Detection of 6’-SL in zebrafish-specific sample provides evidence that the three zebrafish genes GNE, NANS and CMAS are functionally expressed in yeast cells.

Claims

1. A production host comprising a heterologous nucleic acid coding for

d) a fucosyltransferase targeted to a secretion organelle, and/or

e) a sialyltransferase targeted to a secretion organelle.

2. The production host of claim 1 , wherein the production host comprises a metabolic pathway for production, in the secretion organelle, of a human milk oligosaccharide, preferably a fucosylated and/or sialylated oligosaccharide, more preferably,

a fucosylated oligosaccharide, more preferably 1 , 2-fucosyllactose, 2'-fucosyllactose, 3’-fucosyllactose or difucosyllactose, most preferably 2'-fucosyllactose, and/or

a sialylated oligosaccharide, more preferably 3'-sialyllactose or 6'-sialyllactose, or 3'- sialyl-3-fucosyllactose.

3. The production host of any of the preceding claims, wherein the production host further comprises a metabolic pathway for the production of GDP-fucose, preferably for the production of GDP-fucose from GDP-mannose, and wherein preferably the metabolic pathway is in the cytoplasm.

4. The production host of any of the preceding claims, wherein the production host further comprises a metabolic pathway for the production of cytidine 5’- monophosphate N- acetylneuraminic acid, preferably for the production of cytidine 5’- monophosphate N- acetylneuraminic acid from UDP- N-acetyl-glucosamine, and wherein preferably the metabolic pathway is in the cytoplasm.

5. The production host of any of the preceding claims, wherein the secretion organelle is the endoplasmatic reticulum and/or the Golgi apparatus, preferably the Golgi apparatus.

6. The production host of any of the preceding claims, wherein the production host further comprises a lactose transporter targeted to the host cell membrane.

7. The production host of any of the preceding claims, wherein the lactose transporter targeted to the secretion organelle membrane comprises a heterologous localization sequence for integration in the secretion organelle membrane.

8. The production host of any of the preceding claims, wherein the heterologous localization sequence for integration of the lactose transporter in the secretion organelle membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 40, 42, 44, 46, 48, 50, 52, or 54.

9. The production host of any of the preceding claims, wherein

the fucosyltransferase targeted to a secretion organelle, and/or

the sialyltransferase targeted to a secretion organelle

comprises a heterologous localization sequence for localization in the secretion organelle lumen.

10. The production host of any of the preceding claims, wherein the heterologous localization sequence for localization of the fucosyltransferase in the secretion organelle lumen comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38.

1 1 . The production host of any of the preceding claims, wherein the heterologous localization sequence for localization of the sialyltransferase in the secretion organelle lumen comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 38.

12. The production host of any of the preceding claims, wherein

a) the lactose transporter targeted to the secretion organelle membrane and/ or to the host cell membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 3, 22, 24, 26, 28, 30, 32, 34, 36, or 106, and/or b) the GDP-fucose transporter targeted to a secretion organelle membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 12, and/ or

c) the cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ I D No. 82, and/ or

d) the fucosyltransferase targeted to a secretion organelle comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 18 or 20, and/or

e) the sialyltransferase targeted to a secretion organelle comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 98, 100, 102, or 104.

13. The production host of any of the preceding claims, wherein the metabolic pathway for the production of GDP-fucose comprises f) one or more heterologous enzymes, wherein the enzymes are preferably selected from lyase, specifically a hydrolyase, preferably, GDP mannose 4, 6- dehydratase; and oxidoreductase, preferably GDP L- fucose synthase.

14. The production host of any of the preceding claims, wherein the metabolic pathway for the production of cytidine 5’- monophosphate N-acetylneuraminic acid comprises g) one or more of heterologous enzymes, wherein the enzymes are preferably selected from GNE, NANS, NANP, and CMAS.

15. The production host of any of the preceding claims, wherein the production host is a yeast,

preferably of order Saccharomycetales or Schizosaccharomycetales,

Trigonopsidaceae or Schozosaccharomycetaceae

most preferably of genus Saccharomyces.

16. A transporter targeted to a secretion organelle membrane, wherein the transporter is a) a lactose transporter, preferably the lactose transporter comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 3, 22, 24, 26, 28, 30, 32, 34, 36, or 106, and/or

b) a GDP-fucose transporter, preferably the fucose transporter comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 12, and/ or

c) cytidine 5’- monophosphate N-acetylneuraminic acid transporter, preferably the cytidine 5’- monophosphate N-acetylneuraminic acid transporter comprises or consists of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 82.

17. A nucleic acid molecule comprising a nucleic acid sequence encoding

a) a lactose transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 2, 21 , 23, 25, 27, 29, 31 , 33, 35, or 105, and/or

b) a GDP-fucose transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 11 , and/ or

c) a cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to the secretion organelle membrane and having at least 70% sequence identity to SEQ ID No. 81 ,

d) a fucosyltransferase targeted to the secretion organelle and having at least 70% sequence identity to SEQ ID No. 17 or 19, and/or

e) a sialyltransferase targeted to the secretion organelle and having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103 and/ or

f) one or more heterologous enzymes for the production of GDP- fucose, wherein the enzymes are preferably selected from lyase, specifically a hydrolyase, preferably, GDP mannose 4, 6- dehydratase; and oxidoreductase, preferably GDP L- fucose synthase, and/ or

g) one or more heterologous enzymes for the production of cytidine 5’- monophosphate N-acetylneuraminic acid, wherein the enzymes are preferably selected from GNE, NANS, NANP, and CMAS.

18. A nucleic acid construct for the expression of

a) a lactose transporter targeted to the secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 2, 21 , 23, 25, 27, 29, 31 , 33, 35, or 105, and/or

b) a GDP-fucose transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 1 1 , and/or c) a cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 81 , and/or

d) a fucosyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 17 or 19, and/or e) a sialyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103, and/ or

f) one or more heterologous enzymes for the production of GDP- fucose, wherein the enzymes are preferably selected from lyase, specifically a hydro-lyase, preferably, GDP mannose 4, 6- dehydratase; and oxidoreductase, preferably GDP L- fucose synthase, and/ or

19. A nucleic acid construct of claim 18, wherein the nucleic acid construct is an artificial chromosome.

20. An artificial chromosome comprising a nucleic acid sequence for the expression of at least one of

a) the lactose transporter targeted to the secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 2, 21 , 23, 25, 27, 29, 31 , 33, 35, or 105, and/or b) the GDP-fucose transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 11 , and/ or;

c) the cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 81 , and/ or

d) the fucosyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 17 or 19, and/or e) the sialyltransferase targeted to a secretion organelle encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID No. 97, 99, 101 or 103, and/or f) one or more heterologous enzymes for the production of GDP- fucose, wherein the enzymes are preferably selected from lyase, specifically a hydro-lyase, preferably, GDP mannose 4, 6- dehydratase; and oxidoreductase, preferably GDP L- fucose synthase, and/ or

g) one or more heterologous enzymes for the production of cytidine 5’- monophosphate N-acetylneuraminic acid selected from GNE, NANS, NANP, and CMAS.

21 . The artificial chromosome of claim 20, comprising the nucleic acid sequences according to a); b); c); d); e); f); g); a) and b); a) and c); a) and d); a) and e); a) and f); a) and g); b) and d); b) and f); b) and d) and f); c) and e); c) and g); c) and e) and g); a) and b) and d); a) and b) and f); a) and b) and d) and f); a) and c) and e); a) and c) and g); a) and c) and e) and g); or all of a), b), c), d), e), f), g), in any combination.

22. A production host according to any of claims 1-15, comprising a nucleic acid according to claim 17, and/ or a nucleic acid construct according to any of claims 18- 21 , and/or preferably an artificial chromosome according to any of claims 20- 21.

23. A method for production of a human milk oligosaccharide, comprising the steps of

i) providing a production host according to any of claims 1 -15 or 22, and

ii) culturing the production host in the presence of lactose in a fermentation medium comprising an energy source, wherein the energy source is one or more of glycerol, succinate, malate, pyruvate, lactate, ethanol and citrate.

24. The method of production of claim 23, further comprising the step of

iii) obtaining the human milk oligosaccharides from the production host and/or preferably from the fermentation medium.

25. The method of production of any of claims 23-24, wherein the human milk oligosaccharide is a fucosylated and/or sialylated oligosaccharide, more preferably selected from

26. Use of a heterologous localization sequence for targeting a polypeptide to a secretion organelle or a secretion organelle membrane, wherein

the localization sequence is selected from

a’) a sequence for localization of the lactose transporter, and/ or

d’) a sequence for localization of the fucosyl transferase, and/ or

e’) a sequence for localization of the sialytransferase, and the polypeptide is selected from

a) a lactose transporter targeted to a secretion organelle membrane, and/ or b) a GDP- fucose transporter targeted to a secretion organelle membrane, and/ or c) a cytidine 5’- monophosphate N-acetylneuraminic acid transporter targeted to a secretion organelle membrane, and/ or

d) a fucosyltransferase targeted to a secretion organelle, and/ or

e) a sialyltransferase targeted to a secretion organelle.

27. Use of a heterologous localization sequence of claim 28, wherein the localization sequence is selected from,

a) the lactose transporter comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 3, 22, 24, 26, 28, 30, 32, 34, 36 or 106, and/ or

d) the fucosyl transferase comprising or consisiting of an amino acid sequence having at least 70% sequence identity to SEQ ID No. 18 or 20, and/ or

28. The use according to any of claims 26-27, wherein the secretion organelle is the endoplasmatic reticulum and/or the Golgi apparatus, preferably the Golgi apparatus, preferably of a yeast of order Saccharomycetales or Schizosaccharomycetales,

Trigonopsidaceae or Schozosaccharomycetaceae

more preferably of genus Alloascoidea, Ascoidea, Cephaloascus, Babjeviella, Debaryomyces, Enteroramus, Hyphopichia, Kurtzmaniella, Meyerozyma, Millerozyma, Nematodospora, Peterozyma, Priceomyces, Scheffersomyces, Schwanniomyces,

SHEET INCORPORATED BY REFERENCE (RULE 20.6) Spathaspora, Suhomyces, Teunomyces, Wickerhamia, Yamadazyma, Arxula, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete,

Sporopachydermia, Yarrowia, Endomyces, Dipodascopsis, Lipomyces, Zygozyma, Aciculoconidium, Clavispora, Danielozyma, Kodamaea, Metschnikowia, Nectaromyces, Barnettozyma, Cyberlindnera, Komagataella, Phaffomyces, Starmera, Wickerhamomyces, Allodekkera, Brettanomyces, Kregervanrija, Martiniozyma, Nakazawaea, Ogataea, Pichia (synonym: Hansenula), Saturnispora, Arxiozyma, Citeromyces, Cyniclomyces, Eremothecium, Hagleromyces, Issatchenkia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Pachysolen, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Yueomyces, Zygosaccharomyces, Zygotorulaspora, Zygowilliopsis, Hanseniaspora, Saccharomycodes, Arthroascus, Saccharomycopsis, Blastobotrys, Diddensiella, Groenewaldozyma, Spencermartinsiella, Sugiyamaella, Trichomonascus, Wickerhamiella, Zygoascus, Botryozyma, Tortispora, Trigonopsis, Ambrosiozyma, Candida, Deakozyma, Diutina, Kuraishia, Macrorhabdus, Metahyphopichia, Middelhovenomyces, Myxozyma, Nadsonia, Schizoblastosporion, Starmerella, Sympodiomyces or Schizosaccharomyces,

more preferably of species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces hubeiensis, Kluyveromyces lactis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces lactis, Kluyveromyces wickerhamii, Kluyveromyces marxianus, Kluyveromyces nonfermentans, Kluyveromyces siamensis, Kluyveromyces wickerhamii, Pichia barkeri, Pichia bruneiensis, Pichia cactophila, Pichia cecembensis, Pichia cephalocereana, Pichia chibodasensis, Pichia deserticola, Pichia dushanensis, Pichia eremophila, Pichia exigua, Pichia fermentans, Pichia garciniae, Pichia heedii, Pichia insulana, Pichia kluyveri, Pichia kudriavzevii, Pichia manshurica, Pichia membranifaciens, Pichia nakasei, Pichia nongkratonensis, Pichia norvegensis, Pichia occidentalis, Pichia porticicola, Pichia pseudocactophila, Pichia punctispora, Pichia rarassimilans, Pichia scaptomyzae, Pichia scutulata, Pichia sporocuriosa, Pichia terricola, Pichia membranifaciens, Pichia species, Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomyces bayanus, Saccharomyces cf. kudriavzevii, Saccharomyces bayanus, Saccharomyces kudriavzevii, Saccharomyces bayanus, Saccharomyces uvarum, Saccharomyces cariocanus, Saccharomyces cerevisiae (baker's yeast), Saccharomyces bayanus, Saccharomyces

SHEET INCORPORATED BY REFERENCE (RULE 20.6) eubayanus, Saccharomyces eubayanus, Saccharomyces uvarum, Saccharomyces cf. kudriavzevii, Saccharomyces kudriavzevii, Saccharomyces kudriavzevii, Saccharomyces bayanus, Saccharomyces kudriavzevii, Saccharomyces uvarum, Saccharomyces mikatae, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces uvarum, Saccharomyces eubayanus, Saccharomyces cf. cerevisiae/paradoxus, Saccharomyces chevalieri, Saccharomyces douglasii, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces eubayanus, Saccharomyces uvarum,

Saccharomyces jurei, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces paradoxus, Saccharomyces uvarum, Saccharomyces pastorianus, Saccharomyces uvarum, Schizosaccharomyces cryophilus, Schizosaccharomyces japonicus, Schizosaccharomyces kambucha, Schizosaccharomyces kambucha, Schizosaccharomyces pombe, Schizosaccharomyces octosporus,

Schizosaccharomyces pombe (fission yeast), Schizosaccharomyces versatilis, Yarrowia alimentaria, Yarrowia bubula, Yarrowia deformans, Yarrowia divulgata, Yarrowia galli, Yarrowia hollandica, Yarrowia keelungensis, Yarrowia lipolytica, Yarrowia osloensis, Yarrowia parophonii, Yarrowia phangngaensis, Yarrowia porcina or Yarrowia yakushimensis,

most preferably of genus Saccharomyces.

29. The use according to any of claims 26-27, wherein

order Saccharomycetales or Schizosaccharomycetales,

Trigonopsidaceae or Schozosaccharomycetaceae

SHEET INCORPORATED BY REFERENCE (RULE 20.6) Spathaspora, Suhomyces, Teunomyces, Wickerhamia, Yamadazyma, Arxula, Dipodascus, Galactomyces, Geotrichum, Magnusiomyces, Saprochaete, Sporopachydermia, Yarrowia, Endomyces, Dipodascopsis, Lipomyces, Zygozyma, Aciculoconidium, Clavispora, Danielozyma, Kodamaea, Metschnikowia, Nectaromyces, Barnettozyma, Cyberlindnera, Komagataella, Phaffomyces, Starmera, Wickerhamomyces, Allodekkera, Brettanomyces, Kregervanrija, Martiniozyma, Nakazawaea, Ogataea, Pichia (synonym: Hansenula), Saturnispora, Arxiozyma, Citeromyces, Cyniclomyces, Eremothecium, Hagleromyces, Issatchenkia, Kazachstania, Kluyveromyces, Lachancea, Nakaseomyces, Naumovozyma, Pachysolen, Saccharomyces, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Yueomyces, Zygosaccharomyces, Zygotorulaspora, Zygowilliopsis, Hanseniaspora, Saccharomycodes, Arthroascus, Saccharomycopsis, Blastobotrys, Diddensiella, Groenewaldozyma, Spencermartinsiella, Sugiyamaella, Trichomonascus, Wickerhamiella, Zygoascus, Botryozyma, Tortispora, Trigonopsis, Ambrosiozyma, Candida, Deakozyma, Diutina, Kuraishia, Macrorhabdus, Metahyphopichia, Middelhovenomyces, Myxozyma, Nadsonia, Schizoblastosporion, Starmerella, Sympodiomyces or Schizosaccharomyces,

most preferably of genus Saccharomyces.