The present invention relates to a method for producing 2’-fucosyllactose, an enzyme selective ly cleaving the alpha-1, 3-C-O bond of fucosylated carbohydrates and its use for the production of 2’ fucosyllactose, a host cell heterologously expressing the enzyme, a cell free composition comprising at least one enzyme and at least 2’,3-difucoslyllactose (2’,3-DiFL).

2'-fucosyllactose is one major human milk oligosaccharide (HMO) present in human breastmilk. HMOs are endogenous indigestible carbohydrates representing an important compositional dif ference between human breastmilk and infant formula. They are regarded as potent and highly selective modulators of the gut microbiome and accounted for a large number of biological func tions in nutritional metabolism and health issues. Therefore, large efforts are made to manufac ture human identical milk oligosaccharides (HiMOs) in order to improve infant milk formulas and to use HiMOs/HMOs as food ingredients (Phipps et al, Food Chem Tox, 120, 2018, 552-565). HMOs include for example carbohydrates carrying sialyl and/or fucosyl residues.

Production processes for 2’-fucosyllactose (2’-FL) and other HMOs are generally known. For example, 2'-FL and many other HMOs can be produced by biotechnological processes or chem ical synthesis. Biotechnological processes involving fermentation are commonly used for HMO production. Biotechnological production of HMOs often relies on fermentation processes using recombinant Escherichia coli harboring the genes for the respective biosynthetic pathway (Sprenger, G. A. et al; Journal of Biotechnology, 2017, 258, 79). Accordingly, the international application published as WO 2015032412 A1 discloses a fermentative production for HMOs, including 2’-FL and 2’,3-difucosyllactose, using genetically modified E. coli expressing a single recombinant glycosyl transferase, which is an 1 ,2, fucosyltransferase. As examples of further biotechnological processes for HMO production WO 01/04341 and WO 2007/101862 describe how to make core human milk oligosaccharides optionally substituted by fucose or sialic acid, including LNnT, 6'-SL and 3'-0-sialyllactose ("3'-SL") using genetically modified E. coli; and WO 2015/032412 describes making 2'-FL and difucosyllactose or Fuc(a1-2)Gal(pi-4)[Fuc(a1- 3)]Glc (called "DFL" in said patent application) using genetically modified E. coli.

A key step in the biosynthesis of HMOs, in particular of fucosylated lactose species is the trans fer of L-fucose to lactose as depicted in the reaction scheme of Fig. 1. Specifically, fucosyltrans ferase enzymes as e.g. EC 2.4.1.69 move the fucosyl moiety from guanosine diphosphate (GDP) activated-fucose or similar donors to acceptors like lactose.

In the production of 2'-FL this step is particularly crucial as it is observed that alongside 2'-FL also 2’,3-DiFL is produced to a significant extent, depending on the physiological conditions of

OSW 6 FIG/ 20 SEQ the microbes and the process phase. An excess of activated GDP-fucose relative to lactose within the cell may result in the fucosylation of 2'-FL rather than lactose. Besides this imbalance in starting material an insufficient specificity of the fucosyltransferase may also contribute to the formation of 2’,3-DiFL. In any case, 2’,3-DiFL is often regarded as an unwanted by-product and needs to be removed in the downstream processing. Alternatively, even its formation should be prevented. Known options for reducing or preventing 2’,3-DiFL production are modifications of the lactose feed during fermentation, engineering the respective fucosyltransferase enzyme in order to reduce apparent activity or to increase substrate specificity in comparison to currently available fucosyltransferase enzymes. However, the latter options rely on enzyme engineering that requires tedious structure-function analyses and is therefore time-consuming and expen sive.

Alternatively, and as a more immediate remedy, the inventors of the present invention were seeking for an alternative cost- and time-efficient option to solve the problem of 2', 3- difucosyllactose accumulation.

The international application published as WO2015032412 discloses that after production of 2’- FL and 2’,3-DiFL by fermentation, removal of fucosyl residues may be achieved by hydrolysis with strong acids and preferably all fucose residues are removed. A 1,2-a- L- fucosidases hy drolyzing 2’-FL is known for example from Takane Katayama et al. (2004) Journal of Bacteriolo gy, Aug. 2004, p. 4885-4893. The enzyme AfcA from Bifidobacterium bifidum reported within this paper had side activities on other oligosaccharides, but was reported not to act on 3’-FL. According to WO2015032412, complete removal of fucose from 2’-FL and 2’,3-DiFL mixture obtained from the cell-culture medium can also be achieved by sequential enzymatic liberation of the fucose by using 1,2-a- L- fucosidase and 1,3-a- L- fucosidase to remove the fucose from positions 2 and 3, respectively. When a 1,2-a- L- fucosidase were to be used, a cleavage of the 3-O-fucosyl residue would not be expected, and vice versa, according to WO2015032412.

Surprisingly, the inventors of the present invention discovered that the vast majority of fuco sidases tested in contrast to the disclosure of WO2015032412 will release fucose from both positions 2 and 3 or will not show significant activity on fucose in either positions 2 or 3 of 2’,3- DiFL from a fermentation broth, and hence are not suitable for a cost-and time-efficient way to maintain the level of the desired product 2’-FL while reducing the amounts of the undesired side-product 2’,3-DiFL from such mixtures obtained in or by fermentation.

It was therefore an object of the present invention to overcome the disadvantages of the prior art and to provide an improved method of producing HMOs, in particular 2’-FL. Specifically, it is an object of the present invention to provide an efficient way to manufacture 2’-FL on small, medium and/or large scale.

This problem is solved by the invention with the features of the independent patent claims. Ad vantageous developments of the invention, which can be realized individually or in combination, are presented in the dependent claims and/or in the following specification. In particular, this problem is solved by the claimed method for producing 2’-FL, the claimed enzymes, and uses.

As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situa tion in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements.

Further, it shall be noted that the terms “at least one”, “one or more” or similar expressions indi cating that a feature or element may be present once or more than once typically will be used only once when introducing the respective feature or element. In the following, in most cases, when referring to the respective feature or element, the expressions “at least one” or “one or more” will not be repeated, non-withstanding the fact that the respective feature or element may be present once or more than once.

Further, as used in the following, the terms "preferably", "more preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with op tional features, without restricting alternative possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative fea tures. Similarly, features introduced by "in an embodiment of the invention" or similar expres sions are intended to be optional features, without any restriction regarding alternative embodi ments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such a way with other optional or non-optional features of the invention. Accordingly, the present invention relates to a method for producing 2 ^' -fucosyllactose (2’-FL) comprising the steps of: a) contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) with a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL wherein said polypeptide has an amino acid se quence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i); and

(iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); under conditions sufficient for selective cleavage of the alpha-1 , 3-C-O bond of 2’,3-DiFL; and b) obtaining 2’-fucosyllactose generated in step a).

The term "2’,3-difucoslyllactose", abbreviated herein as "2’,3-DiFL", is known to the skilled per son to relate to a lactose molecule comprising an alpha-1 -2 bond between the C2 carbon atom of its galactose moiety and a fucose moiety, and an alpha-1 -3 bond between the C3 carbon of its glucose moiety and a fucose moiety. Correspondingly, "2’-fucoslyllactose" (CAS No. 41263- 94-9), abbreviated herein as "2’-FL", is known to the skilled person to relate to a lactose mole cule comprising an a1-2 bond between the C2 carbon atom of its galactose moiety and a fucose moiety, and “3’-FL” correspondingly to 3’-Fucosyllactose. According to the present invention, 2’,3-DiFL may be obtained by any known process. Preferred are fermentation processes. The use of a fermentation process has the advantage that 2’,3-DiFL may be readily obtained in an aqueous solution and/or fermentation medium. Preferably, the 2’,3-DiFL is provided as an aqueous solution, more preferably comprising additives like at least one buffer, salts, and the like to provide conditions sufficient for selective cleavage of the alpha-1 , 3-C-O bond of 2’,3- DiFL. Also, preferably, the 2’,3-DiFL is provided in a culture medium, e.g. as a spent culture medium from 2’,3-DiFL production, or as a culture medium during 2’,3-DiFL production. A fer mentation process for the production of fucosylated carbohydrates, like mixtures of 2’,3-DiFL and 2’-FL, is disclosed e.g. in WO 2015/032412 A1. Known or conventional fermentation pro cesses for the production of fucosylated carbohydrates usually comprise a step of culturing a genetically modified host cell, typically a bacterial cell, preferably an E. coli cell, in an aqueous culture or fermentation medium containing lactose. Suitable fermentation conditions, fermenta tion media or culture media, and preparation methods for providing a cell free fermentation broth containing 2’,3-DiFL in addition to 2’-FL are known in the art and can be done as dis- closed in the international patent applications published as WO2015032412A1 and WO2010070104A1 as well as in the publication of Baumgaertner and coworkers (Baumgartner et al. (2013), Microb Cell Factories 12:40). Usually, lactose is provided in the culture medium and preferably at least a carbon-based substrate and/or at least an energy source may be pro vided. Suitable carbon-based substrates and energy sources include glucose and glycerol. However, in particular in case lactose-utilizing bacteria, preferably enterobacteria like E. coli or lactic acid bacteria like Lactobacillus spec are used, lactose may be used as carbon and ener gy source.

The term "polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’, 3- DiFL", as used herein, relates to a hydrolase having fucosidase activity, i.e. hydrolyzing a cova lent bond between a fucose moiety and an oligosaccharide, preferably a disaccharide, trisac charide or tetrasaccharide. Preferably, the polypeptide is an alpha-L-fucosidase, EC. 3.2.1.51, more preferably is an 1,3-alpha-L-fucosidase, EC 3.2.1.111. As used herein, the term "selec tive" in the context of "selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL", relates to a cleavage hydrolizing non-alpha-1, 3-C-O bonds of 2’,3-DiFL, in particular the alpha-1, 2-C-O bond of 2’,3-DiFL, at a rate at least 2fold, preferably at least 3fold, more preferably at least 5fold, still more preferably at least 10fold, most preferably at least 100fold lower than the alpha- 1, 3-C-O bond of 2’,3-DiFL. Thus, preferably, the polypeptide being capable of selectively cleav ing the alpha-1, 3-C-O bond of 2’,3-DiFL, preferably, produces from 2’,3-DiFL as a substrate at most 10 mol%, more preferably at most 1 mol%, even more preferably at most 0.1 mol%, most preferably at most 0.01 mol% products different from 2’-FL, based on the sum of all products. “Selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL” preferably refers to hydrolyzing the alpha-1, 3-C-O bond of 2’,3-DiFL, while the alpha-1, 2-C-O bond of 2’,3-DiFL is cleaved to an extent that is statistically significantly lower than the cleavage of the alpha-1, 3-C-O bond. “Sta tistically significantly lower” in this respect, preferably refers to cleaving of the alpha 1, 3-C-O bond of fucosylated lactose to an extent that is a least 2 times higher than cleaving of the 1 ,2-C- O bond, more preferably at least 5 times higher, even more preferably at least 10 times and still even more preferably at least 100 times higher than the cleaving of the alpha 1, 2-C-O bond of 2’,3-DiFL. This means, that preferably the selective cleavage results in a ratio of the amount of 2’-FL to the amount of 3’-FL that is at least 2, more preferably said ratio is at least 3, even more preferably said ratio is at least 5, still even more preferably is at least 10. Thus, preferably, the polypeptides according to the present invention have the advantage that they exhibit a selective activity for cleaving the alpha-1, 3-C-O bond in 2’,3-DiFL under suitable conditions. This can be exploited to reduce the amount of unwanted 2’,3-DiFL, e.g. obtained by conventional fermenta tion processes, and to increase the amount of desired 2’-FL. In accordance, preferably, step b) of the method of obtaining 2’-FL refers to obtaining a mixture of reaction products comprising at least the above described ratio and/or amounts of 2’-FL. Also preferably, the polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL is specific for 2’,3-DiFL; thus, preferably, the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL has an affinity for 2’,3-DiFL at least twofold, preferably at least threefold, more pref erably at least fivefold, still more preferably at least tenfold, most preferably at least 10Ofold, better compared to 2’-FL. As is understood by the skilled person, the affinity of an enzyme for a substrate may be determined and expressed as its K _D value for the substrate.

Preferably, in the method according to the invention the yield of 2’-fucosyllactose is at least 40 % (mol/mol) with respect to the amount of 2’,3-DiFL provided before cleaving. More preferably, the yield of 2’-fucosyl lactose is at least 50 % (mol/mol), still more preferably at least 60 % (mol/mol), even more preferably at least 70 %(mol/mol), even more preferably at least 80 % (mol/mol), most preferably at least 90% (mol/mol) with respect to the amount of 2’,3-DiFL pro vided before cleaving. Also preferably, in particular in case contacting with the polypeptide specified herein is already performed during production, e.g. in the fermentation broth, the molar ratio of 2’-fucosyllactose to 2’,3-DiFL is at least 4, more preferably at least 5, still more prefera bly at least 10, most preferably at least 20. The amounts of 2’,3-DiFL and 2’-FL can be deter mined using high pressure liquid chromatography (HPLC) according to methods known in the art.

Alternatively, the yield of 2’-fucosyllactose can be calculated based on the total amount of car bohydrates in the reaction medium. Preferably, the yield of 2’-FL is at least 30 % with respect to the total amount of carbohydrates in the reaction medium (w/w), more preferably at least 40 %, most preferably at least 50% (w/w).

The polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL has in one embodiment an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under strin gent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the amino acid sequence of (i), wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO: 9, 18 or 19 .

SEQ ID NO: 1 to SEQ ID NO: 8 represent amino acid sequences of putative alpha-L- fucosidases of B. scardovii, strain JCM 12489, DSM 13734 (Toh, H et al; Genome announce ments 2015, Mar-Apr; 3(2)).

SEQ ID NO: 9 represents the amino acid sequence of alpha-1, 3/4-fucosidase of Bifidobacterium longum infantis BiAfcB (Sakumara et al JBC 287 (20) 16709-19, 2012). SEQ ID NO: 10 and SEQ ID NO: 11 each represent an amino acid sequence motif common to group I of the B. scardovii sequences. SEQ ID NO: 13 and SEQ ID NO: 14 each represent an amino acid se quence motif common to group II of the B. scardovii sequences.

SEQ ID NO: 15 to 19 represent artificial fucosidase sequences created based on published fu- cosidase sequences, but skillfully modified in their amino acid sequence length and composition by the inventors and codon optimized on the DNA level for better expression in E. coli.

SEQ ID No. 18 and 19 are artificially short fucosidases to increase the expression level, in crease the solubility of the protein in the host cells, increase activity and reduce protein aggre gation in the host cells.

SEQ ID NO: 20 represents the amino acid sequence of alpha-1, 3/4-fucosidase of Clostridium perfringens ATCC 13124 called CpAfc2 (see Shuquan, F., et al. Journal of Basic Microbiology, 2016. 56(4). Zeuner et al

Table 1: Listing of SEQ ID NOs of putative B. scardovii alpha-L-fucosidase amino acid sequenc- es and B. I. infantis BiAfcB amino acid sequence (SEQ ID NO: 9) and correspondinq Genbank entry codes

It is evident from the alignment in Figure 2 that the amino acid sequences representing putative alpha-L-fucosidases of B. scardovii used herein fall into two groups, group I and group II. Group I consists of SEQ ID NO: 1 to 4, and group II of SEQ ID NO: 5 to 8. Within the groups there is a high degree of sequence identity, i.e. above 90 %. Surprisingly, it was found that within group I SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID N04 share 100 % of sequence identity on amino acid level; and within group II SEQ ID NO: 7 and SEQ ID NO: 8 share 100 % of sequence iden tity on amino acid level. SEQ ID NO: 10 and 11 are amino acid sequence motifs present in group I, while SEQ ID NO: 12 to 14 are amino acid motifs present in group II. Thus, the poly peptide suitable for use in the described method and/or comprised in the enzyme described below preferably comprises at least one amino acid sequence motif selected from the group consisting of the amino acid sequence motifs as shown in SEQ ID NO: 10, or SEQ ID NO: 11 or in any one of SEQ ID NOs: 12 to 14. Alternatively, said polypeptide may comprise an amino acid sequence motif that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence represented by SEQ ID NOs: 10 to 14. In another preferred embodiment the polypeptide suita ble for use in the described method and/or comprised in the enzyme described below comprises the conserved amino acid residues as shown in Figure 2 or 3 marked there by an asterisk in the consensus sequence and at positions corresponding to the positions of these conserved resi dues in any of the sequences of SEQ ID NO: 1 to 8, and more preferably comprise at least one of the motifs shown in SEQ ID NO: 10 to 14, further preferably the motifs as shown in SEQ ID NO 10 and 11 or the motifs of SEQ ID NO: 12 to 14.

In one embodiment the polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL according to the present invention is of bacterial origin. Suitable preferred bacterial species for such polypeptides include those belonging to the following genera: Bifidobacteria, Streptomyces, Lactobacilli, Thermotoga, Sulfolobus, and Xanthomonas. In par ticular, the following bacterial species are included: Bifidobacterium bifidum, B. longum, B. ado lescents, B. animalis, B. breve, B. dentium, B. longum infantis, B. scardovii, Lactobacillus casei, and Sulfolobus solfactaricus. More preferably, the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is derived from a Bifidobacterium species or a Clostridium species or a Bacteroides species, more preferably from Bifidobacterium bifidum, B. longum, B. adolescents, B. animalis, B. breve, B. longum infantis, B. scardovii; Bacteroides ovatus or Clostridium perfringens, more preferably it is derived from Bifidobacterium scardovii, Bacteroides ovatus or Clostridium perfringens. Preferably, the polypeptide suitable in the meth od and/or comprised in the enzyme according to the present invention has an amino acid se- quence selected from group II of the alpha-L-fucosidases of B. scardovii or an amino acid se quence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of group II of B. scardovii alpha-L-fucosidases or an amino acid sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence to the Clostridium perfringens CpAfcp2 fuco- sidase as provided in SEQ ID NO: 20 or an amino acid sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the artificial fucosidases enzymes provided for in SEQ ID NO: 15 to 19, more preferably to those provided as SEQ ID NO: 18 or 19. In one embodiment, the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is the enzyme referred to herein in the Examples as PRO-GH29-004, which is availa ble from Prozomix Limited, Station Court, Haltwhistle, Northumberland, NE499HN, United Kingdom.

In another preferred embodiment, the polypeptide of the invention is an enzyme being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL, is an artificial polypeptide and is preferably:

(i) an amino acid sequence as shown in in any one of SEQ ID NOs:15 to 19, more preferably SEQ ID NO: 18 or 19;

(ii) an artificial amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an artificial amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% but not 100 % identical to the amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NOs: 5 to 7, SEQ ID NO: 9 or SEQ ID NOs: 20;

(iv) an artificial amino acid sequence which is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i) or the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NOs: 5 to 7, SEQ ID NO: 9 or SEQ ID NOs: 20; or

(v) an artificial amino acid sequence which has one or several amino acid changes compared to the amino acid sequences of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii), (iv) and / or (v) for cleaving the alpha-1 , 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to Further embodiments are compositions comprising said artificial enzyme and 2’,3-DiFL, host cells expressing such artificial enzymes, as well as inventive methods and the use of such artifi cial enzymes for the cleaving of 2’,3-DiFL to form 2’-FL.

Preferably, the artificial polypeptide is reducing the amount of 2’,3-DiFL in the methods of the invention by an additional amount compared to the reduction by the corresponding wildtype en zyme. The additional reduction of 2’,3-DiFL compared to the reduction the wildtype enzyme causes is in increasing order of preference at least 10 %, 20 %, 30% or 40 % less of 2’,3-DiFL compared to the control, than the wildtype enzyme achieved compared to the control. The artifi cial enzymes of the invention hence reduce the amount of 2’,3-DiFL by a factor of in increasing order of preference at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or two. For example, as can be seen in Figure 6 B, the artificial enzyme of SEQ ID NO: 18 named CpAFC2-T1 leads to roughly double the reduction in 2’,3-DiFL compared to the wildtype CpAfc2 enzyme under the same conditions.

The polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL ac cording to the present invention may be an isolated enzyme, isolated from its natural context or at least partially purified. In a further preferred embodiment, it is an artificial polypeptide, i.e. a non-naturally occurring polypeptide although derived or related to a naturally occurring polypep tide.

Preferably, variants of the aforesaid polypeptides are also included. As used herein, the term "polypeptide variant" relates to any chemical molecule comprising at least one polypeptide or fusion polypeptide as specified elsewhere herein, having the indicated activity, but differing in primary structure from said polypeptide or fusion polypeptide indicated above. Thus, the poly peptide variant, preferably, is a mutated protein having the indicated biological activity. Prefera bly, the polypeptide variant comprises a peptide having an amino acid sequence corresponding to an amino acid sequence of 100 to 1000, more preferably 200 to 750, even more preferably 300 to 500, or, most preferably, 400 to 500 consecutive amino acids comprised in a polypeptide as specified above. Polypeptide variants referred to herein may be allelic variants or any other species homologs, paralogs, or orthologs. Moreover, the polypeptide variants referred to herein preferably include fragments of the specific polypeptides or the aforementioned types of poly peptide variants as long as these fragments and/or variants have the biological activity as re ferred to above. Such fragments may be or be derived from, e.g., degradation products or splice variants of the polypeptides. Further included are variants which differ due to posttranslational modifications such as phosphorylation, glycosylation, ubiquitinylation, sumoylation, or myristy- lation, by including non-natural amino acids, and/or by being peptidomimetics. Preferably, the polypeptide variant as referred to herein has an amino acid sequence which dif fers due to at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant is still, preferably, at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical with the amino acid sequence of the specific polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the sequence it is compared to for optimal alignment. The percentage is calculated by determining, preferably over the whole length of the polypeptide, the number of positions at which the identical amino acid residue oc curs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the comparison win dow comprises a complete sequence as specified herein. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for simi larity method of Pearson and Lipman (1988), by computerized implementations of these algo rithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wl), or by visual in spection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. More preferably, the program needle from the bioinformatics software package EMBOSS (Ver sion 5.0.0, emboss.source-forge.net/what/) with the default parameters which are, i.e. gap open (penalty to open a gap): 10.0, gap extend (penalty to extend a gap): 0.5, and data file (scoring matrix file included in package): EDNAFUL, is used.

Also, preferably, the polypeptide variant as referred to herein has an amino acid sequence which is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleo tide encoding at least one of the aforesaid specific amino acid sequences.

The term “polynucleotide” is known to the skilled person. As used herein, the term includes nu cleic acid molecules comprising or consisting of a nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20; based on the genetic code, the skilled person is able to provide a polynucleotide encoding a given polypeptide. As the skilled person understands, due to the degeneracy of the genetic code, there usually is more than one polynucleotide encoding a given polypeptide. Preferably, the genetic code is the standard genetic code. The polynucleo tide of the present invention shall be provided, preferably, either as an isolated polynucleotide (i.e. isolated from its natural context) or in genetically modified form. The polynucleotide, prefer ably, is DNA, including cDNA, or is RNA. The term encompasses single as well as double stranded polynucleotides. Preferably, the polynucleotide is a chimeric molecule, i.e., preferably, comprises at least one nucleic acid sequence, preferably of at least 20 bp, more preferably at least 100 bp, heterologous to the residual nucleic acid sequences. Moreover, preferably, com prised are also chemically modified polynucleotides including naturally occurring modified poly nucleotides such as glycosylated or methylated polynucleotides or artificial modified one such as biotinylated polynucleotides.

As used herein, the term polynucleotide, preferably, includes variants of the specifically indicat ed polynucleotides, wherein said variants still encode polypeptides having the activity as speci fied above, i.e. being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL. It is to be understood, however, that a polypeptide having a specific amino acid sequence may be en coded by a variety of polynucleotides, due to the degeneration of the genetic code. The skilled person knows how to select a polynucleotide encoding a polypeptide having a specific amino acid sequence and also knows how to optimize the codons used in the polynucleotide according to the codon usage of the organism used for expressing said polynucleotide. Thus, the term “polynucleotide variant”, as used herein, relates to a variant of a polynucleotide related to herein comprising a nucleic acid sequence characterized in that the sequence can be derived from the aforementioned specific nucleic acid sequence by at least one nucleotide substitution, addition and/or deletion, wherein the polynucleotide variant shall have the activity as specified for the specific polynucleotide, i.e. shall encode a polypeptide according to the present invention. Pref erably, said polynucleotide variant is an ortholog, a paralog or another homolog of the specific polynucleotide. Also, preferably, said polynucleotide variant is a naturally occurring allele of the specific polynucleotide. Polynucleotide variants also encompass polynucleotides comprising a nucleic acid sequence which is capable of hybridizing to the aforementioned specific polynucle otides under stringent hybridization conditions. These stringent conditions are known to the skilled worker and can be found in Current Protocols in Molecular Biology, John Wiley & Sons,

N. Y. (1989), 6.3.1-6.3.6. A preferred example for stringent hybridization conditions are hybridi zation conditions in 6x sodium chloride/sodium citrate (= SSC) at approximately 45°C, followed by one or more wash steps in 0.2x SSC, 0.1% SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depend- ing on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1x to 5x SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridi zation conditions for DNA:DNA hybrids are preferably for example 0.1x SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are pref erably, for example, 0.1x SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sam- brook et al., "Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, ’’Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford.. As a source of polynucleotides for hybridization, DNA or cDNA from viruses, bacteria, fungi, plants, or animals, preferably from a bacterium, more preferably bacteria as specified elsewhere herein, may be used. Further, variants include poly nucleotides comprising nucleic acid sequences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nu cleic acid sequences encoding the polypeptides as specified above. Preferably, also encom passed are polynucleotides which comprise nucleic acid sequences encoding amino acid se quences which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequences specifically indicated. The percent identity values are, preferably, calculated over the entire amino acid or nucleic acid sequence region. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the se quence alignments, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))], which are part of the GCG software packet (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)), are to be used. The sequence identity values recited above in percent (%) are to be determined, preferably, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. A polynucleotide comprising a fragment of any of the specifically indicated nucleic acid se quences is also encompassed as a variant polynucleotide of the present invention. The frag ment shall still encode a polypeptide which still has the activity as specified. Accordingly, the polypeptide encoded may comprise or consist of the domains of the polypeptide of the present invention conferring the said enzymatic activity. A fragment as meant herein, preferably, com prises at least 300, preferably at least 600, more preferably at least 900, most preferably at least 1200 consecutive nucleotides of any one of the specific nucleic acid sequences or en codes an amino acid sequence comprising at least 100, preferably at least 200, more preferably at least 300, most preferably at least 400 consecutive amino acids of any one of the amino acid sequences specified herein above.

An isolated nucleic acid sequence or isolated nucleic acid molecule is one that is not in its na tive surrounding or its native nucleic acid neighbourhood, yet it is physically and functionally connected to other nucleic acid sequences or nucleic acid molecules and is found as part of a nucleic acid construct, vector sequence or chromosome.

The polynucleotides of the present invention either consist, essentially consist of, or comprise the aforementioned nucleic acid sequences. Thus, they may contain further nucleic acid se quences as well. Specifically, the polynucleotides of the present invention may encode fusion proteins wherein one partner of the fusion protein is an immunogenic polypeptide being encod ed by a nucleic acid sequence recited above. Such fusion proteins may comprise as additional part polypeptides for monitoring expression (e.g., green, yellow, blue or red fluorescent proteins, alkaline phosphatase and the like), so called “tags” which may serve as a detectable marker or as an auxiliary measure for purification purposes, and/or scaffold polypeptides such as thiore- doxin, as described herein above. Tags for the different purposes are well known in the art and are described elsewhere herein.

According to the method specified herein, 2’,3-DiFL is contacted with a polypeptide being capa ble of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL. The term "contacting" is under stood by the skilled person to relate to bringing compound into close physical proximity so as to allow the components to interact. Preferably, contacting is admixing 2’,3-DiFL and a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL in the same solution. As is understood by the skilled person, it is not required for the method of the present invention that the contacting is performed under conditions optimal for the aforesaid hydrolysis to occur; preferably, under the conditions of contacting, the polypeptide of the present invention has an activity corresponding to at least 1 %, more preferably at least 5%, even more preferably at least 10 %, still more preferably at least 25 % of the corresponding activity under optimal condi- tions. Conditions sufficient for selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL may in clude the use of an appropriate reaction medium and/or cultivation medium for the host cell and/or buffer. Moreover, said conditions may include adjusting the pH in the medium and/or buffer to a range between 3 and 9, preferably between 4.5 and 7, more preferably between 5.0 and 6.5. Moreover, contacting preferably comprise incubating the mixture at a temperature be ing within the range of from 15°C to 50°C, preferably of from 20°C to 45°C, more preferably 30°C to 40°C, most preferably from 30° to 37 °C. In the situation that the contacting is per formed in a mixture of at least one type of recombinant host cell and cultivation medium the temperature will be the temperature typically used for the cultivation of such a recombinant host cell. For example, for recombinant E. coli cells recombinantly expressing the enzymes to pro duce 2’FL and 2’,3-DiFL as well as one or more polypeptides being capable of selectively cleav ing the alpha-1, 3-C-O bond of 2’,3-DiFL according to the invention, the growth of the E. coli cells and the contacting will typically be done at 37°C. However, in particular in case enzymes from thermophilic or psychrophilic bacteria are used, temperatures outside these ranges may be envisaged. The skilled person knows how to determine the optimal reaction conditions, in par ticular optimal pH, temperature, and salt and ion concentrations for a given enzyme. The con tacting of step a) may be performed without agitation. Preferably, contacting is performed with out agitation, in case of the contacting is performed in the absence of cells. If cells are present during the contacting and continued production by the cells of the substrate 2’3-DiFL and/or of the enzymes to selectively cleave the 2’3-DiFL is desired, agitation is preferably during an incu bation substep. Preferably, the content of 2’,3-DiFL in the aqueous solution and/or fermentation medium containing 2’,3-DiFL is at least 2 g/l, more preferably at least 5 g/l, even more prefera bly at least 10 g/l, still even more preferably at least 20 g/l, wherein the volume is the volume of aqueous solution and/or fermentation medium. Even more preferably, said contacting compris es adding the polypeptide in an amount of preferably 0.01 to 1 mg/ml, more preferably 0.1 mg/ml based on the total volume of the mixture.

In one embodiment an optional incubation substep a2) is part of step a) of the contacting 2’,3- DiFL with a polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3- DiFL. Depending on the amounts of 2’,3-DiFLand of the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL, the type and activity of the said enzyme and the conditions used, an incubation substep a2) may be useful to achieve the desired conversion rates of 2’,3-DiFL. Longer incubation times may increase nonselective cleaving while shorter incubation times may lead to insufficient cleaving of the alpha-1, 3-C-O bond of 2’,3-DiFL. As can be seen from the results shown in Figures 4 and 5 and tables 2 and 3 below, some of the tested enzymes produce very fast results, while others require some time to reach the desired level of conversion of 2’,3-DiFL to 2’-FL, In one embodiment, contacting is performed for at least 1 h, preferably 6 h to 20 h, more preferably for at least 10 to 16 h. In another embodiment the incubation substep a2) may not be longer than 36 h, preferably no longer than 30 h and more preferably no longer than 24 h.

In one embodiment, said contacting comprises adding of the polypeptide to 2’,3-DiFL to obtain a mixture. The skilled person knows how to optimize enzyme activity added, reaction conditions, and reaction time.

Preferably, contacting is contacting a 2’,3-DiFL containing aqueous solution and/or fermentation medium with a polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3- DiFL without the need of pre-purification of the 2’,3-DiFL. The aqueous solution and/or fermen tation medium containing 2’,3-DiFL suitable for contacting 2’,3-DiFL with a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL may be cell-free or it may still contain cells, for example bacterial cells used in the fermentation process for obtaining the aqueous solution and/or fermentation medium containing 2’,3-DiFL. Thus, preferably, contacting is contacting 2’,3-DiFL and the polypeptide being capable of selectively cleaving the alpha-1, 3- C-0 bond of 2’,3-DiFL extracellularly. Accordingly, preferably, step a) of contacting 2’,3-DiFL with a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL, 2’,3-DiFL preferably takes place in a suitable reaction medium containing 2’,3-DiFL, such as an aqueous solution, suspension, emulsion, dispersion, or fermentation medium containing 2’,3- DiFL. Preferably, the reaction medium corresponds to an aqueous solution and/or fermentation medium provided by a conventional fermentation process for the production of fucosylated car bohydrates as described above, and e g. in WO 2015/032412 A1. More preferably, the reaction medium containing 2’,3-DiFL is directly obtained as a product from the fermentation process, i. e. the aqueous solution or fermentation medium containing 2’,3-DiFL is obtained by the cultur ing step of a conventional fermentation process for the production of fucosylated carbohydrates without the need of pre-purification of the 2’,3-DiFL. This is advantageous as the claimed meth od allows for directly processing the aqueous solution and/or fermentation medium obtained by conventional fermentation process in order to increase the amount of 2’-FL without the need of preceding purification steps. Preferably, in step a) of the method specified herein, the polypep tide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL according to the present invention is added to the aqueous solution or fermentation medium containing 2’,3-DiFL in an amount sufficient for mediating cleavage of the 1, 3-C-O bond of 2’,3-DiFL. This means that preferably, the selective cleavage of the 1, 3-C-O bond takes place in the aqueous solution or fermentation medium containing 2’,3-DiFL. Thus, in such case, in step a) of contacting 2 ^' , 3- difucosyllactose (2’,3-DiFL) with a polypeptide being capable of selectively cleaving the alpha- 1, 3-C-O bond of 2’,3-DiFL, the reaction medium may contain cells or may be a cell free fermen tation broth. In case 2’,3-DiFL is comprised in a fermentation broth, step a) is preferably per formed in the presence of cells producing 2’,3-DiFL; thus, step a) is preferably performed at least partly concomitantly to fermentative production of 2’,3-DiFL, in particular towards the end of a fermentation process, or thereafter before removal of cells.

Also preferably, step a) is performed in the absence of cells producing 2’,3-DiFL, i.e. in a cells free fermentation broth. Methods for producing cell free fermentation broths are known in the art and were described herein above.

In a more preferred embodiment, the contacting 2’,3-DiFL with a polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL is performed during part or all of the fermentation process for the production of 2’-FL within the cell-culture-medium, intracellularly and / or extracellularly, thereby allowing fucose captured in 2’,3-DiFL to be re-used for the pro duction of 2’-FL and / or reducing the amount of 2’,3-DiFL present at the end of the fermentation process.

Further, a preferred embodiment of the invention is a method for producing 2'-0-fucosyllactose (2’-FL) comprising the steps of: a) culturing, in an aqueous culture medium containing lactose, a genetically modified cell having one or more recombinant glycosyl transferases, with one or more being a 1,2- fucosyltransferase, capable of modifying lactose or an intermediate in the biosynthetic pathway of 2'-FL or 2’,3-DiFL from lactose and that is necessary for the synthesis of 2'-FL or 2’,3-DiFL from lactose, in a manner that 2’-FL and 2’,3-DiFL are being produced by said genetically modified cell, b) contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) with a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL wherein said polypeptide has an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2,

SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and (iv) an amino acid sequence which has one or several amino acid changes to the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19; under conditions sufficient for selective cleavage of the alpha-1 , 3-C-O bond of 2’,3-DiFL; and c) optionally repeating step a) and b) repeatedly, preferably so that the contacting the 2’,3- DiFL according to step b) is simultaneously to the culturing according to step a); d) obtaining 2’-fucosyl lactose generated in any of steps a), b) or c) or a mixture of 2’- fucosyllactose and 2’,3-DiFL generated in any of steps a), b) or c); e) optionally contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) in a mixture of 2’-fucosyllactose and 2’,3-DiFL generated by step d), preferably a cell-free mixture, with a polypeptide being ca pable of selectively cleaving the apha-1, 3-C-O bond of 2’,3-DiFL wherein said polypeptide has an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19; under conditions suitable for selective cleavage of the alpha-1 ,3-C-O bond of 2’,3-DiFL;

The inventive method for production of 2’-FL wherein the internalization of the lactose takes place via an active transport mechanism under the influence of a lactose permease, is also pre ferred.

The inventive method may further comprise the provision of the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL according to the invention by addition of the polypeptide to the cell-culture medium during fermentation one or more times, by co expression of the genes encoding said polypeptide within the genetically modified cell suitable of 2’-FL and / or 2’,3-DiFL production , and / or by coculturing of the genetically modified cell suitable of 2’-FL and / or 2’,3-DiFL production with a the genetically modified cell producing said polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL according to the invention.

Also, preferably, contacting is contacting 2’,3-DiFL with a polypeptide being capable of selec tively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL within a cell, i.e. intracellularly, more prefer ably within a genetically modified host cell carrying a polynucleotide that encodes a polypeptide as specified herein. More preferably, the genetically modified host cell is a genetically modified microbial host cell expressing an enzyme as described herein below. Suitable genetically modi fied host cells are described herein below.

Also, preferably, contacting is contacting 2’,3-DiFL with a polypeptide being capable of selec tively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL both intracellularly and extracellularly. This may be accomplished by expressing a gene encoding a polypeptide being capable of selective ly cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL inside a 2’,3-DiFL producing cell and / or by (i) adding an export signal to at least a part of the polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL or (ii) lysing a portion of said producer cells in order to re lease the expressed polypeptide.

A "genetically modified" cell or microorganism as understood according to the present invention is genetically different from the wild type cell or microorganism. The genetic difference between the genetically modified microorganism according to any aspect of the present invention and the wild type microorganism is, preferably, the presence of a polynucleotide in the genetically modi- fied microorganism that is absent in the wild type microorganism. In one example, the genetical ly modified microorganism as understood herein may comprise enzymes that enable the micro organism to produce HMOs, preferably including 2’,3-DiFL and/or 2’-FL. The wild type microor ganism relative to the genetically modified microorganism of the present invention may have none or no detectable activity of the enzymes that enable the genetically modified microorgan ism to produce said HMOs. As used herein, the term "genetically modified microorganism" may be used interchangeably with the terms "genetically modified cell" and "genetically modified bacterial cell". The genetic modification as understood according to any aspect of the present invention is usually carried out on the cell of the microorganism which, preferably, is a bacte rium. The genetically modified cells are genetically transformed according to any method known in the art. In particular, the cells may be produced according to the method disclosed in WO 2009/077461 Al .The genetically modified bacterial cells used in the fermentation process for the production of fucosylated carbohydrates, advantageously are genetically modified E. coli cells, preferably LacZY ⁺ E. coli cells, i.e. E. coli cells comprising the lactose permease (LacY) and beta-galactosidase (lacZ) genes; as will be understood by the skilled person, in case a fur ther energy and carbon source other than lactose is provided in the fermentation medium, the bacterial cells may comprise only a lactose permease. Preferably, said cells carry a recombi nant gene encoding a fucosyltransferase. The fucosyltransferase is capable of modifying lac tose or an intermediate in the biosynthetic pathway for 2’-FL synthesis. Advantageously, the fucosyltransferase is a 1,2-fucosyltransferase. Fucosyltransferases and their genes are known in the art. Preferably, the genetically modified microorganism used in the fermentation process export the fucosylated carbohydrates, more preferably a mixture of 2’,3-DiFL and 2’-FL, into the culture medium, e. g. into the extracellular space of the culture medium, during the culturing step. This way a 2’,3-DiFL containing aqueous solution or fermentation medium may be ob tained.

The expression of the gene encoding the fucosyltransferase and, optionally, the gene encoding the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is/are preferably driven by suitable promoter sequences known to the skilled artisan; suitable promot ers include inducible and constitutive promoters. As used herein, the term "promoter" or "tran scription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the di rection of transcription of the transcription initiation site of the coding sequence, and is structur ally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcrip tion initiation sites and any other DNA sequences, including, but not limited to transcription fac tor binding sites, repressor and activator protein binding sites, and any other sequences of nu cleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most cells under most physiological and developmental conditions. An "inducible" promoter is a pro moter that is physiologically or developmental^ regulated, e.g. by the application of a chemical inducer.

In the method of the invention, step (b) of obtaining 2’-FL may comprise a step removing and/or inactivating the polypeptide, e.g. a filtration, precipitation and/or centrifugation step. This en sures that the cleaving activity is removed from the mixture and contributes to accurate termina tion of the cleaving reaction. The filtration step may be carried out by using suitable methods known to the skilled artisan. Preferably, polypeptides and/or related impurities are removed from the reaction medium by ultrafiltration, tangential flow high-performance filtration, tangential flow ultrafiltration, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography and/or gel filtration (i.e., size exclusion chromatography), particularly by chro matography, more particularly by ion exchange chromatography or hydrophobic interaction.

Preferably the method according to the present invention comprises at least one further step c) of separation, drying, purification, packaging, or the like of the 2’-FL produced.

Surprisingly, it has been discovered by the present inventors that specific polypeptides having an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO: 9,

15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19; (ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i); (iii) an ami no acid sequence which is encoded by a polynucleotide that hybridizes under stringent condi tions to a polynucleotide encoding the amino acid sequence of (i); and (iv) an amino acid se quence which has one or several amino acid changes to the amino acid sequence of (i); where in preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19; are capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL under conditions sufficient for selective cleavage of the alpha-1 ,3-C-O bond of 2’,3-DiFL. The use of a polypeptide in a method for producing 2’-FL according to the present invention represents an immediate remedy to reduce the undesired accumulation of 2’,3-DiFL during HMO production processes including biotechnological processes utilizing fucosyltransferase activity. This is advantageous, as it rep- resents a cost- and time-efficient solution to reduce unwanted 2’,3-DiFL accumulation using conventional biotechnological processes like fermentation relying on fucosyltransferase activity. In addition, the selective cleavage of the alpha-1, 3-C-O bond of 2’,3-DiFL has the advantage that the amount of the desired 2’-FL can be increased and the production of undesired 3’-FL is reduced significantly or even prevented. The increased levels of 2’-FL in the product reduce the need for laborious downstream purification and/or isolation steps. Preferably, this advantage is also achieved by the polypeptides comprised in the enzymes described herein.

The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

In a further aspect, the present invention relates to an enzyme selectively cleaving the alpha- 1, 3-C-O bond of fucosylated carbohydrates, preferably the alpha-1, 3-C-O bond of fucosylated oligosaccharides, wherein the enzyme comprises a polypeptide having an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NOs:15 to 19; more preferably those pro vided as SEQ ID NO: 18 or 19;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19;

(iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under strin gent conditions to a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, prefera bly any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19; and

(iv) an amino acid sequence which has one or several amino acid changes to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19; wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1 , 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20; more preferably any one of SEQ ID NO: 9, 18 or 19. Thus, preferably, the enzyme of the present invention is the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond as specified herein above.

In a one aspect of the present invention, the enzyme is a polypeptide having the amino acid sequence selected from the group consisting of: (i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 19; (ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid se quence of (i); and

(iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under strin gent conditions to a polynucleotide encoding the amino acid sequence of (i).

Preferably, the fucosylated carbohydrate is a human milk oligosaccharide (HMO). A preferred HMO is 2’,3-DiFL, preferably produced by the method described above. Preferably, the compo sition comprises at least 2’,3-DiFL and, optionally, 2’-FL. Other suitable but optional compo nents of the compositions include FLU (2'-0-fucosyl-lactulose) and/or FFL (Fuc(cs1 - 2)Fuc(alpha-1 -2)Gal(pi -4)Glc).

The composition of HMOs can be applied for pharmaceutical or nutritional purposes, e.g. as immunomodulatory agent or in baby/infant formulas or supplemented milk products. Thus, the present invention also relates to an infant formula or supplemented milk product comprising said composition of HMOs.

The present invention also relates to a host cell heterologously expressing the enzyme as de scribed above.

The term "host cell", as used herein, relates to any cell capable of heterologously expressing a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL of the present invention. In principle, the host cell can be any bacterial, archeal, or eukaryotic cell. Preferably, the host cell is a bacterial or a yeast cell, more preferably is a bacterial cell. Prefera bly, the host cell is selected from enterobacteriaceae and lactic acid bacteria preferably, the host cell is selected from the list consisting of Escherichia coli, Bacillus spp. (e.g. Bacillus sub- tilis), Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neis seria gonorrhoeae, Neisseria meningitidis, Lactobacillus spp., Lactococcus spp., Enterococcus spp. , Bifidobacterium spp., Sporolactobacillus spp., Micromonospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas spp.. More preferably, the host cell is an E. coli cell. Preferably, the host cell is a genetically modified cell as specified herein above. Suitable ways of obtaining a host cell heterologously expressing the polypeptide as described above are known to the skilled artisan and usually include introducing a nucleic acid sequence or gene encoding the polypeptide as part of a suitable expression construct and, optionally, as part of a nucleic acid vector or nucleic acid construct into the host cell, thereby preferably ob taining a host cell heterologously expressing the enzyme as described above.

Introducing the nucleic acid construct into a host cell to obtain may be achieved, for example by transformation, transduction, conjugation, or a combination of these methods, with a nucleic acid vector or nucleic acid construct comprising a gene encoding for the desired polypeptide, an allele of this gene or parts thereof, and preferably including a gene enabling the expression of the vector/construct, preferably with a polynucleotide as specified herein above. The heterolo gous expression may be achieved in particular by integrating the gene or alleles into the chro mosome of a host cell or preferably an extrachromosomally replicating vector into a host cell.

A "nucleic acid construct" or "nucleic acid vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term "nucleic acid construct" therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms "expression vector" or "expression construct" refer to nucleotide sequences that are ca pable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements.

The expression vector will be introduced into a suitable host cell and be able to effect expres sion of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism of the invention.

Moreover, the present invention relates to the use of an enzyme as described above for the production of 2’-FL. The use of the enzyme according to the invention is advantageous as it mediates the selective cleavage of the 1,3-C-O bond of 2’,3-DiFL and thereby is able to in crease 2’-FL levels.

The present invention moreover relates to a composition comprising at least one enzyme capa ble of selective cleavage of the 1 ,3-C-O bond of 2’,3-DiFL as described above and at least 2’,3- DiFL, preferably a further component is 2’-FL. In a preferred embodiment, the composition comprises said enzyme in an isolated form. In another preferred embodiment the composition is cell-free.

In another preferred embodiment, of the fucose molecules liberated by the enzyme of the inven tion and useful in the methods of the invention, at least 70%, preferably at least 80 %, more preferably at least 90 %, even more preferably at least 95% and yet even more preferably at least 98% of fucose molecules are generated by cleaving an alpha-1, 3-C-O bond.

In a preferred embodiment the contacting is performed in a mixture of at least one type of re combinant host cell and cultivation medium. The polypeptide being capable of selectively cleav ing the alpha-1, 3-C-O bond of 2’,3-DiFL may be added to the cultivation medium, produced by one or more types of host cells in the cultivation medium or produced by the same recombinant host cell that is producing the 2’,3-DiFL. Cultivation medium once inoculated with one or more cells is also referred to as fermentation broth in the art. The two terms are used interchangeably within this application.

Overall, in the context of the present invention, the following embodiments are regarded as pre ferred:

1.A method for producing 2 ^' fucosyllactose (2’-FL) comprising the steps of: a. contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) with a polypeptide being capable of selectively cleaving the alpha-1 , 3-C-O bond of 2’,3-DiFL wherein said polypep tide has an amino acid sequence selected from the group consisting of: i. an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20; ii. an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i); and iii. an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the ami no acid sequence of (i); under conditions sufficient for selective cleavage of the alpha-1, 3-C-O bond of 2’,3-DiFL; and b. obtaining 2’-fucosyllactose generated in step a).

2. The method of embodiment 1, wherein said contacting takes place in an aqueous solu tion, preferably in a cell free fermentation broth. 3. The method of embodiment 1 or 2, wherein said contacting comprises adding of the poly peptide to 2’,3-DiFL to obtain a mixture and wherein said conditions comprise incubating the mixture, at a temperature being within the range of 30°C to 40°C, more preferably at 37°C.

4. The method of any one of embodiments 1 to 3, wherein selectively cleaving the alpha-1, 3-

C-0 bond of 2’,3-DiFL refers to hydrolyzing the alpha-1, 3-C-O bond of 2’,3-DiFL while the alpha-1 ,2-C-O bond of 2’,3-DiFL is cleaved to an extent that is statistically signifi cantly lower than the cleavage of the alpha-1 , 3-C-O bond.

5. The method of any one of embodiments 2 to 4, wherein said contacting comprises adding of the polypeptide in an amount of 0.1 mg/ml based on the total volume of the solution.

6. The method of any one of embodiments 3 to 5, wherein the incubation is performed with out agitation, preferably the incubation is performed for at least 6 h to 20 h.

7. The method of any one of embodiments 1 to 6, wherein said obtaining 2’-FL comprises a filtration step to remove the polypeptide from the mixture.

8. The method of any one of embodiments 1 to 7, wherein the yield of 2’-fucosyllactose is at least 40 % with respect to the amount of 2’,3-DiFL provided before cleaving.

9. The method of any one of embodiments 1 to 8, wherein the polypeptide comprises at least one amino acid sequence motif selected from the group consisting of the amino acid se quence motifs as shown in SEQ ID NO: 10, SEQ ID NO: 11 or in any one of SEQ ID NOs: 12 to 14.

10. The method of any one of embodiments 1 to 9, wherein the polypeptide is derived from a Bifidobacterium species, preferably Bifidobacterium scardovii.

11. An enzyme selectively cleaving the alpha-1, 3-C-O bond of fucosylated carbohydrates, preferably the alpha-1, 3-C-O bond of fucosylated oligosaccharides, wherein the enzyme comprises a polypeptide having an amino acid sequence selected from the group con sisting of: i. an amino acid sequence as shown in SEQ ID NO: 15 to 19; ii. an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO: 9, 15 to 20, more preferably any one of SEQ ID NO: 9, 18 or 19; iii. an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the ami no acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably any one of SEQ I D NO: 9, 15 to 20, more preferably any one of SEQ I D NO:

9, 18 or 19; and iv. an amino acid sequence which has one or several amino acid changes to the one encoded by a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20, preferably any one of SEQ ID NO:

9, 15 to 20; wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha- 1 ,3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19;.

12. The enzyme of embodiment 11, wherein the fucosylated carbohydrate is a human milk oligosaccharide (HMO).

13. The enzyme of embodiment 12, wherein the HMO is 2’,3-difucosyllactose (2’,3-DiFL).

14. The enzyme of any one of embodiments 11 to 13, wherein the polypeptide comprises at least one amino acid sequence motif selected from the group consisting of the amino ac id sequence motifs as shown in SEQ ID NO: 10, SEQ ID NO: 11 or in any one of SEQ ID NOs: 12 to 14.

15. A composition comprising at least one enzyme according to any of embodiments 11 to 14 and at least 2’,3-DiFL, preferably the composition is a cell free composition. 16. Use of an enzyme according to any of embodiments 11 to 14 for the production of 2’ fu- cosyllactose.

17. A host cell heterologously expressing the enzyme according to any of embodiments 11 to 14.

18. The host cell of embodiment 17, wherein the host cell is in addition capable of producing 2’-FL.

19. The host cell of embodiment 17 or 18, wherein the host cell is a bacterial host cell, pref erably selected an Escherichia coli cell.

20. A method for producing 2'-0-fucosyllactose (2’-FL) comprising the steps of: a) culturing, in an aqueous culture medium containing lactose, a genetically modi fied cell having one or more recombinant glycosyl transferases, with one or more being a 1,2-fucosyltransferase, capable of modifying lactose or an intermediate in the biosynthetic pathway of 2'-FL or2’,3-DiFL from lactose and that is necessary for the synthesis of 2'-FL or 2’,3-DiFL from lactose, in a manner that 2’-FL and 2’,3-DiFL are being produced by said genetically modified cell, b) contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) with a polypeptide being capable of selectively cleaving the alpha-1 ,3-C-O bond of 2’,3-DiFL wherein said polypep tide has an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2,

SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the one encoded by a polynucleotide encoding the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19 under conditions sufficient for selective cleavage of the alpha-1, 3-C-O bond of 2’,3-DiFL; and c) optionally repeating step a) and b) repeatedly, preferably so that the contacting the 2’,3-DiFL according to step b) is simultaneously to the culturing according to step a); d) obtaining 2’-fucosyllactose generated in any of steps a), b) or c) or a mixture of 2’-fucosyllactose and 2’,3-DiFL generated in any of steps a), b) or c); e) optionally contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) in a mixture of 2’- fucosyllactose and 2’,3-DiFL generated by step d) , preferably a cell-free mixture, with a polypeptide being capable of selectively cleaving the apha-1, 3-C-O bond of 2’,3-DiFL wherein said polypeptide has an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs:15 to 20;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i);

(iii) an amino acid sequence which is encoded by a polynucleotide that hy bridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the one encoded by a polynucleotide encoding the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19 under conditions sufficient for selective cleavage of the alpha-1, 3-C-O bond of 2’,3-DiFL;

21. The method according to embodiment 20, wherein the internalization of the lactose takes place via an active transport mechanism under the influence of a lactose per mease.

22. The method according to embodiment 20 or 21, wherein the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is produced by the genetical ly modified cell capable of producing 2’-FL and / or 2’,3-DiFL, or said polypeptide is pro duced by a second genetically modified cell.

23. The method according to embodiment 20 or 21, wherein the polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is added to the cell-culture medium during the fermentation one or more times.

24. The method according to any of embodiments 1 to 10 or 20 to 23, or the enzyme accord ing to any of claims 11 to 15, wherein selectively cleaving of the alpha 1, 3-C-O bond re fers to cleaving of the alpha 1 , 3-C-O bond of fucosylated lactose to an extent that is a least 10 times higher than cleaving of the 1 ,2 bond.

25. Any of the previous claims wherein at least one enzyme being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is an isolated enzyme.

Further preferred embodiments:

I. A method for producing 2 ^' fucosyllactose (2’-FL) comprising the steps of: a) contacting 2 ^' ,3-difucosyllactose (2’,3-DiFL) with one or more polypeptides be ing capable of selectively cleaving the alpha-1 , 3-C-O bond of 2’,3-DiFL where in said polypeptide has an amino acid sequence selected from the group con sisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NO: 9 or SEQ ID NOs: 15 to 20;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i); (iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the one encoded by a polynucleotide encoding the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the amino acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the alpha- 1, 3-C-O bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19 under conditions sufficient for selective cleavage of the alpha-1 , 3-C-O bond of 2’,3-DiFL; and b) obtaining 2’-fucosyllactose generated in step a).

II. The method of embodiment I, wherein said contacting of step a) takes place in an aqueous solution.

III. The method of embodiment I or II, wherein said contacting of step a) is at a temper ature being within the range of 30°C to 40°C, more preferably at 30°C to 37°C.

IV. The method of any one of embodiments I to III, wherein selectively cleaving the al- pha-1, 3-C-O bond of 2’,3-DiFL refers to hydrolyzing the alpha-1, 3-C-O bond of 2’,3-DiFL while the alpha-1, 2-C-O bond of 2’,3-DiFL is cleaved to an extent that is statistically significantly lower than the cleavage of the alpha-1, 3-C-O bond.

V. The method of any one of embodiments I to IV, wherein the yield of 2’- fucosyllactose is at least 40 % (mol/mol) with respect to the amount of 2’,3-DiFL provided before cleaving.

VI. The method of any one of embodiments I to V, wherein at least one polypeptide be ing capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL is derived from a Bifidobacterium species or a Clostridium species or Bacteroides species, preferably Bifidobacterium scardovii, Clostridium perfringens or Bacteroides ovatus.

VII. The method of any one of embodiments I to VI, wherein at least one polypeptide be ing capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL comprises at least one amino acid sequence motif selected from the group consisting of the amino acid sequence motifs as shown in SEQ ID NO: 10, SEQ ID NO: 11 or in any one of SEQ ID NOs: 12 to 14. VIII. The method of any one of embodiments I to VII, wherein at least one polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL com prises the polypeptide sequence of the enzyme available from Prozomix Limited, Station Court, Haltwhistle, Northumberland, NE49 9HN, United Kingdom as PRO- GH29-004 (Bifidobacterium scardovii), A0A087DG51 TM734, Lot 2017_1.

IX. The method of any one of embodiments I to VIII, wherein said contacting of step a) takes place in a cell free fermentation broth.

X. The method of any one of embodiments I to IX, wherein the contacting is performed without agitation.

XI. The method of any one embodiment I to X, wherein said contacting of step a) com prises adding of the polypeptide to 2’,3-DiFL to obtain a mixture.

XII. The method of any one of embodiments I to XI, wherein said contacting comprises adding of the one or more polypeptides in an amount of 0.1 mg/ml based on the to tal volume of the solution.

XIII. The method of any one of embodiments I to XII, wherein said obtaining 2’-FL com prises a filtration step to remove the polypeptide from the mixture.

XIV. The method of any one of embodiments I to VIII, X to XIII, wherein the contacting of step a) takes place within a mixture comprising at least one type of recombinant host cell and a cultivation medium.

XV. The method of any one of embodiments I to XIV, wherein the contacting of step a) takes place at a temperature of 37°C.

XVI. The method of any one of embodiments I to XV, wherein the contacting of step a) includes an incubation substep a2) and wherein the incubation is performed for at least 1 h to 30 h.

XVII. Use of a polypeptide being capable of selectively cleaving the alpha-1, 3-C-O bond of 2’,3-DiFL as defined in any one of embodiments I to XVI or of the enzyme as de fined in embodiment XVIII for the production of 2’ fucosyllactose.

XVIII. An artificial enzyme selectively cleaving the alpha-1, 3-C-O bond of a fucosylated carbohydrate, preferably the alpha-1, 3-C-O bond of an fucosylated oligosaccha ride, more preferably of a human milk oligosaccharide (HMO), most preferably of 2’,3-difucosyllactose (2’,3-DiFL), wherein the enzyme comprises a polypeptide having an amino acid sequence selected from the group consisting of:

(i) an amino acid sequence as shown in SEQ ID NO: 15 to 19;

(ii) an amino acid sequence which is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of (i); and (iii) an amino acid sequence which is encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding the amino acid sequence of (i); and

(iv) an amino acid sequence which has one or several amino acid changes to the one encoded by a polynucleotide encoding the amino acid sequence of (i); wherein preferably the selectivity of the enzyme represented by the ami no acid sequences as defined in (ii), (iii) and / or (iv) for cleaving the al- pha-1,3-C-0 bond of 2’,3-DiFL is at least as high as the lowest selectivity of the enzymes represented by any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 9 or in any one of SEQ ID NOs: 5 to 7 or SEQ ID NOs: 15 to 20, preferably 15 to 19.

XIX. A composition comprising at least one artificial enzyme according to embodiment

XVIII and at least 2’,3-DiFL.

XX. The composition of embodiment XIX, wherein the composition is cell free.

XXI. The composition of embodiment XIX or XX, or the enzyme of embodiment XVIII, wherein at least one enzyme is an isolated enzyme.

XXII. A host cell heterologously expressing the enzyme according to embodiment XVIII, preferably wherein the host cell is a bacterial host cell, more preferably is an Esch erichia coli cell.

XXIII. The method according to any of embodiments I to XVI or the enzyme according to embod iment XVIII, wherein selectively cleaving of the alpha 1,3-C-O bond refers to cleaving of the alpha 1 ,3-C-O bond of fucosylated lactose to an extent that is a least 10 times higher than cleaving of the 1,2-C-O bond

Brief description of the figures

Fig. 1 depicts a reaction scheme of 2’-FL. 4 refers to lactose, 2 refers to 2’-FL, 1* refers to 2’, 3- DiFL, 3* refers to 3’-FL

Fig. 2 shows an amino acid sequence alignment of SEQ ID NO: 1 to 8. (SEQ ID NO: 1: BAQ31854.1 , SEQ ID NO: 2: WP_049186088.1, SEQ ID NO: 3: WP_046726010.1, SEQ ID NO: 4: KFI94520.1, SEQ ID NO: 5: WP_082158618.1, SEQ ID NO: 6: WP_081892936.1 SEQ ID NO: 7: BAQ31874.1and SEQ ID NO: 8: KFI94501.1). Conserved residues are marked with a * in the line labelled “consensus”. A dot indicates residues that are semi-conserved be tween the compared sequences and have either of two, rarely three amino acids only. Fig. 3 shows an alignment of the amino acid sequence of selected B. scardovii fucosidases (SEQ ID NO: 1: BAQ31854.1, SEQ ID NO: 2: WP_049186088.1, SEQ ID NO: 5:

WP_082158618.1, and SEQ ID NO: 8: KFI94501.1) and 1, 3-fucosidase of B. longum infestans (SEQ ID NO: 9: pdb|3UES|A). Conserved residues are marked with a * in the line labelled “con sensus”. A dot indicates residues that are semi-conserved between the compared se quences and have either of two, rarely three amino acids only. The location of the motifs as de scribed by SEQ ID NO: 10 to 14 is shown.

Fig.4. Qualitative and quantitative analysis of reaction mixtures.

A. TLC analysis of enzymatic conversions with fermentation broth containing 2',3-DiFL and 2'- FL containing. 1: 2'-FL reference, 2: PRO-GH29-001, 3: PRO-GH29-004, 4: blank (no enzyme), 5: lactose and fucose reference, 2',3-DiFL is indicated by an arrow.

B. Summary of HPLC analysis of testing of the specificity of designated fucosidases in a fer mentation-derived composition comprising 2’-FL and 2’,3-DiFL; A1: a-fucosidase E-FUCTM;

A3: uncharacterised fucosidase PRO-GH29-001, A4: putative a-1 ,3/4-fucosidase PRO-GH29- 002, A5: fucosidase PRO-GH29-004, A6: a-L-fucosidase PRO-GH29-005, A7: typical results for many other enzymes designated as fucosidase but not showing activity on the fucose bonds of either 2’-FL or 2’,3-DiFL in a fermentation derived composition, A12: a blank incubation without enzyme, MIX: Analytical control, an artificial mix of the substances. Black bars represent 2’,3- DiFL, dotted bars represent 2’-FL after incubation with the enzyme.

C. Graphical and numerical results of the HPLC analysis: Comparison of 2',3-difucosyllactose- hydrolysis of two selected fucosidases i.e.PRO-GH29-001 (A3) and PRO-GH29-004 (A5) with a blank incubation without enzyme (A12). Incubation A3 shows predominant alpha-1 -2 hydrolysis while the fucosidase of incubation A5 appears to be highly alpha-1 -3 selective. Black bars rep resent 2’,3-DiFL, dotted bars represent 2’-FL after incubation with the enzyme.

Fig.5 shows the results of in vitro incubation of the fucosidases produced by overexpression in E. coli and subsequent protein extraction. For each part A to C, the top graph shows the results after the start of the incubation with the respective produced protein with the respective sub strate, the bottom graph the results after overnight incubation. Labels: F: BiAfcB (SEQ ID NO:

9), H: CpAfc2-T 1 (SEQ ID NO: 18), L: 4ZRX-T1 (SEQ ID NO: 19). Black bars represent 2’,3- DiFL, diagonally stripped bars represent 2’-FL.

A: Composition of solutions of 25 mM 2’-FL directly after addition of crude cell lysate containing overexpressed fucosidases (top) and after incubation at 30°C overnight (bottom). B: Composition of solutions of 25 mM 3’-FL directly after addition of crude cell lysate containing overexpressed fucosidases (top) and after incubation at 30°C overnight (bottom).

C: Composition of solutions of 25 mM 2’,3’DiFL directly after addition of crude cell lysate con taining overexpressed fucosidases (top) and after incubation at 30°C overnight (bottom).

2’-FL is not a significant substrate for all three enzymes, while 2’, 3’ 2’,3-DiFL is effectively trans formed to 2’-FL and Fucose.

Fig. 6 summarizes the results of the experiment expressing the suitable fucosidases BiAfcB or CpAfc2_T1 or CpAfc2 (SEQ ID NO: 9, 18 and 20, respectively) in host cells producing 2’-FL and 2’,3-DiFL (labelled DiFL) in fermentation after the induction of the expression of the fucosidase. 2’,3-DiFL values relative to the unmodified host cell producing 2’-FL and 2’,3-DiFL during the fermentation run are shown.

A: AMBR ^® results: The grey bar is for cells producing the protein of SEQ ID NO: 9 and the white bar for the cells producing the protein of SEQ ID NO: 18. 2’,3-DiFL is degraded by the two fuco sidases in vivo.

B: Biostat ^® results for the wildtype and the artificially enhanced fucosidases of SEQ ID NO: 20 and 18, respectively. The grey bar is for cells producing the protein of SEQ ID NO: 20 and the white bar for the cells producing the protein of SEQ ID NO: 18. 2’,3-DiFL is degraded by the two fucosidases in vivo, albeit the artificial fucosidase shows double the reduction of the wildtype fucosidases..

Examples

1 Screening of enzymes designated as fucosidases

The following experimental set up was used to analyze the fucosidase activity of the 28 poten tial polypeptides/enzymes:

250 pi buffer (100 mM NaCH3COO, pH 5.5, in double distilled water)

250 mI cell free fermentation broth comprising 2’-FL and 2’,3-DiFL, prepared by standard meth ods for example as disclosed in the international patent application published as WO2015032412 and containing a mixture of 2’-FL and 2’,3-DiFL. Cells were removed from the broth by centrifugation and filtration through a 0.2 pm membrane.

5 pi enzyme preparation (1 % w/w), so that the final concentration was 0.1 mg catalytic prepara tion per mL of total volume.

The cell free fermentation broth contained 12.04 g/l of 2’,3-DiFL and 76.83 g/l of 2'-FL.

The above listed components were mixed to obtain a reaction mixture. The reaction mixture was incubated overnight incubation at 37 °C without agitation. After 1:3 dilution with water, the conversion was terminated by filtration through a 10 kDa membrane or applied directly on a thin layer chromatography (TLC) plate. The reaction mixture was analysed by TLC on a 5x5 cm F254 Silica gel (Merck, Darmstadt). As an eluent n-propanol:water:nitromethane (7:2:1) was used. The detection was performed by dipping in Thymol (2 %, w/v) and H2SO4 (5 % v/v) in EtOH.

Alternatively, the content of individual sugars was determined by HPLC using standard tech niques.

28 enzymes designated as general fucosidases were assayed in the set up described above. Most enzymes showed the undesired strong activity on the alpha-1 -2-C-O bond of fucosylated lactose. In those cases, the TLC analysis revealed lactose and fucose while significant amounts of 2',3-difucosyl lactose were still observed in the reaction mix (see Fig. 4. A). 2'-FL, dominating in the starting material seemed to be diminished as well.

Some assays were further analyzed by HPLC according to standard methods. The results of the qualitative and quantitative analyses are depicted in Fig. 4 B and C

Fig. 4 B. depicts a summary of HPLC analysis of testing of the specificity of designated fuco sidases in a fermentation-derived composition comprising 2’-FL and 2’,3-DiFL. Marked as A1 are the results of suing a a-fucosidase, from Megazyme Ltd. (Megazyme Ltd. Bray Business Park, Bray, Co. Wicklow, A98 YV29, Ireland), order no.: E-FUCTM. Further shown are the re sults for four enzymes obtained obtained from Prozomix Limited, Station Court, Haltwhistle, Northumberland, NE49 9HN, United Kingdom: A3: uncharacterised fucosidase PRO-GH29-001, A4: putative a-1 ,3/4-fucosidase, PRO-GH29-002, A5: fucosidase PRO-GH29-004, A6: a-L- fucosidase, PRO-GH29-005. In assays A1, A3, A4 and A6 the tested enzymes resulted in a loss of 2’-FL while little or no substantial reduction of 2’,3-DiFL.

Shown as A7 are the typical results for many other enzymes designated as fucosidase but not showing substantial activity on the fucose bond of either 2’-FL or 2’,3-DiFL in a fermentation derived composition. The other two columns are a blank incubation without enzyme (A 12) while MIX designates an analytical control, an artificial mix of the substances.

Fig. 4 C. provides the detailed results of the HPLC analysis of assays 3 and 5 and the numerical values.

All in all, the test showed contrary to the expectations, that the vast majority of fucosidases were unsuitable for selective removal of fucose from 2’,3-DiFL without strongly affecting the 2’-FL content in this mix of 2’-FL and 2’,3-DiFL.

From all enzymes screened, PRO-GH29-004 from Bifidobacterium scardovii (Fucosidase PRO- GH29-004 (Bifidobacterium scardovii), A0A087DG51 TM734, Lot 2017_1, obtained from Pro zomix Limited, Station Court, Haltwhistle, Northumberland, NE49 9HN, United Kingdom) showed the best activity (Fig. 4 A, lane 3 - labeled “A5”, columns marked A5 in Fig. 4 B and ). At the end of the incubation apparently no lactose was formed; the small amounts of fucose pro duced can be attributed to the hydrolysis of the alpha-1 -3-C-O bond.

Quantitative analysis by HPLC (Fig. 4B & C) corroborates the above observations. Even though the mass balance was not perfectly closed in this experiment, it can be demonstrated that PRO- GH29-004 removes the undesired bisfucosylated lactose (i.e. 2',3-DiFL) while not hydrolysing 2'-fucosyllactose.

2 Construction of artificial fucosidases and testing for their suitability to specifically degrade 2’,3-DiFL

A number of artificial sequences as candidate fucosidases were generated in silico by the in ventors. These were designed to be of a great variety of sequence diversity, yet maintaining the ability to selectively cleave the alpha-1, 3-C-O bond of 2’,3-DiFL. The resulting artificial se quence share very little sequence identity, for example SEQ ID NO: 18 and SEQ ID NO: 19 have less than 50 % sequence identity, and SEQ ID NO: 15 and SEQ ID NO; 19 less than 30%. Codon optimized DNA sequences for expression in E. coli were generated. Codon-optimized genes encoding the amino acid sequences were ligated into a vector for E. coli expression con taining a C-terminal His-Tag by Biocat (BioCat GmbH, Im Neuenheimer Feld 584,69120 Heidel berg, Germany). The constructs were transformed into a standard strain of E. coli for protein production. The transformants were cultivated in 25 mL EC1 medium supplemented with each 100 pg/mL ampicillin, chloramphenicol, spectinomycin and tetatracycline for selection, and 100 pM IPTG and 1 mg/L rhamnose as inducers and incubated over night at 30°C. Biomass was harvested by centrifugation and suspended 1:4 in 50 mM KPi buffer. 1 mL suspension was lysed using standard techniques. Supernatant and solids were analyzed by SDS-Page for pres ence of proteins of the size expected for the fucosidases.

The artificial fucosidases provided by SEQ ID NO: 18 and designated CpAFc2-T1, and the one provided by SEQ ID NO: 19 and designated 4ZRX-T1 were such produced separately in the E. coli cells, isolated, purified and tested for their action on 2’-FL, 3’FL and 2’,3-DiFL. The se quence of SEQ ID NO: 9 was also included in the experiment.

The proteins of SEQ ID NO 18 and 19 share only 41,8 % and 47,2 % sequence identity, respec tively, with the protein of SEQ ID NO: 9.

In vitro hydrolysis of 2’-FL, 3’-FL and 2’,3-DiFL

25 mM 2’-FL (11.5 g/L), 3’-FL (11.5 g/L) or 2’,3-DiFL (15 g/L) in 100 mM NaPi sodium phos phate buffer, pH 6.5 at 30°C with 20 pL resting cell suspension or crude lysate in a total volume of 370 pL were tested. Samples were taken directly after addition of enzyme (tO), 5, 20, 120 min and overnight by quenching with 1 volume EtOH and analyzed by TLC (n- Propanol:Water:Nitromethane 7:2:1 on Silicagel Platte 60 F254, staining with anisaldehyde, or standard HPLC analysis (a double Rezex ROA-Organic Acid H+ (8%) column and a pre-column of Cation H, an Rl-Detector and 20mM H2SO4 as eluent, a temperature of 50°C and a flow rate of 0.6 ml per minute). Among the tested catalysts, SEQ ID NO: 9 (BiAfcB) displays the best per formance (Figure 5, labelled as experiment F) 3’-FL is hydrolyzed to lactose and fucose (around 7 to 8 g/l and 4 to 5 g/l, respectively), and 2’,3-DiFL to 2’-FL and fucose (around 10 g/l and 3 g/l, respectively) within minutes. After overnight incubation with 11.5 g/L 2’-FL minimal amounts of lactose are found (0.45 g/L vs. 12.7 g/L 2’-FL); overnight incubation of 15 g/L 2’, 3’ 2’,3-DiFL with this enzymes yields 11.6 g/L 2’-FL, 2.5 g/L fucose and 0.03 g/L lactose. Also suitable were SEQ ID NO: 18 and 19 (samples H and L in Figure 5, respectively), albeit slower than SEQ ID NO: 9. Both produced in the overnight treatment a near conversion of 2’,3-DiFL to 2’-FL and fucose without significant production of 3’-FL or lactose (Fig. 5 C, bottom), and only slightly low er amounts of the products as did SEQ ID NO: 9. If 3’FL was supplied as substrate, both SEQ ID NO: 18 and 19 as well as SEQ ID NO: 9 showed activity in cleaving this substrate to fucose and lactose (Figure 5 B, top graph at start, bottom graph after overnight incubation). In contrast to this, very little of 2’-FL as substrate was only converted by these three enzymes (Figure 5 A, top graph at start, bottom graph after overnight incubation).

TLC analysis corroborated the conclusions drawn from the HPLC results: BiAfcB rapidly de- fucosylates 3’-FL and 2’,3-DiFL at the 3’-position. For CpAfc2-T1 the conversion was slower.

Again, the experiments showed that not all fucosidases are suitable for the methods of the pre sent invention. Another artificially created fucosidase showed not the expected activity on any of the three substrates (samples labelled T in Fig. 5), although soluble protein had been produced.

3 Production of 2’-FL with enzymatic removal of 2’,3-DiFL and recycling of fucose

Fermentative production of 2’-FL and 2’,3-DiFL 2’,3-DiFLis conducted according to standard methods, for example according to WO2015032412. Small fermentation vessels like the AMBR® 250 system and Biostat ® fermenters (both from Sartorius AG, Otto-Brenner-Str. 20, D- 37079 Gottingen, Germany) can be used to limit the necessary amounts of materials and en zyme e.g. PRO-GH29-004 .During the fermentation the build-up of 2’,3-DiFL can be monitored or a time point chosen at which 2’,3-DiFL 2’,3-DiFLcan be expected to be present in significant amounts. The enzyme PRO-GH29-004 from Bifidobacterium scardovii is added to the culture medium, and after some incubation, the content of 2’-FL, 2’,3-DiFL 2’,3-DiFLand fucose in the culture medium is analysed. It is shown, that 2’,3-DiFL 2’,3-DiFLis reduced without reducing the amounts of 2’-FL in the culture medium.

4 Expression of 2’,3-DiFL specific fucosidases in host cell during the production of 2’-FL

A typical 2’-FL producing recombinant E. coli strain see for example the ones disclosed in the international patent applications published as WO2015032412A1 and WO2010070104A1 as well as in the publication of Baumgaertner and coworkers (Baumgartner et al. (2013), Microb Cell Factories 12:40) was transformed with a plasmid containing one of the following fucosidas es:

BiAfcB, SEQ ID NO: 9 CpAfc2, SEQ ID NO: 20 and artificial CpAfc2_T1, SEQ ID NO: 18

The two known enzymes BiAfcB (SEQ ID NO: 9) and CpAfc2 (SEQ ID NO: 20) have recently been reported to be useful amongst others as in vitro transfucosidases on oligosaccharides containing single fucose units by Zeuner and co-workers (Zeuner, B., et al., New Biotechnology, 2018. 41 : p. 34-45). For example, the report their use for transfucosylation from 2’-FL or 3’-FL as substrates onto LNT to form LNFPII. The inventors however considered that in vivo not sin gle fucose unit containing oligosaccharides but oligosaccharides containing two fucose unit might be better substrates for these enzymes and choose to express them in a recombinant host cell for the reduction of 2’,3-DiFL, resulting in the reduction of the two-fucose compound 2’,3-DiFL and as a product 2’FL.

Expression of the synthesized genes encoding these three enzymes was regulated by rham- nose inducible promotor rhaBAD (Egan, S.M. and R.F. Schleif, A Regulatory Cascade in the Induction of rhaBAD. Journal of Molecular Biology, 1993. 234(1): p. 87-98):The promoter rha BAD shows little activity in the absence of the inducer; after induction by rhamnose the expres sion is of medium strength compared to the T7 promoter, and results in high protein yields.

The AMBR® 250 system (Sartorius) was used. 139 mL minimal medium (30 ml/L trace element solution, 0.5 mM IPTG, Ampicillin 100 pg/mL for fucosidase strains) were inoculated with 900 pL of a working cell bank culture. When CTR reaches 8 mmol/Lh the glycerol feed (86%) was started. The p02 is controlled at >20% and pH at 6.7 with 15% NH ₄ OH aq. After 50 hours at 37°C fucosidase expression was induced with 5 mM rhamnose.

Minimal medium: citric acid 1.1 g/L, glycerol 10.8 g/L, KH2P04 10.0 g/L, (NH4)2S044.6 g/L, Na2S04 3 g/L, MgS04 * 7H20 1.5 g/L, Thiamin 0.02 g/L, Vitamin 812 0.0001 g/L. Trace element solution: Na2-EDTA*2H204 g/L, CaS04*2H20 1 g/L, ZnS04*7H200.3 g/L, FeS04 ^* 7H20 3.7 g/L, MnS04 ^* H20 0.2 g/L, CuS04 ^* 5H200.15 g/L, Na2Mo04 ^* 2H20 0.04 g/L, Na2Se040.04 g/L.

Table 2 shows the results after the indicated fermentation time at 37°C. After induction the 2’,3- DiFL values for host cells expressing either of the two plasmids experience a dramatic drop compared to the control. Before the induction with rhamnose the 2’,3-DiFL level in those cells carrying the plasmid encoding SEQ ID NO: 9 showed already a significant decrease. This is consistent with the experiments that BiAfcB (SEQ ID NO: 9) is very, very active on 2’,3-DiFL and even the small amounts of this enzyme produced before rhamnose induction results in a significant effect on 2’,3-DiFL.

Table 2

The average 2’,3-DiFL amount relative to the control after the induction is shown in Figure 6 A.

In a further experiment, a Biostat fermenter (Sartorius) with 1900 ml_ minimal medium (30 ml/L trace element solution, 0.5 mM IPTG, Ampicillin 100 pg/mL for fucosidase strains) was inoculat ed with 100 ml_ of an overnight seed culture of host cells comprising the constructs for the wildtype protein CpAfc2 (SEQ ID NO: 20) or the artificial CpAfc2_T1 (SEQ ID NO: 18). When CTR reaches 10 mmol/Lh the glycerol feed (86%) was started. The p02 is controlled at >20% and pH at 6.7 with 15% NH40H aq. After 60 hours at 37°C fucosidase containing strains were induced with 5 mM rhamnose.

Minimal medium: citric acid 1.1 g/L, glycerol 10.8 g/L, KH2P04 10.0 g/L, (NH4)2S044.6 g/L, Na2S04 3 g/L, MgS04 * 7H20 1.5 g/L, Thiamin 0.02 g/L, Vitamin B12 0.0001 g/L.

Trace element solution: Na2-EDTA*2H204 g/L, CaS04*2H20 1 g/L, ZnS04*7H200.3 g/L, FeS04 ^* 7H20 3.7 g/L, MnS04 ^* H200.2 g/L, CuS04 ^* 5H20 0.15 g/L, Na2Mo04 ^* 2H20 0.04 g/L, Na2Se040.04 g/L.

Table 3

Table 3 shows the results after the indicated fermentation time at 37°C. After induction, the 2’,3- DiFL values for host cells expressing either of the two plasmids experience a dramatic drop compared to the control. The skillful optimization resulting in the artificial polypeptide of SEQ ID NO: 18 leads to a much stronger reduction of 2’,3-DiFL compared to the wildtype fucosidase CpAfc2. In the larger BioStat vessel the reduction of 2’-3-DiFL of the artificial CpAfc2_T 1 was higher than in the smaller volume of the Ambr vessel, indicating that the skillfully designed poly peptide of SEQ ID NO: 18 may be well suited for large scale processes.

As can be seen from table 3, some reductions of 2’-FL content were observed in this experi- ment, even temporarily before induction of the fucosidases expression at 24 hours for the wildtype CpAfc2 expressing strain. This may indicate that the reductions of 2-FL content were independent of the fucosidases action.

The average 2’,3-DiFL amount relative to the control after the induction is shown in Figure 6 B.

Literature cited

Baumgartner et al. (2013), Microb Cell Factories 12:40 Phipps et al, Food Chem Tox, 120, 2018, 552-565 Shuquan, F., et al. Journal of Basic Microbiology, 2016. 56(4): p. 347-357.

Takane Katayama et al. (2004) Journal of Bacteriology, Aug. 2004, p. 4885-4893

Sakumara et al JBC 287 (20) 16709-19, 2012

Sprenger, G. A. et al; Journal of Biotechnology, 2017, 258, 79

Toh, H et al; Genome announcements 2015, Mar-Apr; 3(2). WO 01/04341 WO 2007/101862 WO 2010/070104 A1 WO 2015/32412 A1

Zeuner, B., et al., Substrate specificity and transfucosylation activity of GH29 a-l-fucosidases for enzymatic produc-tion of human milk oligosaccharides. New Biotechnology, 2018. 41: p. 34-45. Egan, S.M. and R.F. Schleif, A Regulatory Cascade in the Induction of rhaBAD. Journal of Mo lecular Biology, 1993. 234(1): p. 87-98.