Rhamnose-polysaccharides

Abstract

To provide a method of producing a GAS polysaccharide which results in a homogenous, pure and high yield polysaccharide.SOLUTION: The present invention relates to a method of synthesizing a rhamnose polysaccharide. The invention also relates to a synthetic streptococcal polysaccharide, a streptococcal glycoconjugate, an immunogenic composition or vaccine comprising the streptococcal polysaccharide or glycoconjugate and the polysaccharide, glycoconjugate, immunogenic composition or vaccine for use in raising an immune response in an animal or for use in treating or preventing a disease, condition or infection with a streptococcal etiology.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] (Field) The present invention relates to a method for synthesizing rhamnose polysaccharide. The present invention also relates to synthetic streptococcal polysaccharides, streptococcal glycoconjugates, immunogenic compositions or vaccines containing said streptococcal polysaccharides or glycoconjugates and used to enhance the immune response in animals, or to treat or prevent diseases, conditions or infections associated with streptococcal etiology, said poly saccharides, glycoconjugates, immunogenic compositions or vaccines.

Background Art

[0002] (Background) The genus Streptococci of bacteria is a group of versatile Gram-positive bacteria that infect a wide range of hosts and are the cause of a notable number of diseases.

[0003] Streptococcus pyogenes (Group A Streptococ cus, GAS) is a Gram-positive bacterium restricted to humans that causes various diseases. Presumably the underestimation of the prevalence of this microorganism, more than 700 million people worldwide suffer annually, suggesting that it causes various diseases such as impetigo, pharyngitis, scarlet fever, necrotizing fasciitis, meningitis and toxic shock syndrome among others. Furthermore, post-infectious autoimmune sequelae such as acute rheumatic fever, acute glomerulonephritis or rheumatic heart disease can affect individuals previously affected by GAS infections, expanding the list of clinical symptoms caused by this pathogen. Group A carbohydrate (GAC) is a peptidoglycan-anchored rhamnose-polysaccharide ([[]] derived from Streptococcus pyogenes ([[]] ​It is (RhaPS), which is essential for the survival of bacteria and contributes to the infectivity of Streptococcus pyogenes to the human host. It contributes to the infectivity of Streptococcus pyogenes to the human host.

[0004] Streptococcus agalactiae (Group B Streptococcus, GBS) is an (opportunistic) commensal bacterium, and 20 - 40% of all adult humans carry it. It is an (opportunistic) commensal bacterium, and 20 - 40% of all adult humans carry it. 25% of women carry GBS in the vagina and usually exist asymptomatically. However, in pregnant women, GBS is recognized as a cause of preterm birth, maternal infections, stillbirth, and late miscarriage. However, in pregnant women, GBS is recognized as a cause of preterm birth, maternal infections, stillbirth, and late miscarriage. Despite current preventive measures, 1 in 1000 infants born in the UK develops GBS infection. Preterm infants are known to be at particularly high risk of GBS infection because their immune systems are not fully developed. Preterm infants are known to be at particularly high risk of GBS infection because their immune systems are not fully developed. As a result, in the UK, 1 infant dies and 1 infant survives with long - term disabilities per week due to GBS infection. As a result, in the UK, 1 infant dies and 1 infant survives with long - term disabilities per week due to GBS infection.

[0005] Group C Streptococcus (GCS) can cause pharyngitis and cellulitis that are clinically indistinguishable from gas diseases in humans. Also, in patients with predisposing conditions such as diabetes, cancer, or elderly patients, it is known to cause sepsis, endocarditis, septic arthritis, and necrotizing infections. Group C Streptococcus (GCS) can cause pharyngitis and cellulitis that are clinically indistinguishable from gas diseases in humans. Also, in patients with predisposing conditions such as diabetes, cancer, or elderly patients, it is known to cause sepsis, endocarditis, septic arthritis, and necrotizing infections. In horses, GCS is the cause of a highly contagious and severe upper respiratory infection known as strangles, which is endemic worldwide. In horses, GCS is the cause of a highly contagious and severe upper respiratory infection known as strangles, which is endemic worldwide. In horses, GCS is the cause of a highly contagious and severe upper respiratory infection known as strangles, which is endemic worldwide.

[0006] Group G Streptococcus (GGS) is a major human pathogen that causes skin infections such as, for example, human skin. Group G Streptococcus (GGS) is a major human pathogen that causes skin infections such as, for example, human skin. GGS is present in the oropharynx, gastrointestinal tract, and female reproductive tract. The device is also infected. Other infectious diseases related to GGS include sepsis, endocarditis, meningitis , peritonitis, pneumonia, empyema, septic arthritis, and several other life-threatening infections .

[0007] The options for antibacterial drugs to effectively control, treat, and prevent GAS infection have become more limited . This is due to emerging antibiotic resistance, pandemic development, and the spread of highly pathogenic strains. Therefore, the development of safe and effective vaccine candidates is clearly necessary . For a vaccine that can target most of the more than 120 different GAS serotypes, it is necessary to be based on ubiquitous, conserved, and essential GAS targets . One such target is GAC, which is not only an essential structural component for the pathogen but also a virulence determinant . Summary of the Invention Problems to be Solved by the Invention

[0008] The current form of vaccine development is limited to chemical and enzymatic extraction methods from native bacteria and chemical conjugation to any acceptor compound (e.g., protein or peptide) . This is labor-intensive and results in limiting the yield and quality of the product. There is clearly a need for a method for producing GAS polysaccharides that is not labor-intensive and results in homogeneous, pure, and high-yield polysaccharides . The present invention has been devised with these problems in mind . Means for Solving the Problems

[0009] (Explanation) ​​​​In its broadest sense, the present disclosure relates to methods for synthesizing polysaccharides, particularly rhamnose polysaccharides .

[0010] (Detailed Description) The present invention will be described by way of example with reference to the following drawings:

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] According to a first aspect, a method for synthesizing rhamnose polysaccharide, comprising the following: (i) Using hexose-β-1,4-rhamnosyltransferase, hexose-α-1, 2-rhamnosyltransferase and / or hexose-α-1,3-rhamnosyl transferase, or enzymatically active fragments or variants thereof, to transfer the rhamnose moiety to a hexose monosaccharide, disaccharide or trisaccharide to form a disaccharide, trisaccharide or tetrasaccharide, wherein the rhamnose moiety is included at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide; and (ii) Using the heterologous bacterial enzymes Streptococcus pyogenes group A carbohydrate enzyme C (GacC) and / or Streptococcus pyogenes group A carbohydrate enzyme G (GacG), or enzymatically active homologs, variants or fragments thereof, to extend from the rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide to produce the rhamnose polysaccharide. A method is provided that includes the above steps. The bacterial species from which the enzyme GacC and / or enzyme GacG, or enzymatically active homologs, variants or fragments thereof are derived is heterologous to the bacterial species from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or enzymatically active fragments or variants thereof used in step (i) are derived.

[0013] The bacterial species from which the enzyme GacC and / or enzyme GacG, or enzymatically active homologs, variants or fragments thereof are derived is heterologous to the bacterial species from which the hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase or enzymatically active fragments or variants thereof used in step (i) are derived.

[0014] ​​​​​The inventor of the present invention has found that the Streptococcus p enzyme GacB, which initiates the synthesis of GAC rhamnose polysaccharide, is an α-D-GlcNAc-β-1,4-L rhamnosyl transferase. Quite surprisingly, the inventor has found that these rhamnose polysaccharides can be synthesized using rhamnosyl transferases from bacterial species different from those from which GacB is derived. In other words, the inventors have found that rhamnose polysaccharides can be synthesized using rhamnosyl transferases from bacterial species other than S. pyogenes. This is quite unexpected considering that the function of GacB was previously unknown. It is also surprising that enzymes from different species can act together to synthesize rhamnose polysaccharides. yogenes enzyme GacB is an α-D-GlcNAc-β-1,4-L rhamnosyl transferase. transferase for the first time. Quite surprisingly, the inventor has found that these rhamnose polysaccharides can be synthesized using rhamnosyl transferases from bacterial species different from those from which GacB is derived. transferases. In other words, the inventors have found that rhamnose polysaccharides can be synthesized using rhamnosyl transferases from bacterial species other than S. pyogenes. This is quite unexpected considering that the function of GacB was previously unknown. It is also surprising that enzymes from different species can act together to synthesize rhamnose polysaccharides. rhamnose polysaccharides can be synthesized using rhamnosyl transferases from bacterial species other than S. pyogenes. transferases. This is quite unexpected considering that the function of GacB was previously unknown. It is also surprising that enzymes from different species can act together to synthesize rhamnose polysaccharides. function was previously unknown. It is also surprising that enzymes from different species can act together to synthesize rhamnose polysaccharides. rhamnose polysaccharides.

[0015] In some embodiments, step (ii) comprises producing a rhamnose polysaccharide by extending from the rhamnose moiety at the non-reducing end of a disaccharide, trisaccharide or tetrasaccharide using a heterologous bacterial enzyme GacC or an enzymatically active homolog, variant or fragment thereof. homologs, variants or fragments thereof to produce a rhamnose polysaccharide by extending from the rhamnose moiety at the non-reducing end of a disaccharide, trisaccharide or tetrasaccharide. rhamnose polysaccharide by extending from the rhamnose moiety at the non-reducing end of a disaccharide, trisaccharide or tetrasaccharide. and the like.

[0016] Polysaccharide is a term known in the art used to denote a molecule containing a plurality of identical or different monosaccharides, typically more than four monosaccharides. Thus, the term rhamnose polysaccharide, as used herein, is understood to refer to a molecule containing a plurality of, usually more than four, rhamnose moieties optionally linked to one or more other monosaccharide moieties. For simplicity, a rhamnose polysaccharide is understood to include rhamnose linked to each other by alpha 1,3 or alpha 1,2 linkages. Polysaccharide is a term known in the art used to denote a molecule containing a plurality of identical or different monosaccharides, typically more than four monosaccharides. Thus, the term rhamnose polysaccharide, as used herein, is understood to refer to a molecule containing a plurality of, usually more than four, rhamnose moieties optionally linked to one or more other monosaccharide moieties. As used herein, it is understood to refer to a molecule containing a plurality of rhamnose moieties, usually more than four, optionally linked to one or more other monosaccharide moieties. For simplicity, a rhamnose polysaccharide is understood to include rhamnose linked to each other by alpha 1,3 or alpha 1,2 linkages. rhamnose linked to each other by alpha 1,3 or alpha 1,2 linkages. Each repeat unit may consist of only rhamnose. Or each repeat unit may comprise rhamnose and one or more different monosaccharides. An exemplary repeating unit containing rhamnose is the rhamnose-galactose disaccharide repeating unit. Each / any repeating unit and / or rhamnose moiety may contain any side group. In one embodiment, there are no side groups, and in another embodiment, there are no side groups. serine-phosphate; or one such as a sugar, with or without additional modifications such as phosphate The above side groups may be present.

[0017] In an embodiment, the method is carried out in a bacterium.

[0018] In such an embodiment, the method is understood to be a microbiological method. Other embodiments than those mentioned are understood to be in vitro methods. By "bacteria" is meant: This will be understood to mean bacterial cells. The invention also relates to methods carried out in bacteria. It will be understood that such microbiological methods include the production of large, homogeneous quantities of The enzyme is ideal for the production of a specific product, in this case rhamnose polysaccharide.

[0019] The rhamnose polysaccharide produced by this method is understood to be a synthetic rhamnose polysaccharide. Synthetic rhamnose polysaccharides may be produced as a result of naturally occurring processes, as will be appreciated by those of skill in the art. This will be understood to refer to rhamnose polysaccharides that are not saccharides. This is the first aspect of the method. This is because the combination uses enzymes that do not occur in nature. The bacteria were Streptococcus pyogenes, Escherichia species ( Examples include Streptococcus species other than E. coli or Shigella species (e.g., Shigella dysenteriae or Shigella flexneri). teriae or Shigella flexneri). is a Streptococcus species other than E. coli or Shigella species (e.g., Shigella dysenteriae or Shigella flexneri).

[0020] Typically, the rhamnose polysaccharide produced by this method is a streptococcal polysaccharide. For example, the polysaccharide may include a polysaccharide selected from the group consisting of group A, group B, group C, and group G carbohydrates, or a fragment or variant thereof. The rhamnose moiety, which is understood to refer to rhamnose monosaccharide or a derivative thereof,

[0021] is understood to refer to a rhamnose monosaccharide derivative modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting, rhamnose monosaccharide or a derivative thereof. A derivative of rhamnose monosaccharide is modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. It will be understood that derivatives of rhamnose refer to rhamnose monosaccharides modified by the addition or substitution of one or more groups or elements in the rhamnose monosaccharide such that at least one carbon in the rhamnose monosaccharide can still form a glycosidic bond with at least one other rhamnose monosaccharide or rhamnose moiety. Derivatives of rhamnose may include acetyl or methyl forms of rhamnose, aminorhamnose, carboxyethylrhamnose, halogenated rhamnose, and rhamnose phosphate. Unless otherwise indicated in the context, references to rhamnose moiety herein generally refer to, but should not be construed as limiting,

[0022] Halogenated rhamnose refers to a rhamnose monosaccharide in which one or more groups, e.g., one or more OH groups, in the rhamnose are replaced by a halogen, e.g., fluoride or chloride, to form fluorinated or chlorinated rhamnose, respectively. is understood to refer to a rhamnose monosaccharide in which one or more groups, e.g., one or more OH groups, in the rhamnose are replaced by a halogen, e.g., fluoride or chloride, to form fluorinated or chlorinated rhamnose, respectively. Halogenated rhamnose refers to a rhamnose monosaccharide in which one or more groups, e.g., one or more OH groups, in the rhamnose are replaced by a halogen, e.g., fluoride or chloride, to form fluorinated or chlorinated rhamnose, respectively.

[0023] Amino-rhamnose means that one or more groups of rhamnose are substituted by amine groups. It is understood to refer to rhamnose monosaccharide.

[0024] An example of acetyl-rhamnose may include 2-O-acetyl-α-L-rhamnose. On the other hand, an example of methyl-rhamnose may include 3-O-methyl-L-rhamnose. Another exemplary derivative of rhamnose is carboxyethyl-rhamnose, such as 4-O -(1-carboxyethyl)-L-rhamnose may also be included.

[0025] By an enzymatically active fragment or variant, the sequence of the relevant enzyme may differ from the naturally occurring sequence, provided that the fragment or variant substantially retains the enzyme activity of the enzyme. By retaining the enzyme activity of the enzyme, the fragment and / or variant means retaining at least a part of the enzyme activity compared to the native enzyme. Typically, the fragment and / or variant retains at least 50%, for example 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the activity. In some examples, the fragment and / or variant may have greater enzyme activity than the native enzyme. In some embodiments, the fragment and / or variant may exhibit an increase in another physiological characteristic compared to the native enzyme. For example, this fragment and / or variant may have a longer half-life in vitro and / or in vivo compared to the native enzyme. Tests for determining the half-life of an enzyme, or a fragment or variant thereof, are known to those skilled in the art. Briefly, in vitro tests for the enzyme are known to those skilled in the art. Briefly, in vitro tests for the enzyme fragment and / or variant may have a longer half-life in vitro and / or in vivo compared to the native enzyme. Tests for determining the half-life of an enzyme, or a fragment or variant thereof, are known to those skilled in the art. Briefly, in vitro tests for the enzyme fragment and / or variant may have a longer half-life in vitro and / or It may include incubating the element at different temperatures and pH for different periods. At the end of each period the activity of the enzyme, or its fragment or variant, can be measured using enzyme assays well known to those skilled in the art.

[0026] Enzyme GacC, as used herein, is understood to refer to Streptococcus pyo genes group A carbohydrate enzyme C (UniProtKB - Q9A0G4 (Q9 A0G4_STRP1)). An exemplary amino acid sequence encoding GacC is provided by SEQ ID NO: 1.

[0027] Enzyme GacG, as used herein, is understood to refer to Streptococcus pyo genes group A carbohydrate enzyme G (UniProtKB - Q9A0G0 (Q9 A0G0_STRP1)). In some embodiments, enzyme GacG comprises, or consists of, SEQ ID NO: 2, or an enzymatically active fragment or variant thereof.

[0028] GacG (or an enzymatically active homolog, variant or fragment thereof) is used in the method of the present invention instead of, or in addition to, GacC. GacC is a rhamnose- 1,3α rhamnosyltransferase, while GacG is a predicted bifunctional glycosyltransferase that synthesizes the repeating unit of GAC (α 1,3-α1,2).

[0029] A "homolog" has at least about 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, An enzyme that represents 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity may be included.

[0030] In some embodiments, the enzymatically active homolog is a homolog of GacC.

[0031] The degree (or percentage) of "homology" between two or more amino acid sequences is determined by aligning the sequences and determining the number of aligned residues that are identical, and this can be calculated by adding the number of conservative amino acid substitutions Next, the combined total is divided by the total number of residues compared, and the resulting value is multiplied by 100 to obtain the percentage homology between the aligned sequences.

[0032] Typically, homologs of GacC or GacG include enzymes that substantially retain the enzymatic activity of GacC or GacG.

[0033] In some embodiments, the homolog of GacC comprises or consists of rfbG. RfbG is an alpha-1-3 rhamnosyltransferase derived from Shigella flexneri that has 30% identity with GacC. Thus, in the context of the present disclosure, rfbG is an enzymatically active homolog of GacC. In some embodiments rfbG comprises or consists of SEQ ID NO: 3. RfbG may be identified using UniProtKB - A0A2D0WWB9 (A0A2D0WWB9_9ENTR).

[0034] Homologs of GacC or GacG are from Lancefield group species other than rfbG, S. pyogenes and / or non- S. pneumoniae ​​​​​It may contain or may consist of an enzyme derived from the Lancefield group Streptoccocus species. Or it may consist thereof.

[0035] In some embodiments, homologs of GacC or GacG are enzymes derived from Lancefield group species other than S. pyogenes and / or from non-Lancefield group Streptococcus species other than S. pneumoniae ae. There are.

[0036] As those skilled in the art know, the bacterial Lancefield group refers to a group of different bacterial species that are catalase-negative and coagulase-negative, mainly Streptococcus species. This grouping is based on the carbohydrate composition of the cell wall antigen. Examples of Lancefield group bacteria include:

[0037] ● Group A - Streptococcus pyogenes, Strept ococcus dysgalactiae subsp. equisimilis ● Group B - Streptococcus agalactiae ● Group C - Streptococcus equisimilis, Str eptococcus equi, Streptococcus zooepidemi cus, Streptococcus dysgalactiae, Streptoco ccus dysgalactiae subsp. equisimilis ● Group D - Enterococcus faecalis, Enteroco ccus faecium, Enterococcus durans and Strep tococcus bovis It may contain or may consist of an enzyme derived from the Lancefield group Streptoccocus species. Or it may consist thereof.

[0035] In some embodiments, homologs of GacC or GacG are enzymes derived from Lancefield group species other than S. pyogenes and / or from non-Lancefield group Streptococcus species other than S. pneumoniae ae. There are.

[0036] As those skilled in the art know, the bacterial Lancefield group refers to a group of different bacterial species that are catalase-negative and coagulase-negative, mainly Streptococcus species. This grouping is based on the carbohydrate composition of the cell wall antigen. Examples of Lancefield group bacteria include:

[0037] ● Group A - Streptococcus pyogenes, Strept ococcus dysgalactiae subsp. equisimilis ● Group B - Streptococcus agalactiae ● Group C - Streptococcus equisimilis, Str eptococcus equi, Streptococcus zooepidemi cus, Streptococcus dysgalactiae, Streptoco ccus dysgalactiae subsp. equisimilis ● Group D - Enterococcus faecalis, Enteroco ccus faecium, Enterococcus durans and Strep tococcus bovis ● Group E - Enterococci ● Groups F, G & L - Streptococcus anginosus, S treptococcus dysgalactiae subsp. equisim ilis ● Group H - Streptococcus sanguis ● Group K - Streptococcus salivarius ● Group L - Streptococcus dysgalactiae ● Groups M & O - Streptococcus mitior ● Group N - Lactococcus lactis ● Groups R & S - Streptococcus suis

[0038] The non - Lancefield group Streptococcus species may include Streptococ cus mutans or S. uberis. In some embodiments the non - Lancefield group Streptococcus species may include S. mutans or may consist of S. mutans.

[0039] An enzymatically active homolog of GacC or GacG may be selected from homologs derived from Streptococcus group B, group C, group G, S. mutans, S. uberis or their enzymatically active fragments or variants.

[0040] In some embodiments, an enzymatically active homolog of GacC or GacG is Stre ptococcus group B, group C, group G, S. mutans, or ​​It may be selected from homologs of those enzymatically active fragments or variants. It may be.

[0041] In some embodiments, the enzymatically active homolog of GacC is from Streptococc us group B, group C, group G, S. mutans, S. uberis or a homolog of GacC from those enzymatically active fragments or variants thereof is selected. One skilled in the art will know the streptococcal homologs to GacC . For example, the group B homolog of GacC may be GbcC (UniProtKB_Q8DY Q2 (Q8DYQ2_STRA5)). The group C homolog of GacC may be , GccC (UniProtKB-M4YWQ3 (M4YWQ3_STREQ)) and may be. The group G homolog of GacC is GgcC (UniProtKB-C5WFT 8 (C5WFT8_STRDG)), and the S. mutans homolog of GacC is Sc cC (UniProtKB-A0A0E2EN43 (A0A0E2EN43_STRMG ))). The Suberis homolog of GacC may be SucC (UniProtKB-B 9DU25 (B9DU25_STRU0)). The amino acid sequence of GbcC may include or consist of SEQ ID NO: 4.

[0042] The amino acid sequence of GccC may include or consist of SEQ ID NO: 5, while the amino acid sequence of GgcC may include or consist of SEQ ID NO: 6. In some embodiments, SccC includes or consists of SEQ ID NO: 7. The amino acid sequence of SucC may include or consist of SEQ ID NO: 8.

[0043] In some embodiments, an enzymatically active homolog of GacG is from Streptococ cus group C, group G, S. mutans, S. uberis or an enzymatically active fragment or variant thereof, and is selected from homologs of GacG. Suitable enzymatically active homologs of GacG include, but are not limited to, the group C homolog of GacG, the group G homolog of GacG, GgcG, GgcG, the S. uberis homolog of GacG, and the S. mutans homologs of GacG, SccG.

[0044] In some embodiments, GccG comprises and consists of SEQ ID NO: 9. In some embodiments, GccG comprises or consists of two proteins. The two proteins may comprise or consist of SEQ ID NOs: 10 and 11.

[0045] GgcG may comprise or consist of two proteins. The two proteins may each have UniProtKB C5WFU2 (C5WFU2_STRDG) and C5WFU3 (C5WFU3_STRDG). In some embodiments, GgcG may comprise or consist of SEQ ID NOs: 12 and 13.

[0046] SucG may comprise or consist of the amino acid sequence identified by UniProtKB - B9DU29 (B9DU29_STRU0). For example, SucG may comprise or consist of the amino acid sequence of SEQ ID NO: 14.

[0047] ​SccG may comprise or consist of an amino acid sequence identified by UniProtKB_O82878 (O82878_STRMG). In some embodiments, SccG comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0048] An enzymatically active homolog of GacC or GacG may be selected from homologs from S. mutans, S. uberis or fragments or variants thereof.

[0049] In some embodiments, step (ii) comprises producing a rhamnose polysaccharide by extending from the rhamnose moiety at the non-reducing end of a disaccharide, trisaccharide or tetrasaccharide using an enzymatically active homolog of GacC and / or GacG from S. mutans, or an enzymatically active variant or fragment thereof.

[0050] The invention also encompasses nucleic acid sequences encoding the enzymes (and / or enzymatically active fragments, variants or homologs) of the invention.

[0051] As used herein, when an enzyme is "derived from" a particular bacterial species, this means that the enzyme is naturally occurring in the particular bacterial species. In the context of the present invention, an enzyme "derived from" a particular bacterial species can include an enzyme endogenous to the bacterium in which the method can be carried out, an enzyme or nucleic acid encoding an enzyme isolated from a particular bacterial species, or a variant or fragment thereof. In embodiments where the method is carried out in a bacterium, an enzyme or nucleic acid encoding an enzyme isolated from a particular bacterial species can be introduced into the bacterium in which the method is carried out.

[0052] ​​​​​​​​​​​​​ In embodiments where the method is carried out in bacteria, the enzyme of step (i) and / or the enzyme of step (ii) can be overexpressed in bacteria. By "overexpression", this means an enzyme expression level higher than the level observed for the native enzyme when endogenously expressed in the native bacteria. Various techniques for overexpression are known to those skilled in the art. Further information regarding overexpression techniques can be found in Current Protocols in Molecular Biology( 2019), which is incorporated herein by reference.

[0053] In the context of the present invention, heterologous is used to refer to being different. A heterologous bacterial species is understood to mean a bacterial species different from another bacterial genus, or a bacterial genus different from another bacterial genus.

[0054] In the context of the present invention, it is understood that heterologous does not include a different bacterial strain from another bacterial strain (i.e., for example, two strains of S. mutans).

[0055] "Variants" of an enzyme include insertions, deletions and substitutions of conservative or non-conservative amino acid sequences in which the physiochemical properties of each amino acid do not substantially change (e.g., conservative substitutions such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr). Those skilled in the art will understand that such conservative substitutions should not affect the functionality of each enzyme. Further, small deletions within non-functional regions of the enzyme may also be tolerated, and thus, the present invention's For the purposes, it is considered a "variant". A "variant" also includes recombinant enzyme proteins in which the amino acids are post-translationally modified, for example, by glycosylation or disulfide bond formation. The experimental procedures described herein can be readily adopted by those skilled in the art to determine whether a "variant" can still function as an enzyme.

[0056] Preferably, the variant has an amino acid sequence with at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, 97%, 98% or 99% identity to the "naturally occurring" amino acid sequence of the enzyme.

[0057] It is understood that the variants also include variants of the nucleic acid sequences encoding the enzymes. In particular, the inventors include variants of nucleic acid sequences in which such changes do not substantially change the enzyme activity of the enzyme encoded thereby. Those skilled in the art know that such sequences can be modified without losing enzyme activity. In particular, a single change in a nucleic acid sequence cannot result in an amino acid sequence modified after the expression of the sequence.

[0058] In some embodiments, the method is performed in a bacterial species heterologous to the bacterial species or genus from which the enzymes GacC and / or GacG, or enzymatically active homologs, variants, or fragments thereof are derived. In some embodiments, the method is performed in Gram positive bacteria. The method can be performed in Gram negative bacteria. For example, the method can be performed in Gram negative bacteria (e.g., E. coli or Campylobacter species). ​​​​​​​​​​​It can be carried out. Other suitable Gram-negative bacteria are known to those skilled in the art. In embodiments the bacterial species may be heterologous to the bacterial species or genus from which hexose-β-1,4-rhamnosyltransferase, hexose- α-1,2-rhamnosyltransferase or hexose-α-1,3-rhamnosyl transferase is derived.

[0059] In some embodiments, the method is carried out in E. coli.

[0060] Step ii) of the method may involve using a bacterial enzyme of the Gac cluster, or one or more additional enzymes derived from enzymatically active homologs, variants or fragments thereof.

[0061] As will be appreciated by those skilled in the art, GacB is one of a number of enzymes encoded by a gene cluster of S. pyogenes. The Gac gene cluster (gacA-gac L, MGAS5005_Spy_0602-0613), as defined by van Sorge et al, 2014, is understood to encode 12 different enzymes. The 12 enzymes are GacA, GacB, GacC, Ga cD, GacE, GacF, GacG, GacH, GacI, GacJ, GacK and GacL. Thus, step ii) of the method may further involve using one or more additional enzymes from the Gac cluster of bacterial enzymes, or one or more enzymatically active homologs, variants or fragments thereof. Thus, in some embodiments step ii) of the method is GacA, GacC, GacD, GacE, GacF, Ga cG, GacH, GacI, GacJ, GacK or GacL. cG, GacH, GacI, GacJ, GacK, GacL, or one or more additional enzymes selected from enzymatically active homologs, variants, or fragments thereof including using one or more additional enzymes selected from enzymatically active homologs, variants, or fragments thereof including using one or more additional enzymes selected from enzymatically active homologs, variants, or fragments thereof

[0062] In some embodiments, step ii) of the method further includes using one or more enzymatically active homologs, or enzymatically active variants or fragments thereof, of GacA, GacC, GacD, Ga cE, GacF, GacG, GacH, GacI, GacJ, GacK, GacL In some embodiments, step ii) of the method further includes using one or more enzymatically active homologs, or enzymatically active variants or fragments thereof, of GacA, GacC, GacD, Ga cE, GacF, GacG, GacH, GacI, GacJ, GacK, GacL

[0063] One or more enzymatically active homologs may be from S. mutans and / or S. ube ris

[0064] In some embodiments, one or more enzymatically active homologs are from S. mutans In some embodiments, step ii) may further include using the enzyme GacA or an enzymatically active homolog, fragment, or variant thereof. In some embodiments, step i

[0065] ii) may further include using the enzyme GacA or an enzymatically active homolog, fragment, or variant thereof. In some embodiments, step i ii) may further include using the enzyme GacA or an enzymatically active homolog, fragment, or variant thereof. In some embodiments, step i i) may include using the enzymes GacC and GacG, or one or more enzymatically active homologs variants, or fragments thereof

[0066] In some embodiments, step ii) includes using the enzymes GacC, GacA, and GacG, and one or more enzymatically active homologs, variants, or fragments thereof. Step ii) further includes using the enzymes GacD, GacE, and GacF, or one or more enzymatically active homologs, fragments, or variants thereof including using one or more enzymatically active homologs, fragments, or variants thereof It may also be.

[0067] Step ii) may involve using the enzymes GacC, GacA, GacG, GacD, GacE, and Ga cF, or one or more enzymatically active homologs, fragments, or variants thereof. It may be included.

[0068] In some embodiments, step ii) involves the enzymes GacA, GacC, GacD, GacE , GacF, GacG, GacH, GacI, GacJ, GacK and GacL, and one or more enzymatically active homologs, variants or fragments thereof are used. Including.

[0069] Step ii) may involve using enzymatically active homologs of GacA, GacC, GacD, GacE, GacF, GacG and GacH from Smutans and / or Suberis. It may be included.

[0070] In some embodiments, step ii) involves using enzymatically active homologs of GacA, GacC, GacD, GacE, G acF, GacG and GacH from Smutans. Including using.

[0071] GacA may include or consist of SEQ ID NO: 16. Without wishing to be bound by theory, GacA is thought to function to synthesize the rhamnose moiety necessary for the production of rhamnose polysaccharide. Although not wishing to be bound by theory, GacA is thought to function to synthesize the rhamnose moiety necessary for the production of rhamnose polysaccharide. GacG is thought to be involved in the production of rhamnose polysaccharide by extending from the reducing end rhamnose moiety.

[0072] GacD and GacE function to form an ATP-dependent ABC transporter.​ It is possible. As understood by those skilled in the art, ATP-dependent ABC transporters move substrates across membranes. Therefore, without wishing to be bound by theory, GacD and GacE may then assist in transporting rhamnose polysaccharides across the bacterial membrane so that they can be presented on the bacterial cell wall.

[0073] GacH may comprise or consist of SEQ ID NO: 17. GacH may also be identified using UniProtKB-J7M7C2 (J7M7C2_STRP1).

[0074] In some embodiments, step (ii) further comprises using the enzymes GacH, GacI, GacJ, GacK and GacL, or one or more enzymatically active homologs, variants or fragments thereof.

[0075] GacI and / or GacJ are thought to be able to enhance the catalytic efficiency of a method of synthesizing rhamnose polysaccharides.

[0076] Enzymatically active homologs of GacA can be selected from homologs of GacA from Streptococcus group B, group C, group G, Smutans, Suberis, or enzymatically active fragments or variants thereof. For example, the Streptococcus group B homolog of GacA is RmID. The Streptococcus group C homolog of GacA is RmID, as is the Streptococcus group G homolog of GacA.

[0077] The Streptococcus group B homolog of GacA, RmID, is UniPr It may have otKB-A0A0E1EP43 (A0A0E1EP43_STRAG). Yes In some embodiments, the GacA, RmID Streptococcus group B ho molog comprises or consists of SEQ ID NO: 18.

[0078] The GacA, RmID Streptococcus group C homolog may have UniPr otKB_K4Q921 (K4Q921_STREQ). In some embodiments the GacA Streptococcus group C homolog, where RmID is arr anged to include or consist of SEQ ID NO: 19.

[0079] The GacA, RmID Streptococcus group G homolog may have UniPr otKB A0A2X3AIL5 (A0A2X3AIL5_STRDY). G The GacA Streptococcus group G homolog may include or consist of SEQ ID NO: 20.

[0080] The S. mutans homolog of GacA can be identified using UniProtKB_O33664 (O3 3664_STRMG). In some embodiments, the S. mutans homolog of GacA may include or consist of SEQ ID NO: 21.

[0081] The S. uberis homolog of GacA can be identified using UniProtKB9DU23 (B9DU 23_STRU0). In some embodiments, Ga the S. uberis homolog of GacA may include or consist of SEQ ID NO: 22.

[0082] ​​Enzymatically active homologs of GacD, GacE, and / or GacF may be selected from Streptococcus group C, group G, S. mutans, S. uberis, or homologs from their enzymatically active fragments or variants. Suitable homologs of GacD include, but are not limited to, Streptococcus group C enzyme GccD, Streptococcus group G enzyme GgcD, and S. mutans enzyme SccD. Suitable homologs of GacE include, but are not limited to, Streptococcus group C enzyme GccE, Streptococcus group G enzyme GgcE, and S. mutans enzyme SccE. Suitable homologs of GacF include, but are not limited to, Streptococcus group C enzyme GccF, Streptococcus group G enzyme GgcF, S. mutans enzyme SccF, and S. uberis enzyme SucF. tococcus group C, group G, S. mutans, S. uberis, or homologs from their enzymatically active fragments or variants. Suitable homologs of GacD include Streptococcus group C enzyme GccD, Streptococcus group G enzyme GgcD, and S. mutan s enzyme SccD, but are not limited to these. Suitable homologs of GacE include Streptococcus group C enzyme GccE, Streptococcus group G enzyme GgcE, and S. mutans enzyme SccE, but are not limited to these. Suitable homologs of GacF include Streptococcus group C enzyme GccF, Streptococcus group G enzyme GgcF, S. mut ans enzyme SccF, and S. uberis enzyme SucF, but are not limited to these.

[0083] In some embodiments, GccD comprises, or consists of, the amino acid sequence of SEQ ID NO: 23. GccE may be identified using UniProtKB - A0A380KIL0 (A0A380KIL0_STREQ). In some embodiments, GccE comprises, or consists of, the amino acid sequence of SEQ ID NO: 24. GccF may be identified using UniProt KB - A0A3S4QIR3 (A0A3S4QIR3_STREQ). Optionally, GccF comprises, or consists of, SEQ ID NO: 25. In some embodiments, GccE comprises, or consists of, the amino acid sequence of SEQ ID NO: 24. GccF may be identified using UniProt KB - A0A3S4QIR3 (A0A3S4QIR3_STREQ). Optionally, GccF comprises, or consists of, SEQ ID NO: 25.

[0084] In some embodiments, GgcD comprises or consists of the amino acid sequence of SEQ ID NO: 26. GgcD may be identified using UniProtKB-C5WFT9 (C5WFT9_STRDG ).

[0085] In some embodiments, GgcE is identified by UniProtKB-M4YXS7 (M4YXS 7_STREQ). Optionally, GgcE comprises or consists of SEQ ID NO: 27. GgcF can be identified by UniProtKB_C5WFU1 (C5WFU1_S TRDG). In some embodiments, GgcF comprises or consists of SEQ ID NO: 28.

[0086] SccD may comprise or consist of SEQ ID NO: 29. Optionally, SccD is identified using UniProtKB_I6L8Z4 (I6L8Z4_STRMU) .

[0087] SccE may comprise or consist of SEQ ID NO: 30. Optionally, SccE is identified using UniProtKB_I6L8X8 (I6L8X8_STRMU) .

[0088] SccF may be identified using UniProtKB-O82877 (O82877_STRMG. Optionally, SccF comprises or consists of SEQ ID NO: 31 .

[0089] SucD may be identified using UniProtKB-B9DU26 (B9DU26_STRU0). In some embodiments, SucD comprises or consists of SEQ ID NO: 32 . ​​​​​​​

[0090] SucE may be identified using UniProtKB - B9DU27 (B9DU27_STRU0). In some embodiments, SucE comprises, or consists of, SEQ ID NO: 33.

[0091] SucF may be identified using UniProtKB - B9DU28 (B9DU28_STRU0). In some embodiments, SucF comprises, or consists of, the amino acid sequence of SEQ ID NO: 34.

[0092] The enzymatically active homolog of GacH may comprise, or consist of, the S. mutans enzyme SccH, or an enzymatically active fragment or variant thereof. The enzyme SccH may be identified using UniProtKB_Q8DUS0 (Q8DUS0_STRMU).

[0093] In some embodiments, SccH comprises, or consists of, SEQ ID NO: 35.

[0094] In some aspects, the hexose - β - 1,4 - rhamnosyltransferase is , not N - acetylglucosamine (GlcNAc) - β - 1,4 - rhamnosyltransferase. In some embodiments, the hexose β - 1,4 - rhamnosyltransferase is not GacB.

[0095] By "hexose - β - 1,4 - rhamnosyltransferase" is meant an enzyme that can transfer a rhamnose moiety to a hexose such that a β - 1,4 - linkage is formed between the hexose and the rhamnose moiety. It is understood that the rhamnose moiety moves ​​​​​​​​ and the hexose is at the reducing end and the rhamnose moiety is at the non-reducing end, i.e., the end from which the rhamnose polysaccharide is extended to generate it will be understood.

[0096] Hexose-β-1,4-rhamnosyltransferase may include allose-β-1,4- rhamnosyltransferase, altrose-β-1,4-rhamnosyltransferase, glucose-β-1,4-rhamnosyltransferase, mannose-β-1, 4-rhamnosyltransferase, xylose-β-1,4-rhamnosyltransferase, idose-β-1,4-rhamnosyltransferase, galactose-β-1 4-rhamnosyltransferase, talose-β-1,4-rhamnosyltransferase, diacetylbacillosamine-β-1,4-rhamnosyltransferase or an enzymatically active fragment or variant thereof or may consist of them. ,4-rhamnosyltransferase, talose-β-1,4-rhamnosyltransferase, diacetylbacillosamine-β-1,4-rhamnosyltransferase or an enzymatically active fragment or variant thereof or may consist of them. It may contain or consist of rhamnosyltransferase, or an enzymatically active fragment or variant thereof.

[0097] In some embodiments, hexose-β-1,4-rhamnosyltransferase contains glucose (Glc)-β-1,4-rhamnosyltransferase or an enzymatically active fragment or variant thereof. As will be understood by those skilled in the art, glucose (G lc)-β-1,4-rhamnosyltransferase transfers the rhamnose moiety to glucose, thereby forming a β-1,4 bond between glucose and the rhamnose moiety and is an enzyme capable of doing so. Hexose-β-1,4-rhamnosyltransferase may contain the Wc hF enzyme, or an enzymatically active fragment or variant thereof. Wch The F enzyme is understood to be derived from S pneumoniae and is a glucose (Glc)-β-1,4-rhamnosyltransferase.

[0098] In some embodiments, the WchF enzyme comprises SEQ ID NO: 36, or an enzymatically active fragment or variant thereof.

[0099] An enzymatically active fragment or variant of WchF can have at least 30% amino acid sequence identity to the WchF enzyme.

[0100] In some embodiments, an enzymatically active fragment or variant of WchF has at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% amino acid identity to the WchF enzyme. For example, homologs of WchF from Smitis, Soralis, Spseudopneumoniae and Sperosis share 87%, 93%, 87% and 81% amino acid identity with WchF, respectively. Thus, in the context of the present invention, these specific homologs are understood to be enzymatically active variants of WchF.

[0101] Hexose-α-1,2-rhamnosyltransferase is allose-α-1,2-rhamnosyltransferase, altrose-α-1,2-rhamnosyltransferase, glucose-α-1,2-rhamnosyltransferase, mannose-α-1,2-rhamnosyltransferase, xylose-α-1,2-rhamnosyltransferase, idose-α-1,2-rhamnosyltransferase, a-galactose α- ​​​​​​​​​​​​​1,2-Rhamnosyltransferase, talose-α-1,2-rhamnosyltransferase erase, diacetylbacillosamine-α-1,2-rhamnosyltransferase, Gl cNAc-α-1,2-rhamnosyltransferase or enzymatically active frag ments or variants thereof may be included or may consist thereof.

[0102] In some embodiments, hexose-α-1,2-rhamnosyltransferase is galactose-α-1,2-rhamnosyltransferase or an enzymatically active frag ment or variant thereof, or consists thereof. Hexose-α-1,2-rham nosyltransferase may include the WbbR enzyme, or an enzymatically active fragment or variant thereof. As will be understood by those skilled in the art, the WbbR enzyme (WP_0010 45977.1-UniProtKB-Q32EG0 (Q32EG0_SHIDS)) is derived from Shigella dysenterica and is galactose-α-1,2-rham nosyltransferase.

[0103] The WbbR enzyme may include or may consist of SEQ ID NO: 37.

[0104] Hexose-α-1,3-rhamnosyltransferase is allose-α-1,3- rhamnosyltransferase, altrose-α-1,3-rhamnosyltransferase, glucose-α-1,3-rhamnosyltransferase, mannose-α-1, 3-rhamnosyltransferase, xylose-α-1,3-rhamnosyltransferase, idose-α-1,3-rhamnosyltransferase, galactose-α-1 -3-rhamnosyltransferase, or talose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof, or consists thereof. , 3 - rhamnosyltransferase, talose - α - 1,3 - rhamnosyltransferase ase, diacetylbacillosamine - α - 1,3 - rhamnosyltransferase, Glc NAc - α - 1,3 - rhamnosyltransferase or an enzymatically active frag ment or variant thereof may be included or may consist thereof.

[0105] In some embodiments, the hexose - α - 1,3 - rhamnosyltransferase is GlcNAc - α - 1,3 - rhamnosyltransferase, diNAcBac - α - 1, 3 - rhamnosyltransferase, Glcα - 1,3 - rhamnosyltransferase , galactose - α - 1,3 - rhamnosyltransferase, or a fragment or variant thereof, or consists thereof. The hexose - α - 1,3 - rhamno syltransferase may include GlcNAc - α - 1,3 - rhamnosyltransferase , galactose - α - 1,3 - rhamnosyltransferase, or an enzymatically active fragment or variant thereof, or may consist thereof.

[0106] GlcNAc - α - 1,3 - rhamnosyltransferase may include the WbbL enzyme, or an enzymatically active fragment or variant thereof. The WbbL enzyme is from Escherichia coli. The WbbL enzyme may include SEQ ID NO: 38, or an enzymatically active fragment or variant thereof, or may consist thereof.

[0107] An enzymatically active fragment or variant of WbbL may have at least 20% or at least 25% amino acid sequence identity to the WchF enzyme. For example, WbbL A homologous enzyme of WbbL with 27% amino acid identity has been identified in Mycobacterium tuberculosis. Thus, in the context of the present invention, this homolog would be understood to be an enzymatically active variant of WbbL. This homologous enzyme to WbbL derived from Mycobacterium tuberculosis may contain or consist of SEQ ID NO: 39. Another suitable homolog of WbbL contains or consists of the enzyme rfbF derived from Shigella flexneri. RfbF may contain or consist of SEQ ID NO: 40. RfbF may be identified using UniProtKB - A0A2Y2Z3I0 (A0A2Y2Z3I0_SHIFL).

[0108] Galactose - α - 1,3 - rhamnosyltransferase may include the WsaD enzyme, or an enzymatically active fragment or variant thereof. The WsaD enzyme is derived from Geobacillus stearothermophilus. In some embodiments, the WsaD enzyme contains or consists of SEQ ID NO: 41. An enzymatically active fragment or variant of WsaD may be derived from other Bacilli strains (such as Brevibacillus species and Paenibacillus species). An enzymatically active fragment or variant of WsaD may have at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% amino acid identity to WsaD.

[0109] ​​​​​​​​​​​​​​​​do.

[0110] The inventors have surprisingly found that hexose-β-1,4-rhamnosyltransferase hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3 -Rhamnosyltransferase chimera, or enzymatically active Flag with GacB The enzyme or mutant thereof, or an enzymatically active mutant, fragment or homologue thereof, is They found that the munose moiety could be transferred to a hexose monosaccharide, disaccharide, or trisaccharide. Thus, in some embodiments, the rhamnose moiety is a hexose monosaccharide, disaccharide, or trisaccharide. GacB / hexose-β-1,4-rhamnosyltransferase transfers the sugar hexose-α-1,2-rhamnosyltransferase, hexose-α-1,3 - rhamnosyltransferase, or an enzymatically active fragment or variant In such an embodiment, a chimera is used. It will be understood that spherase is not GacB.

[0111] The chimera is a hexose-β-1,4-rhamnosyltransferase, a hexose-α -1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase linked to the N-terminal region of ferase, or an enzymatically active fragment or variant thereof In some embodiments, the chimera may comprise at least the C-terminal region of GacB. It contains the C-terminal region of GacB linked to the N-terminal region of chF.

[0112] In some embodiments, the chimera corresponds to hexose-β-1,4-rhamnosyltransferase. N-acetylglucosaminyltransferase, hexose-α-1,2-rhamnosyltransferase, hexose α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant amino acid substituted, the first 50, 100, 150, 160, 170, 18 0, 190 or 200 amino acids, excluding the entire amino acid sequence of GacB. Example chimeric may contain the amino acid sequence of GacB, except that the first 178 amino acids of GacB are replaced with the corresponding WchF amino acids (amino acids 1 to 186 ).

[0113] The hexose monosaccharide, disaccharide or trisaccharide to which the rhamnose moiety is transferred can be any hexose . In embodiments, the hexose monosaccharide is not the rhamnose moiety.

[0114] In embodiments where the rhamnose moiety is transferred to a hexose disaccharide or trisaccharide, the monosaccharides of the disaccharide or trisaccharide may be the same or different from each other. For example, the disaccharide may contain two galactose monosaccharides. Alternatively, the disaccharide may contain GlcNAc and galactose. GlcNAc may be at the reducing end of the disaccharide and galactose may be at the non-reducing end.

[0115] The disaccharide may contain one rhamnose moiety. The trisaccharide may contain one or two rhamnose moieties .

[0116] In some embodiments, the monosaccharide at the reducing end of the hexose monosaccharide, disaccharide or trisaccharide to which the rhamnose moiety is transferred (thus, the hexose monosaccharide or the first monosaccharide of the disaccharide or trisaccharide) is glucose or a glucose derivative.

[0117] In the context of the present invention, the glucose derivative refers to GlcNAc or diNAcBac It is understood that. In some embodiments, the hexose monosaccharide, disaccharide or trisaccharide does not contain GlcNA c.

[0118] It will be understood that the monosaccharide at the non-reducing end of the hexose monosaccharide, disaccharide or trisaccharide determines the specificity of the rhamnosyltransferase . This is because the rhamnosyltransferase transfers the rhamnose moiety to the monosaccharide at the non-reducing end of the hexose monosaccharide, disaccharide or trisaccharide . Thus, when the monosaccharide at the non-reducing end is galactose, the hexose rhamnosyl transferase is galactose rhamnosyltransferase.

[0119] The disaccharide or trisaccharide may contain a rhamnose moiety at its non-reducing end.

[0120] Exemplary disaccharides can contain glucose at the reducing end linked to a rhamnose moiety at the non-reducing end. Other exemplary disaccharides include diNAcBac at the reducing end linked to a rhamnose moiety at the non-reducing end, or galactose at the reducing end linked to a rhamnose moiety at the non-reducing end, but are not limited thereto.

[0121] Exemplary trisaccharides include glucose at the reducing end linked to a hexose linked to a rhamnose moiety at the non-reducing end, diNAcBac at the reducing end linked to a hexose linked to a rhamnose moiety at the non-reducing end, or GlcNAc at the reducing end linked to a hexose linked to a rhamnose moiety at the non-reducing end, but are not limited thereto. Optionally, the hexose of the trisaccharide may be a rhamnose moiety or galactose.

[0122] ​​​​​​​​When referring to "linkage" between hexoses, this is understood to refer to a glycosidic bond. In a di- or trisaccharide, the glycosidic bond between two hexoses in the di- or trisaccharide may be an alpha (α) or beta (β) glycosidic bond. An alpha bond may be an alpha 1,3 or alpha 1,2 bond. A beta bond may be a beta 1,4 bond.

[0123] The characteristics of the hexose monosaccharides, disaccharides, and trisaccharides described herein are also applicable, as appropriate, to the hexose monosaccharides, disaccharides, and trisaccharides of the streptococcal polysaccharides of the present invention.

[0124] In step (i) of the method of the present invention, the rhamnose moiety can be transferred, and / or the hexose monosaccharides, disaccharides, or trisaccharides of the streptococcal polysaccharides of the present invention can be included or composed of them. Further examples of monosaccharides, disaccharides, and trisaccharides are provided in Example 2.

[0125] In an embodiment where step (i) involves transferring the rhamnose moiety to a hexose disaccharide or trisaccharide, the method may further include forming a hexose disaccharide or trisaccharide. The hexose disaccharide or trisaccharide can be formed using a hexosyltransferase, i.e., an enzyme capable of transferring a hexose to another hexose. For a hexose trisaccharide, when each monosaccharide of the trisaccharide is the same (e.g., the trisaccharide is formed from three glucoses), one hexosyltransferase can be used to transfer each hexose to the other to form the trisaccharide. However, in an embodiment where the hexose trisaccharide is formed from at least two different hexoses, two different hexosyltransferases are used to form the hexose trisaccharide. Hexosyltransferase is required.

[0126] If the method further comprises forming a hexose disaccharide, the hexose disaccharide can be formed using hexose-α-1,3-hexosyltransferase or an enzymatically active fragment or variant thereof. Hexose-α-1,3-hexosyltransferase is understood to refer to an enzyme capable of transferring a hexose to another hexose to form an α-1,3 bond. In the context of the present invention, the bond can be used to refer to a linkage in other respects. In some embodiments, the hexose disaccharide is formed using hexose-α-1,3-galactosyltransferase. Hexose-α-1,3-galactosyltransferase may comprise, or consist of, GlcNAc-α-1,3-galactosyltransferase, optionally enzyme WbbP, or an enzymatically active fragment or variant thereof. Enzyme WbbP can be identified using UniProt KB-Q53982 (Q53982_SHIDY). -ose-α-1,3-hexosyltransferase or an enzymatically active fragment or variant thereof. Hexose-α-1,3-hexosyltransferase is understood to refer to an enzyme capable of transferring a hexose to another hexose to form an α-1,3 bond. In the context of the present invention, the bond can be used to refer to a linkage in other respects. In some embodiments, the hexose disaccharide is formed using hexose-α-1,3 -galactosyltransferase. Hexose-α-1,3-gal actosyltransferase may comprise, or consist of, GlcNAc-α-1,3-galactosyltransferase, optionally enzyme WbbP, or an enzymatically active fragment or variant thereof. erase, optionally enzyme WbbP, or an enzymatically active fragment or variant thereof. Enzyme WbbP can be identified using UniPro t KB-Q53982 (Q53982_SHIDY). In some embodiments, WbbP may comprise, or consist of, the amino acid sequence of SEQ ID NO: 42. Therefore, in some embodiments, the disaccharide consists of GlcNAc at its reducing end and galactose at its non-reducing end, and the two hexoses are linked via an α- 1,3 bond.

[0127] In some embodiments, the method forms a hexose disaccharide using enzyme WbbP, or an enzymatically active fragment or variant thereof, followed by enzyme WbbR, or Using an enzymatically active fragment or variant, transfer the rhamnose moiety to a hexose to a disaccharide.

[0128] The hexose disaccharide can be formed using a hexose α-1,3-rhamnosyltransferase or an enzymatically active fragment or variant thereof. For example, the hexose disaccharide can be formed using galactose-α-1,3-rhamnosyltransferase, such as WsaD or an enzymatically active fragment or variant thereof. In such embodiments, it is understood that the hexose disaccharide is formed from galactose at the reducing end and a rhamnose moiety at the non-reducing end. When the hexose disaccharide is formed using galactose-α-1,3-rhamnosyltransferase, the enzyme WsaP can also optionally be used in the formation of the disaccharide, for example, to attach a lipid to galactose. The enzyme WsaP is derived from Geobacillus stearothermophilus. WsaP can be identified using UniprotKB-Q7BG44 (Q7BG44_GEO SE). In some embodiments, the WsaP enzyme comprises or consists of SEQ ID NO: 43. The enzymatically active fragment or variant of WsaP can be derived from other Bacilli strains (e.g., Brevibacillus species and Paenibacillus species). The enzymatically active fragment or variant of WsaP is at least 2 0%, 30%, at least 40%, at least 50%, at least 60%, at least 7 0%, at least 80%, at least 85%, at least 90%, at least 95%, at least

[0129] The enzymatically active fragment or variant of WsaP can be derived from other Bacilli strains (e.g., Brevibacillus species and Paenibacillus species). The enzymatically active fragment or variant of WsaP is at least 2 0%, 30%, at least 40%, at least 50%, at least 60%, at least 7 0%, at least 80%, at least 85%, at least 90%, at least 95%, at least may have at least 97%, at least 98% or at least 99% amino acid identity .

[0130] The hexose disaccharide is extended using hexose-α-1,2-hexosyltransferase or an enzymatically active fragment or variant thereof, and before further extension from the rhamnose moiety at the non-reducing end of the trisaccharide or tetrasaccharide, the heterologous bacterial enzymes GacC and / or GacG or an enzymatically active homolog, variant or fragment thereof is used to form a trisaccharide or tetrasaccharide. Exemplary hexose-α-1,2-hexosyltransferases may include, but are not limited to WsaC and WsaE. WsaC can be identified by UniProtKB_Q7BG54 (Q7BG54_GEOSE). Optionally, WsaC comprises, or consists of, SEQ ID NO: 44. WsaE can be identified by UniProtKB_Q7BG51 (Q7BG51_GEOSE E). Optionally, WsaE may comprise, or consist of, SEQ ID NO: 45 . . . . .

[0131] If the method further comprises forming a hexose trisaccharide, as described for the disaccharide, two monosaccharides are linked together and subsequently, a further hexosyltransferase is used to transfer a further hexose to the non-reducing end of the disaccharide. The additional hexosyltransferase can include a hexose-rhamnosyltransferase such that the rhamnose moiety is transferred to the non-reducing end. Suitable hexose-rhamnosyltransferases can be any of the hexose-rhamnosyltransferases described herein . . . . . It can include ka. A suitable hexose-rhamnosyltransferase is rhamnose -α-1,3-rhamnosyltransferase, for example, the enzyme WbbQ or WsaC, or an enzymatically active variant or fragment thereof. It may be possible to identify WbbQ using UniProtKB-A0A0 90NIC3 (A0A090NIC3_SHIDY). In some embodiments, WbbQ contains or consists of SEQ ID NO: 46.

[0132] In some embodiments, the hexose trisaccharide is formed using a rhamnose-α-1,3 -rhamnosyltransferase other than GacC.

[0133] Further information regarding exemplary hexosyltransferases for use in the present invention is provided in the examples.

[0134] The hexose monosaccharide, disaccharide or trisaccharide to which the rhamnose moiety is transferred can be linked to a lipid. Thus, step (i) can include transferring the rhamnose moiety to a lipid-linked hexose monosaccharide, disaccharide or trisaccharide. The bond between the hexose monosaccharide, disaccharide or trisaccharide can include undecaprenyl-diphosphate.

[0135] This method can further include a step (step (iii)) of conjugating a rhamnose polysaccharide to an acceptor molecule using an O-oligosaccharide transferase that can recognize a hexose monosaccharide at the reducing end of the rhamnose polysaccharide to form a rhamnose sugar conjugate.

[0136] O-oligosaccharyltransferase is known as protein glycosylation​​​​​​​​​ In the process, it is used to catalyze the transfer of carbohydrate moieties to the target protein is an enzyme. Protein glycosylation is the process of covalently attaching a carbohydrate moiety, i.e., a polysaccharide, to a protein substrate is. O - oligosaccharide transferase functions by cleaving the phosphate - monosaccharide bond at the reducing end of the polysaccharide. In order to be able to interact with the substrate the O - oligosaccharide transferase must recognize the first two monosaccharides after the phosphate bond. If the substrate does not comply, it may be called an acceptor. Thus, an acceptor molecule can contain a peptide or a protein. This results in the formation of a glyconjugate containing the rhamnose polysaccharide of the present invention. Such glyconjugates are particularly useful as antigens that can be used in immunogenic compositions or vaccines. Furthermore, when this method is carried out in bacteria, the process of glycosylation leads to the presentation of glycoconjugates on the surface of the bacteria. This enables the isolation of the sugar conjugate from the bacteria for further use, or enables the use of the whole bacteria as an antigen that can be used in an immunogenic composition or vaccine In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)). The O - oligosaccharyltransferase is PgIB, PgIL, PgIS or In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)). The O - oligosaccharyltransferase is PgIB, PgIL, PgIS or In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)). In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)).

[0137] In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)). In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)). In some embodiments, the O - oligosaccharyltransferase can recognize glucose or a glucose derivative. In such embodiments, the hexose monosaccharide at the reducing end of the rhamnose polysaccharide is glucose or a glucose derivative (e.g., N - acetylglucosamine (GlcNAc)).

[0138] The O - oligosaccharyltransferase is PgIB, PgIL, PgIS or may comprise WsaB, or an enzymatically active homolog, fragment or variant thereof .

[0139] The PgIB enzyme can be derived from Campylobacter species, such as Campylobacter jejuni or Campylobacter lari. Without wishing to be bound by theory, the PgIB enzyme is thought to be able to recognize any hexose other than glucose .

[0140] The PgIL enzyme may be derived from Neisseria meningitidis. The PgIL enzyme is thought to be able to recognize any hexose other than glucose .

[0141] The PgIS enzyme may be derived from Acinetobacter species. The PgIS enzyme is thought to be able to recognize glucose.

[0142] The WsaB enzyme is derived from Geobacillus stearothermophilus . Enzymatically active variants of the WsaB enzyme may be derived from other Geobacillus species .

[0143] In some embodiments, the O-oligosaccharyltransferase is derived from a bacterial species that is heterologous to the bacterium in which the method is performed .

[0144] The method may further comprise a further step of purifying the rhamnose sugar conjugate. The purification may comprise high performance liquid chromatography (HPLC), such as recycled HPLC, affinity or size exclusion chromatography . Other suitable methods of purification are known to those skilled in the art.

[0145] It will be understood that the method can be carried out on an industrial scale. As is known to those skilled in the art, the bacteria capable of carrying out the method are grown in a liquid medium. Such a liquid medium containing the bacteria can be used to fill a bioreactor on an industrial scale, for example, with a volume of at least 50, 100 or 1000 liters. This advantageously results in the synthesis of a substantial amount of the polysaccharide product of the present invention. A commonly used liquid medium is Luria Broth, which may also be called Lysogeny Broth. Other liquid media are known to those skilled in the art. When this method is carried out in bacteria, the method can be a fed-batch method. "Fed-batch" is a term well known to those skilled in the art. Nevertheless, for the purpose of clarity, "fed-batch" is understood to refer to a synthesis method in which nutrients are supplied to the bacteria through a liquid medium during the culture. Suitable nutrients are known to those skilled in the art. Some exemplary but non-limiting nutrients may include a rhamnose moiety, hexoses other than the rhamnose moiety, and / or divalent cations including but not limited to magnesium and / or manganese. In some embodiments, the rhamnose moiety contains rhamnose. Rhamnose can be supplied to the liquid medium in the D or L isomeric form, preferably the L isomeric form. Which hexose other than the rhamnose moiety is supplied to the liquid medium depends on the composition of the rhamnose polysaccharide produced by the method. The hexose monosaccharide to which the rhamnose moiety is transferred

[0146] When this method is carried out in bacteria, the method can be a fed-batch method. "Fed-batch" is a term well known to those skilled in the art. Nevertheless, for the purpose of clarity, "fed-batch" is understood to refer to a synthesis method in which nutrients are supplied to the bacteria through a liquid medium during the culture. Although "fed-batch" is a term well known to those skilled in the art, for the purpose of clarity, "fed-batch" is understood to refer to a synthesis method in which nutrients are supplied to the bacteria through a liquid medium during the culture. Although "fed-batch" is a term well known to those skilled in the art, for the purpose of clarity, "fed-batch" is understood to refer to a synthesis method in which nutrients are supplied to the bacteria through a liquid medium during the culture.

[0147] Suitable nutrients are known to those skilled in the art. Some exemplary but non-limiting nutrients may include a rhamnose moiety, hexoses other than the rhamnose moiety, and / or divalent cations including but not limited to magnesium and / or manganese. In some embodiments, the rhamnose moiety contains rhamnose. Rhamnose can be supplied to the liquid medium in the D or L isomeric form, preferably the L isomeric form. Which hexose other than the rhamnose moiety is supplied to the liquid medium depends on the composition of the rhamnose polysaccharide produced by the method. The hexose monosaccharide to which the rhamnose moiety is transferred

[0148] In some embodiments, the rhamnose moiety contains rhamnose. Rhamnose can be supplied to the liquid medium in the D or L isomeric form, preferably the L isomeric form. Rhamnose can be supplied to the liquid medium in the D or L isomeric form, preferably the L isomeric form.

[0149] Which hexose other than the rhamnose moiety is supplied to the liquid medium depends on the composition of the rhamnose polysaccharide produced by the method. The hexose monosaccharide to which the rhamnose moiety is transferred ​​​​If the disaccharide or trisaccharide contains glucose, the skilled artisan can determine the appropriate nutrients to be provided in the liquid medium. Hexose monosaccharides, disaccharides, or trisaccharides are glucose. If lactose is included, one skilled in the art will recognize that the appropriate nutrients provided in the liquid medium are galactose. Therefore, the hexose to be supplied to the liquid medium may be, as required, Allose, altrose, glucose, mannose, xylose, idose, galactose one of the following: sucrose, talose, diacetylbacillosamine, GalNAc, or GlcNAc It may be selected from the above.

[0150] The rhamnose moiety and / or other hexoses may be 0.1, 0.25, 0.5, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10 or 15 g / L in liquid medium In some embodiments, the rhamnose moiety and / or The other hexoses were fed to the liquid medium at final concentrations of about 4 g / L of the liquid medium (each). .

[0151] The rhamnose moiety and / or other hexoses may be added at 0.05, 0.1, 0.15, 0. 2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 The compounds may be provided in the liquid medium (each) at a final concentration in the liquid medium of mg / ml.

[0152] In an embodiment, the rhamnose moiety is provided in the liquid medium as L-rhamnose. Munoce is 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5 , at final concentrations of 0.6, 0.7, 0.8, 0.9 or 1.0 mg / mL in liquid medium. The composition may be delivered to a body medium.

[0153] When magnesium is supplied to the liquid medium, this may be supplied in the form of MgSO4 or MgCl2 and may be supplied to the liquid medium at a final concentration in the medium of 0 to 10 mM to form the product.

[0154] Prior to step i), if the method is carried out in bacteria, the method may further comprise introducing into the bacteria one or more nucleic acids encoding one or more of the enzymes described herein. For example, the method may further comprise introducing into the bacteria a nucleic acid encoding O-oligosaccharyltransferase and / or hexose-β-1,4-rhamnosyltransferase, hexose-α1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof. In some embodiments, the method further comprises introducing into the bacteria a nucleic acid encoding the bacterial enzymes GacC and / or the bacterial enzyme GacG or one or more enzymatically active homologs, variants or fragments thereof. The enzymes can then be expressed from their respective nucleic acids. The nucleic acid encoding one or more enzymes may further comprise an endogenous or constitutive promoter and / or a nucleic acid sequence encoding an artificial ribosome binding site.

[0155] Methods for introducing one or more nucleic acids into bacteria are well known to those skilled in the art. One commonly used method is the method of transformation. As used herein, transformation or transfection (which may otherwise be referred to as transfection or transfection) allows the nucleic acid to be introduced into the cell by allowing it to pass through the plasma membrane of the cell. ​Refers to a process. Free nucleic acid is understood to refer to nucleic acid contained within a virus, virus-like particle or other organism; that is, the nucleic acid is independent of the organism (however, it is understood that the nucleic acid may be derived from or isolated from the nucleic acid sequence of an organism).

[0156] Typical methods of transfection include modifying the plasma membrane such that free nucleic acid can cross the plasma membrane (e.g., electroporation), or complexing the free nucleic acid with a reagent that enables it to cross the plasma membrane.

[0157] The nucleic acid for transfection can be in the form of a plasmid, which is understood to be a circular strand of nucleic acid. Thus, a plasmid can contain one or more nucleic acids encoding one or more enzymes.

[0158] The nucleic acid is typically DNA, but RNA can also or alternatively be contemplated.

[0159] Transfection can include methods based on polyethyleneimine, poly-L-lysine, calcium phosphate, electroporation or liposomes. In embodiments, transfection can include methods based on polyethyleneimine, calcium phosphate or liposome-based

[0160] It will be understood that various liposome-based reagents are commercially available for liposome-based transfection methods. The liposome method can be Lipofectamine-based transfection or FuGENE® HD (Promega C may include, but is not limited to, transfection based in Corporation, Wisconsin, USA) including, but not limited to, these.

[0161] Further information regarding transformation / transfection techniques is incorporated herein by reference into Current Protocols in Molecular Bio logy(2019) and can be found therein.

[0162] Plasmids may further include appropriate regulatory sequences including promoter sequences, terminator fragments, enhancer sequences, marker genes and / or other sequences. For further details, see, for example, Sambrook & Russell, Molecular Cl oning: A Laboratory Manual: 3 edition rd for reference.

[0163] Furthermore, plasmids may be engineered to include regulatory sequences that act as enhancers and promoter regions to direct efficient transcription of the fusion protein sequences carried on the construct. Many parts of the regulatory unit are located upstream of the coding sequence of the heterologous gene and are operably linked thereto. Regulatory sequences can direct constitutive or inducible expression of the heterologous coding sequence. Such regulatory sequences are particularly suitable when it is desired that expression occur in a time-specific manner. Induction can be achieved by supplying an inducer to the liquid medium. The inducer may include, or consist of, arabinose, IPTG or rhamnose. Regulatory sequences that can direct inducible expression upon exposure to arabinose, IPTG or rhamnose are known to those skilled in the art. ​​​

[0164] Arabinose can be supplied to the liquid medium at a final concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 g / L in the liquid medium. Optionally, arabinose is supplied to the liquid medium at a concentration of about 2 g / L. in the concentration of.

[0165] IPTG can be supplied to the liquid medium at a final concentration in the liquid medium of 0.1 - 5 mM. In some embodiments, IPTG is supplied to the liquid medium at a final concentration in the liquid medium of 0.1 - 2 mM, preferably at a concentration of about 1 mM.

[0166] L - rhamnose can be supplied to the liquid medium as an inducer at a final concentration of 0.05, 0.1, 0.15, 0.2, 0 .25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / mL in the liquid medium.

[0167] Also provided is a product obtainable using the method according to the first aspect. The product obtainable by the production method according to the first aspect is particularly pure and homogeneous due to its synthesis method. Therefore, the product of the present invention is ideally suitable for commercial use, for example for use as an antigen or for large - scale production for research purposes. for use in research. is suitable for commercial use for large - scale production.

[0168] According to a third aspect, a synthetic streptococcal polysaccharide is provided, the polysaccharide comprising a non - reducing end containing a linear chain of rhamnose moieties and a reducing end containing a hexose monosaccharide, disaccharide or trisaccharide, the hexose monosaccharide, disaccharide or trisaccharide being as described in connection with the aspects of the method. The polysaccharide has an α - 1,3 bond between the hexose monosaccharide, disaccharide or trisaccharide and the linear chain of rhamnose moieties. and contains an α-1,2 bond, or the polysaccharide contains a β-1,4 bond between a hexose monosaccharide, disaccharide or trisaccharide and the linear chain of the rhamnose moiety, and the hexose monosaccharide, disaccharide or trisaccharide does not contain N-acetylglucosamine. As the inventors have found, the naturally occurring GAC from S. pyogenes contains GlcNAc (N-acetylglucosamine) monosaccharides linked by β-1,4 glycosidic bonds to the linear chain of rhamnose monosaccharides. By changing this natural composition of the reducing end sugar, the inventors produced synthetic polysaccharides that retain the chemical composition and antigenic capacity of the α-1,2-α-1,3 rhamnose disaccharide repeating units of GAC, while enabling the production of polysaccharides on an industrial scale with a high level of purity and a tightly regulated

[0169] size distribution, increasing the product length uniformity. Accordingly, typically, the polysaccharide also contains a polysaccharide or a fragment or variant thereof selected from the group consisting of group A, group B, group C and group G carbohydrates. In some embodiments, the polysaccharide contains an α-1,3 bond between a hexose monosaccharide, disaccharide or trisaccharide and the linear chain of the rhamnose moiety. The hexose monosaccharide disaccharide or trisaccharide may contain N-acetylglucosamine, N,N'-diacetylbacillosamine, glucose or galactose. In some embodiments, the polysaccharide contains an α-1,2 bond between a hexose monosaccharide, disaccharide or trisaccharide and the linear chain of the rhamnose moiety. The hexose may contain galactose.

[0170]

[0171]

[0172]

[0173] ​​​​​​​​​​​In some embodiments, the polysaccharide comprises β-1,4 linkages between hexose monosaccharides, disaccharides or trisaccharides, and the linear and hexose of the rhamnose moiety includes glucose. and the linear and hexose of the rhamnose moiety includes glucose.

[0174] According to a fourth aspect, there is provided a streptococcal rhamnose sugar conjugate comprising a streptococcal polysaccharide according to the third aspect bound to an acceptor. The glycoconjugate has a strong antigenic potential and thus the rhamnose glycoconjugate of the present invention is particularly useful in eliciting an immune response, for example as part of an immunogenic composition or vaccine, or as a vaccine. According to a fourth aspect, there is provided a streptococcal rhamnose sugar conjugate comprising a streptococcal polysaccharide according to the third aspect bound to an acceptor. The glycoconjugate has a strong antigenic potential and thus the rhamnose glycoconjugate of the present invention is particularly useful in eliciting an immune response, for example as part of an immunogenic composition or vaccine, or as a vaccine. and thus the rhamnose glycoconjugate of the present invention is particularly useful in eliciting an immune response, for example as part of an immunogenic composition or vaccine, or as a vaccine. and thus the rhamnose glycoconjugate of the present invention is particularly useful in eliciting an immune response, for example as part of an immunogenic composition or vaccine, or as a vaccine. .

[0175] In an embodiment, the polysaccharide is bound to the acceptor at the reducing end of the polysaccharide. The acceptor may comprise a peptide or protein. The acceptor may comprise a peptide or protein.

[0176] In some embodiments, the streptococcal rhamnose sugar conjugate is expressed on the surface of a bacterial host cell, optionally a Gram-negative bacterium such as Escherichia coli. Thus, the present invention also encompasses a bacterial host cell comprising the streptococcal rhamnose sugar conjugate of the fourth aspect on its cell surface. Advantageously, expression on the cell surface of the bacterial host cell allows for ease of immunization of the glycoconjugate. Even more advantageously, this means that a bacterial host cell comprising the streptococcal rhamnose sugar conjugate on its cell surface can be used as a component of an immunogenic composition or vaccine without the need for isolation of the glycoconjugate from the bacterial host cell. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. Thus, the present invention also encompasses a bacterial host cell comprising the streptococcal rhamnose sugar conjugate of the fourth aspect on its cell surface. Advantageously, expression on the cell surface of the bacterial host cell allows for ease of immunization of the glycoconjugate. Even more advantageously, this means that a bacterial host cell comprising the streptococcal rhamnose sugar conjugate on its cell surface can be used as a component of an immunogenic composition or vaccine without the need for isolation of the glycoconjugate from the bacterial host cell. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. Thus, the present invention also encompasses a bacterial host cell comprising the streptococcal rhamnose sugar conjugate of the fourth aspect on its cell surface. Advantageously, expression on the cell surface of the bacterial host cell allows for ease of immunization of the glycoconjugate. Even more advantageously, this means that a bacterial host cell comprising the streptococcal rhamnose sugar conjugate on its cell surface can be used as a component of an immunogenic composition or vaccine without the need for isolation of the glycoconjugate from the bacterial host cell. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. Advantageously, expression on the cell surface of the bacterial host cell allows for ease of immunization of the glycoconjugate. Even more advantageously, this means that a bacterial host cell comprising the streptococcal rhamnose sugar conjugate on its cell surface can be used as a component of an immunogenic composition or vaccine without the need for isolation of the glycoconjugate from the bacterial host cell. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. This reduces the time and cost required to produce the glycoconjugate for downstream use as an immunogenic composition or vaccine. .

[0177] Therefore, according to a fifth aspect, hexose-β-1,4-rhamnosyltransferase Lase, hexose-α-1,2-rhamnosyltransferase or hexose-α -1,3-rhamnosyltransferase, or enzymatically active fragments or variants thereof and heterologous bacterial enzymes GacC and / or GacG or their enzymatically active homologs, variants or frag ments, a bacterial host cell is provided.

[0178] The bacterial host cell may be heterologous to the species from which hexose-β-1,4-rhamnosyltransferase, hexose-α-1,2-rhamnosyltransferase or hexose-α-1,3-rhamnosyltransferase, or enzymatically active fragments or variants thereof are derived. Optionally, the bacterial host cell is a gram-negative bacterium such as Escherichia coli. The bacterial host cell may contain the enzymes described herein and / or nucleic acid

[0179] According to a sixth aspect, an immunogenic composition or vaccine comprising the rhamnose polysaccharide of the second or third aspect or the streptococcal conjugate of the fourth aspect is provided. The immunogenic composition or vaccine may further comprise a pharmaceutically

[0180] acceptable and / or sterile excipient, carrier and / or diluent. / or adjuvant.

[0181] The composition may further comprise a pharmaceutically acceptable The "pharmaceutically acceptable carrier" referred to herein is any physiological vehicle known to those skilled in the art useful for formulating the pharmaceutical composition. The "diluent" referred to herein is any substance known to those skilled in the art useful in diluents for use in pharmaceutical compositions. The medicament can be mixed with, dissolved in, suspended in, or dispersed in a carrier, diluent or excipient.

[0182] The composition can be in the form of capsules, tablets, liquids, ointments, creams, gels, hydrogels, aerosols, sprays, micelles, transdermal patches, liposomes, or any other suitable form that can be administered to animals suffering from or at risk of developing diseases, conditions or infections associated with streptococcal etiology.

[0183] The compositions and / or vaccines of the present invention can be formulated for oral, topical (including skin and sublingual), intramammary, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and / or mucosal administration. In embodiments, the compositions and vaccines of the present invention can be formulated for parenteral administration, optionally subcutaneous, intradermal, intramuscular and / or intravenous administration.

[0184] Also provided are rhamnose polysaccharides of the second or third aspect, streptococcal glycoconjugates of the fourth aspect, or immunogenic compositions or vaccines of the sixth aspect for use in eliciting an immune response in an animal or for use in treating or preventing diseases, conditions or infections caused by streptococcal etiology.

[0185] The animal can be any mammalian subject, e.g., dog, cat, rat, mouse, human, sheep, It can be a goat, a camel, a horse, a cow, a pig and / or a chicken.

[0186] In an embodiment, the animal is an ovine animal, a caprine animal , an equine animal, a porcine animal, a bovine animal or a human. In an embodiment, the animal is an ovine animal. "Ovi" vine animal" is understood to include sheep.

[0187] One of ordinary skill in the art will understand that the term "caprine" includes goats and that "bovine" includes cattle. Equine is a term understood to include horses. As used herein, the term "porcine" includes pigs when used.

[0188] An immune response that contributes to an animal's ability to resolve an infection / infestation and / or helps to reduce the symptoms associated with an infection / infestation may be referred to as a "defensive response." In the context of the present invention, the immune response elicited by the use of the rhamnose polysaccharide described herein may be referred to as a "defensive" immune response. The term "defensive" immune reaction includes any immune reaction that (i) promotes or results in a reduction in the host pathogen load; (ii) reduces one or more of the effects or symptoms of an infection / infestation; and / or (iii) prevents, reduces or limits the occurrence of further (subsequent / secondary) infections. Accordingly, a defensive immune response can prevent an animal from becoming infected / infested with a particular pathogen and / or from developing a particular disease or condition.

[0189]

[0190] "Immune response" can be regarded as any response that induces an antibody (e.g., IgA, IgM, and / or IgG or any other related isotype) response and / or a cytokine or cell-mediated immune response. The immune response can target the rhamnose polysaccharide of the present invention. For example, the immune response can include antibodies that have an affinity for an epitope of the rhamnose polysaccharide or the entire rhamnose polysaccharide.

[0191] Also provided is a method of treating an animal having a disease, condition, or infection associated with a streptococcal etiology, the method comprising administering to the animal a therapeutically effective amount of the rhamnose polysaccharide of the second or third aspect, the streptococcal glycoconjugate of the fourth aspect, or the immunogenic composition or vaccine of the sixth aspect.

[0192] A therapeutically effective amount is understood to refer to an amount sufficient to eliminate, reduce, or prevent a disease, condition, or infection associated with a streptococcal etiology.

[0193] The rhamnose polysaccharide, glycoconjugate, or immunogenic composition or vaccine can be administered as a single dose or as multiple doses. Multiple doses can be administered in a day (e.g., 2, 3, or 4 doses at, for example, 3, 6, or 8-hour intervals). The agent can be administered periodically (e.g., daily, every other day, or weekly) over a period of several days, weeks, or months, as necessary.

[0194] The optimal dose to be administered can be determined by one of ordinary skill in the art and depends on the specific agent used, the strength of the formulation, the mode of administration, and the progression of the It is understood to vary depending on the severity. Additional factors dependent on the individual patients being treated, including the patient's age, weight, gender, diet, and time of administration, will likely require adjustment of the dosage. Known procedures (e.g., procedures commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.)) can be used to establish specific formulations and accurate therapeutic dosing regimens in accordance with the present invention. Additional factors dependent on the individual patients being treated, including the patient's age, weight, gender, diet, and time of administration, will likely require adjustment of the dosage. Known procedures (e.g., procedures commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.)) can be used to establish specific formulations and accurate therapeutic dosing regimens in accordance with the present invention. Known procedures (e.g., procedures commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.)) can be used to establish specific formulations and accurate therapeutic dosing regimens in accordance with the present invention. Known procedures (e.g., procedures commonly used by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.)) can be used to establish specific formulations and accurate therapeutic dosing regimens in accordance with the present invention.

[0195] A kit of parts, comprising: (i) A nucleic acid sequence encoding hexose-β1,4-rhamnosyltransferase, hexose-α-1, 2-rhamnosyltransferase or hexose-α1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof; and A nucleic acid sequence encoding hexose-β1,4-rhamnosyltransferase, hexose-α-1, 2-rhamnosyltransferase or hexose-α1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof; and (ii) A nucleic acid sequence encoding said heterologous bacterial enzymes GacC and / or GacG or enzymatically active homologs, variants or fragments thereof A nucleic acid sequence encoding said heterologous bacterial enzymes GacC and / or GacG or enzymatically active homologs, variants or fragments thereof A kit is also provided that includes.

[0196] Suitable nucleic acid sequences for the kit of parts are as described herein in connection with the methods of the present invention. Suitable nucleic acid sequences for the kit of parts are as described herein in connection with the methods of the present invention.

[0197] In some embodiments, the kit further comprises one or more nucleic acid sequences encoding the O-oligosaccharyltransferase described herein. In some embodiments, the kit further comprises one or more nucleic acid sequences encoding the O-oligosaccharyltransferase described herein.

[0198] Additional nucleic acid sequences that the kit may include are one or more of the following 12 enzymes: GacA, GacD, Gac E, GacF, GacH, GacI, GacJ, GacK and GacL one or more enzymatically active homologs, variants or fragments thereof and may comprise one or more nucleic acid sequences encoding the same.

[0199] In some embodiments, the kit further comprises a nucleic acid sequence encoding GacA, or one or more enzymatically active homologs, variants or fragments thereof. In some embodiments, the kit comprises a nucleic acid sequence encoding GacG, or one or more enzymatically active homologs, variants or fragments thereof.

[0200] In some embodiments, the kit comprises nucleic acid sequences encoding GacG and GacC, or one or more enzymatically active homologs, variants or fragments thereof.

[0201] In some embodiments, the kit further comprises nucleic acid sequences encoding the enzymes GacA, GacD, GacE, and GacF, or one or more enzymatically active homologs, fragments, or variants thereof.

[0202] The kit may further comprise one or more nucleic acid sequences encoding a reporter gene. The reporter sequence may encode a gene or a peptide / protein, the expression of which can be detected by several means. Suitable reporter sequences may encode a gene and / or a protein, the expression of which can be detected, for example, by optical, immunological or molecular means. Exemplary reporter sequences may encode, for example, fluorescent proteins and / or luminescent proteins. Examples include luciferase from firefly (Luc: including codon-optimized forms), green fluorescent protein (GFP), and red fluorescent protein (dsRed). ​​​​​​​​​​​​Arrays that can be mentioned include. One or both of the nucleic acid sequences described in (i) and (ii) of the kit may contain a reporter sequence.

[0203] The kit can optionally further include bacteria, such as Gram-negative bacteria like Escherichia coli. . The bacteria can be heterologous to the bacterial species from which hexose-β-1,4-rhamnosyltransferase, hexose-α- 1,2-rhamnosyltransferase, hexose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant is derived.

[0204] It will be understood that the plurality of nucleic acid sequences may be provided on one or more plasmids.

[0205] All of the features described herein (including any appended claims, abstract, and drawings) ), unless otherwise indicated, can be combined in any combination with any of the above aspects.

Examples

[0206] (Example 1 - GacB is an α-D-GlcNAc β-1,4-L-rhamnosyltransferase) (Introduction) S. pyogenes depends on different mechanisms to withstand host defenses (1 -5). These mechanisms are supported by the synthesis of a wide range of virulence factors among the group A carbohydrate (GAC), which is a surface polysaccharide that constitutes 40% - 60% of the bacterial cell wall (6 -9). GAC consists of a [→3)α-Rha(1→2)α-Rha(1→] rhamnose polysaccharide ( RhaPS) backbone, with β-d-Glc for every α-1,2-linked rhamnose (9 - 11) It has a cNAc(1→3) side chain modification. Recent structural examinations and compositional analyses of GAC have also suggested the presence of glycerol phosphate (GroP)(12), which has been an unobserved observation for over 50 years (13,14). Furthermore, Edgar et al . demonstrated that approximately 25% of the GAC side chain GlcNAc is modified with GroP, imparting a negative charge to this polymer that affects the biology and defense mechanisms of S. pyogene s (12, 13, 15). This feature, previously identified in other surface glycans (16, 17), provided new insights into the structural composition, biosynthesis, and function of GAC.

[0207] GAC is synthesized by 12 proteins, GacABCDEFGHIJKL, encoded by one gene cluster (i.e., MGAS5005_spy0602-06 13) found in all S. pyogenes strains identified to date (1, 18). Through sequencing of a transposon mutant library, Le Breton et al. discovered that 8 of these genes, gacABCDEF G and gacL, are essential for the survival of S. pyogenes (4, 1 9). This information supports the observations of van Sorge et al. who identified the first three genes (gac ABC) of the cluster as essential through insertional mutagenesis (1).

[0208] GAC is involved in (i) lipid - bound receptor initiation, (ii) [→3]α - Rha(1→2)α - R ha(1→]RhaPS backbone synthesis, (iii) membrane translocation, (iv) translocation in the extracellular environment ​formed in five consecutive steps of post-chain modification and (v) ligation to (9) peptidoglycan is currently hypothesized. The cytoplasmic pool of dTDP-L-rhamnose is supplied by the enzymes encoded by two separate gene clusters rmlABC and gacA / rmlD(16).

[0209] Despite recent discoveries, several pressing questions regarding the biosynthesis of GAC remain unanswered. For example, the products of six of the 12 genes that make up the GAC cluster (gacBCDEFG) have not yet been characterized, and the GAC initiation, RhaPS backbone biosynthesis and translocation processes remain unknown.

[0210] As a means of obtaining more information about the GAC initiation step, a detailed examination of the second enzyme encoded by the GAC gene cluster was conducted. In this paper, we demonstrate that GacB is the first enzyme to retain rhamnosyltransferase activity that catalyzes the transfer of L-rhamnose from dTDP-β-L-rhamnose, in disagreement with its preliminary gene annotation and currently proposed function(8). GacB forms a β-1,4-glycosidic bond with lipid-linked GlcNAc diphosphate via a metal-independent mechanism. More importantly, from our studies on phylogenetically related homologs from other important human pathogenic streptococci, particularly Lancefield group B, group C, and group G streptococci, it is revealed that the role of GacB is well conserved within the genus Streptococcus, suggesting that it is a common first step for the production of RhaPS from all Lancefield groups.

[0211] (Experimental procedures) (Bioinformatics analysis) Protein sequence alignment was performed using NCBI BLAST global alignment (h ttps: / / goo.gl / vB9zmD) and Clustal Omega (https : / / goo.gl / 8FbvYP) (49). Molecular weight prediction was performed using the ProtParam tool on the Expa sy server (http: / / www.expasy.org / ). Topology prediction was generated using both SpOctopus (http: / / octo pus.cbr.su.se / ) and the TMHMM algorithm (www.cbs.dt u.dk / services / TMHMM / ).

[0212] Secondary structure prediction was generated using either Phyre2 (https: / / goo.gl / zrGKJ7) or the RaptorX (raptorx.uchicago.edu) homology recognition engine, and these structures were viewed and analyzed using the PyMOL Molecular Graphi cs System (Educational Edition 1.8 Schrodinger, LLC). The Carbohydrate-Active Enzyme Database (http: / / www.cazy.org / )(50) was examined to obtain insights into the classification and characterization of carbohydrate-active enzymes. Phylogenetic relationships were established using Clustal Omega, Clustal X, and the interactive tree of life iTOL (22).

[0213] (Bacterial strains and growth conditions) As host strains for propagation of recombinant plasmids and plasmid integration, E. coli strains DH5α and MC1061 were used interchangeably. For lipopolysaccharide Using E. coli CS2775, a strain lacking Rha modification, as the host strain, RhaP S production was evaluated. E. coli 21548 is an Und-PP -GlcNAc-deficient strain containing a wecA deletion and functions as a negative control for the production of RhaPS . E. coli strain C43(DE3) was used for the production of recombinant proteins. All E. coli strains were grown in LB medium. Unless otherwise indicated, all bacterial cultures were incubated at 37 °C with shaking in an incubator at 200 rpm . If necessary, the medium was supplemented with one or more antibiotics to the following final concentrations: carbenicillin (Amp) 1 00 μg / μL, erythromycin (Erm) 300 μg / μL, or kanamycin ( Kan) 50 μg / mL .

[0214] (Molecular genetics techniques) Table 1 shows the DNA sequences of the forward and reverse oligonucleotide primer pairs used to amplify, delete, or mutagenize the genes of interest. All primers were obtained from Integrated DNA Technologies (IDT) . All PCR reactions were performed using a standard procedure with a SimpliAmp Thermal Cycler from ThermoFisher Scientific . Constructs were cloned using standard molecular biology procedures including restriction enzyme digestion and ligation. All constructs were verified by DNA sequencing .

[0215]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0216] (Determination of RhaPS production) 50 μL of OD of the culture grown at 37 °C 600 The normalized overnight culture was mixed with 50 μL of 6×SDS loading buffer and separated on a 20% Tris-SDS gel (29). The evaluation of RhaPS production was performed via immunoblotting on a PVDF membrane according to conventional immunoblotting techniques Primary antibody: Rabbit anti-Streptococcus pyogenes group A carbohydrate polyclonal antibody (Abcam, ab21034). Secondary antibody: Goat anti-rabbit IgG HRP-conjugated (Biorad, 170-6515). After exposure to Clarity Western ECL (Biorad), the immunoreactive signal was captured using either GENESYS (trademark) 10S UV-Vis spectrophotometer (Thermo Scientific). The immunoreactive signal was captured using either one of them

[0217] (Extraction and radiolabeling of lipid-linked oligosaccharides) The radiolabeled lipid-linked sugars (LLS) of the induced E. coli CS2775 cells with the selected plasmid were extracted using 1:1 CHCl3 / CH3OH and water-saturated butan-1-[[]] ol (1:1 v / v) solution, and the addition of sugar residues in vivo after supplementation with glucose D[6s H](N)(Perkin 3 Elmer) (1 mCi / mL) was determined. The uptake was The rare radioactivity was measured with a Beckman LS6000SE scintillation counter. The organic phase containing LLS was standardized to 0.05 μCi / μL. C:M:AC:A: W mobile phase (180 mL of chloroform + 140 mL of methanol + 9 mL of 1 M ammonium acetate + 9 mL of 13 M aqueous ammonia, 23 mL of distilled water) was used, and the sample was separated by thin-layer chromatography (TLC) on an HPTLC silica gel 60 plate (Merck), then dried and sprayed with En3Hance liquid (Perkin Elmer). The autoradiography image was obtained after 5 - 10 days using Carestream® Kodak® BioMax® XAR Film and MS Intensifying Screens.

[0218] (Purification of Recombinant Expressed Membrane-Associated Protein) Purification was performed according to the protocol established by Waldo et al. (30) with the following modifications. An overnight culture of E. coli C43(DE3) cells expressing the C-terminal GFP fusion protein was diluted 1:100 and incubated for 3 hours until OD = 0.6, then induced with 0.5 mM IPTG and shifted overnight at room temperature with shaking at 200 rpm. 600 The GPF expression was detected by in-gel fluorescence using a Fuji FLA-5000 laser scanner. Cloning, expression, and purification of GacB-WT, GacB-D160N-GFP, and GacB- Y182-GFP: Plasmids containing the GFP-His8-tagged recombinant protein were constructed in the vector pWaldo-E(30 ) as described in Table 1. For protein production and purification, the vector was transformed into E. coli C43(DE ​ 3) Transformed into cells and expressed as described above. Avestin C3 High-P ressure Homogenisator (Biopharma, UK) was used to fractionate the cells, which were then spin-down at 4000×g. The supernatant was centrifuged at 200,000×g for 2 hours to obtain a membrane containing GacB-GFP protein weighing 2 - 3 g. The membrane was solubilized by adding 1% DDM (Anatrace) at 4°C for 2 hours in buffer 1 (500 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2 PO4, 2.7 mM KCl, pH 7.4, 20 mM imidazole, 0.44 mM TCEP ), and then bound to a 1 ml Ni-Sepharose 6 Fast Flow (GE Healthcare) column and pre-washed with buffer 1 + 0.03% DDM. Elution was performed using buffer 1 supplemented with 250 mM imidazole and 0.03 % DDM. Imidazole was removed using a HiPrep 26 / 10 desalting column (GE Healthcare) equilibrated with buffer (PBS , 0.03% DDM, 0.4 mM TCEP). The GFP-His tag was removed by cleavage with Precision Protease at a ratio of 1:100 overnight at 4°C. The cleaved GacB protein was collected after negative IMAC. Protein identity and purity were determined by tryptic peptide mass fingerprinting and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF), respectively, by the (University of Dundee ’Fingerprints’ Proteomics Facility).

[0219] (Synthetic acceptors 1 and 2 of the acceptor) ​Acceptor 2 (P 1 -(11-Phenoxyundecyl)-P 2 -(2-Acetamido -2-deoxy-α-d-glucopyranosyl) diphosphate) was synthesized as the sodium salt from phenoxyundecyl dihydrogen phosphate and 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-α-d -glucopyranosyl dihydrogen phosphate according to the procedure of T.N Druzhini na et al. 2010 (94). Acceptor 1 (P -Tridecyl-P 1 -(2-Acetamido-2-deoxy-α-d-glucopyranosyl 2 ) diphosphate) was synthesized from tridecyl dihydrogen phosphate (obtained in the same manner as phenoxyundecyl dihydrogen phosphate) by the same method as described for Acceptor 2.

[0220] (GacB in vitro enzyme reaction) Purified GacB-WT-GFP, GacB-D160N-GFP, GacB-Y182F -GFP and GacB (tagless) protein (final concentration 0.15 mg / ml) were mixed in 100 μl of TBS buffer supplemented with 1 mM TDP-Rha as the sugar donor and 1 mM acceptor-1 (C -PP-GlcNAc) or 1 mM acceptor-2 (phenol-OOOA-PP-Glc 13 NAc) as the acceptor substrate. Incubated at 30 °C for 3 h to 24 h The assay mixture was adjusted by adding either the exchange of the nucleotide sugar donor to UDP-Rha or UDP-GlcNAc, and either 1 mM MgCl2, 1 mM MnCl2, or 1 mM EDTA to reveal the nature of the metal dependence.

[0221] (Mass spectrometry) ​​​​Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), the acceptor and product of G acB were analyzed in an in vitro assay. 100 5 μl of the reaction sample was purified on a 100 μL Sep-Pak C18 cartridge (W aters, UK) pre-equilibrated with 100% ethanol. The bound sample was washed with 800 μl of H2 0 and 800 μl of 15% ethanol and eluted into two fractions with a) 800 μl of 30% ethanol and b) 800 μl of 60% ethanol. The two eluted fractions were dried in a speed vacuum and resuspended in 20 μl of 50% MeOH, and 1 μl of the sample was mixed with 1 μl of a 2,5-dihydroxybenzoic (DHB) acid matrix (15 mg / mL in 30:70 acetonitrile:0 .1% TFA) and 1 μl was added to the MALDI grid. The sample was analyzed by MALDI on an Autoflex speed mass spectrometer set in the reflectron positive ion mode (Bruker, Germany).

[0222] (NMR analysis) The purified GacB in vitro assay product (0.5 - 2 mg) was dissolved in D2O (550 μL) and measured at 300 K. The spectra were acquired using an automatic matching and tuning on a 4-channel Avance III 800 MHz Bruker NM R spectrometer equipped with a 5 mm TCI CryoProbe ( trademark). The 1D spectra were acquired using relaxation times and acquisition times of 5 s and 1.8 s, respectively. With a spectral width of 11 ppm, 32 - 512 scans were obtained. J connectivities were established in a series of 1D and 2D TOCSY experiments with a mixing time of 20 - 120 ms. Selective 1D T The OCSY spectrum (32) was acquired using 40 ms Gaussian pulses and DIPSI-2 sequence (33) (γB1 / 2π = 10 kHz) for spin locking of 20 - 120 ms. For obtaining 2D TOCSY and ROESY experiments, 2048 and 768 complex points in t2 and t1 respectively, and spectral widths of 11 and 8 ppm in F2 and F1 resulting in t2 and t1 acquisition times of 116 and 60 ms respectively were obtained. Sixteen scans were acquired for each t1 increment using a relaxation delay of 1.5 s. The total acquisition time was 6 - 7 hours per experiment. Forward linear prediction of 4096 points was applied in F1. Zero filling from 0 to 4096 was applied in F2. Cosine squared window function was used for apodization before two - dimensional Fourier transform. The ROESY mixing time was applied with a rectangular pulse formation of γB1 / 2π = 4167 Hz for 250 ms, and the DIPSI - 2 sequence (γB1 / 2π = 10 kHz) was applied for spin locking at 20, 80 and 120 ms. 2D amplitude - mode HMBC experiment: 2048 and 128 complex points in t2 and t1 respectively, spectral widths of 6 and 500 ppm in F2 and F1, and t2 and t1 acquisition times of 0.35 s and 0.6 ms respectively. Two scans were acquired for each 1 increment of 128t using a relaxation delay of 1.2 s. The total acquisition time was 8 minutes. Forward linear prediction up to 512 points was applied in F1 and zero filling up to 4096 was applied in F2. Sine squared window function was used for apodization before Fourier transform in two dimensions. (GacC / Homologous Enzyme Protein Purification) For the production of recombinant proteins, target genes (GacC, GbcC, Cps2F, Sc

[0223] (GacC / Homologous Enzyme Protein Purification) For the production of recombinant proteins, target genes (GacC, GbcC, Cps2F, Sc cC) was synthesized using the IDT gBlock gene fragment synthesis service. GacC and its homologous wild-type sequences were included with an N-terminal device 6x histidine tag for affinity purification and PCR amplified with overhangs designed for cloning into pOPINF pOPINF 1 . Cloning into pOPINF was performed using In-Fusion™ cloning technology (Clontech). Subsequently, the resulting plasmid was transformed into DH5α competent cells for propagation and extraction (miniprep kit; Qiagen) . Positive transformant plasmids were identified by size comparison with non-transformed control pOPINF plasmids using gel electrophoresis and then confirmed by DNA sequencing. For the insertion of point mutants, two overlapping fragments containing the desired point mutants were PCR amplified using the wild-type plasmid as a template. The fragments were designed to contain an overlap of at least 15 bp, cloned into pOPINF, and the sequences were verified as in the case of the wild-type plasmid. The complete list of primers used for both wild-type cloning and mutant cloning can be found in Table A

[0224] Subsequently, the plasmids with verified sequences were transformed into C43 cells for protein expression. For the activity assay, 1 L of E. coli culture typically produced sufficient protein for >50 assays (1 mg L -1 ). The cultures were grown at 37 °C, shaken at 200 rpm to an OD of 0.6 - 1, at which point they were shifted to 18 °C for 1 o hour and then induced with 0.5 mM isopropyl β-d-thiogalactopyranoside (IPTG) Induced. The culture was shaken overnight at 18 o °C. After centrifuging the culture at 3000 × g, , the protein was extracted using an Avestin C3 cell disruptor according to the manufacturer's instructions in buffer A0 (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP) supplemented with protease inhibitor. Then , the lysed culture was subjected to ultracentrifugation at 200,000 × g and the supernatant was collected . Then, wash buffer A (50 mM HEPES pH 7.5, 300 mM Na Cl, 10% glycerol, 2 mM TCEP, 20 mM imidazole) and elution buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2 mM TCEP, 400 mM imidazole) were used according to the manufacturer's instructions to purify the supernatant containing the soluble protein of interest on a nickel affinity (Thermofisher) column . Then, the elution fraction containing the target protein was passed through a desalting column pre-equilibrated with buffer A0 to remove imidazole. The protein sample was concentrated to 0.5 - 1 mg / ml and flash frozen in liquid nitrogen until use.

[0225]

Table 2 - 1

Table 2 - 2

Table 2 - 3

[0226] (HPLC assay) For in vitro enzyme assays, 2.5 mM of the synthetic lipid receptor PH - OOOA - PP - α ​​​-NAG, 12.5 mM TDP-L-rhamnose, 0.5 - 1.5 μM GacB-G FP, and 1.25 - 2.5 μM of the GacC or homolog / mutant of interest were included such that a 50 μL reaction was set up and made up to 50 μL with TBS buffer supplemented with 2 mM MnCl2. The reaction was incubated at 30 o °C and when the desired time point was reached, quenched with 50 μL of acetonitrile and left on ice for 15 minutes. The reaction was spun filtered at 14,000 RPM in a benchtop centrifuge to remove the precipitated protein and then injected onto an Xbridge BEH Amide OBS Prep column (13 0 Å, 5 μM, 10 × 250 mm) connected to an HPLC system equipped with a UV detector set at 270 nm (Ultimate 3000, Thermo). The sample was applied to the column at 4 mL / min over a gradient of increasing concentration of B using running buffer A (95% acetonitrile, 10 mM ammonium acetate, pH 8) and running buffer B (50 % acetonitrile, 10 mM ammonium acetate, pH 8). Increasingly polar products with additional sugar residues eluted later in the gradient and the triply rhamnosylated GacC product typically eluted at ~14 minutes in a 36 minute run. The product purified from the HPLC was dried at reduced pressure to remove excess acetonitrile and then lyophilized to remove residual water and ammonium acetate. The sample could be stored at -20 °C for structural analysis.

[0227] (NMR analysis of GacC product) For NMR analysis at the University of Dundee, the HPLC purified product (0.5 - 2 mg) was resuspended in 600 μL of D2O and the NMR spectrum was recorded at 293 K. The spectrum ​​​​​​​​​​​ The 1H NMR spectra were acquired on a Bruker AVANCE III HD 500 MHz NMR spectrometer equipped with a 5 mm QCPI cryoprobe. The NMR spectra were recorded as described for the GacB reaction product. Spectra were analyzed using Bruker Topspin (4.0.7). D 500MHz NMR spectrometer. The NMR spectra were recorded as described for the GacB reaction product. The spectra were analyzed using Bruker Topsin (4.0.7). For the GacB reaction product. The spectra were analyzed using Bruker Topsin (4.0.7). The spectra were analyzed using Bruker Topsin (4.0.7).

[0228] (Results) (GacB is required for the biosynthesis of the GAC RhaPS chain) To investigate the function of GacB and identify potential catalytic residues, we used Escherichia coli as a heterologous expression system to study GAC RhaPS backbone biosynthesis. We constructed two vectors harboring the homologous genes from S. pyogenes, namely gacACDEFG (gacA-G; ΔgacB) and gacB (Figure 1A). To study the biosynthesis of the GAC RhaPS backbone, we used Escherichia coli as a heterologous expression system. We constructed two vectors harboring the homologous genes from S. pyogenes, namely gacACDEFG (gacA-G; ΔgacB) and gacB (Figure 1A). To study the biosynthesis of the GAC RhaPS backbone, we used Escherichia coli as a heterologous expression system. We constructed two vectors harboring the homologous genes from S. pyogenes, namely gacACDEFG (gacA-G; ΔgacB) and gacB (Figure 1A). To study the biosynthesis of the GAC RhaPS backbone, we used Escherichia coli as a heterologous expression system. We constructed two vectors harboring the homologous genes from S. pyogenes, namely gacACDEFG (gacA-G; ΔgacB) and gacB (Figure 1A).

[0229] The RhaPS chain is presumably translocated to the outer membrane of E. coli, which naturally contains rhamnose linked to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were performed using an rfaS-deficient strain (20). Disruption of the rfaS gene prevents the attachment of rhamnose to LPS on the bacterial outer membrane, creating a strain that lacks endogenous rhamnose on its surface (20). The role of GacB was investigated using the conventional complementation strategy shown in Figure 1. The RhaPS chain is presumably translocated to the outer membrane of E. coli, which naturally contains rhamnose linked to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were performed using an rfaS-deficient strain (20). Disruption of the rfaS gene prevents the attachment of rhamnose to LPS on the bacterial outer membrane, creating a strain that lacks endogenous rhamnose on its surface (20). The role of GacB was investigated using the conventional complementation strategy shown in Figure 1. The RhaPS chain is presumably translocated to the outer membrane of E. coli, which naturally contains rhamnose linked to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were performed using an rfaS-deficient strain (20). Disruption of the rfaS gene prevents the attachment of rhamnose to LPS on the bacterial outer membrane, creating a strain that lacks endogenous rhamnose on its surface (20). The role of GacB was investigated using the conventional complementation strategy shown in Figure 1. The RhaPS chain is presumably translocated to the outer membrane of E. coli, which naturally contains rhamnose linked to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were performed using an rfaS-deficient strain (20). Disruption of the rfaS gene prevents the attachment of rhamnose to LPS on the bacterial outer membrane, creating a strain that lacks endogenous rhamnose on its surface (20). The role of GacB was investigated using the conventional complementation strategy shown in Figure 1. The RhaPS chain is presumably translocated to the outer membrane of E. coli, which naturally contains rhamnose linked to lipopolysaccharide. Therefore, to avoid non-specific binding of anti-GAC antibodies, all transformations were performed using an rfaS-deficient strain (20). Disruption of the rfaS gene prevents the attachment of rhamnose to LPS on the bacterial outer membrane, creating a strain that lacks endogenous rhamnose on its surface (20). The role of GacB was investigated using the conventional complementation strategy shown in Figure 1. The role of GacB was investigated using the conventional complementation strategy shown in Figure 1.

[0230] We investigated the production of RhaPS by gacA-G from our complementary approach using immunoblotting of whole-cell lysates (Figure 1B). If the expression of GacBCDEFG is sufficient to produce the RhaPS chain, synthetic RhaPS should be detectable using specific anti-GAC antibodies. As a result, the gacA-G gene We investigated the production of RhaPS by gacA-G from our complementary approach using immunoblotting of whole-cell lysates (Figure 1B). If the expression of GacBCDEFG is sufficient to produce the RhaPS chain, synthetic RhaPS should be detectable using specific anti-GAC antibodies. As a result, the gacA-G gene FG expression is sufficient to produce the RhaPS chain, synthetic RhaPS should be detectable using specific anti-GAC antibodies. As a result, the gacA-G gene FG expression is sufficient to produce the RhaPS chain, synthetic RhaPS should be detectable using specific anti-GAC antibodies. As a result, the gacA-G gene E. coli cells lacking the cluster (empty vector) did not produce RhaPS (Figure 1, lane 2). Similarly, transformants with the ΔgacB or ΔsccB plasmid lost reactivity with the GAC antibody (Figure 1, lanes 3 and 5). Instead, the co-transformation of sccB + ΔsccB or gacB + ΔgacB restored RhaPS production and emphasized the essentiality of sccB and gacB for the biosynthesis of the GAC backbone (Figure 1, lanes 4 and 6).

[0231] To examine whether gacB and SccB catalyze the same reaction, the inventors tested the ability of gacB to functionally replace SccB and vice versa by co-transforming ΔsccB + GacB and ΔGacB + scB. In all cases SccB and GacB were interchangeable (Figure 2). The predicted start codon of GacB was different from that of Smutans SccB, which used TTG instead of ATG (Figure 2 ). The authors decided to test two versions of SccB; one using TTG as the start codon and the other using ATG. Both versions gave active enzymes capable of complementing either ΔsccB or Δgac B (Figure 2). Unless otherwise specified, all further studies were performed using the sccB construct with the natural TTG start codon.

[0232] (GacB elongates the lipid-bound precursor) It was investigated whether GacB is a GT that uses GlcNAc-PP-Und as an acceptor or not. Released and separated by thin layer chromatography (TLC) after isolation from the bacterial membrane An in vivo experiment was conducted to produce radiolabeled lipid-linked oligosaccharide (LLO). Laminosyl Based on the annotation as a rhamnosyl transferase, radiolabeled dTDP-β-L-rhamnose is a preferred sugar donor for GacB. However, since this compound is not commercially available tritiated glucose was selected as an alternative. Inside bacterial cells, a wide range of organic components including dTDP-L-rhamnose (25) are synthesized using glucose as a substrate.

[0233] We hypothesized that GacB transfers the activated sugar from the (radiolabeled) nucleotide sugar donor to a membrane-bound acceptor monosaccharide-PP-Und, such as GlcNAc-PP-Und. Therefore, the inventors predicted a change in the size of the membrane-bound acceptor compared to the signal of the monosaccharide lipid-linked acceptor after flowing the sample on a TLC plate. As a negative control, Escherichia coli CS2775 (ΔrfaS) transformed with an empty vector was used. This transformant showed a signal consistent with the production of monosaccharide-PP-Und (Figure 3, lane 1) . Upon expression of either the gacB gene or the sccB gene, the inventors observed an accumulation of radioactive signals that migrated more slowly on the TLC plate, suggesting a higher molecular weight for these compounds (Figure 3, lanes 3 and 4). The same shift was observed for the sccAB-DEFG (Δs ccC) construct (Figure 3, lane 2), demonstrating that sccB and ga cB can glycosylate lipid-linked precursors. Based on the literature, the inventors hypothesize that the upper radiolabeled band corresponds to GlcNAc-PP-Und and the lower band corresponds to Rha -GlcNAc-PP-Und (8, 9).

[0234] (GacB is a rhamnosyltransferase that transfers rhamnose from TDP-β-l-Rha to the GlcNAc-PP-lipid receptor ).) The observed band shift suggested that GacB adds a monosaccharide to the lipid-bound precursor, presumably GlcNAc-PP -Und. We investigated this hypothesis using recombinantly produced and purified GacB WT and amino acid variants (variant D 160 N and Y 182 F). We established in vitro assays using the predicted nucleotide sugar donor, TDP-β-1-rhamnose and synthetic acceptor substrates. We tested two of these synthetic substrates designed to mimic the native lipid-linked acceptor: C H -PP-GlcNAc (acceptor 1) or phenyl-O-C1 1H 13 H 27 -PP-GlcNAc (acceptor 2) (Figure 7C). The reactants were purified and characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in positive ion mode 1H 22 . The MALDI-MS spectra of the enzyme reactions (Figure 4) confirmed that GacB catalyzes the addition of one rhamnose to both acceptor substrates when incubated with TDP-β-1-rha (Figure 4B and E). Acceptor 1 has a molecular weight of 563 Da and is detected at both M / z = 608 [M-1H+2Na]

[0235] and M / z = 630 [M-2H+3 Na] (Figure 4A). GacB-GFP lacking the GPF tag and GacB modified the acceptor to M / z = 776 [M-2H+3Na] + and + ​​​​+ resulted in one major peak (Figure 4B, C). In this spectrum, 3 Na + is not on but, 2 Na + is on and corresponds to M / z = 754 [M - 1 H+2Na] + and additional peaks of lower intensity could also be observed. In both cases the product is shifted by m / z = 146 compared to the unmodified acceptor, which is consistent with the addition of one rhamnose via a glycosidic bond. The same mass shift was observed for the second acceptor; M / z = 672 [M - 1H+2Na] and + and M / z = 694 [M - 2H+3Na] + for unmodified acceptor 2 (Figure 4D ) where peaks were detected, but M / z = 818 [M - 1H+2Na] + and M / z = 840 [M - 2H+3Na] + showed overlapping peaks (Figure 4E and 4F). Also, the performance of GacB that catalyzes the rhamnosylation of Gl cNAc-α-1-P was tested, but the product was not detected (data not shown), suggesting that it not only interacts with GlcNAc-P but also requires a second phosphate and lipid to recognize the acceptor substrate.

[0236] We further investigated the specificity of GacB for its sugar-nucleotide donors. In particular, we tested whether G acB is selective for thymidine-based nucleotides and resistant to uridine-based nucleotides such as UDP-Glc, UDP -GlcNAc and UDP-Rha. As shown previously, in the presence of TDP-β-1-Rha, rhamno ... Two products consistent with the uptake of one sulfate and two or three sodium cations were observed in the spectrum (Figure 5A). In contrast, no product peaks were observed with UDP-α-D-Glc or UDP-α-D-GlcNAc as substrates (Figures 5B and C), but residual activity was detected with UDP-β-1-Rha (Figure 5D). This data demonstrates that GacB is not resistant to nucleotide sugars with an α-D configuration. Furthermore, GacB has specificity for deoxyribose (TDP-rhamnose) and / or requires the binding of a thymine methyl group.

[0237] Finally, the metal ion dependence was evaluated in vitro. Compared to the control (Figure 6B), when GacB was supplemented with MgCl2, MnCl2 or EDTA as metal chelating agents (Figures 6C, D, E), no significant difference was observed in the rhamnosylation activity, indicating that GacB does not require divalent metal ions for its activity.

[0238] Together, these data confirm our previous conclusions drawn from the LLS radiolabeling assay (Figure 3). This is the first in vitro evidence demonstrating that GacB is a metal-independent rhamnosyltransferase that catalyzes the initiation step in GAC RhaPS backbone biosynthesis by transferring a single rhamnose to GlcNAc-PP-Und using TDP-β-1-Rha as the exclusive activating nucleotide sugar donor.

[0239] (Study of the catalytic residues of GacB) We were unable to obtain diffraction-quality crystals from the detergent-extracted protein, which is the most Ultimately, it would reveal a detailed insight into the catalytic region. Belonging to the GT-4 family of GTs are two enzymes: the GacB structural model based on BaBshA from Bacillus anthracis (PDB entry 3mbo)(72) and MshA from Corynebacterium glutamicum (PDB ID: 3c4v)(24) was constructed. BaBshA shares 15% identity in 64 out of 424 amino acids with. MshA is a "homologous" GT that shares 16% identical residues in a sequence stretch of 71 out of 446 residues. Based on the poor information provided by the structural model and the multiple sequence alignment described in detail below , the inventors mutated several residues that are highly conserved in over 40 pathogenic streptococcal species .

[0240] Our in vitro E. coli system was the first to enable the study of GacB mutant proteins and enabled the identification of mutants that abolish or reduce the production of the RhaPS backbone . It is impossible to do this with S. pyogenes. This is because deleting the gacB gene results in non-viable cells (1,20). The inventors used the above GT model and the information available from the sequence alignment of multiple streptococci to select residues that may be involved in substrate binding and tend to be conserved between GTs . By in situ mutagenesis, nine recombinant versions of GacB were constructed containing the following amino acid substitutions: D126A, D126N, E222A, E 222Q, D160A, D160N, Y182A, Y182F and K131R. The latter mutation is probably not involved in catalytic activity . ​​​or conserved predicted surface residues that could otherwise inactivate the enzyme. Therefore, it was included as a negative control.

[0241] We found that substitution of D160 with asparagine dramatically reduced the production of RhaPS chains. However, they found that alanine residues did not cause such a significant effect. This suggests that the D160 carboxyl group is hidden by water molecules in the alanine mutant. These results suggest that a cytochrome P450 protein may be required for catalysis that can be selectively substituted. Substitution of Y182 (Y182A) with alanine inhibited RhaPS backbone biosynthesis. Y182F completely inactivated GacB, whereas Y182F significantly inhibited the enzymatic activity of GacB. These results suggest an essential role for the Y182 hydroxyl group in the activation of ribosomal protein.

[0242] We further demonstrated in vitro expression of GacB-GFP using recombinantly expressed and purified GacB-GFP fusions. The mutants D160N and Y182F were examined in the GacB-D160N-GF MALDI-MS analysis of the reaction products from P and GacB-Y182F-GFP showed that both We demonstrated that both mutants lacked enzymatic activity in vitro (Fig. 4G and H). These results suggest that residues D160 and Y182 play roles in substrate binding or catalysis. This supports the hypothesis that

[0243] Finally, we investigated whether the enzyme remains active in the absence of residues predicted to be membrane associated. In an attempt to determine whether this is the case, we created three truncated versions of GacB at the N-terminal As a result, the first 22 (GacB 23-385 ), 75(GacB 76-385 )oh and 118 residues (GacB 119-385) The truncation of was found to lead to its inactivation when evaluated through a complementation assay. The inability to complement their ΔGacB suggests that the N-terminal domain is required for activity and supports the hypothesis that GacB is a membrane-associated rhamnosyltransferase.

[0244] (GacB is a retaining β-1,4-rhamnosyltransferase) Current gene annotation suggests that GacB is a reversing α-1,2 rhamnosyltransferase (1,8). This annotation is not compatible with the acceptor sugar GlcNAc as the carbon at position C2 is already modified with an N-acetyl group. Therefore, GacB can only transfer rhamnose to available hydroxyl groups on C3, C4, or C6. Furthermore, the GAC backbone is composed of repeating units of rhamnose linked via α-1,3-1,2 linkages (9, 12 ), suggesting that GacB is the only rhamnosyltransferase in this pathway that uses a retaining mechanism of action. According to the CAZy database, the GacB sequence is classified as a member of the GT-4 family, which is classified as a retaining GT (27). If this classification is correct for GacB, the stereochemical configuration at the anomeric center of the sugar donor, TDP-β-l-rhamnose, should be retained in the final product. To elucidate whether GacB is an inverting or retaining rhamnosyltransferase, nuclear magnetic resonance (NMR) spectroscopy was performed on purified reaction products 1 and 2.

[0245] Whether GacB is an inverting rhamnosyltransferase or a retaining rhamnosyltransferase To elucidate whether GacB is an inverting or retaining rhamnosyltransferase, nuclear magnetic resonance (NMR) spectroscopy was performed on purified reaction products 1 and 2. 1 The 1H NMR spectra were collected at 800 MHz, and the access The structural integrity of Putters 1 and 2 was established (Figure 7A), and the chemical structures of their products after the enzymatic reaction were determined (Products 1 and 2). The NMR parameters were determined by one-dimensional and two-dimensional (1D and 2D) and two-dimensional total correlation spectroscopy (TOCSY) experiments (Figure 7B) ; their chemical shifts are summarized in Table 2. For both acceptors, the anomeric proton of α-d-Glc NAc appeared as a doublet of doublets with 3J(H1,H2) = 3.4 Hz and 3J(H1, P ) = 7.2 Hz. The proton H2 of α-d-GlcNAc was also split by the 3J(H2,P) = 2.4 Hz coupling with P. A 2D 1H,31P HMQC spectrum (data not shown) revealed the correlation of both of these H-1’ protons with P at -13.5 ppm. Another correlation appeared between 31P at -10.6 ppm and the protons of the adjacent CH2 group of the alkyl chain, confirming the integrity of the acceptor substrate. For acceptor 2, a typical pattern of signals of monosubstituted benzene with an integration intensity of 2:2:1 was observed.

[0246] The addition of rhamnose to both acceptor substrates was accompanied by the appearance of characteristic signals in the anomeric region (4.88 ppm, H1) of the spectrum adjacent to the water signal. The anomeric configuration of this monosaccharide was established in several ways. The measured J(H1, ) = 1.0 Hz 3 coupling constant indicated a β-l configuration for β-l and α-l-Rha (reported 1.1 and 1.8 Hz, respectively). The rotating-frame nuclear Overhauser effect (ROES Y) spectrum (Figure 4B) showed that H1 of rhamnose was spatially close to the other four protons showed this. Among these, there are H2, H3, and H5 protons of rhamnose, and the latter two were confirmed to have a 1,3 axis configuration between H1, H3, and H5 showing a β-l Rha structure Finally, for the 1 H chemical shift of rhamnose and the chemical shifts of α-1 and β-1-rhamnopyranose comparison (Figure 7C) showed good agreement with the chemical shift of β-1-rhamnose (75), thus confirming the configuration of this ring. The fourth rosy cross difference peak of H1 of rhamnose was with H4 of GlcNAc, revealing the presence of a (1→4) linkage between the two monosaccharides This observation was further supported by the comparison of the GlcNAc 1H chemical shifts of the acceptor substrate and the product. Here, the average of the absolute values of the differences between the chemical shifts of the other corresponding protons of GlcNAc was 0.03 ppm, but an increased chemical shift (+0.21 ppm) was observed for H4 upon glycosylation. As expected, the signals of the alkyl and aryl side chains did not substantially change in each acceptor-product pair.

[0247] In conclusion, 1 H-NMR spectroscopy revealed the formation of the β-l-Rha (1→4) d-GlcNAc moiety and the integrity of the formed product.

[0248] (Group A, B, C, G streptococci share the RhaPS initiation stage) In addition to S. mutans SccB, highly sequence-identical GacB homologs are found in other clinically important Streptococcus species such as those from Group B (GBS), Group C (GCS ) and Group G (GGS). ​​​All homologous enzymes are located in the corresponding gene classes encoding the biosynthesis of Lancefield antigens, i.e., group B, group C, and group G carbohydrate chains (15). ter, i.e., in group B, group C, and group G carbohydrate chains (15). The homologous gene products share 67%, 89%, and 89% amino acid identity with GacB, respectively (Table 2, Figure 8). There is varying degrees of evidence depending on the bacterial species, and the chemical structure of RhaPS in these streptococci is generally understood (9). The currently accepted structures for GAC, GBC, G CC, GGC, and SCC are summarized in Figure 8. Notably, none of the studies leading to the understanding of the surface carbohydrate structures included data describing the mechanism of action of the enzymes involved in the priming step of each RhaP S biosynthesis. The currently accepted structures for GAC, GBC, G CC, GGC, and SCC are summarized in Figure 8. Notably, none of the studies leading to the understanding of the surface carbohydrate structures included data describing the mechanism of action of the enzymes involved in the priming step of each RhaP S biosynthesis. are included.

[0249] Based on the high sequence identity to GacB, the authors hypothesized that the carbohydrate biosynthesis of group A, group B, group C, and group G streptococci has a conserved starting step where the first rhamnose residue is transferred onto a lipid-linked acceptor to form Rha-β-1,4-GlcNAc-PP-Und. The inventors tested the ability of the homologs from GBS, GCS, and GGS ( GbsB, GcsB, and GgsB, respectively) to functionally replace GacB in the production of the RhaPS chain (Figure 9). All of the authors' results showed that all homologous proteins were able to restore the RhaP S backbone when co-expressed with their genes in a ΔgacB expression plasmid, suggesting that these enzymes can perform the same enzymatic reaction. S backbone when co-expressed with their genes in a ΔgacB expression plasmid, suggesting that these enzymes can perform the same enzymatic reaction. able.

[0250] We have shown that GacB requires GlcNAc-PP-Und as an acceptor, Enzymes from GBS, GCS, and GGS can use different lipid-linked acceptor substrates such as Glc-PP-Und. Therefore, to determine whether the GacB homolog requires GlcNAc-PP-Und as a lipid receptor, the inventors performed complementation assays using E. coli ΔwecA cells lacking GlcNAc-PP-Und (23). As a positive control, Spneumoniae WchF, a Glc-1,4-β-rhamnosyltransferase that uses only Glc-PP-Und as a substrate, was identified (28). As expected, GacB was unable to restore the RhaPS chain when co-transformed with the ΔGacB vector in the absence of GlcNAc-PP-Und (Figure 9A, lane 2). GacB homologs from GBS, GCS, and GGS were also unable to produce the RhaPS backbone (Figure 9A, lanes 4-6), but were able to replace GacB function in the ΔrfaS strain (Figure 9B). Only WchF, which uses the Glc-PP-Und receptor for the transfer of rhamnose residues, restored RhaPS biosynthesis in the absence of GlcNAc-PP-Und (Figure 9A, lane 3). Combined with the data from the inventors' in vitro enzyme reactions, these results suggest that GacB homologs from GBS, GCS, and GGS are also GlcNAc-1,4-β-rhamnosyltransferases that require GlcNAc-PP-Und as a membrane-bound acceptor. (Most streptococcal pathogens are predicted to have GlcNAc-1,4-β-rhamnosyltransferase.)

[0251] (Most streptococcal pathogens are predicted to have GlcNAc-1,4-β-rhamnosyltransferase.) ​​​​​​​​​​​​​​S. pneumoniae wchF uses Glc-PP-Und as a receptor for Gl c-β-1,4-rhamnosyltransferase (28). This is 67-89% compared to homologous enzymes from GBS , GCS, GGS and Smutans, and shares 51% amino acid identity with GacB. For a better understanding of the conservation of GacB in the genus Streptococcus, we extended our bioinformatics analysis and searched for other strains carrying Gac B homologous genes. We found 48 human / veterinary pathogenic Streptococcus species with a single GacB homolog, sharing 50-94% sequence identity (Table 2, Figure 10). Five of the 48 species we identified had an identity below 51% (S. mitis, S. pneumoniae, S. oralis subsp. tigurinus, S. peroris and S. pseud opneumoniae), while all other proteins showed more than 65% homology with GacB. For simplicity, the five Streptoco ccus strains with low amino acid identity were called the "low identity" subgroup, and the remaining species were called the "high identity" subgroup. From sequence analysis paired with a complementation assay, the hypothesis was derived that all GacB homologs included in the "high identity" subgroup have GlcNAc-β-1,4rhamnosyltransfe rase activity. In contrast, the "low identity" subgroup includes Spneum oniae WchF, a known Glc-1,4-β-rhamnosyltransferase (28). All five members of the "low identity group" are WchF

[0252] From sequence analysis paired with a complementation assay, the hypothesis was derived that all GacB homologs included in the "high identity" subgroup have GlcNAc-β-1,4rhamnosyltransfe rase activity. In contrast, the "low identity" subgroup includes Spneum oniae WchF, a known Glc-1,4-β-rhamnosyltransferase (28). All five members of the "low identity group" are WchF ​​​​​When compared, it shows very high sequence identity (above 90%).

[0253] GacO from S. pyogenes, a WecA homolog, is a substrate of GacB and has been shown to be responsible for the biosynthesis of a certain GlcNAc-PP-Und(8,9). Thus assuming that the "low" and "high identity" groups utilize different substrates, and thus, when comparing the sequence identity of G acO homologs, it was investigated whether equivalent discrepancies should be observed. Among 48 pathogenic streptococcal genomes (Table 2, Figure 10), all strains from the "high identity" sub -group share gacO homologs with 63 - 92% sequence identity . Importantly, none of the genomes of the "low identity" subgroup contain gene products with sequence identity equal to or less than 30% of G acO. This group presents gene products with high homology to Spneumoni ae Cps2E that transfers Glc-1-P to P-Und to generate Glc-PP-Und . S. miti s, S. oralis subsp. tigurinus, S. peroris and S. pseudopneumoniae homologs share 98% sequence identity with Cps2E .

[0254] The degree of phylogenetic conservation of GacB in the genus Streptococcus emphasizes the importance of this gene for the survival and pathogenesis of streptococcal pathogens. Overall, these results indicate that streptococcal species with GacB homologs having a high degree of identity (>65%) transfer rhamnose from TDP-β-l-rhamnose to membrane-bound GlcNAc-PP-Und . ​By transferring, the first directed step in the biosynthesis of their surface RhaPS is catalyzed by GlcNAc-β-1,4-rhamnosyltransferase is proposed. In contrast, following the function of S. pneumoniae serotype 2 WchF, it is hypothesized that species within the "low identity" subgroup contain rhamnosyltransferases that act on lipid-linked Glc-PP-Und

[0255]

Table 3

[0256] (The N-terminal region of GacB encodes specificity for GlcNAc acceptor) We performed a multiple sequence alignment of GacB homologs from all 48 streptococcal pathogens to identify the most variable and conserved regions in the protein sequence We observed a higher divergence between the "high identity" and "low identity" subgroups in their N-terminal domains (Table 2). More precisely, a low sequence conservation region can be identified between GacB amino acid residues 40 and 80, suggesting that this part of the domain is involved in either GlcNAc acceptor sugar recognition or essential protein-protein interactions

[0257] We previously knew from experiments that GacB could not initiate the biosynthesis of RhaPS in the wecA deletion background (Figure 9A, lane 2). Based on this finding, mutations were introduced into the GacB amino acid sequence to identify residues involved in sugar acceptor recognition The aim was that the GacB mutant could utilize lipid-linked sugars other than GlcNAc-PP-Und ​​​​​​​​​​In the process of the wecA-deficient Escherichia coli strain recognizing the acceptor, this was able to rescue the initiation of RhaPS.

[0258] Therefore, we investigated a structural model based on the GacB homolog from Bacillus anthracis, BaBshA (PDB entry 3mbo), which suggested that residues L128, R131, and GNT100 were potentially involved in sugar receptor recognition. We mutated these residues to mimic those found in WchF. The complementation assay using GacB L128H_R131L did not complement ΔGacB in the ΔwecA background (Figure 11, lane 2). Following a sequential approach, additional amino acid substitutions corresponding to those found in WchF: L128H_R131L_GNT1 00ARC and L128H_R131L_GNT100ARC_A105P were introduced to modify the GacB primary sequence. None of these mutants recognized glucose and initiated the rhamnose chain, and thus did not restore the activity of GacB. Finally, the first 178 residues of GacB were replaced with the corresponding WchF amino acids (1 - 186). When expressed in the wecA deletion background, this WchF-GacB chimera was able to synthesize the RhaPS backbone on the alternative acceptor substrate Glc-PP-Und. (Figure 11, lane 5).

[0259] (Discussion) This study revealed the first step involved in GAC biosynthesis and the first reported metal-independent standalone, retaining, and non-processive α-D-GlcNAc β-1,4-L-rhamnosyltransferase Provides insights into the function of GacB, a lanceolase. This insight is shown in Figure 12 Schematically shown, which shows the elucidated structure of GAC and the endogenous S involved in the synthesis of each part .mutans enzymes. Other enzymes from Gram-negative and Gram-positive bacteria involved in polysaccharide biosynthesis Use lipid-linked GlcNAc as an acceptor and use either dTDP-L- or GDP-D-rhamnose sugar nucleotides, but they are α-1,3 or Or α-1,4 glycosidic bonds result (29-31). Also, the GAC backbone is α-1,3- Composed of repeating units of rhamnose linked via 1,2 bonds (9, 13), Suggests that GacB is the only rhamnosyltransferase in this pathway that uses a retention mechanism of action .

[0260] We also showed that Streptococcus RhaPS can be synthesized in a recombinant expression system, namely Escherichia coli, on a different receptor, Und-PP-Glu, using an enzyme called WchF . Specifically, this is schematically shown in Figure 13. Figure 13 shows how the rhamnose moiety is transferred to a glucose monosaccharide using the enzyme WchF to form a disaccharide having glucose at the reducing end and a rhamnose moiety at the non-reducing end . The enzyme WchF promotes the formation of a β-1,4 Glycosidic bond between two monosaccharides. Then, using the bacterial enzyme GacC or its enzymatically active Homolog GbcC, extend from the rhamnose moiety at the non-reducing end of the disaccharide To produce rhamnose polysaccharide. WchF is derived from S pneumoniae, which is Heterologous to the bacteria from which GacC or GbcC is derived (S. mutans and S. agalactiae) . In this particular embodiment, this method involves WchF, GacC and Gbc C or GbcC is derived from bacteria (S. mutans and S. agalactiae) Is heterologous. In this particular embodiment, this method involves WchF, GacC and Gbc It was carried out in Escherichia coli, which is also a species different from the bacteria from which C is derived.

[0261] As a result, a synthetic streptococcal polysaccharide having a non-reducing end containing a linear chain of the rhamnose moiety and a reducing end containing a glucose monosaccharide was formed, and the polysaccharide contains a β-1,4 bond between glucose and the linear chain of the rhamnose moiety. As will be understood by those skilled in the art, this is because the monosaccharide at the reducing end is glucose rather than Glc NAc, which is different from the naturally occurring GAC (shown in FIG. 12). different.

[0262] (Example 2) To further illustrate the present invention, this example is directed to further exemplary synthetic methods of the present invention as well as rhamnose polysaccharides.

[0263] FIG. 14 is another exemplary embodiment of the present invention. FIG. 14 shows how the enzyme W bbL derived from Escherichia coli can be used to transfer the rhamose moiety to a GlcNAc monosaccharide. This forms a disaccharide having GlcNAc at its reducing end and a rhamnose moiety at its non-reducing end, and having an α-1,3 glycosidic bond between the rhamnose moiety and GlcNAc. Next, the rhamnose polysaccharide is produced by elongation from the rhamnose moiety at the reducing end of the disaccharide using the bacterial enzyme GacC or its enzymatically active homolog GbcC. Since W bbL is derived from Escherichia coli, GacC and GbcC are derived from bacterial species heterologous to the bacterial species from which they are derived. from.

[0264] In this particular example, this method is carried out in Escherichia coli, but other bacteria can be envisioned for this purpose. Therefore, in this particular embodiment, WbbL is specific to Escherichia coli. ​​It can be endogenous or overexpressed in E. coli.

[0265] As shown in Figure 14, this method results in the production of a synthetic streptococcal polysaccharide having a non-reducing end containing the linear chain of the rhamnose moiety and a reducing end containing the Glc NAc monosaccharide, and this polysaccharide contains an α-1,3 bond between G lcNAc and the linear chain of the rhamnose moiety. This is different from the endogenous GAC( shown in Figure 12) because GAC contains a β-1,4 bond between GlcNAc and the linear chain of rhamnose. As schematically shown in Figure 15, other enzymes that are hexose-α-1,3-rhamnosyltransferases can be used instead of WbbL, and Figure 15 is different from Figure 14 in that the monosaccharide is glucose instead of GlcNAc. Therefore, the product of Figure 14 is a synthetic streptococcal polysaccharide having a non-reducing end containing the linear chain of the rhamnose moiety and a reducing end containing a glucose monosaccharide, and the polysaccharide contains an α-1,3 bond between glucose and the linear chain of the rhamnose moiety. This is different from the endogenous GAC (shown in Figure 12) in that it contains glucose and an α-1,3 bond. Other synthetic methods are also within the scope of the present invention. Figure 16 shows such an exemplary method. In this method, diNAcBac-α-1,3 rhamnosyltransferase is used to transfer the rhamnose moiety to the diNAcBac monosaccharide. Therefore, a disaccharide having diNA cBac at its reducing end and a rhamnose moiety at its non-reducing end is formed. The two monosaccharides are linked by an α-1,3 glycosidic bond. Then, the bacterial enzyme GacC or its enzymatically active homolog GbcC is used to extend from the rhamnose moiety at the non-reducing end of the disaccharide and is different from the endogenous GAC (shown in Figure 12) in that it contains glucose and an α-1,3 bond.

[0266] Other synthetic methods are also within the scope of the present invention. Figure 16 shows such an exemplary method. In this method, diNAcBac-α-1,3 rhamnosyltransferase is used to transfer the rhamnose moiety to the diNAcBac monosaccharide. Therefore, a disaccharide having diNA cBac at its reducing end and a rhamnose moiety at its non-reducing end is formed. The two monosaccharides are linked by an α-1,3 glycosidic bond. Then, the bacterial enzyme GacC or its enzymatically active homolog GbcC is used to extend from the rhamnose moiety at the non-reducing end of the disaccharide and is different from the endogenous GAC (shown in Figure 12) in that it contains glucose and an α-1,3 bond. By doing so, rhamnose polysaccharide is produced. diNAcBac α-1,3-rhamnosyl transferase is derived from a bacterial species different from the bacterial species from which GacC or its enzymatically active homolog GbcC is derived.

[0267] The method of Figure 16 results in the production of a synthetic Streptococcus polysaccharide having a non-reducing end containing a linear chain of rhamnose moieties and a reducing end containing diNAcBac monosaccharides, and the polysaccharide contains an α-1,3 bond between diNAcBac and the linear chain of rhamnose moieties. This is different from the endogenous GAC (shown in Figure 12) in that GAC contains a β-1,4 bond between GlcNAc and the linear chain of rhamnose. )

[0268] Figure 17 shows another exemplary method and product. In this method, a disaccharide, trisaccharide or tetrasaccharide can be formed before elongation from the rhamnose moiety. For the disaccharide, the rhamnose moiety is transferred to a galactose monosaccharide using galactose α-1,2-rhamnosyltransferase WbbR. This forms a disaccharide having galactose at the reducing end and a rhamnose moiety at the non-reducing end. Then, the rhamnose polysaccharide is produced by elongating from this rhamnose moiety to form a linear chain of rhamnose moieties. In this example, the elongation uses the enzymes GacC, GacG, or GbcC (see the final schematic in Figure 17 and the schematic above). WbbR is derived from Shigella, a bacterial species different from the Streptococcus species from which GacC, GacG or GbcC are respectively derived. This method results in the production of a synthetic Streptococcus polysaccharide having a non-reducing end containing a linear chain of rhamnose moieties and a reducing end containing galactose monosaccharides, and the polysaccharide contains an α-1,2 bond between diNAcBac and the linear chain of rhamnose moieties. ​​​​​​​​​​​​​​It includes a combination.

[0269] As also shown by the schematic view of the top and vicinity of FIG. 17, another embodiment is the formation of a trisaccharide before elongation from the rhamnose -ose moiety. For the trisaccharide, the enzyme WbbP is used to transfer the galactose monosaccharide to GlcNAc and form an α-1,3 glycosidic bond between the two monosaccharides. Next, the enzyme WbbR is used as described above for the disaccharide so that the rhamnose moiety is transferred to galactose. After this, elongation can be carried out as detailed above for the disaccharides.

[0270] On the left of FIG. 17, there is a spot blot (positive antibody blot). Each blot represents a sample from one experiment, and each column represents a triple under the same conditions. For each experiment, the sample from the reaction was added as a spot, and an anti-GAC antibody was used to determine whether the reaction was successful in forming the rhamnose polysaccharide. The middle column shows a triple of samples obtained from a reaction in which the galactose monosaccharide was transferred to GlcNAc using the enzyme WbbP, followed by the enzyme WbbR and then GacG. The dot plot on the left shows that this reaction can produce the rhamnose polysaccharide of the present invention.

[0271] WbbP can alternatively be used to form a disaccharide (i.e., a galactose monosaccharide at its non-reducing end linked by an α-1,3 glycosidic bond to GlcNAc at its reducing end), and then the rhamnose polysaccharide is produced by elongation from the rhamnose moiety at the non-reducing end of the disaccharide (see the figure below FIG. 17). The column of dot plots on the left of this schematic shows that this reaction can also produce the rhamnose polysaccharide of the present invention. ​​​​​​​​​​​​​​

[0272] Optionally, one or two additional rhamnose moieties may be added to the rhamnose linked to the galactose. and transferring the tetra or pentane to the base portion prior to the elongation step as detailed above. One or two additional rhamnose moieties can be synthesized using the enzyme WbbQ to form saccharides. and subsequently transcribed using GbcC, as shown in the third schematic diagram of FIG. GacC, and the left column of dot plots in this figure shows the The reaction involving P, WbbR, WbbQ and GacC produces the rhamnose polysaccharide according to the invention. Verify that you have been successful in doing so.

[0273] Regarding the tri-, tetra- or pentasaccharide methods, these methods are A synthetic peptide having a linear reducing end and a non-reducing end containing GlcNac and galactose. This results in the production of adult streptococcal polysaccharides, which are composed of a linear chain of rhamnose moieties and galactose. It contains an α-1,2 bond and an α-1,3 bond between galactose and GlcNAc.

[0274] In embodiments in which a rhamnose moiety is transferred to a disaccharide or trisaccharide, any of the hexoses may be Combinations can be used to synthesize disaccharides or trisaccharides using alpha or beta linkages as described herein. This is shown in FIG. 18. Similarly, the rhamnose moiety To extend the rhamnose polysaccharide from If a bond is formed between each pair of rhamnose moieties, GacC, GacG, or Any enzymatically active homologue of a fragment or mutant of It is possible.

[0275] Figure 19 confirms that WbbL can be used in place of GacB or SccB to produce rhamnose polysaccharide. The figure shows an anti-GAC Western blot of total E. coli lysates from cells expressing the empty plasmid control or WbbL-complemented gene clusters RmlD-SccC-SccD-SccE-SccF-SccG(deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG(deltaGacB). Lane 1 is the ladder. Lane 2 confirms that no GAC was produced in E. coli cells with an RgpA deletion, while lane 3 confirms that expression of WbbL alone in RgpA-deficient cells did not restore GAC synthesis. The third column shows a lysate from E. coli cells that have an RgpA deletion but also express the gene cluster GacA-GacC-GacD-GacE-GacF-GacG(ΔGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. Similar results are observed when rgpA-deficient cells express the gene cluster RmlD-SccC-SccD-SccE-SccF-SccG(deltaSccB) together with WbbL (see the last two columns for replicates). This data confirms that WbbL can be used with heterologous enzymes from other species to produce this rhamnose polysaccharide. Figure 20 confirms that GacC can introduce up to five rhamnose sugars onto the product generated from GacB. Figure 20 shows in vivo (E. coli) lipid-linked oligosaccharides complemented with the empty plasmid control or WbbL, gene clusters RmlD-SccC-SccD-SccE-SccF- SccG(deltaSccB) and GacA-GacC-GacD-GacE-Ga cF-GacG(deltaGacB) expressing cells. The first column is the ladder. The second column confirms that no GAC was produced in E. coli cells with an RgpA deletion, while the third column confirms that expression of WbbL alone in RgpA-deficient cells did not restore GAC synthesis. The third column shows a lysate from E. coli cells that have an RgpA deletion but also express the gene cluster GacA-GacC- GacD-GacE-GacF-GacG(ΔGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. The rgpA-deficient cells expressing the gene cluster RmlD-SccC-SccD-SccE- SccF-SccG(deltaSccB) together with WbbL (see the last two columns for replicates) show similar results. This data confirms that WbbL can be used with heterologous enzymes from other species to produce this rhamnose polysaccharide. Figure 20 confirms that GacC can introduce up to five rhamnose sugars onto the product generated from GacB. Figure 20 shows in vivo (E. coli) lipid-linked oligosaccharides complemented with the empty plasmid control or WbbL, gene clusters RmlD-SccC-SccD-SccE-SccF- SccG(deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG(ΔGacB) expressing cells. The first column is the ladder. The second column confirms that no GAC was produced in E. coli cells with an RgpA deletion, while the third column confirms that expression of WbbL alone in RgpA-deficient cells did not restore GAC synthesis. The third column shows a lysate from E. coli cells that have an RgpA deletion but also express the gene cluster GacA-GacC- GacD-GacE-GacF-GacG(ΔGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. The rgpA-deficient cells expressing the gene cluster RmlD-SccC-SccD-SccE- SccF-SccG(deltaSccB) together with WbbL (see the last two columns for replicates) show similar results. This data confirms that WbbL can be used with heterologous enzymes from other species to produce this rhamnose polysaccharide. Figure 20 confirms that GacC can introduce up to five rhamnose sugars onto the product generated from GacB. Figure 20 shows in vivo (E. coli) lipid-linked oligosaccharides complemented with the empty plasmid control or WbbL, gene clusters RmlD-SccC-SccD-SccE- SccF-SccG(deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG(ΔGacB) expressing cells. The first column is the ladder. The second column confirms that no GAC was produced in E. coli cells with an RgpA deletion, while the third column confirms that expression of WbbL alone in RgpA-deficient cells did not restore GAC synthesis. The third column shows a lysate from E. coli cells that have an RgpA deletion but also express the gene cluster GacA-GacC- GacD-GacE-GacF-GacG(ΔGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. The rgpA-deficient cells expressing the gene cluster RmlD-SccC-SccD-SccE- SccF-SccG(deltaSccB) together with WbbL (see the last two columns for replicates) show similar results. This data confirms that WbbL can be used with heterologous enzymes from other species to produce this rhamnose polysaccharide.

[0276] Figure 20 confirms that GacC can introduce up to five rhamnose sugars onto the product generated from GacB. Figure 20 shows in vivo (E. coli) lipid-linked oligosaccharides complemented with the empty plasmid control or WbbL, gene clusters RmlD-SccC-SccD-SccE-SccF-SccG(deltaSccB) and GacA-GacC-GacD-GacE-GacF-GacG(deltaGacB) expressing cells. The first column is the ladder. The second column confirms that no GAC was produced in E. coli cells with an RgpA deletion, while the third column confirms that expression of WbbL alone in RgpA-deficient cells did not restore GAC synthesis. The third column shows a lysate from E. coli cells that have an RgpA deletion but also express the gene cluster GacA-GacC-GacD-GacE-GacF-GacG(ΔGacB). No GAC was found in these cells. However, the fourth column shows that GAC is produced when WbbL is expressed in the cells of the third column. The rgpA-deficient cells expressing the gene cluster RmlD-SccC-SccD-SccE-SccF-SccG(deltaSccB) together with WbbL (see the last two columns for replicates) show similar results. This data confirms that WbbL can be used with heterologous enzymes from other species to produce this rhamnose polysaccharide. shows radiolabeled LOS. TLC plates with radiolabeled LOS from E. coli CS2775 having gacB (lane 1) or gacBC (lane 2) were film-exposed.

[0277] Homologs to GacC may function in a similar manner. Figure 21 shows similar results as shown in Figure 20, except using GbcC, GccC, and GgcC from homologous enzymes from group B, C, and G streptococci. Figure 21 shows film-exposure of TLC plates with radiolabeled LOS from E. coli CS2775 having gacB and gac C (lane 1), gacB alone (lane 2), gacB and gbcC (lane 3), g acB and gcC (lane 4), gacB and ggcC (lane 5). G acC, GbcC, GccC, GgcC are homologous enzymes from group A, B, C, and G streptococci, and the figure shows that all 3 - 5 rhamnose sugars are transferred onto the product of GacB. acC, GbcC, GccC, GgcC are homologous enzymes from group A, B, C, and G streptococci, and the figure shows that all 3 - 5 rhamnose sugars are transferred onto the product of GacB.

[0278] Similarly, the inventors have shown that the GacC enzyme function is conserved among streptococci and can complement the SccC enzyme of E. coli. Shown in Figure 22: A) Gene complementation strategy. The sccC gene was replaced with the homologous genes gacC, gbcC, gcC, g gcC. B) Immunoblot of whole cell lysates for bacterial complementation assay was probed with anti-group A antibody.

[0279] Complementation tests confirmed that the function of the GacC enzyme is conserved among Streptococci from group B, C, G, and S. mutan s.

[0280] Phylogenetic analysis of the GacO, GacB and GacC enzymes shows a high degree of similarity and thus , the function is conserved in Streptococcus - all pathogenic strains are predicted to produce RhaPS with the same adapter / stem, and as such, all are suitable for use according to the present invention.

[0281] Figure 23 shows A) a phylogenetic tree based on the GacB ortholog protein sequences identified from 48 pathogenic streptococci. The asterisk after the species name indicates that the ortholog sequence was not searched from the whole genome that was sequenced. The sequences were aligned using the Clustal Omega default neighbor - joining clustering method and then plotted using the iTOL online tool. B) The bar graph shows the percentage of the degree of homology to Spyogenes GacO (red), GacB (blue) or GacC (green). The figures next to the GacO, GacB and GacC labels represent the steps catalyzed by S pyogenes. The indented figure in the center of the figure is based on our current knowledge regarding the roles of Streptococcus pneumoniae Cps2E, Cps2T (WchF) and Cps 2F (James 2013). GacB (blue) or GacC (green). The figures next to the GacO, GacB and GacC labels represent the steps catalyzed by S pyogenes. The indented figure in the center of the figure is based on our current knowledge regarding the roles of Streptococcus pneumoniae Cps2E, Cps2T (WchF) and Cps 2F (James 2013).

[0282] Figure 24 shows that GacC rhamnosylates the synthetic LLO substrate (GacB product) in vitro. A) From HPLC analysis, it was shown that GacC extends the chemoenzymatic lipid - linked disaccharide using three types of rhamnose residues generated using GacB. Subsequently, the chemical bonds were analyzed by NMR. B) Chemical depiction of the GacB / C reaction with the in vitro acceptor substrate

[0283] ​​​​​​​​Furthermore, in all the data presented by the inventors using NMR and mass spectrometry techniques although not, GacC can also be a 4-rhamnose sugar, and GacC is considered to be an invert alpha-1,3 r hamnosyltransferase. Figure 25 shows the complete assignment of proton and carbon sugar signals. 1 The H assignment was based on the analysis of some F1 band selective 2D TOCSY spectra. 13 The C signals were assigned using 2D 1 H, 13 C HSQC. The linkage was assigned using a 2D NOESY experiment. The chemical shifts for each of the sugar residues are in good agreement with the published data for the 1H signals and 13 C signals for glucopyranose.

[0284] The inventors further showed that the rhamnose polysaccharide according to the present invention can be produced using different enzyme combinations. Figure 26 shows that the rhamnose polysaccharide according to the present invention can be produced using the enzymes from Escherichia coli combined with Streptoc cous mutans and Shigella dysent eriae combined with Shigella dysenteriae. Figure 26 shows a whole cell western blot using an anti-group A carbohydrate antibody. Total E. coli cell lysates were separated by SDS-PAGE. NewR haPS is constructed by combining the gene product of Shigella dysenteriae and the gene product of Smutans / group A streptococcus. RmlD_GacD _E_F_G + WbbP_Q_R is sufficient to build NewRhaPS. NewRhaPS is RmlD_SccC_D_E_F_G and WbbP_Q_R It can also be built by combining them.

[0285] From the above, as schematically shown in Fig. 27, Shigella spp. is expected to be further available to provide adapter / stem and GAC repeat units. In the natural system, before GacG introduces immunogenic repeat units, GacB and Gac C enzymes introduce the adapter / stem region (red frame). The figure shows, as an example, three α1,3-rhamnose sugars installed by GacC. Substitution of GacB / C enzymes to generate NewRhaPS (substitution of GlcNAc-β1,4- rhamnose-α1,3-rhamnose adapter / stem) provides an alternative method to maintain the immunogenic repeat units (proposed to be introduced by GacG enzyme activity). Replacing the adapter region (green box) with O-otase-compatible polysaccharides / oligosaccharides is sufficient to construct immunogenic polysaccharides (α1,2-α1,3 rhamnose). (proposed to be introduced by GacG enzyme activity). Replacing the adapter region (green box) with O-otase-compatible polysaccharides / oligosaccharides is sufficient to construct immunogenic polysaccharides (α1,2-α1,3 rhamnose). .

[0286] As described herein, the rhamnose polysaccharide of the present invention can be conjugated to a suitable protein and presented on the surface of bacteria. Fig. 28 shows that the rhamnose polysaccharide prepared according to the present invention is a suitable substrate for use in the E. coli glycosylation system. Re glinski et al., npj Vaccines (2108)3:53 According to the procedure described in , a periplasmic expression test system was set up. Fig. 28 shows that NewRhaPS is a compatible substrate for O-Otase (PglB) / protein glycosylation technology (PGCT). Test protein NanA (conforming to Reglinski) + / active / inacti ​ Periplasmic expression of the NewRhaPS system (1-8). Lanes 5 and 7 show that two different expression conditions for the NewRhaPS system are Nan A - positive for NewRhaPS glycosylation. Lane 9: GAC chemically extracted from Streptococcus pyogenes (GAC positive control for the antibody).

[0287] This description should not be construed as limiting, and other variations and their actual embodiments will be understood to be within the scope of the present invention.

[0288] (References) JPEG2025108479000011.jpg91166JPEG2025108479000012.jpg230166JPEG2025108479000013.jpg226166JPEG2025108479000014.jpg205166

[0289] (Sequences) Sequence number 1 GacC [Chemistry]

[0290] Sequence number 2 GacG [Chemistry]

[0291] Sequence number 3 RfbG [Chemistry]

[0292] Sequence number 4 GbcC [Chemistry]

[0293] Array number 5 GccC

Chem.

[0294] Array number 6 GgcC

Chem.

[0295] Array number 7 SccC

Chem.

[0296] Array number 8 SucC

Chem.

[0297] Array number 9 GccG

Chem.

[0298] Array number 10 GccG protein 1

Chem.

[0299] Array number 11 GccG protein 2

Chem.

[0300] Array number 12 GgcG protein 1

Chem.

[0301] Array No. 13 GgcG Protein 2

Chem.

[0302] Array No. 14 SucG

Chem.

[0303] Array No. 15 SccG

Chem.

[0304] Array No. 16 GacA

Chem.

[0305] Array No. 17 GacH

Chem.

[0306] Array No. 18 Group B RMID

Chem.

[0307] Array No. 19 Group C RMID

Chem.

[0308] Array No. 20 Group G RMID

Chem.

[0309] Array No. 21 RmID S. mutans [Chemical formula]

[0310] Accession number 22, RmID, S. uberis [Chemical formula]

[0311] Accession number 23, GccD [Chemical formula]

[0312] Accession number 24, GccE [Chemical formula]

[0313] Accession number 25, GccF [Chemical formula]

[0314] Accession number 26, GgcD [Chemical formula]

[0315] Accession number 27, GgcE [Chemical formula]

[0316] Accession number 28, GgcF [Chemical formula]

[0317] Accession number 29, SccD [Chemical formula]

[0318] Array number 30 SccE

Chem.

[0319] Array number 31 SccF

Chem.

[0320] Array number 32 SucD

Chem.

[0321] Array number 33 SucE

Chem.

[0322] Array number 34 SucF

Chem.

[0323] Array number 35 SccH

Chem.

[0324] Array number 36 WchF_pHD0486

Chem.

[0325] Array number 37 WbbR

Chem.

[0326] Accession No. 38 WbbL_pHD0480

Chem.

[0327] Accession No. 39 WbbL

Chem.

[0328] Accession No. 40 RfbF

Chem.

[0329] Accession No. 41 WsaD

Chem.

[0330] Accession No. 42 WbbP

Chem.

[0331] Accession No. 43 WsaP

Chem.

[0332] Accession No. 44 WsaC

Chem.

[0333] Accession No. 45 WsaE

Chem.

[0334] Accession No. 46 WbbQ

Chem.

Claims

1. A method for synthesizing rhamnose polysaccharide, comprising the following steps: (i) using hexose-β-1,4-rhamnosyltransferase, hexose-α-1, 2-rhamnosyltransferase and / or hexose-α-1,3-rhamnosyl transferase, or enzymatically active fragments or variants thereof, to transfer the rhamnose moiety to a hexose monosaccharide, disaccharide or trisaccharide, thereby forming a disaccharide, trisaccharide or tetrasaccharide containing the rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide; (ii) using the heterologous bacterial enzyme Streptococcus pyogenes group A carbohydrate enzyme C (GacC) and / or Streptococcus pyogenes group A carbohydrate enzyme G (GacG) or enzymatically active homologs, variants or fragments thereof to extend from the rhamnose moiety at the non-reducing end of the disaccharide, trisaccharide or tetrasaccharide to produce the rhamnose polysaccharide. A method comprising the above steps.

2. The method according to claim 1, wherein the method is carried out in a bacterial species heterologous to the bacterial species from which the enzyme GacC and / or GacG, or enzymatically active homologs, variants or fragments thereof are derived.

3. The method according to claim 1 or 2, wherein the hexose-β-1,4-rhamnosyltransferase is not GlcNAc-β- 1,4-rhamnosyltransferase.

4. The method according to any one of the preceding claims, wherein the hexose-β-1,4-rhamnosyltransferase is Glc-β-1,4- rhamnosyltransferase or an enzymatically active fragment or variant thereof.

5. The method according to claim 4, wherein the Glc-β-1,4-rhamnosyltransferase comprises the WchF enzyme, or an enzymatically active fragment or variant thereof.

6. The method according to claim 5, wherein the WchF enzyme comprises SEQ ID NO: 36, or an enzymatically active fragment or variant thereof.

7. The method according to any one of the preceding claims, wherein the hexose-α 1,2-rhamnosyltransferase is galactose-α- 1,2-rhamnosyltransferase or an enzymatically active fragment or variant thereof. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​

8. The galactose-α-1,2-rhamnosyltransferase comprises the WbbR enzyme, or an enzymatically active fragment or variant thereof, and the method according to claim 7

9. 。 The method according to claim 8, wherein the WbbR enzyme comprises SEQ ID NO: 37, or an enzymatically active fragment or variant thereof

10. The hexose-α-1,3-rhamnosyltransferase is GlcNAc-α-1,3-rhamnosyltransferase, diNAcBac-α-1,3-rhamnosyltransferase, Glc-α-1,3-rhamnosyltransferase, galactose-α-1,3-rhamnosyltransferase, or an enzymatically active fragment or variant thereof, and the method according to any one of the preceding claims

11. The method according to claim 10, wherein the GlcNAc-α-1,3-rhamnosyltransferase comprises the WbbL enzyme, or an enzymatically active fragment or variant thereof, and the galactose-α-1,3-rhamnosyltransferase comprises the WsaD enzyme, or an enzymatically active fragment or variant thereof

12. The method according to claim 11, wherein the WbbL enzyme comprises SEQ ID NO: 38, or an enzymatically active fragment or variant thereof

13. The method according to claim 11, wherein the WsaD enzyme comprises SEQ ID NO: 41, or an enzymatically active fragment or variant thereof

14. The enzymatically active homolog of GacC and / or GacG is selected from homologs from Streptococcus group B, group C, group G, S. mutans, S. uberis, or an enzymatically active fragment or variant thereof, and the method according to any one of the preceding claims

15. The method according to any one of the preceding claims, wherein the method is carried out in Gram-negative bacteria

16. The method according to any one of the preceding claims, wherein the method is carried out in E. coli

17. Step ii) uses one or more additional enzymes derived from the bacterial enzymes of the Gac cluster, or one or more enzymatically active homologs, variants or fragments thereof ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 。 ​ ​ ​ The method according to any one of the preceding claims, further comprising

18. The method, comprising: (iii) conjugating the rhamnose polysaccharide to an acceptor molecule using an O-oligosaccharyltransferase capable of recognizing the hexose monosaccharide at the reducing end of the rhamnose polysaccharide to form a rhamnose conjugate

19. The method according to claim 18, wherein when dependent on claim 2, 15 or 16, the O-oligosaccharyltransferase is heterologous to the bacterium in which the method is performed

20. The method according to claim 16 or 17, wherein the O-oligosaccharyltransferase comprises PgIB, PgIL, PgIS or WsaB or enzymatically active homologs, fragments or variants thereof

21. The method according to any one of claims 18 to 20, wherein the acceptor molecule comprises a peptide or a protein

22. The method according to any one of claims 18 to 21, further comprising purifying the rhamnose conjugate, optionally by affinity or size exclusion chromatography

23. A product obtainable by using the method according to any one of claims 1 to 22

24. A synthetic streptococcal polysaccharide having a non-reducing end comprising a straight chain of rhamnose moieties and a reducing end comprising a hexose monosaccharide, disaccharide or trisaccharide, wherein the polysaccharide comprises an α-1,3 bond or an α-1,2 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of rhamnose moieties; or the polysaccharide comprises a β-1,4 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of rhamnose moieties, and the hexose monosaccharide, disaccharide or trisaccharide does not contain N-acetylglucosamine, A synthetic streptococcal polysaccharide

25. The synthetic streptococcal rhamnose polysaccharide according to claim 24, wherein the polysaccharide comprises an α-1,3 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of rhamnose moieties, and the hexose comprises N-acetylglucosamine, N,N'-diacetylbacillosamine, glucose or galactose

26. The polysaccharide comprises an α-1,3 bond between the hexose monosaccharide, disaccharide or trisaccharide and the straight chain of rhamnose moieties, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ which contains an α-1,2 bond and wherein said hexose contains galactose, according to claim 24 The synthetic Streptococcal rhamnose polysaccharide described in.

27. The polysaccharide contains a β-1,4 bond between the linear chain of the hexose monosaccharide, disaccharide or trisaccharide and the rhamnose moiety, and the hexose contains glucose, as described in claim 24 The synthetic Streptococcal rhamnose polysaccharide described in.

28. The polysaccharide contains a polysaccharide, a fragment or a variant thereof selected from the group consisting of Group A, Group B, Group C and Group G carbohydrates, according to claim 24 The synthetic Streptococcal rhamnose polysaccharide according to any one of claims 24 to 27.

29. A Streptococcal rhamnose complex carbohydrate containing the rhamnose polysaccharide according to any one of claims 24 to 28 conjugated to an acceptor.

30. The Streptococcal complex carbohydrate according to claim 29, wherein the polysaccharide is conjugated to the acceptor at the reducing end of the polysaccharide.

31. The acceptor contains a peptide or a protein, according to claim 29 or 30 The Streptococcal complex carbohydrate described in.

32. An immunogenic composition or vaccine containing the product according to claim 23, the synthetic Streptococcal rhamnose polysaccharide according to any one of claims 24 to 28 or the Streptococcal complex carbohydrate according to any one of claims 29 to 31.

33. The immunogenic composition or vaccine further contains a pharmaceutically acceptable and / or sterile excipient, carrier and / or diluent, according to claim 32 The immunogenic composition or vaccine described in.

34. The immunogenic composition or vaccine further contains an antigen, a polypeptide and / or an adjuvant, according to claim 32 or 33 The immunogenic composition or vaccine described in.

35. Used to increase the immune response in animals or to treat or prevent diseases, conditions or infections associated with Streptococcal etiology, the product according to claim 23, the synthetic Streptococcal rhamnose polysaccharide according to any one of claims 24 to 28, the Streptococcal complex carbohydrate according to any one of claims 29 to 31, the immunogenic composition or vaccine according to any one of claims 32 to 34.

36. A bacterial host cell, hexose-β-1,4-rhamnosyltransferase, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Hexose-α-1,2-rhamnosyl transferase or hexose-α-1,3 -rhamnosyl transferase, or enzymatically active fragments or variants thereof and heterologous bacterial enzymes GacC and / or GacG or their enzymatically active homologs, variants or fragments, a bacterial host cell.

37. A kit of parts, comprising: (i) a nucleic acid sequence encoding hexose-β-1,4-rhamnosyl transferase, hexose-α-1, 2-rhamnosyl transferase or hexose-α 1,3-rhamnosyl trans ferase, or enzymatically active fragments or variants thereof; and (ii) a nucleic acid sequence encoding heterologous bacterial enzymes GacC and / or GacG or their enzymatically active ho mologs, variants or fragments A kit comprising. ​

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