NZ796027A - In vivo synthesis of sialylated compounds - Google Patents

Abstract

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered microorganisms. The present invention describes engineered micro-organisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These micro-organisms can dephosphorylate N- acetylglucosamine-6-phopshate to N-acetyl glucosamine and convert the N-acetylglucosamine to N- acetylmannosamine. These micro-organisms also have the ability to convert N-acetylmannosamine to N-acetyl- neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose , N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosamine-6-phopshate to N-acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

Description

The present invention is in the technical field of tic biology and metabolic engineering.

More particularly, the present invention is in the technical field of fermentation of metabolically ered microorganisms. The present invention describes engineered micro-organisms able to synthesize sialylated compounds via an intracellular biosynthesis route. These microorganisms can phorylate N-acetylglucosaminephopshate to yl glucosamine and convert the N-acetylglucosamine to N-acetylmannosamine. These micro-organisms also have the ability to convert N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the present invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a e medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lactoNbiose , N-acetyllactosamine and/or an aglycon, wherein said rganism intracellularly dephosphorylates N-acetylglucosaminephopshate to N- acetylglucosamine, converts N-acetylglucosamine to N-acetylmannosamine and convert the latter further to N-acetyl-neuraminate.

NZ 796027 [Link] http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.183 [Link] http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.1.60 In vivo synthesis of sialylated compounds The t ation is a divisional application of New Zealand ation No. 755558, which is incorporated in its entirety herein by reference.

The present invention is in the cal field of tic biology and metabolic engineering. More particularly, the present ion is in the technical field of fermentation of metabolically ered microorganisms. The present invention describes engineered micro-organisms able to synthesize sialylated nds via an intracellular biosynthesis route. These micro-organisms can dephosphorylate N-acetylglucosaminephosphate to N-acetyl glucosamine and convert the N- acetylglucosamine to N-acetylmannosamine. These micro-organisms also havethe ability to t N-acetylmannosamine to N-acetyl-neuraminate. Furthermore, the t invention provides a method for the large scale in vivo synthesis of sialylated compounds, by culturing a microorganism in a culture medium, optionally comprising an exogenous precursor such as, but not limited to lactose, lacto-N-biose, N-acetyllactosamine and/or an aglycon, wherein said microorganism intracellularly dephosphorylates N-acetylglucosaminephosphate to N-acetylglucosamine, converts N- acetylglucosamine to ylmannosamine and convert the latter further to N-acetyl-neuraminate.

Background ated compounds such as sialic acid and sialylated oligosaccharides have gained attention the last years, because of their broad application range. For e, sialic acid is considered as an anti-viral precursor. Sialylated oligosaccharides form an essential part of human milk and are ed antiadhesive and immunomodulatory properties; others described them to be involved in brain development. Sialylation, in general, of ns, lipids or aglycons are used in anti-cancer medicine and in the treatment of neurological diseases.

Sialic acid is a general term used to describe a large family of acidic sugars that are predominantly found on the cell surface of eukaryotic cells. The most common sialic acid is N-acetylneuraminic acid or Neu5Ac, an acidic nine-carbon sugar that undergoes several modifications to generate the members of the sialic acid family. As seen in e.g. Fig. 1 of WO2008097366, the diversity of the sialic acid family is represented with over 50 known members. Sialic acid represents a large family of cellsurface ydrates that are derived from an acidic, nine-carbon parent compound called N- acetylneuraminic acid or . Neu5Ac is often decorated with acetyl, phosphate, methyl, sulfate and lactyl groups, which are described to be required for desirable cell signalling and cell adhesion events mediated by sialic acid.

Sialic acids and sialylated compounds are common in higher eukaryotic organisms which produce them in a conserved biosynthetic route. This route starts from endogenic UDP-N-acetylglucosamine which is converted to sialic acid through the action of a UDP-N-acetylglucosamine 2-epimerase (hydrolysing) (EC 3.2.1.183), a N-acylmannosamine kinase (EC 2.7.1.60), a N-acylneuraminate phosphate synthase (EC 2.5.1.57) and a P phosphatase (EC 3.1.3.29). This sialic acid can 40 subsequently be activated and transferred to the desired acceptor via a CMP-sialic acid synthase (EC 2.7.7.43) and e.g. a sialyltransferase.

Efforts have been made to express this biosynthetic route in other eukaryotic organisms, s yotic systems were not ed. The y was functionally expressed in yeast a pastoris) and plant (Arabidopsis thaliana) to e sia|y|ated N-glycans.

However, large scale production of sia|y|ated oligosaccharides was never reported. The functional overexpression of eukaryotic genes in prokaryotic systems remains a daunting task without certain e due to the lack of specific chaperones, faulty enzyme folding and missing cell lles. On top ofthat remains the huge energy requirement ofthe pathway and the depletion of intercellulair UDP-GlcNAc (UDP-N-acetylglucosamine), necessary for cell growth.

Processes based on enzymatic, chemical as well as fermentative production of sia|y|ated compounds exist. However, all of them have significant disadvantages. For instance, chemical synthesis requires many sequential chemical steps and enzymatic synthesis es ive precursors, whereas the fermentative process is still under heavy development. Nonetheless, the latter has the highest industrial production potential.

One type of described fermentative production process uses a thesis route that originates from prokaryotes like obacter jejuni that naturally produces sialic acid or sia|y|ated nds. This biosynthesis route starts from endogenous UDP-N-acetylglucosamine which cells use for their cell wall. This is converted to N-acetylmannosamine and N-acetylneuraminate by the action of an UDP-N-acetylglucosamine epimerase (generally named neuC) and a sialic acid synthase (generally named neuB).

Using only part of this prokaryotic biosynthesis route, Priem et al. biology 12, 2002, 235- 240) describe the use of living bacterial cells to produce sialyloligosaccharides. In this , sialyllactose was directly produced by growing cells of metabolically engineered Escherichia coli strains which overexpressed the Neisseria meningitidis genes for alpha-2,3-sialyltransferase and for CMP-Neu5Ac synthase, these strains were further devoid of beta-galactosidase and N- acetylneuraminic acid c) aldolase activities. These microorganisms were grown at high cell density with glycerol as the carbon and energy source, while exogenous lactose and Neu5Ac were supplied as precursors for sialyllactose synthesis. During the growth, lactose and Neu5Ac were internalized by the induction of the expression of an E. coli galactoside and an exogenous Neu5Ac permease. Lactose and Neu5Ac accumulate in the cytoplasm where Neu5Ac was then converted into u5Ac to be further transferred on lactose to form sialyllactose. Large scale production of sialyloligosaccharides by this microbiological method requires ant amounts of Neu5Ac as a precursor.

Another microbial system was developed for production of sialyloligosaccharides without the need of an exogenous supply of sialic acid. W02007101862 describes such method for producing sia|y|ated oligosaccharides with rganisms comprising heterologous genes encoding a CMP-Neu5Ac synthetase, a sialic acid synthase, an UDP-GlcNAc-6—phosphate 2- epimerase and a sialyltransferase, and wherein the endogenous genes coding for sialic acid aldolase (NanA) and for ManNAc kinase (NanK) have been deleted or inactivated. The use of 4o this prokaryotic biosynthesis route is very energy intensive for the cell. Furthermore, the described route for producing the sia|y|ated oligosaccharides competes for the UDP-GlcNAc which is essential for the cells own peptidoglycan synthesis. Building on this concept, Kang et al. have created a production host that does not use a sialic acid synthase, but the endogenous sialic acid aldolase, which has a less favourable chemical equilibrium (Metabolic engineering 14, 2012, 9).

EP1484406 describes the production of Neu5Ac using E. coli overexpressing N- acetylglucosamine 2-epimerase and Neu5Ac synthase, but needs N-acetylglucosamine (GlcNAc) as al precursor. In the described method, GlcNAc needs to be used as such. Therefore, the cells in EP1484406 need to be disrupted such that the GlcNAc can be used directly by the GlcNAcepimerase. As described by Lundgren et al. (Org. Biomol. Chem., 2007, 5, 1903 - 1909) intact cells will convert the incoming GlcNAc to N-acetylglucosaminephosphate (GlcNAcP) which will be used by the cell for cell growth. This GlcNAcP is not available intercellular and can therefore not be used for the epimerase which needs a non-phosphorylated GlcNAc for isation to ManNAc. This explains why permeabilization of the cells of EP1484406 is necessary. As explained by Lundgren et al., the GlcNAcP can be used for making Neu5Ac but this requires another synthesis pathway comprising UDP-GlcNAc as an intermediate, which is described above in W02007101862. The resulting pathway further increases energy demand compared to the one described in the latter patent because uridylation of GlcNAc requires an extra ATP.

Deng et al. (Metabolic Engineering 7 (2005), 201-214) describes the production of GlcNAc via intracellular production of P which is then efficiently dephosphorylated and ed into the medium as GlcNAc. According to Deng et al., this dephosphorylation s upon export, more specifically in the periplasm of Escherichia coli. The extracellular produced GlcNAc described in this method, is not available for ellular conversion. This method to produce GlcNAc requires a two-phase fed batch process, i.e. a cell growth phase followed by a GlcNAc production phase which is only induced after the culture had reached a high cell y, to minimize inhibitory effects of phosphorylated amino sugars.

Others have attempted the same by logously expressing phosphatases and encountered the problem of reduced growth and strong metabolic burden (Lee and Oh, Metabolic engineering, 2015, 143-150). The main reason for said reduction in growth/biomass ion is the non-specificity of the phosphatase that is introduced, which dephosphorylates other essential phosphorylated compounds. Such modifications hence lead to reduced fitness and lower specific productivity. It furthermore leads to ive pressure to mutate the production pathway during production, which reduces the l process ity.

The production pathways of sialic acid and sialylated oligosacharides require the formation of high level of phosphorylated (e.g. GlcNAcP) and nucleotide pathway ediates. It is commonly understood that such formation leads to aspecific degradation of these intermediates by activation of aspecific phosphatases, which in turn leads to reduced s. In order to circumvent the effect of the expression of metabolic pathways on the growth of the production hosts, it is standard to use inducible sion systems. In this method first biomass is formed and later in the production process the production pathway is activated by for instance IPTG. This was applied by others for the production of sialic acid and sialylated 4o oligosaccharides (W02007101862; Priem et al. iology 12, 2002, 235-240; Kang et al., Metabolic engineering 14, 2012, 623-629; Yang et al., Metabolic engineering 43, 2017, 21-28).

Apart from losing tivity and titer, another downside in the use of ble systems is the excretion of intermediate pathway metabolites such as GlcNAc and ManNAc. This leads to the requirement of extra ream processing steps for the purification, hence a higher production cost in the production of sia|ic acid, sia|y||actose or other sia|y|ated compounds.

The methods for producing sia|y|ated compounds, discussed hereabove, are still insufficient in meeting the large demand of the biotechnological, pharmaceutical and medical ries. A metabolic engineering approach that successfully overcomes the problems referred to above, would represent a significant and long d advance in the field.

Summary Surprisingly, we have been able to create a production pathway that does not e induction, and does not e a UDP-GlcNAc epimerase, but allows constitutive expression which also allows better tuning of the metabolic y improving production and reducing byproduct formation during the production s.

According to one embodiment of the present invention, there is provided a method for sia|y|ated compound production with rganisms which does not require induction.

According to a r embodiment of the present ion, there is provided a production pathway that does not require a UDP-GlcNAc epimerase, and comprising modulating sion of phosphatase which does not pose a metabolic burden to the cell as was shown previously in the art. Said further embodiment of the present invention provides also an increased sia|y|ated compound production by modulating the expression of phosphatase.

In another further embodiment, the above method, when combined with the constitutive expression of the genes of the metabolic pathway, also allows better tuning of the metabolic pathway reducing byproduct formation during the tion process.

Description The present invention bes an economical, more efficient and alternative biosynthesis route for the production of sia|y|ated compounds using micro-organisms.

The present invention provides a method of producing sia|y|ated compounds by fermentative growth of microorganisms.

In ular, the invention relates to a method for the production of sia|y|ated compounds, wherein the method comprises culturing a microorganism in a culture medium. The microorganism intracellularly converts following reactions: N-acetylglucosaminephosphate to N-acetylglucosamine, N-acetylglucosamine to N-acetylmannosamine, and N- acetylmannosamine to N-acetyl-neuraminate. Furthermore, this microorganism is unable to: i) convert N-acetylglucosamine-6—P to glucosamineP, ii) convert N-acetylglucosamine to N- acetylglucosamineP, and iii) convert N-acetyl-neuraminate to N-acetyl-mannosamine.

Preferably, the conversion of N-acetylglucosaminephosphate to N-acetylglucosamine is obtained by the action of an ellularly sed phosphatase. In r preferred embodiment the N-acetylglucosamine is converted to N-acetylmannosamine by an intracellularly expressed N-acetylmannosamine epimerase. In an alternative preferred embodiment the ylmannosamine is converted by an intracellular expressed sia|ic acid synthase to N-acetyl-neuraminate. Even more preferably, the rganism comprises all three enzymes such that the microorganism converts i) N-acetylglucosaminephosphate to N- acetylglucosamine by action of an intracellularly expressed phosphatase, ii) the N- acetylglucosamine to N-acetylmannosamine by an intracellularly expressed N- acetylmannosamine epimerase; and iii) the N-acetylmannosamine to N-acetyl-neuraminate by an intracellular expressed sialic acid synthase.

Preferably, the microorganism used in the method of the invention is unable to produce following enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a N-acetylglucosamine kinase, and iii) a N-acetylneuraminate aldolase.

The present invention also provides a microorganism which expresses i) a phosphatase to dephosphorylate N-acetylglucosaminephosphate to ylglucosamine (EC 3.1.3.), ii) a GlcNAc 2-epimerase to convert N-acetylglucosamine (GlcNAc) to N-acetylmannosamine (manNac) (EC 5.1.3.8), and iii) a sialic acid synthetase to sise N-acetyl-neuraminate (Neu5Ac) from N-acetylmannosamine (ManNAc) (EC 2.5.1.56). Furthermore, this microorganism is unable to: i) convert ylglucosamine-6—P to glucosamineP, ii) convert yl-glucosamine to N-acetyl-glucosamineP, and iii) convert N-acetyl-neuraminate to N- acetyl-mannosamine.

In one , the invention provides a micro-organism that is enabled to catalyse the following reactions: the intracellular conversion of ylglucosaminephosphate to N- glucosamine, the intracellular conversion of N-acetylglucosamine to N- acetylmannosamine and, the intracellular conversion of ylmannosamine to sialic acid.

It is generally accepted that ylglucosaminephosphate is naturally efficiently excreted out of the cell and meanwhile dephosphorylated by phosphatases in the periplasm (see p. 212, second column, Deng et 0]., Metabolic Engineering 7 (2005), 201-214). Therefore, without the present invention, this excreted product would be unavailable for conversion to sialic acid.

Furthermore, re-internalization occurs through transport proteins which orylate the N- acetylglucosamine.

The use of an intracellular N-acetylglucosamineepimerase ensures lower energy (ATP) consumption than the classical prokaryotic route (via UDP-N-acetylglucosamine). This enables a more efficient production of sialic acid, sialylated oligosaccharides and/or sialylated products with a healthier and more efficient strain. By optimizing expression levels, the unfavourable chemical brium is overcome and no need of large amounts of free N-acetylglucosamine are necessary, as is in literature. Indeed, in the art, this enzyme is solely used in tic ons which use high concentrations of N-acetylglucosamine to produce N- acetylmannosamine. It would be hence logical that the use of an epimerase would require large amounts of ellular formed GlcNAc which is shown to be released in the medium (see Deng as described supra), r, the present ion has proven this can be avoided. Another advantage of the present invention over enzymatic s, is that nsive substrates can be used in the present invention, as for example a monosaccharide such as for example glucose, 4o galactose or fructose, a disaccharide such as for example sucrose or maltose or a polyol, such as, but not limited to, glycerol. This enables an economic production method by fermentation.

Different phosphatases (EC 3.1.3.) that convert N-acetylglucosaminephosphate into N- acetylglucosamine are described in the art and can be used in the present invention.

Phosphatases from the HAD superfamily and the ke family are described in the art.

Examples from these families can be found in the enzymes expressed from genes yan, inhX, yniC, ybiV, yidA, ybjl, yigL or cof from Escherichia coli. One phosphatase that catalyzes this reaction is identified in cladiella emersonii. Phosphatases are generally fic and the activity is generally not related to the family or structure. Other examples can thus be found in all phosphatase families. ic phosphatases are easily identified and screened by well- known methods as described by Fahs et 0/. (ACS Chem. Biol., 2016, 11 (11), 2944-2961).

Preferably, the phosphatase of the present invention is a HAD-alike atase. A HAD-alike phosphatase as defined herein refers to any phosphatase polypeptide which comprises: - any one or more of the following motifs as defined below: Motif 1: thDx[TV] (SEQ ID NO: 73), or Motif 2: [GSTDE][DSEN]x(1—2)[hP] x(1-2) [DGTS] (SEQ ID N05: 74, 75, 76, 77) wherein h means a hydrophobic amino acid (A, I, L, M, F, V, P, G) and x can be any distinct amino acid.

In another preferred embodiment, HAD-alike polypeptides typically have in increasing order of preference at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % overall sequence identity to any one of the polypeptides represented by SEQ ID N05: 43 ,44, 45, 47, 48, 50, 51, 52, 54, 55 or 57. Preferably, those polypeptides also comprise at least one of the above identified Motifs. More preferably, they comprise both motifs.

The l sequence identity is determined using a global ent algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and ably with sequences of mature proteins (i.e. t taking into account secretion signals or transit peptides). Compared to l sequence identity, the sequence identity will generally be higher when only ved domains or motifs are considered.

In a preferred embodiment, the HAD-alike ptide comprises any one of SEQ ID N05: 43 ,44, 45, 47, 48, 50, 51, 52, 54, 55 or 57.

In another preferred embodiment, the phosphatase is chosen from the HAD superfamily or the HAD-like phosphatase family. More preferably, the phosphatase is chosen from the group comprising: i) enzymes expressed by the genes yan, inhX, yniC, ybiV, yidA, ybjl, yigL or coffrom Escherichia coli, ii) the phosphatase of cladiella emersonii and iii) other phosphatase families. es of N-acetyl-D-g|ucosmineepimerase (EC 5.1.3.8) can be found in prokaryotes and eukaryotes. Examples for yotes are found in cyanobacteria like for example Acaryochloris marina, Anabaena variabilis, Anabaena marina, Nostoc punctiforme, Acaryochloris species, Anabaena species, Nostoc species and Synechocystis species. They are also found in Bacteroides 4o species like for example Bacteroides ovatus and oides thetaiotaomicron and in Capnocytophaga canimorsus and Mobiluncus mulieris. In eukaryotics, N-acetyl-D-glucosmine epimerase is found in Glycin max, Mus musculus, Homo sapiens, Rattus norvegicus, Bos Taurus, Sus scrofa, Canis lupus. Preferably, in the method and microorganism of the present invention, N-acetylmannosamineepimerase is chosen from the group comprising i) N- mannosamineepimerase from cyanobacteria, more in particular from chloris marina, Anabaena ilis, Anabaena , Nostoc punctiforme, Acaryochloris species, Anabaena species, Nostoc species and Synechocystis species; ii) N-acetylmannosamine epimerase from Bacteroides species, more in particular from Bacteroides ovatus, Bacteroides thetaiotaomicron, ytophaga canimorsus and Mobiluncus mulieris; iii) N-acetyl-D- glucosmineepimerase from Glycin max, Mus musculus, Homo sapiens, Rattus norvegicus, Bos Taurus, Sus scrofa or Canis lupus.

N-acetyl neuraminate synthase (also called sialic acid synthase in the art) (EC 2.5.1.56) activity is found in several prokaryotic organisms like for example Streptococcus agalatiae, Bacillus subtilis, Legionella pneumophilla, Campy/obacterjejuni, Idiomarina loihiensis, Moritella viscosa, brio salmonicida, Escherichia coli, Methanocaldococcus jannaschi, Clostridium sordellii, vibrio proteoclasticus, Micromonas a or Neisseria meningitis. Preferably, in the method and microorganism of the invention, the sialic acid (or N-acetyl neuraminate) synthase is chosen from the group comprising: sialic acid se from Streptococcus agalatiae, Bacillus subtilis, Legionella philla, Campy/obacterjejuni, Idiomarina loihiensis, Moritella viscosa, Aliivibrio salmonicida, Escherichia coli, Methanocaldococcus jannaschi, Clostridium sordellii, Butyrivibrio proteoclasticus, Micromonas commoda or Neisseria meningitis.

In one preferred aspect, any one or more of the phosphatase, N-acetylmannosamine ase and sialic acid synthase is overexpressed in the microorganism. In an alternative preferred aspect, any one or more of the phosphatase, N-acetylmannosamine epimerase and sialic acid synthase is introduced and expressed in the microorganism.

In another , the rganism lacks the genes encoding for following enzymes i) a N- acetylglycosaminephosphate deacetylase, ii) a N-acetylglucosamine , and iii) a N- acetylneuraminate aldolase. In another preferred aspect, the genes encoding for following enzymes i) a N-acetylglycosaminephosphate ylase, ii) a N-acetylglucosamine , and iii) a N-acetylneuraminate aldolase are reduced in activity, preferably said genes are deleted or knocked-out, in the microorganism.

In another preferred aspect, the microorganism further encodes a n that facilitates uptake of lactose and lacks enzymes that metabolize lactose. Methods to produce microorganisms which resist lactose killing and the resulting microorganisms are described in W02016/075243 which is herein incorporated by reference.

In a preferred aspect the microorganisms of, and used in the method of, the invention also express a CMP-sialic acid synthase (EC 43) and a sialyltransferase (EC 2.4.99.1) in order to activate the sialic acid and transfer it to a desired compound.

In a preferred aspect, the N-acetylglucosaminephosphate is obtained by introducing a glucosamine-phosphate N-acetyltransferase (EC 2.3.1.4) which uses intracellular glucosamine- 40 6-phosphate as a substrate. In most micro-organisms, glucosaminephosphate is naturally present in the cell, but the intracellular tion can be elevated by expressing a L- glutamine:D-fructosephosphate aminotransferase without inhibition, obtained either through protein engineering or by screening natural enzymes, such as present in gram positive bacteria (Deng et 0]., lic ering 7 (2005), 201-214).

In the present invention, the expression of the genes to convert N-acetylglucosamine-6— phosphate to N-acetyl-neuraminate or sia|ic acid are optimized in a way that enables intracellular dephosphorylation of N-acetylglucosaminephosphate, prevents toxic accumulation of N-acetylglucosaminephosphate and prevents excretion of N- acetylglucosamine and/or N-acetylmannosamine. Said optimization is the result of the use of constitutive sion of the genes of the production pathway. In a preferred embodiment, the present ion prevents the excretion of at least 10%, 20%, 30%, 35%, 40%, 45%, 50%, or 60% of the formed N-acetylglucosamine and/or ylmannosamine. In a further preferred embodiment, the microorganism produces less extracellular N-acetylglucosamine and/or N- acetylmannosamine than sialylated compound. More preferably, the microorganism produces less than 50%, 40%, 30%, 20%, 10%, 5%, 2% extracellular ylglucosamine and/or N- acetylmannosamine than sialylated compound. In another preferred embodiment of the present invention the rganism produces equal or more than 50%, 60%, 70%, 80%, 90%, 95%, 98% extracellular sialylated compound on total extracellular carbohydrate.

In a particular aspect, the invention s to a method for synthesis of sialylated compounds, without any ous sia|ic acid addition to the culture medium.

The sialylated compound can be N-acetylneuramic acid, a sialylated oligosaccharide, a ated lipid, sialylated glycolipids (such as, but not limited to gangliosides, ceramides), a sialylated protein or a sialylated n.

A sialylated oligosaccharide is a charged sia|ic acid containing oligosaccharide, i.e. an oligosaccharide having a sia|ic acid residue. It has an acidic nature. Some examples are 3-SL (3- sialyllactose), 3-sialyllactosamine, 6—SL (6-sialyllactose or n- acetylneuraminate alfa 2,6 galactosyl beta 1,4 Glucose), 6-sialyllactosamine, oligosaccharides comprising 6-sialyllactose, SGG ccharide (Neu5Ac ,3Gal beta -1,3GalNac beta -1,3Gala-1,4Gal beta -1,4Ga|), sialylated tetrasaccharide (Neu5Ac-alfa-2,3Gal beta -1,4GlcNAc beta -14GlcNAc), pentasaccharide LSTD (Neu5Ac alfa-2,3Gal beta cNAc beta -1,3Gal beta -1,4Glc), sialylated lacto-N-triose, ated lacto-N-tetraose, sialyllacto-N-neotetraose, monosialyllacto-N- hexaose, disialyllacto-N-hexaose |, monosialyllacto-N-neohexaose |, monosialyllacto-N- aose ||, disialyllacto-N-neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose ||, sialyllacto-N-tetraose a, disialyllacto-N-hexaose |, sialyllacto-N-tetraose b, 3-sialyl fucosyllactose, disialomonofucosyllacto-N-neohexaose, monofucosylmonosialyllacto-N- octaose (sialyl Lea), sialyllacto-N-fucohexaose ||, disialyllacto-N-fucopentaose ||, monofucosyldisialyllacto-N-tetraose and oligosaccharides bearing one or several sia|ic acid residu(s), including but not limited to: oligosaccharide moieties of the osides selected from GM3 (3sialyllactose, Neu5Aca-2,3Gal beta-4Glc) and accharides comprising the GM3 motif, GD3 Neu5Aca-2,8Neu5Aca-2,3Gal beta -1,4Glc GT3 (Neu5Aca-2,8Neu5Aca-2,8Neu5Aca- 2,3Gal beta -1,4Glc); GM2 GalNAc beta -1,4(Neu5Aca-2,3)Gal beta -1,4Glc, GMl Gal beta - 4o 1,3GalNAc beta -1,4(Neu5Aca-2,3)Gal beta c, GD1a a-2,3Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,3)Gal beta -1,4Glc GT1a Neu5Aca-2,8Neu5Aca-2,3Gal beta -1,3GalNAc beta - 1,4(Neu5Aca-2,3)Gal beta -1,4Glc GD2 GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta - 1,4Glc GT2 GspalNAc beta -1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gal beta -1,4Glc GD1b, Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta -1,4Glc GT1b Neu5Aca-2,3Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta-1,4Glc Gle Neu5Aca- 2,8Neu5Aca-2,3Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca2,3)Gal beta -1,4Glc GT1c Gal beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gal beta -1,4Glc GQlc, Neu5Aca-2,3Gal beta -1,3GalNAc beta eu5Aca-2,8Neu5Aca-2,8Neu5Aca2,3)Gal beta - 1,4Glc GP1c Neu5Aca-2,8Neu5Aca-2,36al beta -1,3GalNAc beta -1,4(Neu5Aca-2,8Neu5Aca- 2,8Neu5Aca2,3)Gal beta c GD1a Neu5Aca-2,3Gal beta -1,3(Neu5Aca-2,6)GalNAc beta - 1,4Gal beta -1,4Glc Fucosyl-GM1 Fuca-1,ZGal beta -1,3GalNAc beta -1,4(Neu5Aca-2,3)Gal beta -1,4Glc; all of which may be ed to the tion of the ponding gangliosides by ng the above oligosaccharide moieties with ceramide or synthetizing the above oligosaccharides on a ceramide.

The term micro-organism or organism or cell as indicated above refers to a microorganism chosen from the list comprising a bacterium, a yeast, or a , or, refers to a plant or animal cell. The latter ium preferably belongs to the phylum of the Proteobacteria or the phylum of the Firmicutes or the phylum of the Cyanobacteria or the phylum Deinococcus-Thermus. The latter bacterium belonging to the phylum Proteobacteria belongs ably to the family Enterobacteriaceae, preferably to the species Escherichia coli. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such as but not limited to Escherichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term s to cultivated Escherichia coli strains - designated as E. coli K12 s - which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, M61655, M182, , MC1060, MC1061, MC4100, JM101, NZN111 and AA200. Hence, the present invention specifically relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said E. coli strain is a K12 strain. More specifically, the t invention relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said K12 strain is E. coli M61655. The latter bacterium belonging to the phylum Firmicutes belongs preferably to the Bacilli, preferably Lactobacilliales, with members such as acillus Iactis, Leuconostoc mesenteroides, or Bacillales with members such as from the species Bacillus, Bacillus is or, B. amyloliquefaciens. The latter Bacterium belonging to the phylum bacteria, preferably belonging to the family of the Corynebacteriaceae, with members Corynebacterium glutamicum or C. ntans, or belonging to the family of the of the Streptomycetaceae with members Streptomyces griseus or 5. fradiae. The latter yeast preferably belongs to the phylum of the Ascomycota or the phylum of the Basidiomycota or the phylum of the Deuteromycota or the phylum of the Zygomycetes. The latter yeast belongs preferably to the genus Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia or Starmerella. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or illus. 40 The culture medium for the production host can optionally comprise an exogenous precursor or this precursor can be produced by the strain itself, such as a glycan like for example lactose, lactosamine, lacto-N-triose, lacto-N-tetraose, lacto-N-neotetraose; an oligosaccharide; a e; a lipid or an aglycon. In one particular aspect, the process of the invention is based on the active uptake of an exogenous precursor, such as for example a mono, di or tri-saccharide, more particularly an exogenous precursor selected from lactose, N-acetyllactosamine, lacto-N- biose, galactose, beta-galactoside, and alpha-galactoside such as but not limited to globotriose (Gal-alpha-1,4Gal-beta-1,4Glc), while the microorganisms are growing on an inexpensive carbon substrate, such as a disaccharide such as sucrose or maltose. Moreover, these microorganisms are also able to grow on glucose, fructose or glycerol. The expression exogenous precursor is intended to denote a compound involved in the biosynthetic pathway of the product according to the invention that is alized by the rganism.

In one , the invention provides for method for production of sialylated forms of lacto-N- triose, lacto-N-tetraose or lacto-N-neotetraose. Any one of these three molecules are synthetized by the organism via the activity of a galactosyltransferase (EC 2.4.1.38), preferably originating from the group comprising Homo sapiens, Bos taurus, Mus mulatta, Gallus , Danio rerio, Helicobacter pylori and hilus ducrey and/or a N- acetylglucosaminyltransferase (EC 2.4.1.90) preferably originating from the group comprising 305 Taurus, Homo Sapiens and Mus Musculus. To enhance the formation of these oligosaccharides the genes coding for UDP sugar ase and galactosephosphate uridylyltransferase are lacking, reducing in activity or knocked out in the rganism.

In another aspect a method for producing a ated oligosaccharide is provided in which the method ses culturing a microorganism as described above and wherein the microorganism produces internally, activated N-acetylneuraminate as donor substrate for a sialyltransferase; and wherein the method further comprises ing the microorganism in a culture medium which comprises an exogenous precursor selected from the group consisting of lactose, N-acetyllactosamine, lacto-N-biose, ose, alactoside, and alpha-galactoside such as but not limited to globotriose (Gal-alpha-1,4Gal-beta-1,4Glc)galactose. The exogenous precursor is actively taken up into the microorganism and the exogenous precursor is the acceptor substrate for the sialytransferase for producing the sialylated accharide.

In a further aspect, the method according to the invention provides for the tion of 35ialyllactose or 6sialyllactose. In this method the microorganism is ated at high cell density on a carbon substrate, such as glucose or glycerol, and fed with lactose. The lactose is internalized by the lactose permease and sialylated by the recombinant sialyltransferase using the CMP- N-acetyl-neuraminate endogenously generated from N-acetylglucosamine.

The microorganism or cell of the invention is capable to grow on a ccharide, disaccharide, oligosaccharide, polysaccharide, , a x medium or a mixture thereof as the main carbon source. With the term main is meant the most important carbon source for biomass formation, carbon dioxide and/or by-products formation (such as acids and/or alcohols, such as acetate, lactate, and/or ethanol), i.e. 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, 99 % of all the required carbon is derived from the above-indicated carbon source. In one embodiment of the invention, said carbon source is the sole carbon source for said organism, i.e. 100 % of all the required carbon is derived from the indicated carbon source. 40 In a further preferred embodiment, the microorganism or cell of the invention is using a split metabolism having a production pathway and a biomass pathway as described in W02012/007481, which is herein incorporated by reference. Said organism can, for example, be genetically modified to accumulate fructosephosphate by altering the genes selected from the phosphoglucoisomerase gene, phosphofructokinase gene, fructosephosphate aldolase gene, fructose isomerase gene, and/or fructose:PEP phosphotransferase gene.

With the term ccharide is meant a sugar that is not decomposable into simpler sugars by hydrolysis, is classed as either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Examples are glucose, fructose, galactose, mannose, ribose and/or ose.

With the term disaccharide is meant a sugar that is composed of two monosaccharides that are chemically bound. Examples are maltose, sucrose, lactose, trehalose, cellobiose and/or chitobiose.

With the term oligosaccharide is meant a sugar that is composed of three to ten monosaccharides that are chemically bound. Examples are maltotriose, -oligosaccharides, o-oligosaccharides, mannan oligosaccharides, isomaltooligosaccharide, human milk oligosaccharides and/or glucooligosaccharides.

With the term polyol is meant an alcohol containing multiple hydroxyl groups. For example glycerol, sorbitol, or ol.

With the term complex medium is meant a medium for which the exact constitution is not determined. Examples are molasses, corn steep liquor, peptone, tryptone or yeast extract.

Production of sialylated compounds can be sed by adding precursors to the medium, such as N-acetylglusosamine, N-acetylmannosamine, glutamine, glutamate, phosphoenolpyruvate and/or pyruvate.

The sialylated compounds produced in the method of the invention as described above may be recovered using various methods, or a combination thereof, known in the art. Depending on the produced sialylated nd, the compound is available in the extracellular fraction or retained in the cells. When the produced sialylated compound is retained in the cells, the sialylated compound will first be ed from the cells by cell tion. Again depending on the produced sialylated compound, the cells may be separated from the extracellular fraction.

In the other case, cells are disrupted without first separation from the extracellular fraction, wherein cells are disrupted by techniques such as, but not d to, heating, freeze thawing and/or shear stress through tion, mixing and/or French press. The extracellular and/or intracellular fraction may be separated from the cells and/or cell debris by centrifugation, filtration, microfiltration, and nanofiltration. Flocculating agents may be used to aid in product separation. The sialylated compounds in the extracellular or intracellular fraction may be extracted by ion ge, ultra-or nanofiltration or electrodialysis, chromatography such as size exclusion, ion chromatography and simulated moving bed. Another example of filtering the sialylated compounds from liquid phase is by tion using a deep bed filter with cotton and ted carbon or carbon , where after the permeate is passed through a carbon polisher ed by e.g. a 0.2 micron microfiltration membrane system to remove color, micro- organisms and suspended carbon particles. Thereafter the sialylated compound may be concentrated in a vacuum evaporator to obtain a trate. The concentrate can be 40 precipitated and/or dried through heat drying, spray drying and/or lyophilization to obtain high purity sialylated compound. An amorphous form powder can then be obtained. This ous powder may further be crystallised to obtain crystalline sialylated compound.

In exemplary embodiment, sialylated compounds may be isolated from the culture medium using methods known in the art for fermentations. For e, cells may be removed from the culture medium by centrifugation, filtration, flocculation, decantation, or the like. Then, the sialylated compounds may be ed from the extracellular fraction using methods such as ion- ge. A further purification of said sialylated compounds may be accomplished, for example, by ltration or ultrafiltration or ion ge to remove any remaining DNA, protein, LPS (endotoxins), or other impurity.

In another exemplary ment, sialyllactose may be isolated from the culture medium using methods known in the art for fermentations. For example, cells may be removed from the culture medium by centrifugation, filtration, flocculation, decantation, or the like. Then, the sialyllactose may be isolated from the extracellular fraction using methods such as ion- ge. A further purification of said sialyllactose may be accomplished, for example, by nanofiltration or ultrafiltration or ion exchange to remove any remaining DNA, protein, LPS (endotoxins), or other ty. Another purification and ation step is accomplished by crystallization or precipitation of the product. Another formulation step is to spray dry or lyophilize sialyllactose.

The sialylated compound may contain a counter ion, such as, a monovalent ion, such as a proton, sodium ion, potassium, a divalent ion, such as m magnesium, iron, or, a trivalent ion such as iron, or a combination of ions.

Throughout the disclosure of the present disclosure the term sialic acid, N-acetyl neuraminate and yl neuraminic acid are used interchangeably.

As used herein, the term intracellular or intracellularly in e.g. intracellularly converting, ellularly tion, intracellularly expressed, ellular formed must be understood to mean within the cell of the microorganism. The term extracellular must be understood to mean e of the cell.

Further definitions used throughout the present specification Homologue(s) "Homologues“ of a protein encompass peptides, oligopeptides, ptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the fied protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence ions of single or le amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)tag, glutathione S- transferase-tag, protein A, 4o maltose-binding protein, dihydrofolate reductase, Tag»100 epitope, c-myc epitope, FLAG(R)- epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.

A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as r hydrophobicity, hydrophilicity, antigenicity, propensity to form or break cal structures or beta -sheet structures). Amino acid substitutions are typically of single es, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative tution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).

Table 1: Examples of ved amino acid substitutions Substitutions Substitutions Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by inant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, ques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, 17- Gen in vitro mutagenesis (USB, Cleveland, OH), hange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR- mediated site-directed mutagenesis or other site- directed mutagenesis ols.

Derivatives "Derivatives" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally ing amino acid residues. "Derivatives" of a protein also encompass peptides, oligopeptides, ptides which comprise naturally ing altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non- naturally d amino acid residues compared to the amino acid sequence of a naturally- occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid ce from which it is derived, for e a reporter le or other ligand, covalently or non-covalently bound to the amino acid ce, such as a reporter molecule which is bound to facilitate its detection, and non- naturally occurring amino acid residues ve to the amino acid sequence of a naturally- ing protein. Furthermore, "derivatives" also e fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or doxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Orthologue(s)/Para|ogue(s) Orthologues and paralogues encompass evolutionary concepts used to describe the ral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from ent organisms that have originated through speciation, and are also derived from a common ancestral gene.

Domain, Motif/Consensus sequence/Signature The term "domain" refers to a set of amino acids conserved at ic positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the ure, stability or function of a protein.

Identified by their high degree of vation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.

The term "motif or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain). list databases exist for the identification of s, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242- 244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized e syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., g D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 ). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

Methods for the ent of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and . GAP uses the algorithm of 40 Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990)] Mol Biol 215: 403- ) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the al Centre for Biotechnology Information (NCBI). Homologues may readily be fied using, for example, the ClustalW multiple sequence ent algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage.

Global percentages of similarity and ty may also be determined using one of the methods available in the MatGAT software e (Campanella et al., BMC Bioinformatics. 2003 Jul ,4:29. : an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be med to optimise alignment between conserved motifs, as would be nt to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, ic domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is ularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1);195—7).

Reciprocal BLAST Typically, this involves a first BLAST ing BLASTing a query sequence (for example using any of the sequences listed in Table A of the Examples section) against any ce database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST s may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) t sequences from the organism from which the query sequence is derived. The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is d, a BLAST back then y results in the query sequence amongst the highest hits; an orthologue is identified if a anking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the e, the more significant the score (or in other words the lower the chance that the hit was found by chance).

Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of cal nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, ed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.

Construct 4o Additional regulatory elements may include transcriptional as well as ational enhancers.

Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. An intron sequence may also be added to the 5 untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol, as described in the definitions section. Other l sequences (besides er, enhancer, silencer, intron sequences, 3UTR and/or 5UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a ic cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule).

For the detection ofthe successful transfer ofthe nucleic acid ces as used in the methods of the invention and/or selection of transgenic microorganisms comprising these nucleic acids, it is advantageous to use marker genes (or er genes). Therefore, the genetic construct may ally comprise a selectable marker gene. The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker removal are known in the art, useful ques are described above in the definitions section. tory element/Control ce/Promoter The terms atory element", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "promoter" lly refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other ns, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is ed for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream ting sequences, ers and ers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences. The term "regulatory element" also asses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.

Constitutive promoter A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ.

Transgenic/Transgene/Recombinant For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid ce or an organism transformed with the nucleic acid 40 sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either (a) the nucleic acid sequences ng proteins useful in the methods of the invention, or (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or somal locus in the original rganism or the presence in a c library. In the case of a genomic library, the natural genetic environment of the nucleic acid ce is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the lly occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid ce encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is ed by non-natural, synthetic ficial") methods such as, for example, mutagenic ent. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815.

A transgenic microorganism for the purposes of the invention is thus understood as meaning, as above, that the c acids used in the method of the invention are not present in, or originating from, the genome of said rganism, or are present in the genome of said rganism but not at their natural locus in the genome of said rganism, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural on in the genome of a microorganism, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been ed. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic microorganism are mentioned herein.

It shall further be noted that in the context of the present invention, the term "isolated c acid" or "isolated polypeptide" may in some instances be considered as a synonym for a "recombinant nucleic acid" or a "recombinant polypeptide", respectively and refers to a nucleic acid or polypeptide that is not located in its natural genetic environment and/or that has been modified by recombinant methods.

Modulation The term "modulation" means in relation to expression or gene expression, a process in which the expression level is d by said gene expression in comparison to the control microorganism, the expression level may be sed or decreased. The original, unmodulated expression may be of any kind of sion of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. For the purposes ofthis invention, the al lated expression may also be absence of any expression. The term ating the activity" shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased production yield and/or increased growth of the microorganisms. The expression can increase from zero (absence of, or immeasurable expression) to a certain amount, or can decrease from a certain amount to immeasurable small amounts or zero.

Expression The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific c construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

Increased expression/overexpression The term "increased expression" or "overexpression" as used herein means any form of expression that is onal to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level might also be zero, i.e. absence of expression or urable expression.

Methods for increasing sion of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as er or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a cleotide so as to upregulate sion of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by on, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443), or ed promoters may be introduced into a microorganism cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. |f polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3-end of a polynucleotide coding . The polyadenylation region can be derived from the natural gene, from a variety of other microorganism genes, or from T-DNA.

Moreover, the present invention relates to the following specific embodiments: 1. Method for the production of sialylated compounds, the method comprising: - culturing a rganism in a culture medium, said culture medium optionally comprising an 40 ous precursor, - wherein said rganism intracellularly converts N-acetylglucosaminephosphate to N- acetylglucosamine, said N-acetylglucosamine to N-acetylmannosamine and said N- acetylmannosamine to N-acetyl-neuraminate; and - wherein said microorganism is unable to i) convert N-acetylglucosamineP to glucosamine- 6-P, ii) convert yl-glucosamine to N-acetyl-glucosamineP, and iii) convert N-acetyl- neuraminate to N-acetyl-mannosamine. 2. The method according to embodiment 1 wherein: i) said conversion of N-acetylglucosaminephosphate to ylglucosamine is obtained by the action of an intracellularly expressed phosphatase, ii) said N-acetylglucosamine to N-acetylmannosamine conversion is performed by an intracellularly expressed N-acetylmannosamine epimerase; and iii) intracellular expressed sialic acid synthase converts said N-acetylmannosamine to yl- neuraminate. 3. The method according to any one of embodiment 1 or 2 wherein said organism is unable to produce following enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a N- acetylglucosamine kinase, and iii) a N-acetylneuraminate aldolase. 4. The method according to any one of embodiment 1 to 3, wherein all said conversions are catalysed by enzymes encoded by constitutively expressed genes.

. The method according to embodiment 2 wherein the phosphatase is chosen from the HAD superfamily or the HAD-like phosphatase family, preferably said phosphatase is chosen from the group comprising: i) s expressed by the genes yan, inhX, yniC, ybiV, yidA, ybjl, yigL or cof from Escherichia coli, ii) the phosphatase of Blastocladiella nii and iii) other phosphatase families, more preferably said phosphatase is a ike phosphatase polypeptide as defined in the claims. 6. The method according to any one of the embodiments 2, 3, 4 or 5, wherein the N- acetylmannosamineepimerase is chosen from the group comprising i) N- acetylmannosamineepimerase from cyanobacteria, more in particular from Acaryochloris marina, Anabaena variabilis, na marina, Nostoc punctiforme, Acaryochloris species, Anabaena species, Nostoc species and Synechocystis species; ii) N-acetylmannosamine epimerase from Bacteroides species, more in particular from Bacteroides ovatus, Bacteroides otaomicron, Capnocytophaga canimorsus and Mobiluncus is; iii) N-acetyl-D- glucosmineepimerase from Glycin max, Mus us, Homo sapiens, Rattus norvegicus, Bos Taurus, Sus scrofa or Canis lupus. 7. The method according to any one of the embodiments 2, 3, 4, 5 or 6, wherein the sialic acid synthase is chosen from the group comprising: sialic acid synthase from Streptococcus agalatiae, Bacillus subtilis, ella pneumophilla, Campy/obacterjejuni, Idiomarina loihiensis, Moritella viscosa, brio salmonicida, ichia coli, Methanocaldococcus jannaschi, Clostridium sordellii, Butyrivibrio proteoclasticus, onas commoda or Neisseria meningitis. 8. The method according to any one of the preceding embodiments, n said sialylated compound is selected from the group consisting of N-acetylneuramic acid, ated o|igosaccharide, sialylated lipids, sialylated protein, sialylated aglycon. 9. The method ing to the previous embodiment, wherein said sialylated compound is a sialylated o|igosaccharide.

. The method according to embodiment 9, n said sialylated o|igosaccharide is sia|y||actose, preferably any one of 3-SL or 6—SL. 11. The method according to embodiment 9, wherein said sialylated o|igosaccharide is disialyl lacto-N-tetraose. 12. The method according to embodiment 8, wherein said sialylated compound is N- acetylneuraminic acid. 13. The method according to any one of ment 1 to 10 wherein said ated nd is a sialylated lacto-N-triose, lacto-N-tetraose or a lacto-N-neotetraose, and wherein said microorganism further comprises the activity of a galactosyltransferase (EC 2.4.1.38), ably said galactosyltransferase originates from the group comprising Homo sapiens, Bos taurus, Mus mulatta, Gallus gallus, Danio rerio, Helicobacter pylori and Haemophilus ducrey; and/or said microorganism comprises the activity of a N-acetylglucosaminyltransferase (EC 90), preferably said N-acetylglucosaminyltransferase originates from the group sing Bos taurus, Homo sapiens and Mus musculus. 14. The method ing to embodiment 13 wherein said microorganism is unable to express the genes coding for UDP sugar hydrolase and galactose-1—phosphate uridylyltransferase.

. The method according to any one of ments 1 to 14, wherein said microorganism produces less than 50%, 40%, 30%, 20%, 10%, 5%, 2% extracellular N-acetylglucosamine and/or N-acetylmannosamine than sialylated compound and/or said micro-organism produces equal or more than 50%, 60%, 70%, 80%, 90%, 95%, 98% sialylated compound on total carbohydrate 16. A method for producing a sialylated o|igosaccharide, comprising: a) culturing a microorganism according to the method of any one of ments 1 to 7, 14 and 15, and wherein said microorganism produces internally, activated N-acetylneuraminate as donor substrate for a sialyltransferase; and b) culturing said microorganism in a culture medium comprising an ous precursor selected from the group consisting of lactose, N-acetyllactosamine, lacto-N-biose, galactose, beta-galactoside, and alpha-galactoside such as but not limited to globotriose lpha- 1,4Gal-beta-1,4Glc)galactose, wherein active uptake into the microorganism of said exogenous precursor occurs and wherein said exogenous precursor is the acceptor substrate for said sialytransferase for producing the sialylated o|igosaccharide. 17. The method according to embodiment 2, wherein any one or more of said phosphatase, N-acetylmannosamine epimerase and sialic acid synthase is overexpressed in the microorganism. 18. The method according to embodiment 2, wherein any one or more of said phosphatase, N-acetylmannosamine epimerase and sialic acid synthase is introduced and expressed in the microorganism. 19. The method according to embodiment 3, wherein said microorganism lacks the genes encoding for following enzymes i) a ylglycosaminephosphate deacetylase, ii) a N- acetylglucosamine kinase, and iii) a N-acetylneuraminate a|do|ase.

. The method according to embodiment 3, n in said microorganism the genes encoding for ing enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a N- acetylglucosamine kinase, and iii) a N-acetylneuraminate aldolase are reduced in activity, preferably said genes are deleted or knocked-out. 21. The method according to any one of the embodiments 1 to 20, wherein said rganism further encodes a protein that facilitates uptake of lactose and lacks enzymes that metabolize lactose. 22. The method according to any one of embodiments 1 to 21, wherein said microorganism is a bacteria, preferably an Escherichia coli strain, more preferably an Escherichia coli strain which is a K12 strain, even more ably the Escherichia coli K12 strain is Escherichia coli M61655. 23. The method according to any one of embodiments 1 to 21, wherein said microorganism is a yeast. 24. The method according to any one of embodiments 1 to 23, wherein the exogenous precursor is chosen from the group comprising lactose, galactose, beta-galactoside, and alpha- galactoside, such as riose lpha-1,4Gal-beta-1,4Glc).

. A microorganism for the production of sialylated compounds, said microorganism - intracellularly converts ylglucosaminephosphate to N-acetylglucosamine, said N- glucosamine to N-acetylmannosamine and said N-acetylmannosamine to N-acetyl- neuraminate; and - is unable to i) convert N-acetylglucosamineP to glucosamineP, ii) t N-acetyl- glucosamine to N-acetyl-glucosamineP, and iii) convert yl-neuraminate to N-acetyl- mannosamine. 26. A microorganism for the production of a sialylated compound, said microorganism being defined in any one of embodiments 2 to 24. 27. A cell culture medium comprising lactose as sor and the microorganism of any one of embodiments 25 or 26. 28. The method ing to one of embodiments 1 to 24, for the production of 35ialyllactose or 6sialyllactose, wherein the microorganism is ated at high cell density on a carbon substrate, such as glucose or glycerol, and fed with lactose which is internalized by the lactose permease and sialylated by said recombinant sialyltransferase using the CMP- N-acetyl- neuraminate endogenously generated from N-acetylglucosamine. 29. The method according to any one of embodiments 1 to 24, wherein said sialylated compound is isolated from said culture medium by means of a unit ion selected from the group centrifugation, filtration, microfiltration, iltration, ltration, ion exchange, electrodialysis, chromatography, simulated moving bed, evaporation, precipitation, llization, lyophilization and/or spray drying . A sialylated compound produced according to the method described in any one of embodiments 1 to 24, wherein said sialylated compound is purified by centrifugation and/or filtration, ion-exchange, concentration through ation or nanofiltration, formulation through crystallization or spraydrying or lyophilization. 31. A sialylated compound produced according to the method described in any one of embodiments 1 to 24, wherein said sialylated compound is added to food formulation, feed formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation. 32. The method according to any one of embodiments 1 to 24, wherein said culture medium comprises any one or more of the following: a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, a complex medium as the main carbon . 33. The method according to embodiment 32, wherein said main carbon source provides at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of all required carbon for the growth of said rganism. 34. The method according to embodiment 32, wherein said monosaccharide is chosen from the group comprising glucose, fructose, galactose, mannose, ribose or arabinose.

. The method ing to embodiment 32, n said disaccharide is chosen from the group comprising maltose, e, lactose, trehalose, cellobiose or chitobiose. 36. The method according to embodiment 32, wherein said oligosaccharide is chosen from the group comprising maltotriose, fructo-oligosaccharides, galacto-oligosaccharides, mannan oligosaccharides, isomaltooligosaccharide or glucooligosaccharides. 37. The method according to embodiment 32, wherein said polyol is chosen from the group comprising glycerol. 38. The method according to embodiment 32, wherein said complex medium is chosen from the group comprising molasses, corn steep liquor, peptone, tryptone or yeast extract.

In a preferred aspect, the present invention s to the following preferred specific embodiments: 1. A method for the production of a ated nd in a microorganism, the method comprising: - ing a microorganism in a culture medium, said e medium optionally comprising an exogenous precursor, wherein said microorganism comprises at least one c acid encoding a phosphatase, at least one nucleic acid encoding an N-acetylmannosamine ase; and at least one nucleic acid encoding a sialic acid synthase, and wherein said microorganism is unable to i) convert N-acetylglucosamineP to glucosamine P, ii) convert N-acetyl-glucosamine to yl-glucosamineP, and iii) convert N-acetyl- inate to N-acetyl-mannosamine; and - ting expression in said microorganism of a nucleic acid encoding a HAD-alike phosphatase polypeptide, wherein said HAD-alike phosphatase polypeptide comprises: - at least one of the following motifs: Motif 1: thDx[TV] (SEQ ID NO: 73), or Motif 2: [GSTDE][DSEN]x(1-2)[hP] x(1-2) [DGTS] (SEQ ID N05: 74, 75, 76, 77) wherein h means a hydrophobic amino acid (A, I, L, M, F, V, P, G) and x can be any distinct amino acid; - or a homologue or derivative of any one of SEQ ID N05: 43 ,44, 45, 47, 48, 50, 51, 52, 54, 55 or 57 having at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % overall sequence identity to said polypeptide. 2. The method according to preferred embodiment 1, wherein said HAD-alike polypeptide comprises any one of SEQ ID N05: 43 ,44, 45, 47, 48, 50, 51, 52, 54, 55, 57. 3. Method according to preferred embodiment 1, wherein said modulated expression is effected by introducing and expressing in a microorganism a nucleic acid encoding a HAD-alike ptide. 4. Method ing to preferred embodiment 1, wherein said modulated expression is effected by the action of a constitutive promoter. 5. The method according to any one of the preceding preferred embodiments, wherein said sialylated compound is selected from the group consisting of N-acetylneuramic acid, ated oligosaccharide, sialylated lipids, sialylated protein, sialylated aglycon. 6. The method according to the previous preferred embodiment, n said sialylated nd is a sialylated oligosaccharide. 7. The method according to preferred embodiment 8, wherein said sialylated oligosaccharide is sialyllactose. 8. The method according to preferred embodiment 8, wherein said sialylated oligosaccharide is disialyl lacto-N-tetraose. 9. The method according to red embodiment 7, wherein said sialylated compound is N-acetylneuraminic acid.

. The method according to any one of preferred embodiment 1 to 9 wherein said sialylated compound is a sialylated lacto-N-triose, lacto-N-tetraose or a N-neotetraose, and wherein said microorganism further comprises the activity of a galactosyltransferase (EC 2.4.1.38), preferably said ga|actosy|transferase originates from the group comprising Homo sapiens, Bos taurus, Mus mulatta, Gallus gallus, Danio rerio, Helicobacter pylori and Haemophilus ducrey; and/or said microorganism comprises the activity of a N- acetylglucosaminyltransferase (EC 2.4.1.90), preferably said N-acetylglucosaminyltransferase originates from the group sing Bos taurus, Homo sapiens and Mus musculus. 11. The method according to preferred embodiment 12 wherein said microorganism is unable to express the genes coding for UDP sugar hydrolase and ga|actosephosphate uridylyltransferase. 12. The method according to any one of preferred embodiments 1 to 13, wherein said microorganism produces less than 50%, 40%, 30%, 20%, 10%, 5%, 2% extracellular N- acetylglucosamine and/or ylmannosamine than sialylated compound and/or said micro- organism produces equal or more than 50%, 60%, 70%, 80%, 90%, 95%, 98% sialylated compound on total ydrate 13. A method for producing a sialylated oligosaccharide, comprising: a) culturing a microorganism according to the method of any one of preferred embodiments 1 to 12, and wherein said microorganism produces internally, activated N-acetylneuraminate as donor substrate for a sialyltransferase; and b) culturing said microorganism in a culture medium comprising an exogenous precursor ed from the group consisting of lactose, N-acetyllactosamine, lacto-N-biose, ga|actose, a|actoside, and alpha-ga|actoside such as but not limited to globotriose (Gal-alpha- 1,4Gal-beta-1,4Glc)galactose, wherein active uptake into the microorganism of said exogenous precursor occurs and wherein said exogenous sor is the acceptor substrate for said ransferase for producing the sialylated oligosaccharide. 14. The method according to preferred embodiment 1, wherein any one or more of said N- acetylmannosamine epimerase and sialic acid synthase is overexpressed in the microorganism.

. The method according to preferred embodiment 1, n any one or more of said N- acetylmannosamine epimerase and sialic acid synthase is introduced and sed in the microorganism. 16. The method according to preferred embodiment 1, wherein said microorganism lacks the genes encoding for following enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a ylglucosamine kinase, and iii) a ylneuraminate aldolase. 17. The method ing to preferred embodiment 1, n in said microorganism the genes encoding for following enzymes i) a N-acetylglycosaminephosphate deacetylase, ii) a N-acetylglucosamine , and iii) a N-acetylneuraminate aldolase are reduced in activity, preferably said genes are deleted or knocked-out. 18. The method according to any one of the preferred embodiments 1 to 17, wherein said microorganism further s a protein that facilitates uptake of e and lacks enzymes that metabolize lactose. 19. The method ing to any one of preferred embodiments 1 to 18, wherein said microorganism is a bacterium, preferably an Escherichia coli strain, more preferably an Escherichia coli strain which is a K12 strain, even more preferably the Escherichia coli K12 strain is Escherichia coli M61655.

. The method according to any one of preferred embodiments 1 to 18, wherein said microorganism is a yeast. 21. The method ing to any one of preferred embodiments 1 to 20, wherein the exogenous sor is chosen from the group comprising lactose, galactose, beta-galactoside, and alpha-galactoside, such as globotriose (Gal-alpha-1,4Gal-beta-1,4Glc). 22. Microorganism, obtainable by a method according to any one of claims 1 to 21, wherein said microorganism comprises a recombinant nucleic acid encoding a HAD-alike polypeptide. 23. A microorganism for the production of sialylated compounds wherein said microorganism comprises at least one nucleic acid ng a phosphatase, at least one nucleic acid encoding an N-acetylmannosamine epimerase; and at least one c acid encoding a sialic acid synthase, and wherein said microorganism is unable to i) convert N-acetylglucosamine-6—P to glucosamineP, ii) convert N-acetyl-glucosamine to N-acetyl-glucosamineP, and iii) convert N-acetyl-neuraminate to N-acetyl-mannosamine; characterised in that said microorganism comprises a modulated expression of a nucleic acid encoding a HAD-alike phosphatase polypeptide as defined in preferred embodiment 1. 24. Construct comprising: (i) c acid encoding a HAD-alike polypeptide as defined in preferred embodiment 1 or 2; (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally (iii) a transcription termination sequence.

. Construct according to preferred embodiment 24, wherein one of said control ces is a constitutive promoter. 26. Use of a construct according to preferred embodiment 24 or 25 in a method for producing sialylated compounds. 27. A sialylated compound produced according to the method described in any one of preferred embodiments 1 to 21, wherein said sialylated compound is added to food ation, feed formulation, pharmaceutical formulation, cosmetic ation, or agrochemical ation. 28. A microorganism for the production of a sialylated compound, said microorganism being defined in any one of embodiments 2 to 21. 29. A cell culture medium sing lactose as sor and the microorganism of any one of embodiments 22, 23 or 28.

. The method according to one of embodiments 1 to 21, for the tion of llactose or 6sialyllactose, wherein the microorganism is ated at high cell density on a carbon substrate, such as glucose or glycerol or sucrose, and fed with lactose which is internalized by the lactose permease and sialylated by said inant sialyltransferase using the CMP- N-acetyl-neuraminate endogenously generated from N-acetylglucosamine. 31. The method according to any one of embodiments 1 to 21, wherein said sialylated compound is isolated from said culture medium by means of a unit ion selected from the group centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, ion exchange, electrodialysis, chromatography, simulated moving bed, evaporation, precipitation, crystallization, lyophilization and/or spray drying 32. A ated nd produced according to the method described in any one of embodiments 1 to 21, wherein said sialylated compound is purified by centrifugation and/or filtration, ion-exchange, concentration through evaporation or nanofiltration, formulation through crystallization or spraydrying or lyophilization. 33. A sialylated compound produced according to the method described in any one of embodiments 1 to 21, wherein said sialylated compound is added to food formulation, feed ation, pharmaceutical ation, cosmetic formulation, or agrochemical formulation. 34. The method according to any one of embodiments 1 to 21, wherein said culture medium comprises any one or more of the following: a monosaccharide, disaccharide, oligosaccharide, polysaccharide, polyol, a complex medium as the main carbon source.

. The method ing to embodiment 34, n said main carbon source provides at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of all ed carbon for the growth of said microorganism. 36. The method ing to embodiment 34, wherein said monosaccharide is chosen from the group comprising glucose, fructose, galactose, mannose, ribose or arabinose. 37. The method according to embodiment 34, wherein said disaccharide is chosen from the group comprising maltose, sucrose, lactose, trehalose, cellobiose or chitobiose. 38. The method according to embodiment 34, wherein said oligosaccharide is chosen from the group sing maltotriose, fructo-oligosaccharides, galacto-oligosaccharides, mannan oligosaccharides, isomaltooligosaccharide or glucooligosaccharides. 39. The method according to embodiment 34, wherein said polyol is chosen from the group comprising glycerol. 40. The method according to embodiment 34, wherein said complex medium is chosen from the group comprising molasses, corn steep liquor, e, tryptone or yeast extract.

The following drawings and examples will serve as r ration and clarification of the present invention and are not intended to be limiting.

Brief description of the drawings Fig. 1 shows an exemplary pathway as used in example 2 for the production of sialic acid according to the t invention. Fig. 1A shows the pathway without all K0 and pression signs. Fig. 1B shows the pathway as used in e 2 with the knock-out indicated with a cross and overexpression with an upgoing arrow next to the indicated enzyme.

Fig. 2 shows the production results of the Escherichia coli strain capable of producing sialic acid as described in example 2.

Fig. 3 shows examples of different sialylated compounds which can be produced in the method of the present invention.

Fig. 4 shows the l density and sialic acid production of strains supplemented with the indicated phosphatases.

Fig. 5 shows the growth rates of strains supplemented with the ted phosphatases.

Fig. 6 shows the parts of an ent of the phosphatases tested in the examples.

Example 1: Materials and methods Method and materials Escherichia coli Media Three different media were used, namely a rich Luria Broth (LB), a minimal medium for shake flask (MMsf) and a minimal medium for fermentation (MMf). Both minimal media use a trace element mix.

Trace element mix consisted of 3.6 g/L FeC|2.4H20, 5 g/L CaC|2.2H20, 1.3 g/L MnC|2.2H20, 0.38 g/L CuCI2.2H20, 0.5 g/L CoC|2.6H20, 0.94 g/L ZnCI2, 0.0311 g/L H3B04, 0.4 g/L Na2EDTA.2H20 and 1.01 g/L thiamine.HC|. The molybdate solution ned 0.967 g/L Na2MoO4.2H20. The selenium solution contained 42 g/L Se02.

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5 % yeast extract (Difco) and 0.5% sodium chloride (VWR, Leuven, Belgium).

Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar , Erembodegem, Belgium) added.

Minimal medium for shake flask experiments (MMsf) contained 2.00 g/L NH4CI, 5.00 g/L (NH4)2S04, 2.993 g/L KH2P04, 7.315 g/L , 8.372 g/L MOPS, 0.5 g/L NaCI, 0.5 g/L MgS04.7H20. A carbon source chosen from, but not limited to glucose, fructose, e, glycerol and maltotriose, was used. The concentration was default 15 g/L, but this was subject to change depending on the experiment. 1 mL/L trace element mix, 100 uL/L molybdate solution, and 1 mL/L selenium solution. The medium was set to a pH of 7 with 1M KOH. ing on the experiment lactose could be added as a precursor.

The minimal medium for fermentations contained 6.75 g/L NH4CI, 1.25 g/L (NH4)2S04, 1.15 g/L KH2P04 (low phosphate medium) or 2.93 g/L KH2P04 and 7.31 g/L KH2P04 (high phosphate medium), 0.5 g/L NaCI, 0.5 g/L 7H20, a carbon source including but not limited to glucose, sucrose, fructose, maltose, glycerol and riose, 1 mL/L trace element mix, 100 uL/L molybdate solution, and 1 mL/L selenium solution with the same composition as described above. x medium, e.g. LB, was sterilized by autoclaving (121 °C, 21) and minimal medium (MMsf and MMf) by filtration (0.22 um Sartorius). If necessary the medium was made selective by adding an antibiotic (e.g. ampicillin (100mg/L), chloramphenicol (20 mg/L), icillin /L), spectinomycin (40mg/L) and/or kanamycin (50mg/L)).

Strains Escherichia coli MG1655 [lambda', F', rph-1] was obtained from Coli Genetic Stock Center (US), CGSC Strain#: 7740 in March 2007. Mutant strains were constructed using the homologous recombination, as described by Datsenko and Wanner (PNAS 97 (2000), 6640-6645).

Plasmids pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT-flanked kanamycin ance (kan) gene), and pCP20 (expresses FLP recombinase activity) plasmids were obtained from Prof.

R. Cunin (Vrije siteit Brussel, Belgium in 2007).

Plasmid pCX-CjneuB was constructed using Gibson ly. The gene CjneuBl was expressed using the expression vector as described by Aerts et. al (Eng. Life Sci. 2011, 11, No. 1, 10-19). d pCX-CjneuB-NmneuA-dest was constructed using Gibson assembly. The genes CjneuBl, NmneuA and dest were expressed using the expression vector as described by Aerts et. al (Eng. Life Sci. 2011, 11, No. 1, 10-19).

Plasmids for phosphatase expression were constructed using Golden Gate assembly. The phosphatases (EcAphA, EcCof, EcHisB, EcOtsB, EcSurE, EcYaed, EcchU, EcYedP, EchbT, EchdA, EchgB, EchhX, EcYniC, EchaB, EchbL and PsMupP) were sed using promoters apFAB87 and apFAB346 and UTRs gene10_SD2-junction_HisHA and UTR1 AA'I'I'CGCCGGAGGGATA'I'I'AAAAtgaatggaaaattgAAACATC'I'I'AATCATGCTAAGGAGG'I'ITI'CTAATG (SEQ ID NO: 41). All promoters and UTRs except UTR1 are described by Mutalik et. al (Nat.

Methods 2013, No. 10, 354-360). Also phosphatases EcAppA, Epoh, EcSerB, EcNagD, EcthA, EcYbiV, EcijL, EchbR, EcheH, EchgL, Ec ijG, EchfG, , ScDOG1 and BsAraL are expressed using the same ers and UTRs.

Plasmid pBR322-NmneuB was constructed using a pBR322 vector via Golden Gate assembly.

The promoter and UTR used for the sion of NmNeuB are promoter apFAB299 and UTR galE_SD2-junction_BCD12. d pSC101-NmneuA-dest was constructed using a pSC101 vector via Golden Gate assembly. The promoters and UTRs used for the expression of NmneuA are promoter apFAB37 and UTR galE_SD2-junction_BCD18. The promoters and UTRs used for the expression of dest are promoter apFAB339 and UTR galE_SD2-junction_BCD12. All promoters and UTRs are described by k et. al (Nat. Methods 2013, No. 10, 354-360).

Plasmids were maintained in the host E. coli DH5alpha (F', phi80dlache/taM15, delta(lacZYA- argF) U169, deoR, recAl, endAl, hst17(rk', mk+), phoA, supE44, lambda', thi-1, gyrA96, reIA1). 40 Bought from |nvitrogen.

Gene disruptions Gene tions as well as gene introductions were performed using the technique published by Datsenko and Wanner (PNAS 97 (2000), 6640-6645). This technique is based on antibiotic selection after homologous recombination performed by lambda Red recombinase. Subsequent catalysis of a flippase recombinase ensures removal of the antibiotic ion cassette in the final production strain.

In Table A the necessary primers for the construction of the gene disruption cassette are listed.

Table A: Lists of s to construct disruption cassette for the target gene. lacZYA C'I'I'GTAGGCCTGATAAGCGCA GCGCAACGCAATTAATGTGAGTI'AGCT GCGTATCAGGCAA'IT'I—I—I'ATAATCTI'CAT CACTCATTAGGCACCCCAGGCTI'CGCCT TI'AAATGGCGCGC (SEQ ID NO: 1) ACCTGTGACGGAAG (SEQ ID NO: 2) nagABCDE CGCTI'AAAGATGCCTAATCCGCCAACGG GGCGTI'I'GTCATCAGAGCCAACCACGT CTfACAI I I IACTTA'I'I'GAGGTGAATAGT CCGCAGACGTGGTTGCTATCATATGAAT GTAGGCTGGAGCTGCTI'C (SEQ ID NO: ATCCTCCTI'AG (SEQ ID NO: 4) nanA TEK TAATGCGCCGCCAGTAAATCAACATGAA CCAACAACAAGCACTGGATAAAGCGAG ATGCCGCTGGCTCCGTGTAGGCTGGAG TCTGCGTCGCCTGGTI'CAGTI'CACATAT CTGC'I'I'C (SEQ ID NO: 5) GAATATCCTCCTI'AG (SEQ ID NO: 6) AAAATACATCTGGCACGTFGAGGTGTTA CCTCCAGATAAAAAAACGGGGCCAAAA ACGATAATAAAGGAGGTAGCAAGTGTA GGCCCCGGTAGTGTACAACAGTCCATA AGCTGCTI'C (SEQ ID NO: 7) TGAATATCCTCCTTAG (SEQ ID NO: 8) For the genomic integration of the necessary genes into the production hosts genome based on the same technique used for the gene disruption, sed , with specific alterations to the disruption te. n a homology site and the FRT site of the tion cassette, the to be integrated constructed is located. This allows for elegant integration of the constructed in the region dictated by the homology sites.

Using this workflow, a direct gene disruption and genomic integration is possible. Primers that were used for target integration are at specific sites are listed in Table B.

Table B: Primers used for genomic integration Integration location nagABCDE GT'ITGGCGTI—I'GTCATCAGAGCCAA TTGTCATTG'I'I'GGATGCGACGCTCAA CCACGTCCGCAGACGTGGTI'GCTAT GCGTCGCATCAGGCATAAAGCAGAC GCTGGAGCTGC'I'I'C (SEQ TTAAGCGAC'I'I'CATTCACC (SEQ ID ID NO: 9) NO: 10) CATGGCGGTAATGCGCCGCCAGTA CCAACAACAAGCACTGGATAAAGCG AATCAACATGAAATGCCGCTGGCTC AGTCTGCGTCGCCTGG'I'I'CAGTTCAC CGTGTAGGCTGGAGCTGCTTC (SEQ TTAAGCGAC'I'I'CATTCACC (SEQ ID ID NO: 11) NO: 12) AAAATACATCTGGCACG'I'I'GAGGTG CCTCCAGATAAAAAAACGGGGCCAA TI'AACGATAATAAAGGAGGTAGCA AAGGCCCCGGTAGTGTACAACAGTC AGTGTAGGCTGGAGCTGC'ITC (SEQ C'I'I'AAGCGAC'I'I'CA'I'I'CACC (SEQ ID ID NO: 13) NO: 14) lacZYA GCGCAACGCAATTAATGTGAGTI'AG GCTGAAC'I'I'GTAGGCCTGATAAGCG CTCACTCA‘I‘I'AGGCACCCCAGGCTI' CAGCGTATCAGGCAAT'ITI'TATAATC GCTGGAGCTGC'I'I'C (SEQ TTAAGCGAC'I'I'CATTCACC (SEQ ID ID NO: 15) NO: 16) ath-gidB CAAAAAGCGGTCAAATTATACGGTG ATAACGTGGCT'ITT'ITI'GGTAAGCAG CGCCCCCGTGA'ITI'CAAACAATAAG AAAATAAGTCATTAGTGAAAATATCT GTGTAGGCTGGAGCTGC'I'I'C (SEQ TAAGCGACTTCA'I'I'CACC (SEQ ID ID NO: 17) NO: 18) Clones carrying the temperature sensitive pKD46 helper plasmid were grown in 10 mL LB media with ampicillin (100 mg/L) and L-arabinose (10 mM) at 30 °C to an ODeoOnm of 0.6. The cells were made electro competent by sequential washing, once with 50 mL, and once with 1 mL ice-cold deionized water. Next, the cells were resuspended in 50 uL of ice-cold water. Finally, 10-100 ng of disruption/integration cassette was added to 50 uL of the washed cell solution for electroporation. Electroporation was med using a Gene Pulser (trademark of BioRad) (600 Ohm 25 uFD, and 250 V).

After oporation, cells were resuscitated in 1 mL LB media for 1 h at 37 °C, and finally plated out onto LB-agar containing 25 mg/L of chloramphenicol or 50 mg/L of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with primers upstream and ream of the modified region and were subsequently grown on LB-agar at 42 °C for the loss of the pKD46 helper plasmid. The mutants were finally tested for ampicillin sensitivity.

The selected mutants (chloramphenicol or kanamycin resistant) were transformed with pCP20 plasmid, which is an llin and mphenicol resistant plasmid that shows temperature- sensitive replication and thermal induction of FLP synthesis. The ampicillin-resistant transformants were selected at 30 °C, after which a few were colony purified in LB at 42 °C and then tested for loss of all antibiotic ances and thus also of the FLP helper plasmid. The gene tions and/or gene ation are checked with control primers and sequenced. These primers are listed in Table C.

Table C: Primers to validate either gene tion and/or genomic integration for specific gene ta rgets.

Gene targets lacZYA CAGGT'I'I'CCCGACTGGAAAG (SEQ TGTGCGTCG'I'I'GGGCTGATG (SEQ ID NO: 19) ID NO: 20) nagABCDE CGCTTGTCATTG'I'I'GGATGC (SEQ GCTGACAAAGTGCGA'ITI'GTTC (SEQ ID NO: 21) ID NO: 22) nanA TEK GTCGCCCTGTAA'I'I'CGTAAC (SEQ C'I'I'TCGGTCAGACCACCAAC (SEQ ID ID NO: 23) NO: 24) manXYZ ACGCCTCTGA'I'I'TGGCAAAG (SEQ AGCCAGTGCGCTTAATAACC (SEQ ID ID NO: 25) NO: 26) atpl-gidB GCTGAACAGCAATCCACTI'G (SEQ TGAACGATATGGTGAGCTGG (SEQ ID NO: 27) ID NO: 28) Heterologous and homologous expression Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the ing companies: DNA2.0, Gen9 or IDT.

Escherichia coli native genes, as e.g., atases, were picked from the E. coli K-12 MG1655 genome. The origin of other genes are indicated in the relevant table.

Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Gene were optimized using the tools of the er. ation conditions A preculture of 96well microtiter plate experiments was started from single colony on a LB plate, in 175 (AL and was incubated for 8h at 37 °C on an orbital shaker at 800 rpm. This culture was used as inoculum for a 96well microtiter plate, with 175 (AL MMsf medium by diluting 300x.

These cultures in turn, were used as a preculture for the final experiment in a 96well plate, again by diluting 300x. The 96well plate can either be microtiter plate, with a culture volume of 175 (AL or a 24well deepwell plate with a culture volume of 3mL.

A preculture for shake flask experiments was started from a single colony on a LB-plate, in 5 mL LB medium and was incubated for 8 h at 37 °C on an orbital shaker at 200 rpm. From this e, 1 mL was transferred to 100 mL minimal medium (MMsf) in a 500 mL shake flask and incubated at 37 °C on an orbital shaker at 200 rpm. This setup is used for shake flask experiments.

A shake flask experiment grown for 16h could also be used as an inoculum for a bioreactor. 4% of this cell solution was to inoculate a 2L Biostat Dcu-B with a 4 L working volume, controlled by MFCS control re rius Stedim h, Melsungen, Germany). Culturing condition were set to 37 °C, 800 rpm stirring, and a gas flow rate of 1.5 L/min. The pH was controlled at 7 using 0.5 M H2S04 and 25% NH4OH. The exhaust gas was cooled. 10% solution of silicone antifoaming agent was added when foaming raised during the tation (approximately 10 BL). The use of an inducer is not required as all genes are tutively expressed.

Material and methods Saccharomyces cerevisae Media Strains are grown on Synthetic Defined yeast medium with Complete ment e (SD CSM) or CSM drop-out (SD CSM-Ura) containing 6.7 g/L Yeast Nitrogen Base without amino acids (YNB w/o AA, Difco), 20 g/L agar (Difco) (solid cultures), 22 g/L glucose monohydrate or 20 g/L lactose and 0.79 g/L CSM or 0.77 g/L CSM-Ura (MP Biomedicals).

Saccharomyces cerevisiae BY4742 d by Bachmann et 0]. (Yeast (1998) 14:115-32) was used available in the Euroscarf culture collection. All mutant strains were created by homologous recombination or plasmid transformation using the method of Gietz (Yeast 11:355- 360, 1995). Kluyveromyces marxianus lactis is ble at the LMG culture collection (Ghent, Belgium).

Plasmids Yeast expression plasmid p2a_2u_sia_GFA1 (Chan 2013 (Plasmid 70 (2013) 2-17)) was used for expression of foreign genes in Saccharomyces cerevisae. This plasmid contains an ampicillin resistance gene and a bacterial origin of replication to allow for selection and maintenance in E. coli. The plasmid further contains the 2p yeast ori and the Ura3 selection marker for ion and maintenance in yeast. Finally, the plasmid can contain a beta-galactosidase expression cassette. Next, this plasmid also contains a N-acetylglucosamineepimerase (for example from Bacteroides ovatus (BoAGE)) and a sialic acid synthase (for example from Campy/obacterjejuni (CjneuB)). Finally, it also contains the fructoseP-aminotransferase from romyces cerevisiae, ScGFAl.

Yeast sion plasmid p2a_2u_sia_glmS is based on p2a_2u_sia but modified in a way that also glmS*54 oseP-aminotransferase from Escherichia coli) is expressed.

Yeast expression plasmids p2a_2u_sia_glmS_phospha is based on p2a_2u_sia_glmS but modified in a way that also EcAphA (SEQ ID NO: 42), EcCof (SEQ ID NO: 43), EcHisB (SEQ ID NO: 44), EcOtsB (SEQ ID NO: 45), EcSurE (SEQ ID NO: 46), EcYaed (SEQ ID NO: 47), EcchU (SEQ ID NO: 48), EcYedP (SEQ ID NO: 49), EchbT (SEQ ID NO: 50), EchdA (SEQ ID NO: 51), EchgB (SEQ ID NO: 52), EchhX (SEQ ID NO: 53), EcYniC (SEQ ID NO: 54), EchaB (SEQ ID NO: 55), EchbL (SEQ ID NO: 56), PsMupP (SEQ ID NO: 57), EcAppA (SEQ ID NO: 58), Epoh (SEQ ID NO: 59), EcSerB (SEQ ID NO: 60), EcNagD (SEQ ID NO: 61), EcthA (SEQID NO: 62), EcYbiV (SEQ ID NO: 63), EcijL (SEQ ID NO: 64), EchbR (SEQ ID NO: 65), EcheH (SEQ ID NO: 66), EchgL (SEQ ID NO: 67), Ec ijG (SEQ ID NO: 68), EchfG (SEQ ID NO: 69), EcYbiU (SEQ ID NO: 70), ScDOGl (SEQ ID NO: 71) and BsAraL (SEQ ID NO: 72) are expressed.

Yeast expression plasmid p2a_2u_SL-glmS is based on p2a_2u_sia but modified in a way that also 2 (lactose permease from Kluyveromyces lactis), NmneuA (CM P-sialic acid synthase from Neisseria meningitides) and dest (sialyltransferase acterium damselae) are expressed.

Plasmids were maintained in the host E. coli DH5alpha (F', phi80dlacheltaM15, delta(lacZYA- argF)U169, deoR, recAl, endAl, hst17(rk', mk+), phoA, supE44, lambda', thi-1, gyrA96, reIA1).

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Gene SlOH promoters Genes are expressed using tic constitutive promoters, as bed in by k (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012).

Heterologous and homologous expression Genes that needed to be expressed, be it from a d or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9 or IDT Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Gene were optimized using the tools of the supplier.

Cultivations conditions In general, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown for 2-3 days at 30 °C.

Starting from a single colony, a preculture was grown over night in 5 mL at 30 °C, shaking at 200rpm. Subsequent 500 mL shake flask experiments were inoculated with 2% of this preculture, in 100 mL media. These shake flasks were incubated at 30 °C with an l g of 200 rpm. The use of an inducer is not required as all genes are constitutively sed.

Material and methods Bacillus subtilis Media Two different media are used, namely a rich Luria Broth (LB), a minimal medium for shake flask (MMsf). The minimal medium uses a trace element mix.

Trace element mix consisted of 0.735 g/L CaCI2.2H20, 0.1 g/L MnC|2.2H20, 0.033 g/L CuC|2.2H20, 0.06 g/L COC|2.6H20, 0.17 g/L ZnCl2, XX g/L H3BO4, XX g/L NaZEDTA.2H20 and 0.06 g/L Na2MoO4. The Fe-citrate solution contained 0. 135 g/L FeCI3.6H20, 1 g/L Na-Citrate (Hoch 1973 2887).

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR, Leuven, Belgium).

Luria Broth agar (LBA) plates consisted of the LB media, with 12 g/L agar (Difco, Erembodegem, Belgium) added.

Minimal medium for shake flask experiments (MMsf) contains 2 g/L (NH4)2S04, 7.5 g/L KH2P04, 17.5 g/L K2HP04, 1.25 g/L Na-Citrate, 0.25 g/L MgS04.7H20, 0.05g/L tryptophan, from 10 up to g/L glucose or another carbon source including but not limited to glucose, fructose, maltose, glycerol and maltotriose, 10 mL/L trace element mix, and 10 mL/L Fe-citrate solution. The medium was set to a pH of 7 with 1M KOH.

Complex medium, e.g. LB, was sterilized by autoclaving (121 °C, 21) and minimal medium (MMsf) by filtration (0.22 um Sartorius). If ary, the medium was made selective by adding an antibiotic (e.g. zeocin (20mg/L)).

Strains Bacillus subtilis 168, available at Bacillus Genetic Stock Center (Ohio, USA).

Plasmids and gene pression Plasmids for gene on via Cre/lox are constructed as described by Yan et al. (Appl & environm microbial, sept 2008, p5556-5562).

Expression vectors can be found at Mobitec (Germany), or at ATCC (ATCC® number 87056). The genes BsglmS, SCGNAl and CjneuB are cloned in these expression vectors. A suitable promoter for expression can be derived from the part repository (iGem): sequence id: BBa_K143012, 23000, BBa_K823002 or BBa_K823003. Cloning can be performed using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

Plasmids are maintained in the host E. coli DH5a|pha (F', phi 80dlacheltaM15, delta(lacZYA- argF)U169, deoR, recAl, endAl, hst17(rk', mk+), phoA, supE44, lambda', thi-1, gyrA96, relA1).

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Gene disruptions ting of genes is done via homologous ination with linear DNA and ormation via the electroporation as described by Xue et al. (J. . Meth. 34 (1999) 183-191). The method of gene knock-outs is described by Liu et al. (Metab. . 24 (2014) 61-69). This method uses 1000bp homologies up- and downstream of the target gene. The homologies to be used in this invention, are listed in table D. After the modification, the mutants are verified using primers upstream and downstream of the modified region. These primers are given in table E. Next, the modification is confirmed by sequencing (performed at LGC Genomics (LGC group, y)).

Table D Gene to be Upstream homology Downstream homology disrupted nagA-nagB Gactgcaagatttcggcctgggcggacggga at Aaggaacatgctgacttatgaatatcaataaaca cgtcagttttgtaatttctgtatcaatgattttcat atcgcctattccgatttactatcagattatggagca ggtctcttcctcaagtccgagccggtcgtattgct attaaaaacccaaattaagaacggagagctgcag gctcccagagttcaagattcatgacaat ccggatatgcctcttccttctgagcgcgaatatgcc cgtgattcgtttattgcttctgaccgcgccagcgc gaacaattcgggatcagccggatgacagttcgcc caaatagcgtcatcacattgataatgccaaggcc aggcgctttctaatttagttaatgaaggcttgctct cctgatctcaagaaggtgctcaattaattccgga atcgcctgaaagggcggggcacctttgtcagcaa cccacaagagtatcctgatcctcctgccg aatggaacaagcacttcaagggctgaca tatttcaacgcaatcatcggcaacaaggcgatgc agctttaccgaggatatgaaaagccgcgggatga cctcttttcacaagctctagcgctgtttcgctttttc caccgggcagcaggctcattgattatcagcttatt cgacgccgctttttcctgtgatcagcacgccgac gattcaactgaggagctcgcggctatattaggctg accatatatatcgacaagaacgccatgaattgct cgggcacccctcctctatccataaaatcactcggg gtggtaggcgccagcctgctctcaaggaagttgg tgcggctggcaaatgatattccgatggcgattgag ttaaacggcttgacagtcttgtcgttttcagcggc tcctcacatattccgtttgagcttgcgggtgaattg gatctgaggacaggcaccccatttttctcggagg aacgaatcgcattttcagtcgtcgatctatgatcat cgtcaatcagctcctgcgggatgggcatatctct attgaaaggtacaacagcataccgatttcccgtgc agaaagaataatagctggtgttacatcagtgcac ggagcttgagccaagcgctgccaccacg agagaatccattcgctgctttttctcctcttcagga gaagaagcgaatattcttggtattcaaaagggag agctgttcaaagaaagaaagctctgtttttccga cgcctgtcctattaattaaacgaacaacatatctgc gaagctgcacgcgctccctcgggtaatatgtaaa agaacggaactgcttttgagcatgcaaaatccgta ggcaatttcaatacctggtcttgataggt ggcgaccgttatacatttgtccactatatg cactcattgtaatcgggcggttaattccttcttctc gatcgtctttcataaaaaaagcctccaacccttttt cgctgattaattccaaattgaactgttccattacg aaggattggagacatggcgaaaatcaaactggtc tcttttgtgcgaacctttgccacgatatgttcctcc tggtgccggacgatatgtttcttttttcgtcttgaac tgttccgggctgccccgagcttgctcacaatactt ttccagatcggtgatttcgttttgccgttaaaactgt tcattttatcactttcgggcttgaacctaaaacag cttccactataatgtaccaataataaacagactgc attttataaaaggggggaaaacacctcagctggt ggttcaagatgatcccagcggaattcagctgtgtc ctagatcactagtctgaaaaagagtaaaataaa cccgctcttcacttgctcccgttttccgagctcttca ggtattcaaattccagaaaggcggatcatct tatacgtta (SEQ ID NO: 34) (SEQ ID NO: 33) ngcggacatggaataaatcacaaacgacaaa Gtgacaccccctcaaagagatagacaagcaccat gatgacgccggcaagaatagagttaatcaaata atttgttatgaccaatttatgatacttgtcattacga gagcacgggcgcaacgaacaagaaagaaaact atttagcaccgcccttatcaaactgtcaatattaat caaccggttctgtaattccggtcagcatagatgt ttctgaaaatttgttataaaagaaggatacaaatc gagcgccgcagaaatcatcacgccggagatcat tttcatattgggagggcaaatggtattatggtctca cttttccggacgcgcggtatggataatggc atgaaaaagaacggattgcatacagaatgggga aagagcaacggccggcagacagaaaatcatgt gaatgaaatgacagctttatattctgttatcaagtt aagggaaatcccccatcataaagcgcccggctg taaaatcattgagttaattaaatcgggcaaatatc tcgggtctcccgcgaaaaaccttgtcaggtcgcc aggcgaatgatcagctgccgacggagagtgagtt ggtta cggtgttgcctgttgatgggtctgtgtattc ttgcgaacaatatgatgtcagcagaacaactgtga tcccatcataaaatagaaaggcgtataaaaaat ctctgcagcagctagagcttgagggatat atgatgcaggccaaaaggaatcagcaaacgat attaaaagaattcaaggaaaagggacatttgtat agatcgttgcataaaagaacaggccgactgttg cggcggccaaaatacaaacgccgattccgcataa aatcggcaattaaactgctggctgcgttaattcc gattacgagctttgcagaacaaatgagaggacttc gttttggatcagcggccaaacgaatgagaaaat gttctgaatcaaaagtgcttgagcttgtggtgattc gacgccgatcaccaatgaactgacggaagtaat ctgccgatcattccatcgccgagcttttgaaaatga gatcgggacaaagcgttttccagagaaaaatcc aagagaatgaacctgtcaacaagcttgtcagagt aaggaccggatgcagctcgattgatgaaaatcg cagatacgccgagggggaacctttgcagtatcat cttatataaataggcggcgagaagcccgataat acctcatatattccctggaaggcggcaccggggct gattcctccgaaaacccccatatcaatcaggtgc ggcgcaggaggaatgcaccggctcgctgtttgaat tcggctccttcata cggaggctgaaggccgagta tgttaaggacaaaatacaatattgaaatcagcag attttcccatattgtcgagggtgacggttaaaatt gggcacggaatcgatcgaaccgattttaacggat aagtatccgatgacagcggcaagtccggctaca gaaacgatcagcggacacttattaaccaatgtcg ccttctccgccggctaatccgatcgcgaccccca ctgcgtttttatcagaatcccttacctatg cggcgaaaatcagcggaaggttatcgaatacaa ataaaaatgaagaagtggtggaatatgcgcaaat cgccgcccgcatcctttataatagggatgttcagt tattacacggggagaccgaacgaaattcaccgta ttgtctccgaaacggagcaaaagacct gaacagtcatatcattcataaagcaatgtgttttaa gctgccggcaggacggcaaccggagtcatcaac gaagggaatggtggttctatgtttttatttacgaat ccaagctgctgcagaatttgaaatgcct ggaaaagtgctgtggggagcagt (SEQ ID NO: ttttaaacatgacagtctccttttattgtg (SEQ 36) ID NO: 35) Table E nagA-nagB Tgtaatcgggcggttaattc (SEQ ID Gccctttcaggcgatagag (SEQ ID gamA Acggcgaaaatcagcggaag (SEQ Tcactctccgtcggcagctg (SEQ ID ID NO: 39) Heterologous and homologous expression Genes that needed to be expressed, be it from a plasmid or from the genome were synthetically synthetized with one of the following companies: DNA2.0, Gen9 or IDT.

Expression could be further facilitated by optimizing the codon usage to the codon usage of the expression host. Gene were zed using the tools of the supplier.

Cultivations conditions A preculture, from a single colony on a LB-plate, in 5 mL LB medium was incubated for 8 h at 37 °C on an orbital shaker at 200 rpm. From this culture, 1 mL was transferred to 100 mL l medium (MMsf) in a 500 mL shake flask and incubated at 37 °C on an l shaker at 200 rpm.

This setup is used for shake flask experiments. The use of an inducer is not required as all genes are constitutively expressed.

Analytical methods Optical density Cell density of the culture was ntly monitored by measuring optical density at 600 nm (Implen Nanophotometer NP80, rg, Belgium). Cell dry weight was obtained by centrifugation (10 min, 5000 g, Legend X1R Thermo Scientific, Belgium) of 20 g reactor broth in pre-dried and weighted falcons. The pellets were subsequently washed once with 20 mL physiological solution (9 g/L NaCI) and dried at 70 °C to a constant weight. To be able to convert OD6oonm measurements to biomass concentrations, a correlation curve of the OD6oonm to the biomass concentration was made.

Measurement of cell dry weight From a broth sample, 4 x 10 g was transferred to centrifuge tubes, the cells were spun down (5000g, 4 °C, 5 min), and the cells were washed twice with 0.9% NaCI solution. The centrifuge tubes containing the cell pellets were dried in an oven at 70 °C for 48 h until constant weight.

The cell dry weight was ed gravimetrically; the tubes were cooled in a desiccator prior to weighing.

Liguid chromatography The concentration of ydrates like, but not limited to, e, fructose and lactose were determined with a Waters Acquity UPLC H-class system with an ELSD detector, using a y UPLC BEH amide, 130 A, 1.7 um, 2.1 mm x 50 mm heated at 35 °C, using a 75/25 acetonitrile/water solution with 0.2% ylamine (0.130 mL/min) as mobile phase.

Sialyllactose was quantified on the same machine, with the same column. The eluent however was modified to 75/25 acetonitrile/water solution with 1% formic acid. The flow rate was set to 0.130 mL/min and the column temperature to 35 °C.

Sialic acid was quantified on the same machine, using the REZEX ROA column (300 x 7.8 mm ID).

The eluent is 0.08% acetic acid in water. The flow rate was set to 0.5 mL/min and the column temperature to 65 °C. GlcNAc and ManNAc were also measured using this method.

Growth rate measurement The maximal growth rate (uMax) was calculated based on the ed optical densities at 600nm using the R package .

Example 2: production of sialic acid in Escherichia coli A first example provides an Escherichia coli strain e of producing N-acetylneuraminate (sialic acid) (see figure 1B).

A strain capable of accumulating aminephosphate using sucrose as a carbon source was further ered to allow for N-acetylneuraminate production. The base strain overexpresses a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP), a fructokinase from nas mobilis (merk), a mutant fructoseP-aminotransferase (Ecglm$*54, as described by Deng et al. (Biochimie 88, 419-429 (2006))). To allow for gene sialic acid production the operons nagABCDE, nanATEK and manXYZ were disrupted. BaSP and merk were introduced at the location of nagABCDE and Ecglm$*54 was introduced at the location of nanATEK. These modifications were done as described in example 1 and are based on the principle of Datsenko & Wanner (PNAS USA 97, 6640-6645 (2000)).

In this strain, the biosynthetic pathway for producing sialic acid as bed in this invention, was implemented by overexpressing a glucosamineP-aminotransferase from Saccharomyces cerevisiae l), a N-acetylglucosamineepimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from obacterjejuni (CjneuB). SCGNAl and BoAGE were expressed on locations nagABCDE and manXYZ, respectively. CjneuB was expressed using the high copy plasmid pCX-CjneuB.

The strain was cultured as described in example 1 (materials and methods). Briefly, a 5mL LB preculture was inoculated and grown overnight at 37 °C. This e was used as inoculum in a shake flask experiment with 100mL medium which contains 10g/L sucrose and was made as bed in example 1. Regular samples were taken and analyzed as described in example 1.

The evolutions of the concentrations of biomass, sucrose and sialic acid are easily followed and an end concentration of 0.22g/L N-acetylneuraminate was produced extracellularly, as can be seen in figure 2.

The same organism also produces N-acetylneuraminate based on glucose, maltose or glycerol as carbon source.

Example 3: production of 6-sialyllactose in Escherichia coli Another example ing to present invention is the use of the method and strains for the production of 6-sialyllactose.

The strain of example 3 is a daughter strain of the strain used in example 2. The strain is further modified by overexpressing a lactose se EclacY from Escherichia coli (as described and demonstrated in example 1 of a CMP-sialic acid synthethase from Neisseria meningitides A) and a sialyltransferase from Photobacterium damselae (dest). On top of that lacZ is ted.

The genes NmneuA and dest, are expressed from a plasmid, together with CjneuB. This d is pCX-CjneuB-NmneuA-dest, and is made as described in example 1.

Said strain is inoculated as a preculture ting of 5m| LB medium as described in e 1.

After growing overnight at 37°C in an incubator. 1% of this preculture is inoculated in a shake flask containing 100ml medium (MMsf) containing 10g/l sucrose as carbon source and 10 g/l e as precursor. The strain is grown for 300h at 37°C.

This strain produces quantities of 6-sialyllactose.

Example 4: production of sialic acid in Saccharomyces cerevisiae using heterologous fructoseP-aminotransferase Another example provides use of an eukaryotic organism, in the form of Saccharomyces cerevisae, for the invention. This method utilizing the pathway of the invention shall be obtained in Saccharomyces siae by introducing and expressing a N-acetylglucosamineepimerase (for example from Bacteroides ovatus (BoAGE)) and a sialic acid synthase (for example from Campy/obacterjejuni (CjneuB)).

As starting point, a strain with increased lic flux s N-acetylglucosamine phosphate is needed. This is achieved by overexpressing the fructoseP-aminotransferase mutant from Escherichia coli (Ecglm$*54).

To create a N-acetylneuraminate producing Saccharomyces cerevisiae ing to this invention, the genes are introduced via a 2-micron plasmid (Chan 2013 (Plasmid 70 (2013) 2- 17)) and the genes are expressed using synthetic constitutive promoters (Blazeck 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11)) as also described in example 1. The specific plasmid used in this embodiment is p2a_2u_sia_glmS. This d is introduced into Saccharomyces cerevisae using the transformation que described by Gietz and Woods (2002, PMID 12073338) and a mutant strain is ed Said strain is capable of converting fructosephosphate into aminephosphate, followed by glucosaminephosphate conversion in N-acetylglucosaminephosphate. This N- acetylglucosaminephosphate moiety is further converted to N-acetylglucosamine, said N- acetylglucosamine into N-acetylmannosamine and finally this N-acetylmannosamine is converted into N-acetylneuraminate.

A ture of said strain is made in 5mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30°C as described in example 1. This preculture is inoculated in 100mL medium in a shakeflask with 10g/L sucrose as sole carbon source and grown at 30°C.

Regular samples are taken and the production of N-acetylneuraminate is measured as described in example 1. This strain and method produces quantities of N-acetylneuraminate.

The same organism also produces N-acetylneuraminate based on glucose, maltose or glycerol as carbon source.

Example 5: production of 6-sialyllactose in Saccharomyces siae Another e provides use of an otic organism, in the form of Saccharomyces cerevisae, for the invention. This method utilizing the pathway of the invention shall be obtained in Saccharomyces cerevisiae by introducing and expressing a N-acetylglucosamineepimerase (for example from Bacteroides ovatus (BoAGE)) and a sialic acid synthase (for example from Campy/obacterjejuni (CjneuB)).

On top of that, further modifications are made in order to produce 6sialyllactose. These modifications comprise the addition of a lactose permease, a CMP-sialic acid synthase and a sialyltransferase. The preferred lactose permease is the KILAC12 gene from Kluyveromyces lactis tively NmneuA from Neisseria meningitides and dest from acterium ae, as also described in example 3.

As starting point, a strain with increased metabolic flux towards N-acetylglucosamine phosphate is needed. This is achieved by overexpressing the fructoseP-aminotransferase mutant from Escherichia coli (Ecglm$*54).

To create a N-acetylneuraminate producing Saccharomyces cerevisiae according to this invention, the genes are uced via a 2-micron plasmid (Chan 2013 (Plasmid 70 (2013) 2- 17)) and the genes are expressed using tic tutive promoters (Blazeck 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11)) as also described in example 1. The ic plasmid used in this embodiment is _sia_glmS. This plasmid is introduced into Saccharomyces cerevisae using the transformation technique described by Gietz and Woods (2002) and a mutant strain is obtained Said strain is e of ting sephosphate into aminephosphate, said aminephosphate into N-acetylglucosaminephosphate, said N-acetylglucosamine-6— phosphate into N-acetylglucosamine, said N-acetylglucosamine into N-acetylmannosamine and finally said N-acetylmannosamine into N-acetylneuraminate. Said N-acetylmannosamine is then ted to CM P-sialic acid and erred to lactose to obtain 6sialyllactose.

A preculture of said strain is made in 5mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30°C as described in example 1. This preculture is inoculated in 100mL medium in a shakeflask with 10g/L sucrose as sole carbon source and grown at 30°C.

Regular samples are taken and the production of N-acetylneuraminate is measured as described in example 1. This strain and method produces quantities of 6sialyllactose.

The same organism also produces N-acetylneuraminate based on e, maltose or glycerol as carbon source.

Example 6: production of sialic acid in Saccharomyces cerevisiae using autologous fructoseP-aminotransferase Another example provides use of an eukaryotic organism, in the form of Saccharomyces cerevisae, for the invention. This method utilizing the pathway of the invention shall be obtained in romyces cerevisiae by introducing and expressing a N-acetylglucosamineepimerase (for example from Bacteroides ovatus (BoAGE)) and a sialic acid synthase (for example from Campy/obacterjejuni (CjneuB)).

As starting point, a strain with increased metabolic flux towards N-acetylglucosamine phosphate is needed. This is achieved by overexpressing the native fructoseP- aminotransferase ScGFAl.

To create a N-acetylneuraminate producing Saccharomyces cerevisiae according to this invention, the genes are introduced via a 2-micron plasmid (Chan 2013 (Plasmid 70 (2013) 2- 17)) and the genes are expressed using tic constitutive promoters (Blazeck 2012 (Biotechnology and ineering, Vol. 109, No. 11)) as also described in example 1. The specific plasmid used in this embodiment is p2a_2u_sia_GFA1. This plasmid is introduced into Saccharomyces cerevisae using the transformation technique described by Gietz and Woods (2002) and a mutant strain is obtained Said strain is capable of converting fructosephosphate into glucosaminephosphate, said glucosaminephosphate into N-acetylglucosaminephosphate, said N-acetylglucosamine-6— phosphate into N-acetylglucosamine, said N-acetylglucosamine into N-acetylmannosamine and y said N-acetylmannosamine into N-acetylneuraminate.

A preculture of said strain is made in 5mL of the synthetic defined medium SD-CSM containing 22 g/L glucose and grown at 30°C as bed in example 1. This preculture is inoculated in 100mL medium in a shakeflask with 10g/L sucrose as sole carbon source and grown at 30°C.

Regular samples are taken and the production of N-acetylneuraminate is measured as described in e 1. This strain and method produces quantities of ylneuraminate.

The same organism also es N-acetylneuraminate based on glucose, maltose or glycerol 4o as carbon source.

Example 7: production of sialyllactoses and other sialylated compounds In an alternative embodiment of example 3, the sialyltransferase is changed to another sialyltransferase with different activity. This can be an alpha-2,3-sialyltransferase alpha-2,6- sialyltransferase, an alpha-2,8-sialyltransferase or a combination thereof. These sialyltransferases are widely available in nature and well annotated.

In this way, production of different sialyllactoses like for e 6—sialyllactose, 3-sialyllactose or a mixture thereof can be obtained.

The s are cultivated as stated in example 1 and example 3.

The pathways created in examples 2 to 7 can also be ed with other pathways for the synthesis of larger oligosaccharides, e.g. sialyl-lacto-N-triose, sialyllacto-N-tetraose, disialyllactose-N-tetraose, sialyllacto-N-neotetraose, and disialyllactose-N-neotetraose. To this end, the transferases to synthetize these glycosidic bonds are co-expressed with the pathway genes to form CMP-sialic acid and the transferase (as described above) to sialylate said oligosaccharide.

Examples of such transferases are ST6Ga|I, ST6Ga|II, ST3Ga|I until VI, ST6Ga|NAc I until VI and ST8Sia I until VI, as bed by Datta (Current Drug Targets, 2009, 10, 483-498) and Harduin-Lepers (Biochimie 83 (2001) 727-737). Further examples originating from marine sms are described by to (Mar. Drugs 2010, 8, 2781-2794).

Example 8: production of sialylated N-neotetraose The aim of this ment was to demonstrate the functionality of presented invention of the production of other sialylated oligosaccharides, in this case sialyltated lacto-N-neotetraose.

A lacto-N-neotetraose producing strain was developed following the protocol described in example 1. For production, the expression of a N-acetylglucosaminyltransferase and a galactosyltransferase are needed, this is ed by introduction of the genes legtA and legtB respectively, both from Neisseria meningitides. Next, the lactose importer Eclachrom Escherichia coli is (as described and demonstrated in example 1 of here also incorporated by reference). Finally, the genes ushA and galT are knocked out. In this way, a lacto-N-neotetraose producing strain is obtained.

To be able to grow on lactose and produce N-acetylglucosaminephosphate, a sucrose orylase from Bifidobacterium adolescentis (BaSP), a kinase from Zymomonas mobilis (frk) and a mutant fructoseP-aminotransferase (Ecglm$*54, as bed by Deng et al (Biochimie 88, 419-429 (2006))) were pressed as described in example 1.

In this , the method for producing sialic acid as described in this invention, was implemented by overexpressing a glucosamineP-aminotransferase from Saccharomyces cerevisiae (ScGNAl), a N-acetylglucosamineepimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from Campy/obacterjejuni (CjneuB). SCGNAl and BoAGE are expressed on locations nagABCDE and manXYZ, respectively. CjneuB is expressed from plasmid pCX-CjneuBNmneuA-dest.

Sialylation of the lacto-N-neotetraose moiety is performed by the conversion of sialic acid to CMP-salic acid by a CMP sialic acid synthethase, e.g. NmneuA from ria meningtides, subsequently followed by a sialyl transferase, e.g. dest, from Photobacterium damselae. These genes (NmneuA and dest) are expressed from the high copy plasmid pCX-CjneuB-NmneuA- dest.

The strain is cultured as described in example 1 (materials and methods). Briefly, a 5mL LB preculture is inoculated and grown overnight at 37°C. This culture was used as inoculum in a shake flask experiment with 100mL medium which contains 10g/L sucrose as carbon and energy source, 10g/L e as precursor and was made according to the description in example 1. r s are taken and analyzed. This strain produces quantities of sialylated lacto-N- neotetraose.

Alternative glycosyltransferases are possible. If EchbO (from Escherichia coli O55:H7) is expressed instead of legtB for example, tion of sialylated N-tetraose is obtained.

Example 9: Production of sialic acid with us subtilis In another embodiment, this invention can be used for production of N-acetylneuraminate in Bacillus subtilis, yet another bacterial production host.

A N-acetylneuraminate producing strain is obtained through this invention by starting with a strain, capable of overproducing glucosaminephosphate intracellularly. For this, the native fructoseP-aminotransferase (BsglmS) is overexpressed. The ing enzymatic activities are disrupted by knocking out the genes nagA, nagB and gamA: N-acetylglucosaminephosphate deacetylase and aminephosphate isomerase.

In this strain, the method for producing sialic acid as described in this invention, is implemented by overexpressing a glucosamineP-aminotransferase from Saccharomyces cerevisiae (ScGNAl), a N-acetylglucosamineepimerase from Bacteroides ovatus ) and a sialic acid synthase from Campy/obacterjejuni (CjneuB). These genes are introduced via a plasmid, as described in example1.

The strain is cultured as described in example 1 (materials and methods). Briefly, a 5mL LB preculture is ated and grown ght at 30 °C. This culture is used as inoculum in a shake flask experiment with 100mL medium which contains 10g/L sucrose and is made according to the description in example 1. This strain produces quantities of N-acetylneuraminic acid.

Example 10: Fermentations of 6-sialyllactose producing strain with no excretion of , ManNAc or sialic acid Another example according to the present invention provides use of the method and strains for the production of 6—sialyllactose.

An Escherichia coli strain capable of accumulating aminephosphate using sucrose as a carbon source was further engineered to allow for N-acetylneuraminate production. This base strain presses a sucrose phosphorylase from Bifidobacterium adolescentis (BaSP), a fructokinase from nas mobilis (merk), a mutant fructoseP-aminotransferase (Ecglm$*54, as described by Deng et al. (Biochimie 88, 419-429 (2006)). To allow for 6- 40 sialyllactose tion the operons nagABCDE, nanATEK and manXYZ were disrupted. BaSP and merk were introduced at the on of nagABCDE, EcglmS*54 was introduced at the location of nanATEK. These modifications were done as described in e 1 and are based on the principle of Datsenko & Wanner (PNAS USA 97, 6640-6645 (2000)).

In this strain, the biosynthetic pathway for producing 6—sialyllactose as described in this invention, was implemented by overexpressing a glucosamineP-aminotransferase from Saccharomyces cerevisiae (ScGNAl), a N-acetylglucosamineepimerase from Bacteroides ovatus (BoAGE) and a sialic acid synthase from Neisseria meningitides B). ScGNAl and BoAGE were expressed on ons nagABCDE and manXYZ, respectively. NmNeuB was expressed using the high copy d pBR322-NmNeuB. The strain is further modified by pressing a lactose permease EclacY from Escherichia coli (as described and demonstrated in example 1 of acid synthethase from Neisseria meningitides A) and a sialyltransferase from acterium damselae (dest). On top of that, lacZ is disrupted. NmNeuA and dest were expressed using the low copy plasmid pSC101-NmneuA-dest.

The strain was cultured in a bioreactor as described in example 1 (materials and methods).

Briefly, a 5mL LB preculture was inoculated and grown overnight at 37 °C. This culture was used as inoculum in a shake flask ment with 500mL medium which contains 10g/L sucrose and was made as described in example 1. This e was used as inoculum in a 2L bioreactor experiment. Regular s were taken and analyzed as described in example 1. The final concentration of 6-sialyllactose was 30.5 g/L. No ellular GlcNAc, ManNAc and sialic acid was detected during the fermentation and in the final broth.

The same organism also produces 6—sialyllactose based on e, maltose or glycerol as carbon source.

Example 11: Effect of phosphatase on growth and production of sialic acid A further example provides growth results and sialic acid production of several Escherichia coli strains capable of producing N-acetylneuraminate c acid) wherein the strains are expressing an extra phosphatase as indicated der.

The base strain overexpresses a mutant fructoseP-aminotransferase (EcglmS*54, as described by Deng et al. (Biochimie 88, 419-429 (2006)), a glucosamineP-aminotransferase from Saccharomyces cerevisiae (ScG NA1), a N-acetylglucosamineepimerase from Bacteroides ovatus (BoAGE) and a sialic acid se from Campy/obacterjejuni (CjneuB). To allow for gene sialic acid production the operons nagABCDE and nanATEK. The lacYZA operon was replaced by only a single gene operon, the native [0ch which is required for the production of sialyllactose as described in example 10. These modifications were done as described in example 1 and are based on the principle of Datsenko & Wanner (PNAS USA 97, 6640-6645 (2000)).

This base strain was then supplemented with different phosphatase bearing ds for ing the effect of the phosphatase on growth and sialic acid production. The base strain was used as blank in the comparison. These plasmids consisted of, besides the phosphatase and 4o a promoter driving expression of the phosphatase, a pSC101 ori and a spectomycin resistance marker. The following phosphatases were expressed: EcAphA (SEQ ID NO: 42), EcCof (SEQ ID NO: 43), EcHisB (SEQ ID NO: 44), EcOtsB (SEQ ID NO: 45), EcSurE (SEQ ID NO: 46), EcYaed (SEQ ID NO: 47), EcchU (SEQ ID NO: 48), EcYedP (SEQ ID NO: 49), EchbT (SEQ ID NO: 50), EchdA (SEQ ID NO: 51), EchgB (SEQ ID NO: 52), EchhX (SEQ ID NO: 53), EcYniC (SEQ ID NO: 54), EchaB (SEQ ID NO: 55), EchbL (SEQ ID NO: 56) and PSMupP (SEQ ID NO: 57). Other phosphatases that are expressed are EcAppA (SEQ ID NO: 58), Epoh (SEQ ID NO: 59), EcSerB (SEQ ID NO: 60), EcNagD (SEQ ID NO: 61), EcthA (SEQ ID NO: 62), EcYbiV (SEQ ID NO: 63), EcijL (SEQ ID NO: 64), EchbR (SEQ ID NO: 65), EcheH (SEQ ID NO: 66), EchgL (SEQ ID NO: 67), Ec ijG (SEQ ID NO: 68), EchfG (SEQ ID NO: 69), EcYbiU (SEQ ID NO: 70), ScDOGl (SEQ ID NO: 71) and BsAraL (SEQ ID NO: 72).

In a first experiment a subset of the above described strains was used. In a second ment a second subset of the above described strains were tested.

Each strain was cultured as described in example 1 (materials and methods). Briefly, the workflow consists of 3 growth steps: first growth on LB, followed by growth on MMsf with 15 g/L glycerol, and finally a growth stage using 15g/L glycerol MMsf. The first step is performed in a 96well plate, using 175 uL LB per well, and incubated overnight at 37 °C. The second step is performed in a 96well plate using 175 uL medium, incubated for 24 h at 37 °C. The final growth step was performed in: i) in a 96well plate using 175 uL , incubated at 37 °C to determine the uMax for the first ment (see figure 5) and ii) in a 24well deepwell plates using 3 mL do determine sialic acid production and optical ies for the second experiment (see figure Reference table for Figure 4 and 5: man——Promoter blank NA 1 46 2 apFABS7 3 apFABS7 apFABs46 —_apFABs46 apFABs46 —_apFABs46 —_apFABS7 apFABs46 apFABS7 11 —_apFABS7 12 —_apFABS7 13 apFABS7 14 apFABs46 apFABS7 16 apFABs46 17 apFABs46 18 apFABS7 19 apFABs46 7 21 apFABS7 22 apFABs46 Based on s 4 and 5 phosphatases enabling strains to grow better than the blank strain (no crippled ) and producing more sialic acid than the blank strain, can be chosen.

Based on the above, it was found that phosphatases comprising at least Motif 1 and Motif 2 provide a strain which is not crippled and produces more sialic acid than the blank strain.

Example 12: Identification of further seguences related to the atases used in the methods of the invention Sequences (polypeptides) related to SEQ ID N05: 43, 44, 45, 47, 48, 49, 50, 51, 52, 54, 55 and 57 were identified t those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using se sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to ce databases and by calculating the statistical significance of matches. The output of the analysis was viewed by se comparison, and ranked according to the ility score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to es, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical amino acids between the two compared polypeptide ces over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example the E-value may be increased to show less stringent s. This way, short nearly exact matches may be identified.

Table 1A to 1K provides a list of homologue polypeptide sequences related to SEQ ID NO: 43, 44, 45, 47, 48, 50, 51, 52, 54, 55 and 57, respectively.

Table 1A: Examples of polypeptides related to Ec Cof (SEQ ID NO: 43), showing sequence identity to SEQ ID 43: % identity t) short genbank fier SEQ ID NO 99,6 Shigella flexneri WP_095762248.1 78 99,3 Shigella boydii WP_095785299.1 79 98,2 Escherichia fergusonii WP_024256925.1 80 89,3 Staphylococcus aureus WP_094409981.1 81 89 Escherichia albertii WP_000113024.1 82 81,6 Citrobacter amalonaticus WP_046476411.1 83 81,6 Salmonella enterica WP_023234244.1 84 80,5 Escherichia coli WP_088543831.1 85 Table 18: Examples of polypeptides related to Ec HisB (SEQ ID NO: 44), showing sequence identity to SEQ ID 44: % identity (matgat) short genbank identifier SEQ ID NO 99,4 Shigella flexneri K-315 EIQ21345.1 86 99,2 Escherichia albertii WP_059217413.1 87 98,9 Shigella flexneri 085559.1 88 98,6 Shigella sonnei WP_077125326.1 89 98,6 Escherichia coli WP_088129012.1 90 98 Shigella dysenteriae 080078.1 91 98 Escherichia ae WP_038355110.1 92 94,6 Salmonella bongori WP_000080052.1 93 Table 1C: es of polypeptides related to Ec OtsB (SEQ ID NO: 45), showing sequence identity to SEQ ID 45: % identity (matgat) short genbank identifier SEQ ID NO 99,6 Shigella sonnei WP_077124555.1 94 99,6 ichia coli WP_032172688.1 95 99,2 Shigella flexneri 198868.1 96 85,7 Escherichia albertii WP_059227241.1 97 83,1 Escherichia fergusonii WP_000165652.1 98 Table 1D: Examples of polypeptides related to Ec Yaed (SEQ ID NO: 47), showing sequence identity to SEQ ID 47: SEQ ID % identity (matgat) short genbank identifier NO 99,5 Escherichia fergusonii WP_001140180.1 99 99,5 Shigella sonnei WP_047565591.1 100 99 Escherichia coli WP_061103769.1 101 95,8 Escherichia albertii WP_001140171.1 102 93,2 Kluyvera intermedia 371746.1 103 93,2 Citrobacter koseri WP_047458784.1 104 89 Kosakonia arachidis WP_090122712.1 105 85,9 Kluyvera cryocrescensWP_061282459.1 106 85,9 Leclercia adecarboxylata WP_039030283.1 107 Table 1E: Examples of polypeptides related to Ec chUB (SEQ ID NO: 48), showing sequence identity to SEQ ID NO: 48: % ty (matgat) short genbank identifier SEQ ID NO 99,5 Shigella sonnei WP_094313132.1 108 97,7 Escherichia coli 775764.1 109 95,4 Escherichia coli WP_032302947.1 110 92,7 Shigella flexneri OUZ88260.1 111 Table 1F: Examples of ptides related to Ec beT (SEQ ID NO: 50), showing sequence identity to SEQ ID NO: 50: % identity (matgat) short genbank identifier SEQ ID NO 99,1 Shigella sonnei WP_094323443.1 112 87,5 Citrobacter werkmanii NBRC 105721 GAL43238.1 113 86,6 Citrobacter freundii KGZ33467.1 114 86,6 Citrobacter amalonaticus Y19 06.1 115 85,6 Salmonella enterica WP_080095242.1 116 85,6 Escherichia fergusonii WP_001203376.1 117 ella enterica subsp. enterica serovar 118 85,6 Hadar KKD79316.1 Table 16: Examples of polypeptides d to Ec YidA (SEQ ID NO: 51), showing sequence identity to SEQ ID NO: 51: SEQ ID % identity (matgat) short genbank identifier NO 99,6 Escherichia coli WP_053263719.1 119 99,3 Escherichia onii WP_000985562.1 120 99,3 Shigella sonnei WP_094337696.1 121 94,4 Trabulsiella guamensis WP_038161262.1 122 94,1 Citrobacter amalonaticus WP_061075826.1 123 93,7 Klebsiella pneumoniae WP_048288968.1 124 93,3 siella odontotermitis 178096.1 125 90 Enterobacter kobei WP_088221256.1 126 Table 1H: Examples of polypeptides related to Ec YigB (SEQ ID NO: 52), showing sequence identity to SEQ ID NO: 52: % identity (matgat) short genbank identifier SEQ ID NO 99,6 Shigella sonnei WP_094322240.1 127 93,7 Shigella sonnei WP_052962467.1 128 87 Salmonella ca WP_079797638.1 129 85,7 Citrobacter braakii WP_080625916.1 130 81,9 Enterobacter hormaechei WP_047737367.1 131 81,1 Lelliottia amnigena WP_059180726.1 132 80,3 cia adecarboxylata WP_039031210.1 133 Table 1|: Examples of polypeptides related to Ec YniC (SEQ ID NO: 54), showing sequence identity to SEQ ID NO: 54: % ty (matgat) short genbank identifier SEQ ID NO 85,6 Shigella flexneri 1235-66 E|Q75633.1 134 85,1 Kosakonia sacchari WP_074780431.1 135 85,1 Enterobacter mori WP_089599104.1 136 84,7 Lelliottia amnigena WP_064325804.1 137 84,7 Enterobacter sp. 638 017112.1 138 84,2 Kosakonia radicincitans WP_071920671.1 139 Salmonella enterica subsp. enterica serovar 140 84,2 Newport str. CDC 2010K-2159 AKD18194.1 Table 1]: Examples of polypeptides related to Ec anB (SEQ ID NO: 55), showing sequence identity to SEQ ID NO: 55: % identity (matgat) short genbank identifier SEQ ID NO 97,9 Shigella flexneri K-315 E|Q18779.1 141 93,6 Escherichia albertii WP_059215906.1 142 88,3 Salmonella enterica WP_079949947.1 143 85,6 Kluyvera intermedia 006827.1 144 85,1 Trabulsiella termitis WP_054177678.1 145 84,6 Yokenella regensburgei 817298.1 146 84,6 Raoultella terrigena WP_045857711.1 147 83,5 Klebsiella pneumoniae 190334.1 148 Table 1K: Examples of polypeptides related to Ps MupP (SEQ ID NO: 57), showing sequence identity to SEQ ID NO: 57: % identity (matgat) short genbank identifier SEQ ID NO 94,6 monas putida group WP_062573193.1 149 94,6 Pseudomonas sp. GM84 WP_008090372.1 150 93,3 Pseudomonas entomophila 151 92,4 monas vranovensis WP_028943668.1 152 83,9 Pseudomonas cannabina WP_055000929.1 153 93,3 Pseudomonas monteilii WP_060480519.1 154 Sequences have been tentatively assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such d ces, either by keyword search or by using the BLAST algorithm with the c acid sequence or polypeptide sequence of interest.

Special nucleic acid sequence databases have been created for particular sms, such as by the Joint Genome Institute.

Example 13: Identification of domains and motifs comprised in polypeptide sequences useful in performing the methods ofthe invention The Integrated Resource of Protein Families, s and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence- based searches. The ro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized ns to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families.

Pfam is hosted at the Sanger Institute server in the United Kingdom. ro is hosted at the European Bioinformatics Institute in the United m.

The results of the |nterPro scan of the polypeptide sequences as represented by SEQ ID N05: 43, 44, 45, 47, 48, 49, 50, 51, 52, 54 and 55 are ted in Table 2.

Table 2: |nterPro scan results (major accession numbers) of the polypeptide sequence as represented by SEQ ID N05: 43, 44, 45, 47, 48, 49, 50, 51, 52, 54 and 55.

Data base Accession number Accession name |nterpro |PR023214 HAD superfamily Alignment of the tested phosphatase polypeptides was done and figure 6 shows part of the alignment. Motif 1 and motif 2 are indicated with boxes. Alignment was made using clustalomega.

Example 14: Effect of phosphatase on growth and production of sialic acid in Saccharomyces siae A further example of sialic acid production of several Saccharomyces cerevisiae strains e of producing N-acetylneuraminate (sialic acid) wherein the strains are expressing an extra phosphatase as indicated hereunder.

The strain used here is derived from the strain described in example 4. To e growth and production of sialic acid in Saccharomyces cerevisiae according to this invention, the phosphatase genes are introduced via a 2-micron plasmid (Chan 2013 id 70 (2013) 2-17)) and the genes are expressed using synthetic constitutive promoters ck 2012 (Biotechnology and Bioengineering, Vol. 109, No. 11)) as also described in example 1. The specific plasmids used in this embodiment is p2a_2u_sia_glmS-phospha. This plasmid based on the d p2a_2u_sia_glmS plasmid is described in e 1. It is introduced into Saccharomyces cerevisae using the transformation que described by Gietz and Woods (2002, PMID 12073338) and a mutant strain is obtained. The effect of phosphatase expression on growth and production of sialic acid of these mutants are evaluated as described in example Example 15: Effect of phosphatase on growth and production of sialic acid in Bacillus subtilis In r embodiment, this invention can be used to enhance growth and production of sialic acid in Bacillus subtilis, yet another bacterial production host.

The strain used here is derived from the strain described in example 9. Additionally to the tions described in e 9, phosphatase genes EcAphA (SEQ ID NO: 42), EcCof (SEQ ID NO: 43), EcHisB (SEQ ID NO: 44), EcOtsB (SEQ ID NO: 45), EcSurE (SEQ ID NO: 46), EcYaed (SEQ ID NO: 47), EcchU (SEQ ID NO: 48), EcYedP (SEQ ID NO: 49), EchbT (SEQ ID NO: 50), EchdA (SEQ ID NO: 51), EchgB (SEQ ID NO: 52), EchhX (SEQ ID NO: 53), EcYniC (SEQ ID NO: 54), EchaB (SEQ ID NO: 55), EchbL (SEQ ID NO: 56), PsMupP (SEQ ID NO: 57), EcAppA (SEQ ID NO: 58), Epoh (SEQ ID NO: 59), EcSerB (SEQ ID NO: 60), EcNagD (SEQ ID NO: 61), EcthA (SEQ ID NO: 62), EcYbiV (SEQ ID NO: 63), EcijL (SEQ ID NO: 64), EchbR (SEQ ID NO: 65), EcheH (SEQ ID NO: 66), EchgL (SEQ ID NO: 67), Ec ijG (SEQ ID NO: 68), EchfG (SEQ ID NO: 69), EcYbiU (SEQ ID NO: 70), ScDOGl (SEQ ID NO: 71) and BsAraL (SEQ ID NO: 72) are overexpressed on a plasmid, as described in example 1. Subsequently, this plasmid is introduced in us subtilis. The effect of phosphatase expression on growth and production of sialic acid of the created mutants are evaluated as described in example 11.

Claims (6)

1. Claims 1. A lically engineered microorganism for the production of a sialylated compound wherein said sialylated compound is chosen from N-acetylneuraminate (sialic acid) or a sialylated oligosaccharide, said microorganism - intracellularly converting N-acetylglucosaminephosphate to N-acetylglucosamine, said N- acetylglucosamine to N-acetylmannosamine and said ylmannosamine to N-acetylneuraminate ; and - a) having a reduced or abolished sion of at least one nucleic acid ce ng a polypeptide that converts i) N-acetylglucosamineP to glucosamineP, ii) N-acetyl-glucosamine to N-acetyl-glucosamineP, or iii) N-acetyl-neuraminate to N-acetyl-mannosamine; or b) being unable to convert any one or more of i) N-acetylglucosamineP to glucosamineP, ii) N- acetyl-glucosamine to N-acetyl-glucosamineP, or iii) N-acetyl-neuraminate to N-acetylmannosamine - and wherein at least one phosphoenolpyruvate:sugar phosphotransferase system for the import of a ride that is not used as a carbon source during fermentative production of the sialylated compound has been disabled.
2. 2. Microorganism according to claim 1, wherein said at least one phosphoenolpyruvate: sugar otransferase system is encoded by at least one of the genes chosen from the list of the genes encoding manX, manY, manZ, nagE.
3. 3. Microorganism according to claim 1, wherein said rganism can e an exogenous carbon source present in the fermentation broth as sole carbon source without using a phosphoenolpyruvate:sugar phosphotranferase system for the acquisition of said exogenous carbon source, preferably said carbon source is sucrose, e, maltose, glycerol.
4. 4. A metabolically engineered microorganism for the tative production of a sialylated oligosaccharide, said microorganism- intracellularly converts N-acetylglucosaminephosphate to N- acetylglucosamine, said N-acetylglucosamine to N-acetylmannosamine, said N-acetylmannosamine to N-acetyl-neuraminate and said N-acetyl-neuraminate to CMP-sialic acid; and -a) having a reduced or abolished expression of at least one nucleic acid encoding or driving expression of a polypeptide that converts i) N-acetylglucosamineP to glucosamineP, ii) N-acetyl- glucosamine to N-acetyl-glucosamineP, or iii) N-acetyl-neuraminate to N-acetyl-mannosamine, or b) being unable to convert any one or more ofi) N-acetylglucosamineP to glucosamineP, ii) N- acetyl-glucosamine to N-acetyl-glucosamineP, or iii) yl-neuraminate to N-acetyl- mannosamine.
5. 5. A metabolically engineered microorganism for the fermentative production of a sialylated oligosaccharide, said microorganism - ellularly converts N-acetylglucosaminephosphate to N- acetylglucosamine, said ylglucosamine to N-acetylmannosamine, said N-acetylmannosamine to N-acetyl-neuraminate and said yl-neuraminate to CMP-sialic acid; and - having a reduced or abolished activity of at least one enzyme chosen from i) a N-acetylglycosamine- 6-phosphate deacetylase, ii) a N-acetylglucosamine kinase, and iii) a N-acetylneuraminate aldolase, preferably the gene coding for any one of said enzymes is deleted or knocked-out.
6. 6. Microorganism according to any one of claim 4 or 5 for the production of a sialylated oligosaccharide wherein said rganism comprises at least one nucleic acid encoding a HAD-alike phosphatase, at least one nucleic acid encoding an N-acetylmannosamine epimerase, at least one nucleic acid encoding a sialyltransferase, at least one nucleic acid ng a CMP-sialic acid synthethase; and at least one nucleic acid encoding a sialic acid synthase. 7. rganism according to any one of claim 4 to 6 wherein said microorganism comprises a nucleic acid encoding a sialic acid synthase polypeptide originating from Campylobacter jejuni or Neisseria meningitides. 8. Microorganism according to any one of claim 1 to 7, wherein said microorganism r comprises an increased expression of a nucleic acid encoding a HAD-alike phosphatase, wherein said HAD-alike phosphatase comprises: - at least one of the following motifs: Motif 1: TV] (SEQ ID NO: 73), or Motif 2: [GSTDE][DSEN]x(1-2)[hP]x(1-2)[DGTS] (SEQ ID NOs: 74, 75, 76, 77) wherein h means a hydrophobic amino acid (A, I, L, M, F, V, P, G) and x can be any distinct amino acid; or - any one of SEQ ID NOs: 46, 52, 69, 70, 57, 42, 47, 43, 50, 59, 65, 54, 58, 60, 45, 67, 44, 48, 51, or 55. 9. Microorganism according to claim 8, wherein said HAD-alike atase comprises a homologue or derivative of any one of SEQ ID NOs: 46, 52, 69, 70, 57, 42, 47, 43, 50, 59, 65, 54, 58, 60, 45, 67, 44, 48, 51, or 55 having at least 80 %, 81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % overall sequence identity to said polypeptide and wherein said homologue or derivative increases one or more of sialic acid, biomass production and maximal growth rate ed to a reference strain having the same genetic make-up but lacking the increased expression of said HAD-alike phosphatase. 10. Microorganism according to any one of claim 1 to 9, wherein said ated accharide is chosen from the list consisting of sialyllactose, disialyl lacto-N-tetraose, sialylated lacto-N-triose, sialylated lacto-N-tetraose and sialylated lacto-N-neotetraose. 11. Microorganism according to claim 10, wherein said sialylated oligosaccharide is sialyllactose. 12. Microorganism according to any one of claim 1 to 9, wherein said sialylated oligosaccharide is a sialylated lacto-N-triose, sialylated N-tetraose or sialylated lacto-N-neotetraose, said microorganism further comprises the activity of a galactosyltransferase (EC 2.4.1.38) ), preferably said galactosyltransferase originates from the group comprising Homo sapiens, Bos taurus, Mus mulatta, Gallus gallus, Danio rerio, Helicobacter pylori and Haemophilus ducrey; and/or said microorganism comprises the ty of a N-acetylglucosaminyltransferase (EC 2.4.1.90), preferably said N- acetylglucosaminyltransferase originates from the group comprising Bos taurus, Homo s and Mus us. 13. Microorganism according to claim 12, wherein said microorganism is unable to s the genes coding for UDP sugar hydrolase and galactosephosphate uridylyltransferase. 14. Microorganism according to any one of claim 1 to 13, wherein any one or more of said N- acetylmannosamine epimerase and sialic acid synthase is overexpressed in the microorganism or is introduced and expressed in the rganism. 15. Microorganism according to any one of claim 1 to 14, wherein said microorganism further s a protein that facilitates uptake of lactose and lacks s that metabolize lactose. 16. Microorganism according to any one of claim 1 to 15, wherein said microorganism is a bacterium, ably an Escherichia coli strain, more preferably an Escherichia coli strain which is a K12 strain, even more preferably the Escherichia coli K12 strain is Escherichia coli , or wherein said microorganism is a yeast. 17. A metabolically engineered microorganism for the fermentative production of a sialylated oligosaccharide, said microorganism comprising: i) a sialic acid biosynthesis pathway comprising a glucosaminephosphate N-acetyltransferase for converting aminephosphate to N- acetylglucosaminephosphate, an N-acetylglucosaminephosphate phosphatase for converting N- acetylglucosaminephosphate to N-acetylglucosamine, an N-acetylmannosamine epimerase (also known as N-acetylglucosamine 2-epimerase) for ting said N-acetylglucosamine to N- acetylmannosamine, ii) a cytidine 5’-monophospho--N-acetylneuraminic acid synthetase for converting N-acetyl neuraminate to CMP-and said N-acetylmannosamine to N-acetyl-neuraminate and iii) a sialyltransferase. 18. Microorganism according to claim 17 wherein said sialyltransferase is originating from Photobacterium ae and/or wherein said microorganism ses a nucleic acid encoding a sialic acid se polypeptide originating from Campylobacter jejuni or Neisseria itides. 19. A sialylated compound ed by a microorganism according to any one of claim 1 to 18, wherein said sialylated compound is purified by any one or more of centrifugation and/or filtration, ion-exchange, concentration through evaporation or nanofiltration, ation through crystallization or spraydrying or lyophilization. 20. A sialylated compound produced by a microorganism according to any one of claim 1 to 18, wherein said sialylated compound is added to food formulation, feed formulation, pharmaceutical formulation, cosmetic formulation, or agrochemical formulation. 22. A method for the production of a sialylated compound wherein said sialylated compound is chosen from N-acetylneuraminate (sialic acid) or a sialylated oligosaccharide, comprising the steps of: i) cultivating the metabolically engineered microorganism according to any one of claims 1 to 18, and ii) ting and purifying the sialylated oligosaccharide. 004-PCT seqlist