Patent Application
20210087599
The present invention relates to sialyltransferases, their use in producing sialylated oligosaccharides, and to the use of said sialylated oligosaccharides in providing nutritional formulations.
More than 150 structurally distinct human milk oligosaccharides (HMOs) have been identified to date. Although HMOs represent only a minor amount of total human milk nutrients, their highly beneficial effect on the development of breast fed infants became evident over the past decades.
Up to 20% of the total HMO content in human milk is acidic. Thus, these HMO molecules possess at least one sialic acid moiety. While only 3% of the sialic acid contained in human milk is available in free form, 23% and 74% are bound to (glyco-)proteins and oligosaccharides, respectively. The most common member of the sialic acid family is N-acetylneuraminic acid (Neu5Ac). As part of an oligomeric saccharide, N-acetylneuraminic acid often accounts for the saccharide's biological activity.
Sialylated HMOs (SHMOs) were observed to support the resistance to pathogens as well as gut. Interestingly, recent studies further demonstrated the protective effect of long-chained SHMO against necrotizing enterocolitis, which is one of the most common and lethal diseases in preterm infants. In addition, SHMOs are believed to support an infant's brain development and its cognitive capabilities.
Although extensive variations in the HMO profile between different donors hamper an absolute quantification of acidic oligosaccharides, especially affecting the structural isomers of sialyllacto-N-tetraose, the most abundant acidic HMOs are 3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL), sialyllacto-N-tetraose a (LST-a), sialyllacto-N-tetraose b (LST-b), sialyllacto-N-tetraose c (LST-c) and disialyllacto-N-tetraose (DSLNT).
Regarding the structural complexity of the sialylated HMOs (
Generally speaking, metabolic engineering of microorganisms to produce HMOs represents the most promising approach for producing HMOs in an industrial scale, and was already developed for 2′-fucosyllactose, 3-fucosyllactose and 3′-sialyl-lactose.
Nevertheless, engineering biosynthetic pathways for the production of HMOs is often limited by the specificity and activity of the glycosyltransferases which are involved in the biosynthesis of the desired HMO, e.g. fucosyl-, galactosyl-, N-acetyl-glucosaminyl- or sialyltransferases, especially within a heterologous expression system such as a recombinant bacterial cell.
Unfortunately, genes encoding human sialyltransferases are barely expressed in prokaryotic microorganisms. Thus, these genes and enzymes are inapplicable in biotechnological processes using genetically engineered bacterial strains such as Escherichia coli for producing sialylated HMOs.
Several sialyltransferases (SiaTs) from bacterial species have been identified and characterized so far, e. g. from Neisseria, Campylobacter, Pasteurella, Helicobacter and Photobacterium, as well as from mammals and viruses. Sialyltransf erases have been generally classified into six glycosyltransferase (GT) families, based on protein sequence similarities. Therein, all eukaryotic and viral sialyltransferases are grouped into the GT family 29, whereas bacterial SiaTs are comprised in the groups GT4, GT38, GT42, GT52 or GT80. Besides, sialyltransferases and polysialyltrans-ferases can be distinguished due to the linkages that they form, e. g. into α-2,3-, α-2,6- and α-2,8-sialyltransferases. All these sialyltransferases transfer the sialic acid residue from cytidine 5′-monophosphate sialic acid (e. g. CMP-Neu5Ac) to a variety of acceptor molecules, usually galactose-(Gal), N-acetylgalactosamine-(GalNAc) or N-acetylglucosamine (GlcNAc) moieties or another sialic acid (Sia) moieties.
Several bacterial sialyltransferases were well characterized in the past and are already proven to be suitable for the production of 3′-SL or 6′-SL. Hardly any knowledge could have been achieved about sialyltransferases enabling the synthesis of sialylated penta- or hexasaccharides such as LST-a, LST-b or DSLNT, thereby limiting the establishment of a production process for any one of these SHMOs. As a consequence, the unavailability of highly pure amounts of these desired oligosaccharides impedes an extensive scientific evaluation of their health beneficial properties.
Therefore, there is a need for a cost-efficient process for producing one or more SHMOs, especially tetrasacharides, pentasaccharides and hexasaccharides possessing one or two sialic acid residues, in high amounts and high purity.
The object is solved, inter alia, by the identification and characterization of new sialyltransferases and their use in the production of sialylated human milk oligosaccharides by means of whole cell fermentation or biocatalysis.
According to a first aspect, provided is a method for producing sialylated oligo-saccharides, wherein a genetically engineered cell is used for producing said sialylated oligosaccharide. Said genetically engineered cell comprises at least one heterologous sialyltransferase which is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides.
According to a second aspect, provided is a genetically engineered cell for use in a method for producing sialylated oligosaccharides, wherein said genetically engineered cell has been genetically engineered to express a heterologous sialyltransferase which is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides.
According to a third aspect, provided is a recombinant nucleic acid molecule for expressing a heterologous sialyltransferase when propagated in a cell, wherein said sialyltransferase is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides.
According to a fourth aspect, provided are sialyltransferases being capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides.
According to a fifth aspect, provided is the use of a sialyltransferase being capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides, for producing sialylated oligosaccharides.
According to a sixth aspect, provided is a method for producing sialylated oligosaccharides by in vitro biocatalysis, wherein a sialyltransferase is used, said sialyltransferase being capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, LNT-II and human milk oligosaccharides.
According to a seventh aspect, provided are sialylated oligosaccharides being produced by a method according to the first aspect or by a method according to the sixth aspect.
According to an eight aspect, provided is the use of sialylated oligosaccharides according to the seventh aspect for manufacturing a nutritional composition.
According to a ninth aspect, provided is a nutritional composition containing at least one sialylated oligosaccharide according to the seventh aspect.
According to a tenth aspect, provided is an infant formula containing at least one sialylated human milk oligosaccharide.
In an attempt to identify sialyltransferases which are suitable for use in a process of manufacturing a sialylated HMO, nucleic acid databases and protein databases were searched. One hundred putative sialyltransferases were identified by means of sequence similarities to known glycosyltransferases. Said putative sialyltransferases were assessed for sialyltransferase activity.
According to the first aspect, provided is a method for producing a sialylated oligosaccharide, the method comprising the steps of
The method is a method for producing a sialylated oligosaccharide.
The term “oligosaccharide” as used herein refers to polymers of monosaccharide residues, wherein said polymers comprise at least three monosaccharide residues, but no more than 10 monosaccharide residues, preferably no more than 7 monosaccharide residues. The oligosaccharides are either a linear chain of monosaccharides or are branched. In addition, the monosaccharide residues of the oligosaccharides may feature a number of chemical modifications. Accordingly, the oligosaccharides may comprise one or more non-saccharide moieties.
The term “sialylated oligosaccharide” as used herein refers to oligosaccharides comprising one or more sialic acid residues. In a preferred embodiment, the sialic acid residue is an N-acetylneuraminic acid (Neu5Ac) residue. The N-acetyl-neuraminic acid residue is typically transferred from CMP-Neu5Ac as donor substrate to an acceptor molecule.
The method for producing a sialylated oligosaccharide comprises the step of providing a genetically engineered cell comprising a heterologous sialyltransferase which is capable of possessing an α-2,3-sialyltransferase activity and/or an α-2,6-sialyltransferase activity.
The genetically enginieered cell is a prokaryotic cell or a eukaryotic cell. Preferably, the genetically engineered cell is a microbial cell. Appropriate microbial cells include yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.
In an additional and/or alternative embodiment, the microbial cell is a prokaryotic cell, preferably a bacterial cell, more preferably a bacterial cell selected from the group consisting of Bacillus, Lactobacillus, Lactococcus, Enterococcus, Bifido-bacterium, Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas. Suitable bacterial species are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter freundii, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Enterococcus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acido-philus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenfi, Lactococcus lactis, Pantoea citrea, Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermophiles and Xanthomonas campestris.
In an alternative embodiment, the eukaryotic cell is a yeast cell, an insect cell, a plant cell or a mammalian cell. The yeast cell is preferably selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp., in particular Pichia pastoris, Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp.
The genetically engineered cell has been genetically engineered to comprise a heterologous sialyltransferase.
The term “genetically engineered” as used herein refers to the modification of the cell's genetic make-up using molecular biological methods. The modification of the cell's genetic make-up may include the transfer of genes within and/or across species boundaries, inserting, deleting, replacing and/or modifying nucleotides, triplets, genes, open reading frames, promoters, enhancers, terminators and other nucleotide sequences mediating and/or controlling gene expression. The modification of the cell's genetic make-up aims to generate a genetically modified organism possessing particular, desired properties. Genetically engineered cells can contain one or more genes that are not present in the native (not genetically engineered) form of the cell. Techniques for introducing exogenous nucleic acid molecules and/or inserting exogenous nucleic acid molecules (recombinant, heterologous) into a cell's hereditary information for inserting, deleting or altering the nucleotide sequence of a cell's genetic information are known to the skilled artisan. Genetically engineered cells can contain one or more genes that are present in the native form of the cell, wherein said genes are modified and re-introduced into the cell by artificial means. The term “genetically engineered” also encompasses cells that contain a nucleic acid molecule being endogenous to the cell, and that has been modified without removing the nucleic acid molecule from the cell. Such modifications include those obtained by gene replacement, site-specific mutations, and related techniques.
The genetically engineered cell comprises a heterologous sialyltransferase.
The term “sialyltransferase” as used herein refers to polypeptides being capable of possessing sialyltransferase activity. “Sialyltransferase activity” refers to the transfer of a sialic acid residue, preferably of an N-acetylneuraminic acid (Neu5Ac) residue, from a donor substrate to an acceptor molecule. The term “sialyltransferase” comprises functional fragments of the sialyltransferases described herein, functional variants of the sialyltransferases described herein, and functional fragments of the functional variants. “Functional” in this regard means that the fragments and/or variants possess sialyltransferase activity. Functional fragments of a sialyltransferase encompass truncated versions of a sialyltransferase as encoded by it naturally occurring gene, which truncated version is capable of possessing sialyltransferase activity. Examples of truncated versions are sialyltransferases which do not comprise a so-called leader sequence which typically directs the polypeptide to a specific subcellular localization. Typically, such leader sequences are removed from the polypeptide during its subcellular transport, and are also absent in the naturally occurring mature sialyltransferase.
The heterologous sialyltransferase is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule. The term “capable of” with respect to the heterologous sialyltransferase refers to the sialyltransferase activity of the heterologous sialyltransferase and the provision that suitable reaction conditions are required for the heterologous sialyltransferase to possess its enzymatic activity. In the absence of suitable reaction conditions, the heterologous sialyltransferase does not possess its enzymatic activity, but retains its enzymatic activity and possesses its enzymatic activity when suitable reaction conditions are restored. Suitable reaction conditions include the presence of a suitable donor substrate, the presence of suitable acceptor molecules, the presence of essential cofactors such as—for example—monovalent or divalent ions, a pH value in an appropriate range, a suitable temperature and the like. It is not necessary that the optimum values for each and every factor effecting the enzymatic reaction of the heterologous sialyltransferase is met, but the reaction conditions have to be such that the heterologous sialyltransferase performs its enzymatic activity. Accordingly, the term “capable of” excludes any conditions upon which the enzymatic activity of the heterologous sialyltransferase has been irreversibly impaired, and also excluded exposure of the heterologous sialyltransferase to any such condition. Instead, “capable of” means that the sialyltransferase is enzymatically active, i.e. possesses its sialyltransferase activity, if permissive reactions conditions (where all requirements being necessary for the sialyltransferase to perform its enzymatic activity) are provided to the sialyltransferase.
Sialyltransferases can be distinguished on the type of sugar linkage they form. As used herein, the terms “α-2,3-sialyltransferase” and “α-2,3-sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an alpha-2,3 linkage to galactose or a galactose residue of the acceptor molecule. Likewise, the terms “α-2,6-sialyltransferase” and “α-2,6-sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an alpha-2,6 linkage to galactose, N-acetylgalactosamine and/or N-acetylglucosamine, a galactose residue or a N-acetylgalactosamine residue and/or a N-acetylglucosamine residue of an acceptor molecule. Likewise, the terms “α-2,8-sialyltransferase” and “α-2,8-sialyltransferase activity” refer to polypeptides and their enzymatic activity which add a sialic acid residue with an alpha-2,8 linkage to galactose, N-acetylgalactosamine and/or N-acetylglucosamine, a galactose residue or a N-acetylgalactosamine residue and/or a N-acetylglucosamine residue of an acceptor molecule.
The term “heterologous” as used herein refers to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that is foreign to a cell or organism, i.e. to a polypeptide, amino acid sequence, nucleic acid molecule or nucleotide sequence that does not naturally occurs in said cell or organism. A “heterologous sequence” or a “heterologous nucleic acid” or “heterologous polypeptide”, as used herein, is one that originates from a source foreign to the particular host cell (e.g. from a different species), or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is modified from its original form. The heterologous sequence may be stably introduced, e.g. by transfection, transformation, conjugation or transduction, into the genome of the host microbial host cell, thus representing a genetically modified host cell. Techniques may be applied which will depend on the host cell the sequence is to be introduced. Various techniques are known to a person skilled in the art and are, e.g., disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Accordingly, a “heterologous polypeptide” is a polypeptide that does not naturally occur in the cell, and a “heterologous sialyltransferase” is a sialyltransferase that does not naturally occur in the cell.
The heterologous sialyltransferase is capable of transferring a sialic acid residue, e.g. a N-acetylneuraminic acid (Neu5Ac) residue, from a donor substrate, e.g. CMP-Neu5Ac, to an acceptor molecule. The acceptor molecule is lactose, lacto-N-triose II (LNT-II) or an oligosaccharide selected from the group consisting of human milk oligosaccharides.
In an additional and/or alternative embodiment, the acceptor molecule is a human milk oligosaccharide selected from the group consisting of trisaccharides, tetrasaccharides and pentasaccharides.
In an additional and/or alternative embodiment, the acceptor molecule is a human milk oligosaccharide selected from the group consisting of lacto-N-tetraose, lacto-N-neotetraose, LST-a and LST-b.
In an embodiment, the heterologous sialyltransferase is selected from the group consisting of
In an additional and/or alternative embodiment, the genetically engineered cell has been transformed to contain a nucleic acid molecule which comprises a nucleotide sequence encoding the heterologous sialyltransferase. Preferably, the nucleotide sequence is selected from the group consisting of
The expression “any one of SEQ ID NOs: 1 to 33” refers to any one of the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33. The same principle applies to the expression “any one of SEQ ID NOs: 34 to 66”. Generally speaking, the expression “any one of SEQ ID NOs: X to Z”, wherein “X” and “Z” represent a natural number, refers to all sequences (nucleotide sequences or amino acid sequences) represented by any one of the “SEQ ID NOs” comprising an identification number from X to Z.
In addition, the genetically engineered cell has been genetically engineered to express the nucleotide sequence encoding the heterologous sialyltransferase. To this end, the nucleotide sequence encoding the heterologous sialyltransferase is operably linked to at least one expression control effecting transcription and/or translation of said nucleotide sequence encoding the heterologous sialyltransferase in the genetically engineered cell.
The term “operably linked” as used herein, refers to a functional linkage between the nucleotide sequence encoding the heterologous sialyltransferase and a second nucleotide sequence, the nucleic acid expression control sequence (such as promoter, operator, enhancer, regulator, array of transcription factor binding sites, transcriptional terminator, ribosome binding site), wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the nucleotide sequence encoding the heterologous sialyltransferase. Accordingly, the term “promoter” designates DNA sequences which usually “precede” a gene in a DNA polymer and provide a site for initiation of the transcription into mRNA. “Regulator” DNA sequences, also usually “upstream” of (i.e., preceding) a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as “promoter/regulator” or “control” DNA sequence, these sequences which precede a selected gene (or series of genes) in a functional DNA polymer cooperate to determine whether the transcription (and eventual expression) of a gene will occur. DNA sequences which “follow” a gene in a DNA polymer and provide a signal for termination of the transcription into mRNA are referred to as transcription “terminator” sequences.
In an embodiment, the genetically engineered cell comprises a heterologous sialyltransferase being capable of possessing α-2,3-sialyltransferase activity, and the human milk oligosaccharide is LNT. The thus produced sialylated oligosaccharide is LST-a.
In an additional and/or alternative embodiment, the heterologous sialyltransferase being capable of possessing α-2,3-sialyltransferase activity is selected from the group consisting of
In an additional and/or alternative embodiment, the genetically engineered cell comprises a recombinant or synthetic nucleic acid molecule which comprises at least one nucleotide sequence encoding said heterologous sialyltransferase being capable of possessing α-2,3-sialyltransferase activity, wherein said at least one nucleotide sequence is selected from the group consisting of
In an additional and/or alternative embodiment, the heterologous sialyltransferase being capable of possessing α-2,3-sialyltransferase activity has a relative efficacy of at least 100-fold, at least 200-fold, at least 300-fold, at least 1000-fold, at least 10,000-fold, as compared to the relative efficacy of SiaT16 as represented by SEQ ID NO: 27 by means of quantitative analysis of LNT sialylation using LC-MS/MS following the method as described in example 5.
In another embodiment, the heterologous sialyltransferase is capable of possessing α-2,6-sialyltransferase activity, and the human milk oligosaccharide is LNT. The thus produced sialylated oligosaccharide is LST-b.
In an additional embodiment, the heterologous sialyltransferase being capable of possessing α-2,6-sialyltransferase activity is selected from the group consisting of
In an additional and/or alternative embodiment, the genetically engineered cell comprises a recombinant or synthetic nucleic acid molecule which comprises at least one nucleotide sequence encoding said heterologous sialyltransferase being capable of possessing α-2,6-sialyltransferase activity, wherein said at least one nucleotide sequence is selected from the group consisting of
In an additional and/or alternative embodiment, the heterologous sialyltransferase being capable of possessing α-2,3-sialyltransferase activity has a relative efficacy of at least 100-fold, more preferably of at least 200-fold, most preferably of at least 300-fold, as compared to the relative efficacy of SiaT5 as represented by SEQ ID NO: 33 by means of quantitative analysis of LNT sialylation using LC-MS/MS following the method as described in example 5.
In an additional and/or alternative embodiment, the at least one genetically engineered cell possesses an increased production of one or more nucleotide-activated sugars selected from the group consisting of CMP-N-acetylneuraminic acid, UDP-N-acetylglucosamine, UDP-galactose and GDP-fucose. Preferably, the at least one genetically engineered cell has been further genetically engineered to possess an increased production of one or more of said nucleotide-activated sugars. The production of the at least one of said nucleotide activated sugars is increased in the further genetically engineered cell as compared to the production of the same nucleotide-activated sugar(s) in the progenitor cell of the further genetically engineered cell prior to being further genetically engineered to possess an increased production of at least one of said nucleotide-activated sugars.
In an additional and/or alternative embodiment, the at least one cell has been further genetically engineered to overexpress one or more genes encoding for a polypeptide being capable of possessing an enzymatic activity selected from the group consisting of L-glutamine:D-fructose-6-phosphate aminotransferase, N-acetylglucosamine-1-phosphate uridyltransferase, glucosamine-1-phosphate acetyl transferase, phosphoglucosamine mutase, glucosamine-6-phosphate-N-acetyl-transferase, N-acetylglucosamine-2-epimerase, UDP-N-acetylglucosamine-2-epimerase, sialic acid synthase, phosphoenolpyruvate synthase, CMP-sialic acid synthetase, UDP-galactose-4-epimerase, galactose-1-phosphate uridylyl-transferase, phosphoglucomutase, glucose-1-phosphate uridylyltransferase, phosphomannomutase, mannose-1-phosphate guanosyltransf erase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase and fucose kinase/L-fucose-1-phosphate-guanyltransferase. Said overexpression of the one or more genes is an overexpression as compared to the progenitor cell of the further genetically engineered cell prior to being further genetically engineered to possess overexpression of said one or more genes.
Overexpression of one or more of said genes increases the amount of the corresponding enzyme(s) in the genetically engineered cell, and hence increases the corresponding enzymatic activity in the cell to enhance intracellular production of at least one of said nucleotide-activated sugars.
In an additional and/or alternative embodiment, the at least one genetically engineered cell lacks or possesses a decreased activity of one or more enzymatic activities selected from the group consisting of β-galactosidase activity, gluco-samine-6-phosphate deaminase, N-acetylglucosamine-6-phosphate deacetylase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase and N-acetylneuraminic acid aldolase as compared to the cell prior to be genetically engineered.
In an additional and/or alternative embodiment, one or more of the genes encoding a β-galactosidase, a glucosamine-6-phosphate deaminase, a N-acetylglucosamine-6-phosphate deacetylase, a N-acetylmannosamine kinase, a N-acetylmanno-samine-6-phosphate epimerase and a N-acetylneuraminic acid aldolase has/have been deleted from the genome of the genetically engineered cell or the expression of one or more of the genes encoding a β-galactosidase, a glucosamine-6-phosphate deaminase, a N-acetylglucosamine-6-phosphate deacetylase, a N-acetylmannosamine kinase, a N-acetylmannosamine-6-phosphate epimerase and a N-acetylneuraminic acid aldolase has/have been inactivated or at least decrease in the genetically engineered cell by further genetically engineering of cell. The expression of said genes is decreased in the further genetically engineered cell as compared to the progenitor cell of the further genetically engineered cell prior to being further genetically engineered to possess a decreased expression of said genes.
In an additional and/or alternative embodiment, the at least one genetically engineered cell comprises at least one selected from the group consisting of a functional lactose permease, a functional fucose permease and a functional sialic acid transporter (importer), preferably comprises and expresses at least one nucleotide sequence encoding one selected from the group consisting of a functional lactose permease, a functional fucose permease and a functional sialic acid transporter (importer).
In an additional and/or alternative embodiment, the genetically engineered cell possesses activity of at least one glycosyltransferase selected from the group consisting of a β-1,3-N-acetylglucosaminyltransferase, a 3-1,3-galactosyltrans-ferase, a β-1,4-galactosyltransferase, a α-2,3-sialyltransferase and a α-2,6-sialyl-transferase.
In an additional and/or alternative embodiment, the at least one genetically engineered cell is cultivated in a fermentation broth and under conditions permissive for the production of the sialylated oligosaccharide.
The fermentation broth contains at least one carbon source for the genetically engineered cells. The at least one carbon source is preferably selected from the group consisting of glucose, fructose, sucrose, glycerol, and combinations thereof.
In an additional and/or alternative embodiment, the fermentation broth contains at least one selected from the group consisting of N-acetylglucosamine, galactose and sialic acid.
In an additional and/or alternative embodiment, wherein the at least one genetically engineered cell is cultivated in the absence of and/or without addition of one or more selected from the group consisting of N-acetylglucosamine, galactose and sialic acid, the at least one genetically engineered cell is cultivated in the presence of lactose, lacto-N-triose II (LNT-II) or at least one HMO, preferably an HMO selected from the group consisting of trisaccharides, tetrasaccharides and pentasaccharides, more preferably an HMO selected from the group consisting, LNT and LNnT.
The method comprises the optional step of recovering the sialylated oligosaccharide that has been produced by the at least one genetically engineered cell during its cultivation in the fermentation broth. The sialylated oligosaccharide can be recovered from the fermentation broth after the genetically engineered cell have been removed, for example by centrifugation, and/or can be recovered from the cells, for example in that the cells are harvested from the fermentation broth by centrifugation, and are subjected to a cell lysis step. Subsequently, the sialylated oligosaccharides can be further purified from the fermentation broth and/or cell lysates by suitable techniques known to the skilled artisan. Suitable techniques include microfiltration, ultrafiltration, diafiltration, simulated moving bed type chromatography, electrodialysis, reverse osmosis, gel filtration, anion exchange chromatography, cation exchange chromatography, and the like.
According to a second aspect, provided is a genetically engineered cell for use in a method for producing sialylated oligosaccharides. Said genetically engineered cell and preferred embodiments of said genetically engineered cell has/have been described herein before in connection with the method. Hence, the genetically engineered cell comprises a heterologous sialyltransferase, said heterologous sialyltransferase being capable of possessing an α-2,3-sialyltransferase activity and/or an α-2,6-sialyltransferase activity for transferring a sialic acid residue, e.g. N-acetylneuraminic acid (Neu5Ac) residue from a nucleotide-activated form as donor substrate, e.g. CMP-Neu5Ac, to an acceptor molecule, wherein the acceptor molecule is selected from the group consisting of lactose, lacto-N-triose II and human milk oligosaccharides.
According to the third aspect, provided are recombinant nucleic acid molecules for expressing a sialyltransferase when propagated in a cell, said sialyltransferase being a heterologous sialyltransferase when expressed in the cell. The recombinant nucleic acid molecule(s) comprise(s) a nucleotide sequence encoding a sialyltrans-ferase which is capable of transferring a sialic acid residue, e.g. a N-acetyl-neuraminic acid residue, from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, lacto-N-triose II and human milk oligosaccharides.
Preferred embodiments of the nucleotide sequences encoding a sialyltransferase which is capable of transferring a sialic acid residue from a donor substrate to an acceptor molecule, wherein said acceptor molecule is selected from the group consisting of lactose, lacto-N-triose II and human milk oligosaccharides, such as preferred nucleotide sequences, are disclosed herein before in connection with the method of producing sialylated oligosaccharides. For example, the sialyltransferase is capable of transferring a N-acetylneuraminic acid residue from CMP-Neu5Ac to lactose, lacto-N-triose II or a human milk oligosaccharide.
The nucleotide sequence encoding the sialyltransferase is operably linked to at least one expression control sequence. Thus, in an additional and/or alternative embodiment, the recombinant nucleic acid molecule comprises at least one expression control sequence mediating transcription and/or translation of the nucleotide sequence encoding the sialyltransferase when said recombinant nucleic acid molecule is propagated in the cell.
According to the fourth aspect, provided are sialyltransferases being capable of possessing an α-2,3-sialyltransferase activity and/or an α-2,6-sialyltransferase activity transferring a sialic acid residue, e.g. a N-acetylneuraminic acid residue from a donor substrate, e,g. CMP-Neu5Ac, to an acceptor molecule, wherein said acceptor molecule is lactose, lacto-N-triose II or a human milk oligosaccharide.
In an embodiment, the acceptor molecule is selected from the group consisting of trisaccharides, tetrasaccharides and pentasaccharides. In an additional and/or alternative embodiment, the acceptor molecule is selected from the group consisting of LST-a and LST-b.
In an additional and/or alternative embodiment, the sialyltransferase is selected from the group consisting of
According to the fifth aspect, provided is the use of the sialyltransferases described herein before and being capable of transferring a sialic acid residue from a donor substrate, e.g. a N-acetylneuraminic acid residue from CMP Neu5AC, to an acceptor molecule, wherein said acceptor molecule is lactose, lacto-N-triose II or a human milk oligosaccharide, for producing sialylated oligosaccharides.
Said sialyltransferases are capable of transferring a sialic acid residue to an acceptor molecule, said acceptor molecule being a human milk oligosaccharide, thereby producing a sialylated oligosaccharide.
The human milk oligosaccharide may be a neutral oligosaccharide or an acidic oligosaccharide, i.e. a human milk oligosaccharide comprising at least one sialic acid residue.
The sialylated oligosaccharide produced by using the sialyltransferases as described herein before, may be a human milk oligosaccharide or may be a sialylated oligosaccharide not found in naturally occurring human milk.
According to the sixth aspect, provided is a method for producing sialylated oligosaccharides by in vitro biocatalysis, wherein a sialyltransferase is used, said sialyltransferase being capable of transferring a sialic acid residue from a donor substrate, e.g. a N-acetylneuraminic acid residue from CMP-Neu5Ac, to an acceptor molecule, wherein said acceptor molecule is a human milk oligosaccharide.
The method comprises the steps of:
According to a seventh aspect, provided are sialylated oligosaccharides being produced by a method according to the first aspect or by a method according to the sixth aspect.
In an embodiment, the sialylated oligosaccharide is a human milk oligosaccharide, preferably a tetrasaccharide, a pentasaccharide or a hexasaccharide, more preferably a sialylated oligosaccharide selected from the group consisting of LST-a, LST-b and DSLNT.
According to the eight aspect, provided is the use of a sialylated oligosaccharide being produced by a whole cell fermentation approach or an in vitro biocatalysis as described herein before for manufacturing a nutritional composition. Said nutritional composition contains at least one sialylated oligosaccharide which has been produced by a method as disclosed herein before.
Thus, according to the ninth aspect, provided is a nutritional composition containing at least one sialylated oligosaccharide which has been produced by a method as disclosed herein before. Preferably, the at least one sialylated oligosaccharide is 3′-sialyllactose, 6′-sialyllactose, LST-a, LST-b, LST-c or DSLNT.
In an additional and/or alternative embodiment, the nutritional composition further contains at least one neutral HMO, preferably 2′-FL.
In an additional and/or alternative embodiment, the nutritional composition contains 3-SL, 6-SL and 2′-FL.
In an additional embodiment, the nutritional composition is selected from the group consisting of medicinal formulations, infant formula and dietary supplements.
The nutritional composition may be present in liquid form or in solid form including, but not limited to, powders, granules, flakes and pellets.
According to the tenth aspect, provided is an infant formula containing at least one sialylated HMO. Said sialylated HMO is a HMO selected from the group of sialylated oligosaccharides that have been produced by a method as described herein before.
In an embodiment, the at least one sialylated HMO that is contained in the infant formula is selected from the group consisting of 3-SL, 6-SL, LST-a, LST-b, LST-c and DSLNT.
In an additional and/or alternative embodiment, the infant formula contains the at least one sialylated HMO and one or more neutral HMOs.
In an additional and/or alternative embodiment, the infant formula contains 3-SL, 6-SL and 2′-FL.
The present invention will be described with respect to particular embodiments and with reference to drawings, but the invention is not limited thereto but only by the claims. Furthermore, the terms first, second and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequence, either temporally, spatially, in ranking or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
It is to be noticed that the term “comprising”, used in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It is thus to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a device comprising means A and B” should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.
Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.
Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.
Furthermore, some of the embodiments are described herein as a method or combination of elements of a method that can be implemented by a processor of a computer system or by other means of carrying out the function. Thus, a processor with the necessary instructions for carrying out such a method or element of a method forms a means for carrying out the method or element of a method. Furthermore, an element described herein of an apparatus embodiment is an example of a means for carrying out the function performed by the element for the purpose of carrying out the invention.
In the description and drawings provided herein, numerous specific details are set forth. However, it is understood that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.
The invention will now be described by a detailed description of several embodiments of the invention. It is clear that other embodiments of the invention can be configured according to the knowledge of persons skilled in the art without departing from the true spirit or technical teaching of the invention, the invention being limited only by the terms of the appended claims.
Metabolic engineering included mutagenesis and deletions of specific genes, respectively, and genomic integrations of heterologous genes. The genes lacZ and araA were inactivated by mutagenesis using mismatch-oligonucleotides as described by Ellis et al., (Proc. Natl. Acad. Sci. USA 98: 6742-6746 (2001)).
Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). To prevent intracellular degradation of N-acetylneuraminic acid the genes encoding N-acetylglucosamine-6-phosphate deacetylase (nagA) and glucosamine-6-phosphate deaminase (nagB) as well as the whole N-acetylneuraminic acid catabolic gene cluster, encoding N-acetylmannosamine kinase (nanK), N-acetylmannosamine-6-phopsthate epimerase (nanE), N-acetylneuraminic acid aldolase (nanA) and the sialic acid permease (nanT) were deleted from the genome of the E. coli strain BL21 (DE3) strain. Also genes wzxC-wcaJ were deleted. WcaJ encodes an UDP-glucose:undecaprenyl phosphate glucose-1-phosphate transferase catalysing the first step in colanic acid synthesis (Stevenson et al., J. Bacteriol. 1996, 178:4885-4893). In addition, the genes fucl and fucK, coding for L-fucose isomerase and L-fuculose kinase, respectively, were removed.
Genomic integration of heterologous genes was performed by transposition. Either the EZ-Tn5™ transposase (Epicentre, USA) was used to integrate linear DNA-fragments or the hyperactive C9-mutant of the mariner transposase Himar1 (Lampe et al., Proc. Natl. Acad. Sci. 1999, USA 96:11428-11433) was employed for transposition. To produce EZ-Tn5 transposomes the gene of interest together with a FRT-site flanked antibiotic resistance marker was amplified with primer 1119 and 1120 (all primers used are listed in table 3 below); the resulting PCR-product carried on both sites the 19-bp Mosaic End recognition sites for the EZ-Tn5 transposase. For integration using Himar1 transposase expression constructs (operons) of interest were similarly cloned together with a FRT-site flanked antibiotic resistance marker into the pEcomar vector. The pEcomar vector encodes the hyperactive C9-mutant of the mariner transposase Himar1 under the control of the arabinose inducible promoter ParaB. The expression fragment <Ptet-/acY-FRT-aadA-FRT> (SEQ ID NO: 67) was integrated by using the EZ-Tn5 transposase. After successful integration of the gene for the lactose importer LacY from E. coli K12 TG1 (acc. no. ABN72583) the resistance gene was eliminated from streptomycin resistant clones by the FLP recombinase encoded on plasmid pCP20 (Datsenko and Wanner, Proc. Natl. Acad. Sci. 2000, USA 97:6640-6645), generating strain #534. Besides, the csc-gene cluster of E. coli W (acc. no. CP002185.1) comprising the genes for sucrose permase, fructokinase, sucrose hydrolase, and a transcriptional repressor (genes cscB, cscK, cscA, and cscR, respectively), that enable the strain to grow on sucrose as sole carbon source, was inserted in the genome. This csc-cluster was integrated into the genome of the E. coli BL21(DE3) strain by transposition using plasmid pEcomar-cscABKR. To enhance de novo synthesis of UDP-N-acetyl-glucosamine, genes encoding L-glutamine:D-fuctose-6-phosphate aminotrans-ferase (glmS), phosphoglucosamine mutase from E. coli K-12 substr. MG1655 (glmM) and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli K-12 substr. MG1655 (acc. no. NP_418185, NP_417643, NP_418186, respectively) were codon-optimized and obtained by gene synthesis. The operon glmUM was cloned under the control of the constitutive tetracyclin promoter Ptet while glmS was cloned under the constitutive PT5 promoter. The transposon cassette <Ptet-g/mUM-PT5-g/mS-FRT-dhfr-FRT> (SEQ ID NO: 68), flanked by the inverted terminal repeats specifically recognized by the mariner-like element Himar1 transposase was inserted from pEcomar-g/mUM-glmS. Altogether, the described genome modifications were leading to the E. coli BL21(DE3) strain #942, which represents the chassis for strain development. Tables 1, 2 and 3 comprise all strains, oligonucleotides used for cloning as well as general plasmids used within this study, respectively.
Strain #942 was modified for the production of sialic acid by the genomic integration of the expression cassettes <PtetglmSm-gna1-FRT-aacC1-FRT> (SEQ ID NO: 69), <Ptet-slr975-FRT-cat-FRT> (SEQ ID NO: 70), <Ptet-neuBC-FRT-kan-FRT> (SEQ ID NO: 71) and <Ptet-ppsA-FRT-aad1-FRT> (SEQ ID NO: 72). All genes were codon-optimized for expression in E. coli and prepared synthetically by GenScript cooperation. GlmSm represents a mutagenized version of GImS, thus, eliminating the feed-back inhibition by glucosamine-6-phosphate. The gene gnal encodes a glucosamine-6-phosphate acetyltransferase originating from Saccharomyces cerevisiae. Genes were subcloned as an operon behind the constitutive promotor Ptet and fused to the FRT-site flanked gentamycin resistance gene using primers glmSm/gna1_1-8. Similarly, the genes neuB (acc. no. AF305571), encoding a sialic acid synthase, and neuC (acc. no. AF305571), encoding an UDP-N-acetylgluco-samine-2-epimerase, both originating from Campylobacter jejuni, were subcloned as an operon behind the constitutive promotor Ptet and fused to the FRT-site flanked kanamycin resistance gene using primers neuBC_1-6. The gene s/r1975 (acc. no. BAL35720), also cloned behind the constitutive promotor Ptet and fused to the FRT-site flanked chloramphenicol resistance gene using primers slr_1-4, encodes a N-acetylglucosamine 2-epimerase from Synechocystis sp. PCC6803. The gene ppsA (acc. no. ACT43527) encoding the phosphoenolpyruvate synthase of E. coli BL21(DE3) was similarly cloned for constitutive expression and fused to the FRT-site flanked streptomycin resistance gene using primers ppsA_1-4. The genomic integrations finally led to the Neu5Ac producing strain #1363, which was used for the screening of the sialyltransferases 1 to 26.
E. coli BL21(DE3)
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ Δara
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ Δara
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ Δara
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ Δara
E. coli BL21(DE3)
E. coli BL21(DE3) ΔlacZ Δara
Escherichia coli BL21(DE3) strain #942 was used to build up a screening strain for the plasmid encoded sialyltransferases 27 to 100. Therefore, to enable the uptake and nucleotide-activation of sialic acid, the genes nanT and neuA, respectively, were integrated. The nanT gene (acc. no. B21_03035), encoding the E. coli Neu5Ac major facilitator superfamily transporter, was amplified from genomic DNA of E. coli BL21(DE3) and the neuA gene, originating from Campylobacter jejuni (acc. no. AF305571), was codon-optimized and obtained by synthesis. The genes were cloned as an operon under the control of the constitutive tetracyclin promoter Ret and the resulting expression fragment <Ptet-neuA-nanT-lox66-kan-lox72> (SEQ ID NO: 73) was integrated by using the EZ-Tn5 transposase, yielding the screening strain #1730.
Escherichia coli K12 integrated into vector
coli K12 integrated into vector pEcomar; cat,
Gene sequences of characterized or putative sialyltransferases were received from the literature and public databases. Since sialyltransferases are often described to exhibit higher activity when their signal peptide is deleted, we analyzed the corresponding protein sequences by the on-line prediction tool SignalP (Petersen et al., Nature Methods, 2011 Sep. 29; 8(10):785-6). Genes were synthetically synthesized by GenScript cooperation either, as annotated, in a full length form or, when a signal peptide is predicted, as a truncated variant lacking the N-terminal signal peptide (Table 4).
The sialyltransferases 1 to 26 were each subcloned as an operon with neuA into pDEST14 by SLIC using gene specific primers (Table 2), yielding plasmids of the general kind: pDEST14-siaT-neuA. The remaining sialyltransferases 27 to 100 were directly subcloned by GenScript cooperation into plasmid pET11a using restriction sites NdeI and BamHI. Both expression systems allow the IPTG-inducible gene expression (
Campylobacter jejuni. Cloning of sialyltransferases 30 to 32, 34, 37, 39, 41, 42, 51,
Neisseria meningitidis
Campylobacter jejuni strain OH4384
Campylobacter jejuni strain OH4384
Helicobacter acinonychis
Helicobacter acinonychis
Photobacterium sp. JT-ISH-224
Pasteurella dagmatis strain DSM 22969
Photobacterium sp. JT-ISH-224
Vibrio sp. JT-FAJ-16
Pasteurella multocida PM70
Photobacterium damselae JT0160
Streptococcus agalactiae
Haemophilus-somnus-2336
Haemophilus ducreyi 35000HP
Haemophilus ducreyi 35000HP
Photobacterium phosphoreum JT-ISH-
Photobacterium leiognathi JT-SHIZ-119
Photobacterium leiognathi JT-SHIZ-145
Campylobacter coli
Vibrio harveyi
Streptococcus entericus
Avibacterium paragaffinarum
Haemophilus parahaemolyticus HK385
Alistipes sp. CAG:268
Alistipes sp. AL-1
Pasteurella multocida PM70
Campylobacter jejuni strain 81-176
Alistipes shahii WAL 8301
Actinobacillus suis ATCC 33415
Actinobacillus capsulatus DSM 19761
Bibersteinia trehalosi USDA-ARS-USMARC-189
Photobacterium damselae subsp.
damselae CIP 102761
Haemophilus somnus 2336
Escherichia coli BL21(DE3) #1363 and #1730 harbouring plasmids encoding for 100 sialyltransferases were grown at 30° C. in 100 ml shake flasks filled with 20 ml of mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (wt/vol) glucose, ampicillin 100 μg ml−1, kanamycin 15 μg ml−1 and zeocin 40 μg ml−1. When the cultures reached an OD600 of 0.1 to 0.3, gene expression was induced by addition of 0.3 mM IPTG. After an incubation for one hour, 1.5 mM lactose was added to #1363 cultures whereas 1.5 mM lactose as well as 1.5 mM sialic acid was added to #1730 cultures. The incubation continued for 72 to 96 hours. Then, cells were harvested by centrifugation and mechanically disrupted in a defined volume using glass beads. Subsequently, samples were applied to thin layer chromatography (TLC) on Silica Gel 60 F254 (Merck KGaA, Darmstadt, Germany). A mixture of butanol:acetone:acetic acid:H20 (35/35/7/23 (v/v/v/v)) was used as mobile phase. For detection of the separated substances, the TLC plate was soaked with thymol reagent (0.5 g thymol solved in 95 ml ethanol, 5 ml sulfuric acid added) and heated.
The results are summarized in Table 5. In the entire screening, thirty two genes were identified to encode for α-2,3-sialyltransferases, thus producing 3′-SL. 19 enzymes synthesized 6′-SL and were depicted as α-2,6-sialyltransferases. An α-2,3- as well as an α-2,6-sialyltransferase activity could only be observed for 3 enzymes. Accordingly, the expression of 46 enzymes did not result in sialyllactose formation. The screening appeared highly accurate since the described activities of extensively characterized sialyltransferases, e. g. SiaT1 (Gilbert et al., J Biol Chem. 1996 Nov. 8; 271(45):28271-6; Gilbert et al., Eur J Biochem. 1997 Oct. 1; 249(1):187-94), SiaT6 (Tsukamoto et al., J Biochem, 2008 February; 143(2):187-97) and SiaT11 (Yamamoto et al., J Biochem 1996 July; 120(1):104-10), could be confirmed. Regarding the product formation, the experimental setting allowed a semi-quantitative comparison of the screened enzymes. But to achieve deeper knowledge about their kinetic properties, the 3 potentially best performing α-2,3- and α-2,6-sialyltransferases were applied to in vitro assays.
Escherichia coli BL21(DE3) harbouring plamids encoding for 100 sialyltransferases were grown at 30° C. in 100 ml shake flasks filled with 20 ml of 2YT medium supplemented with ampicillin 100 μg ml−1. When the cultures reached an OD600 of 0.1 to 0.3, gene expression was induced by addition of 0.3 mM IPTG and the incubation was continued for 12 to 16 hours. Cells were harvested by centrifugation and mechanically disrupted in a defined volume of 50 mM Tris-HCl pH7.5 using glass beads. The protein extract was kept on ice until the assay started. The in vitro assay was carried out in a total volume of 25 μl including 50 mM Tris-HCl pH7.5, 5 mM MgCl2, 10 mM CMP-Neu5Ac and 5 mM LNT. The assay started with the addition of 3 μl protein extract and continued for 16 hours. Product formation was determined by mass spectrometry.
Mass spectrometry analysis was performed by MRM (multiple reaction monitoring) using a LC Triple-Quadrupole MS detection system. Precursor ions are selected and analyzed in quadrupole 1, fragmentation takes place in the collision cell using argon as CID gas, selection of fragment ions is performed in quadrupole 3. Chromatographic separation of lactose, 3′-Sialyllactose and 6′-Sialyllactose after dilution of culture supernatant 1:100 with H2O (LC/MS Grade), was performed on a XBridge Amide HPLC column (3.5 μm, 2.1×50 mm (Waters, USA) with a XBridge Amide guard cartridge (3.5 μm, 2.1×10 mm) (Waters, USA). Column oven temperature of the HPLC system was 50° C. The mobile phase was composed of acetonitrile:H2O with 10 mM ammonium acetate. A 1 μl sample was injected into the instrument; the run was performed for 3.60 min with a flow rate of 400 μl/min. 3′-Sialyllactose and 6′-Sialyllactose were analyzed by MRM in ESI positive ionization mode. The mass spectrometer was operated at unit resolution. Sialyllactose forms an ion of m/z 656.2 [M+Na]. The precursor ion of Sialyllactose was further fragmented in the collision cell into the fragment ions m/z 612.15, m/z 365.15 and m/z 314.15. Collision energy, Q1 and Q3 Pre Bias were optimized for each analyte individually. Chromatographic separation of lactose, LNT-II, LNT and LST-a or -b after dilution of particle-free biocatalysis-reaction or crude extract, respectively, 1:50 with H2O (LC/MS Grade), was performed on a XBridge Amide HPLC column (3.5 μm, 2.1×50 mm (Waters, USA) with a XBridge Amide guard cartridge (3.5 μm, 2.1×10 mm) (Waters, USA). Column oven was run at 35° C. The mobile phase was composed of acetonitrile:H2O with 0.1% ammonium hydroxide. A 1 μl sample was injected into the instrument; the run was performed for 3.50 min with a flow rate of 300 μl/min. Lactose, LNT-II, LNT as well as LST-a and -b were analyzed by MRM in ESI negative ionization mode. The mass spectrometer was operated at unit resolution. Lactose forms an ion of m/z 341.00 [M−H]. The precursor ion of Lactose was further fragmented in the collision cell into the fragment ions m/z 179.15, m/z 161.15 and m/z 101.05. LNT-II forms an ion of m/z 544.20 [M−H]. The precursor ion of LNT-II was further fragmented into the fragment ions m/z 382.10, m/z 161.00 and m/z 112.90. LNT forms an ion of m/z 706.20 [M−H]. The precursor ion of LNT was further fragmented into the fragment ions m/z 382.10, m/z 202.10 and m/z 142.00. LST-a and -b forms an ion of m/z 997.20 [M−H]. The precursor ion of LST-a and-b was further fragmented into the fragment ions m/z 290.15, m/z 202.15 and m/z 142.15. Collision energy, Q1 and Q3 Pre Bias were optimized for each analyte individually. Quantification methods were established using commercially available standards (Carbosynth, Compton, UK).
The results of the in vitro screening are summarized in Table 5. Twenty eight genes were identified to produce LST-a whereas only 6 enzymes synthesized LST-b. Accordingly, the expression of 66 enzymes did not result in the formation of either LST-a or LST-b. The assay is regarded to be accurate since the activity of SiaT1, which was already described to sialylate LNT (Gilbert et al., J Biol Chem. 1996 Nov. 8; 271(45):28271-6; Gilbert et al., Eur J Biochem. 1997 Oct. 1; 249(1):187-94), could be verified. Irrespectively of the protein overexpression level, sialyltransferases that produced best were selected for determination of Km and Vmax.
To rank the best performing sialyltransferases, their Km values for donor and acceptor substrates were determined in vitro. Escherichia coli BL21(DE3) strain #287 was used for overproduction of the enzymes. Cells were incubated in 100 ml 2YT medium in shaking flasks supplemented with 100 μg m−1 ampicillin at 30° C. until an OD600 of 0.3 was reached. Then, 0.3 mM IPTG was added and the incubation was continued for 12 to 16 hours. Cells were harvested by centrifugation and mechanically disrupted in a defined volume of 50 mM Tris-HCl pH7.5 using glass beads. The protein extract was kept on ice until the assay started. The in vitro assay was carried out in a total volume of 50 μl including 50 mM Tris-HCl pH7.5, 5 mM MgCl2 and varying concentrations of CMP-Neu5Ac (0.05-30 mM) as well as of lactose or LNT (0.1-50 mM). The assay started with the addition of 35 to 750 μg protein extract. After 1 to 10 minutes of incubation at 30° C., the assay was inactivated at 95° C. for 5 minutes. Product formation was determined by mass spectrometry. Data were evaluated using the enzyme kinetic module of SigmaPlot v12.5 to calculate Km and Vmax.
During screening, the best performing α-2,3-sialyltransferases for LST-a production appeared to be SiaT8, SiaT9 and SiaT20. In contrast, SiaT6, SiaT18 and SiaT19 were observed to sialylate LNT most efficiently among the tested α-2,6-sialyltransferases. Their kinetic parameters for CMP-Neu5Ac and LNT as well as lactose are depicted in Table 6. Solely SiaT20 does not follow a Michaelis-Menten kinetic.
Escherichia coli BL21(DE3) strain #534 was used to construct a lacto-N-tetraose (LNT) producing strain. The β-1,3-N-acetylglucosaminyltransferase gene IgtA from Neisseria meningitidis MC58 (acc. no. NP_274923) was codon-optimized for expression in E. coli and prepared synthetically by gene synthesis. Together with the gene galT, encoding a galactose-1-phosphate uridylyltransferase from E. coli K-12 substr. MG1655 (acc. no. NP_415279), that was similarly obtained by gene synthesis, IgtA was inserted by transposition (SEQ ID NO: 188) using plasmid pEcomar-IgtA-ga/T. To enhance de novo synthesis of UDP-N-acetylglucosamine, genes encoding L-glutamine:D-fuctose-6-phosphate aminotransferase (glmS), phosphoglucosamine mutase from E. coli K-12 substr. MG1655 (glmM) and N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU) from E. coli K-12 substr. MG1655 (acc. no. NP_418185, NP_417643, NP_418186, respectively) were codon-optimized and obtained by gene synthesis. The operon glmUM was cloned under the control of constitutive tetracyclin promoter Ptet while glmS was cloned under the constitutive PT5 promoter. The transposon cassette <Ptet-glmUM-PTT5-glmS-FRT-dhfr-FRT>, flanked by the inverted terminal repeats specifically recognized by the mariner-like element Himar1 transposase was inserted from pEcomar-glmUM-glmS revealing a lacto-N-triose II production strain. Metabolic engineering further included the genomic integration of the transposon cassettes <Ptet-wbdO-PT5-galE-FRT-cat-FRT> (SEQ ID NO: 187), flanked by the inverted terminal repeats specifically recognized by the mariner-like element Himar1 transposase, which was inserted from pEcomar-wbdO-ga/E. To prevent intracellular degradation of N-acetylneuraminic acid the nanKETA gene cluster was deleted from the genome of the E. coli strain BL21(DE3) strain according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). To provide sufficient donor substrate (CMP-Neu5Ac) for the sialylation of LNT, the uptake mechanism for sialic acid as well as the capability for its nucleotide-activation was implemented into the E. coli strain. As described above, the genes nanT and neuA were cloned as an operon (using primers neuA/nanT_1-6) under the control of the constitutive tetracyclin promoter Ptet and the resulting expression fragment <Ptet-neuA-nanT-lox66- kan-lox72> was integrated by using the EZ-Tn5 transposase, finally generating strain #2130.
Escherichia coli BL21(DE3) #2130 cells harbouring expression plamids encoding for the sialyltransferases SiaT9 or SiaT19 were grown at 30CC in 100 ml shake flasks filled with 25 ml of mineral salts medium (Samain et al., J. Biotech. 1999, 72:33-47) supplemented with 2% (wt/vol) glucose, 5g/l NH4Cl, ampicillin 100 μml−1, kanamycin 15 μg ml−1 and gentamycin 5 μg ml−1. When the cultures reached an OD600 of 0.5 to 1, 3 mM lactose was added. After 24 hours of incubation sialyltransferase gene expression was induced by addition of 0.3 mM IPTG. Concomitantly, 3 mM of sialic acid was added to the cultures. The incubation continued for 48 hours. Then, cells were harvested by centrifugation and mechanically disrupted in a defined volume using glass beads. Subsequently, thin layer chromatography (TLC) was performed to confirm the intracellular formation of the sialyllacto-N-tetraoses-a and -b. As shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 17183391.6 | Jul 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/070214 | 7/25/2018 | WO | 00 |
