Specific α-1,2-fucosyltransferases for the biocatalytic synthesis of 2'-fucosyllactose

Abstract

The present invention relates to an enzyme characterized in that it is a fusion protein, which (i) comprises an N-terminal domain of a fucosyltransferase, and (ii) comprises at least amino acids 155 to 286 of SEQ ID NO: 5 or an amino acid sequence at least 80% identical thereto as a C-terminal domain, and has fucosyltransferase activity, the N-terminal domain and the C-terminal domain being derived from two different fucosyltransferases. The present invention further relates to a method for producing 2'-fucosyllactose, characterized in that lactose is present in a reaction mixture in the presence of at least one substance selected from the group of substances consisting of glucose, glycerol, sucrose, fucose, and GDP-, ADP-, CDP- and TDP-fucose that reacts with the enzyme.

Description

Technical Field

[0001] The present invention relates to an enzyme characterized by being a fusion protein, comprising (i) the N-terminal domain of fucosyltransferase and (ii) at least amino acids 155 to 286 of SEQ ID NO: 5, or an amino acid sequence having at least 80% identity thereto as the C-terminal domain, having fucosyltransferase activity, and the N-terminal domain and the C-terminal domain being derived from two different fucosyltransferases. The present invention further relates to a method for producing 2'-fucosyllactose, characterized in that lactose is present in a reaction mixture in the presence of at least one substance selected from the group consisting of glucose, glycerol, sucrose, fucose, and GDP-, ADP-, CDP-, and TDP-fucose.

Background Art

[0002] To date, approximately 200 different complex oligosaccharides, referred to as human milk oligosaccharides, or HMOs (plural) or HMO (singular) for short, have been identified in human milk. The high diversity results from various combinations of five monosaccharides, D-glucose, D-galactose, N-acetyl-D-glucosamine, L-fucose, and N-acetylneuraminic acid, resulting in simple and sometimes very complex oligosaccharides. Depending on which monosaccharides HMOs are formed from, fucosylated neutral HMOs, non-fucosylated neutral HMOs, and sialylated acidic HMOs are distinguished (Petschacher and Nidetzky 2016, J. Biotechnol. 235, pp. 61-83).

[0003] Unlike other important components of human milk, namely sugars such as lactose, lipids, and proteins, HMOs are not metabolized by infants. Rather, they play a crucial role in the development of a healthy gut microbiota, the prevention of infections, and the development of a healthy immune system. HMOs achieve these effects by providing beneficial bacteria that can metabolize HMOs, giving pathogens that cannot metabolize HMOs a growth advantage. Furthermore, they prevent pathogens from adhering to the intestinal wall by mimicking the sugar structure of epithelial cells to which pathogens bind, thereby saturating the pathogen's surface and ultimately leading to its excretion. Finally, after absorption from the intestines, HMOs also directly influence the gene regulation of intestinal epithelial cells and immune cells, thereby exerting systemic anti-inflammatory effects, particularly through cytokine expression (Faijes et al. 2019, Biotechnology Advances 37, pp. 667-697; Petschacher 2018, Die Hebamme 31, pp. 409-414).

[0004] Human milk is characterized by its high content of HMOs and their complex composition. In some cases, certain HMOs are present in significant amounts only in human milk. Therefore, although HMOs are also detected in other mammals, they are found only at very low concentrations. Thus, to achieve the beneficial properties mentioned above, HMOs are supplemented in human milk substitutes. Another emerging area of ​​use is as a dietary supplement for adults (Elison et al. 2016, Br.J.Nutr. 116, pp. 1356-1368).

[0005] The most common HMO in human milk is the trisaccharide 2'-fucosyl lactose, or 2'-FL for short (Deng et al. 2020, Syst. Microbiol. and Biomanuf. 1, pp. 1-14). 2'-FL consists of the monosaccharides D-glucose, D-galactose, and L-fucose, with D-galactose covalently bonded to D-glucose via a β-1,4-glycosidic bond and to L-fucose via an α-1,2-glycosidic bond (Fuc-α1,2-Gal-β1,4-Glc).

[0006] Enzymatic processes are an attractive option for the synthesis of HMOs such as 2'-FL, because the necessary selective bond formation would make chemosynthesis uneconomical. The regioselectivity and stereoselectivity of enzymes enable synthesis without protecting groups, which is economically advantageous, especially for more complex structures.

[0007] In the enzymatic catalysis of fucosylated HMOs, fucosyltransferases are commonly used. These belong to the glycosyltransferase enzyme family (GT; EC 2.4.) and catalyze the transfer of fucose units from a donor, usually guanosine diphosphate fucose, or simply GDP-fucose, to an acceptor, which can be an oligosaccharide, glycoprotein / protein, or glycolipid / lipid. The reactive group of the acceptor to which the fucosyltransferase transfers fucose determines the class of the fucosyltransferase. α-1,2-, α-1,3 / 4-, and α-1,6-fucosyltransferases are distinguished. The enzymatic synthesis of 2'-FL uses α-1,2-fucosyltransferase, which transfers the fucose from GDP-fucose to the 2'-hydroxyl group of a lactose, more precisely a galactose unit, resulting in the formation of an α-1,2-glycosidic bond. When using a nonspecific 1,2-fucosyltransferase for this purpose, 2'-FL is formed according to the following scheme (1), and in addition to the 3'-hydroxyl group of the glucose unit, fucosylation can also occur in a nonspecific manner, resulting in the formation of the byproduct 2,3-difucosyllactose, more precisely Fuc-α1,2-Gal-β1,4-(Fuc-α1,3-)Glc: (1) Lactose + GDP-fucose → 2'-FL + difucosyl lactose. In contrast, with specific 1,2-fucosyltransferases, 2'-FL is formed according to scheme (2) without the formation of undesirable byproducts. (2) Lactose + GDP-Fucose → 2'-FL

[0008] Fucosyltransferases belong to the glycosyltransferase class (EC 2.4), but these are just examples of enzymes in this class. Generally, glycosyltransferases catalyze the transfer of sugar molecules from a donor to an acceptor. Although sequence homology between different GTs is low, most GTs can be assigned to one of two structural superfamilies, namely GT-A and GT-B. What the two superfamilies have in common is that the enzyme consists of two domains linked to each other by a linker structure / sequence. The active site of the enzyme is formed from the region of the two domains and is located between them.

[0009] Each enzyme in the GT-A family has an N-terminal domain consisting of a β-pleated sheet surrounded by an α-helix (known as a Rothman fold), which is the domain that recognizes the donor, while the C-terminal domain mainly consists of a mixed β-pleated sheet and binds to the acceptor.

[0010] In contrast, GT-B family enzymes possess two Rossmann fold structures. The N-terminal domain forms the acceptor binding site, while the C-terminal structure is responsible for donor binding. Perhaps due to the lower variability of donor sugars compared to the broad range of acceptor sugars, the C-terminal domains of different glycosyltransferases in the GT-B family are more conserved than the N-terminal domains (Albesa-Jove et al. 2014, Glycobiology 24, pp. 108-124).

[0011] Due to conserved folding within structural superfamilies such as the GT-B family, it is possible to combine the domains of two different glycosyltransferases from the same structural superfamily. The exchange of similarly folded protein domains from different origins, also known as domain swapping, is a commonly used method in enzyme characterization and metabolic engineering, enabling the production of hybrid enzymes with novel properties (e.g., altered activity or substrate specificity) (Schmid et al. 2016, Front.Microbiol.7, 182, pp.1-7; Hansen et al. 2009, Phytochemistry 70, pp.473-482; Park et al. 2009, Biotechnol.Bioeng.102, pp.988-994; Truman et al. 2009, Chem.Biol.16, pp.676-685). However, the results of previous domain swapping experiments in glycosyltransferases have been inconsistent. On the one hand, the acceptor or donor specificity of glycosyltransferases was altered by the exchange of the corresponding domains (Truman et al. 2009, Chem. Biol. 16, pp. 676-685). On the other hand, both the C-terminal and N-terminal domains influence acceptor specificity, and as a result, predicting substrate specificity is usually impossible (Hansen et al. 2009, Phytochemistry 70, pp. 473-482). This is also because the active site of this enzyme class is certainly formed by the placement of the two domains adjacent to each other, and as a result, slight differences in the three-dimensional structure often lead to an inactive enzyme or at least a distorted binding site. Therefore, such experiments can, in principle, be expected to yield an enzyme that is inactive or lacks the desired specificity and reactivity.

[0012] In addition to specificity and activity, protein stability and solubility play a major role in fucosyltransferases. For example, in fucosyltransferase expression in E. coli, the formation of inclusion bodies (Lee et al. 2015, Microbiology and Biotechnology Letters 43, pp. 212-218) and low protein stability (Wang et al. 1999, Microbiology (Reading) 145, pp. 3245-3253) are frequently observed. Therefore, repeated attempts have been made to increase the solubility / stability and folding of fucosyltransferases. Similar to the co-expression of chaperones (Lee et al. 2015, Microbiology and Biotechnology Letters 43, pp. 212-218), there is the option of translational fusion of fucosyltransferase with quick-folding and highly soluble proteins, such as glutathione-S-transferase (GST) (Albermann et al. 2001, Carbohydr. Res. 334, pp. 97-103). Alternatively, solubility can be increased by adding charged amino acids, such as negatively charged aspartate tags (Chin et al. 2015, J. Biotechnol. 210, pp. 107-115). There are also approaches aimed at improving protein stability by using amino acid consensus sequences formed from multiple homologous proteins (Porebski and Buckle 2016, Protein Eng. Des. Sel. 29, pp. 245-251). This approach is based on the hypothesis that conserved amino acids contribute more to stability than unconserved amino acids, taking into account the evolutionary information represented by homologous sequences (Steipe et al. 1994, J. Mol. Biol. 240, pp. 188-192).

[0013] Prior to the present invention as described herein, the synthesis of 2'-FL using various α-1,2-fucosyltransferases had already been demonstrated by the time it was industrially implemented. These include, in particular, α-1,2-fucosyltransferase from Helicobacter pylori UA802 (futC, GenBank AF076779; European Patent No. 1243674, Kyowa Hakko, 1990), α-1,2-fucosyltransferase from Helicobacter mustelae NCTC12198 / ATCC43772 (futL, GenBank CBG40460.1; European Patent No. 1426441, Kyowa Hakko, 2001), α-1,2-fucosyltransferase from serotype O126 E. coli (wbgL, Engels and Elling 2014, Glycobiology 24, pp.170-178), and Bacteroides fragilis. This includes α-1,2-fucosyltransferase (wcfB, Chin et al. 2017, J. Biotechnol. 257, pp. 192-198) derived from H. fragilis. When comparing the above enzymes in terms of 2'-FL yield in batch fermentation of E. coli strains for 2'-FL production, the highest yield was obtained with futC derived from H. pylori UA802, thus suggesting the high activity of this enzyme (Huang et al. 2017, Metab. Eng. 41, pp. 23-38).

[0014] Several sequence modifications were also performed for more accurate characterization and optimization of the enzyme. Analysis of futC from H. pylori UA802 showed that shortening the N-terminus by using two alternative start codons (Δaa1-15, i.e., starting from amino acid M16, or Δaa1-46, i.e., starting from amino acid M47) resulted in complete loss of activity (Wang et al. 1999, Microbiology (Reading) 145, pp. 3245-3253). Similarly, inserting a resistance gene upstream of the conserved region aa163-173, more precisely downstream of aa152 in the futC gene, resulted in loss of function in the production of fucosylated Lewis Y antigen in H. pylori UA802 (Wang et al. 1999, Mol. Microbiol. 31, 1265-1274). These experiments demonstrated that the entire coding sequence is necessary for fucosyltransferase activity, and that even minor manipulations of the amino acid sequence can lead to complete inactivation of the enzyme.

[0015] Since futC derived from H. pylori UA802 accepts not only lactose but also monofucosylated sugars as substrates (Wang et al. 1999, Microbiology (Reading) 145, pp. 3245-3253), the synthesis of 2'-FL involves the formation of the byproduct difucosyl lactose (DFL) or lactodifucotetraose (LDFT), more precisely Fuc-α1,2-Gal-β1,4-(Fuc-α1,3-)Glc, (Yu et al. 2018, Microb. Cell. Fact. 17, 101, pp. 1-10) as a result of further fucosylation of the 3'-hydroxyl group of the glucose unit. This was also demonstrated for α-1,2-fucosyltransferase derived from H. pylori strain 26695 (Chin et al. 2017, J. Biotechnol. 257, pp. 192-198).

[0016] DFL formation in such production batches has a double negative impact on the efficiency of 2'-FL synthesis: not only is 2'-FL lost as a result of conversion to DFL, but the required activated fucose (GDP-fucose) is also consumed. Subsequently, the latter can no longer be used for 2'-FL synthesis. Furthermore, because the physical and chemical properties of 2'-FL and DFL are very similar, the formed DFL makes it difficult to process the fermentation culture into pure 2'-FL.

[0017] Therefore, in preferred embodiments, it is preferable to use a site- and substrate-specific α-1,2-fucosyltransferase for the specific fucosylation of the 2'-hydroxyl group of the galactose unit of lactose. An example that has been identified is the α-1,2-fucosyltransferase futL from H. mustelae NCTC12198 / ATCC43772, which specifically forms 2'-FL with a significant reduction in the synthesis of the byproduct DFL (European Patent No. 2877574, Glycosyn, 2015). Compared with futC from H. pylori UA802, it has also been used for the synthesis of 2'-FL as a result of the described high activity and specificity for lactose (European Patent No. 1426441, Kyowa Hakko, 2001). On the other hand, when futL and futC, which is similar to H. pylori UA1210, were directly compared in terms of 2'-FL yield under the same batch fermentation conditions (Huang et al. 2017, Metab. Eng. 41, pp. 23-38), it was shown that futL achieved only about 75% of the yield obtained with futC.

[0018] In the CAZY database (www.cazy.org / GT11_bacteria.html), both 1,2-fucosyltransferases, namely futC from H. pylori UA802 and futL from H. mustelae NCTC12198 / ATCC43772, are assigned to glycosyltransferase family 11 (GT-11) (Ma et al. 2006, Glycobiology 16, pp. 158-184). Regarding the GT-11 family, it was previously predicted by Breton et al. 2012, Curr. Opin. Struct. Biol. 22, pp. 540-549, that the enzymes classified within it could exhibit GT-B folding, and that this should remain a hypothesis (Petschacher and Nidetzky 2016, J. Biotechnol. 235, pp. 61-83). However, to date, it has been impossible to clearly assign members of the GT11 family to either the GT-B or GT-A folding family (Schmid et al. 2016, Front. Microbiol. 7, 182, pp. 1-7), which further makes accurate prediction of donor / acceptor specificity in domain swapping experiments impossible.

[0019] According to the presented prior art (see Scheme 2 for the definition of specific ones), the N-terminal domain of a particular futL from H. mustelae NCTC12198 / ATCC43772 is expected to be involved in acceptor binding and therefore in the fucosylation of lactose to 2'-FL without forming a byproduct DFL, given its proposed assignment to the GT-B superfamily. [Prior art documents] [Patent Documents]

[0020] [Patent Document 1] European Patent No. 1243674 [Patent Document 2] European Patent No. 1426441 [License 3] European Patent No. 2877574 [Non-licensed literature]

[0021] [Non-licensed Document 1] Petschacher and Nidetzky 2016,J.Biotechnol.235,pp.61-83 [Non-licensed Document 2] Faijes et al. 2019, Biotechnology Advances 37, pp. 667-697 [Non-licensed Document 3] Petschacher 2018, Die Hebamme 31, pp. 409-414 [Non-licensed Document 4] Elison et al. 2016, Br.J.Nutr.116, pp. 1356-1368 [Non-licensed Document 5] Deng et al. 2020, Syst.Microbiol.and Biomanuf.1, pp. 1-14 [Non-licensed Document 6] Albesa-Jove et al. 2014, Glycobiology 24, pp. 108-124 [Non-licensed Document 7] Schmid et al. 2016, Front.Microbiol.7, 182, pp. 1-7 [Non-licensed Document 8] Hansen et al. 2009, Phytochemistry 70, pp. 473-482 [Non-licensed Document 9] Park et al. 2009, Biotechnol.Bioeng.102, pp. 988-994 [Non-licensed Document 10] Truman et al. 2009, Chem.Biol.16, pp. 676-685

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[0022] The objective of the present invention was to improve the efficiency of biocatalytic synthesis of 2'-FL, that is, to achieve an increase in the yield of 2'-FL, while simultaneously avoiding the formation of a very similar by-product organism DFL, thereby facilitating the post-processing of 2'-FL and thus enabling the establishment of an economical industrial process. [Means for solving the problem]

[0023] This objective is achieved by providing an enzyme characterized by being a fusion protein, wherein (i) it comprises the N-terminal domain of a fucosyltransferase, and (ii) it comprises, as a C-terminal domain, an amino acid sequence identical to at least amino acids 155-286 of SEQ ID NO: 5, or at least 80%, preferably at least 90%, more preferably at least 95%, thereto, and it possesses fucosyltransferase activity, and the N-terminal domain and the C-terminal domain are derived from two different fucosyltransferases. Surprisingly, this fusion protein succeeded in combining the substrate specificity and positional specificity of α-1,2-fucosyltransferase futL with the potentially higher activity of enzyme futC. This was unpredictable from the prior art, because it was expected that the N-terminal domain of SEQ ID NO: 5, and not the C-terminal domain of SEQ ID NO: 5, would be involved in the specific binding of the substrate lactose to the fusion protein, preventing further fucosylation of 2'-FL to DFL. This provides an enzyme that can be used in an economically effective method for specifically fucosyling lactose to 2'-fucosyllactose. [Brief explanation of the drawing]

[0024] [Figure 1] Vector map of the expression plasmid pWC1. Summary of individual elements of the expression vector. Restriction sites of the enzymes EcoRI and XbaI used are also indicated. [Figure 2] E. coli K12 strain for producing 2-fucosyllactose. For the enzymatic synthesis of 2'-fucosyllactose using a specific 1,2-fucosyltransferase, the E. coli K12 strain was genetically modified to accept the substrate lactose via the transporter lacY but be unable to metabolize it. For this purpose, cds for lacA and lacZ were deleted from the genome. To enhance intracellular production of GDP-fucose from glucose in the de novo synthesis pathway, cds for the lon protease were deleted from the genome to prevent the proteolytic degradation of rcsA, the transcriptional activator of the gene for this specific de novo synthesis pathway. The level of rcsA can be further increased by plasmid overexpression. Deletion of wcaJ prevents the consumption of GDP-fucose for colonic acid synthesis, and as a result, activated fucose can be transferred to endogenous lactose by plasmid-encoded 1,2-fucosyltransferase. Expression of a specific 1,2-fucosyltransferase prevents the formation of the undesirable byproduct difucosyllactose (DFL) via fucosylation of 2'-fucosyllactose. [Figure 3] HPLC analysis of 2'-FL and DFL synthesis. HPLC analysis of the supernatant after fermentation shows the production of 2'-FL and, if present, DFL using FutC* / FutL, FutC, and FutL. Chromatograms of 2'-FL, lactose, and DFL standards are shown for comparison. [Figure 4] HPLC analysis of the synthesis of 2'-FL after complete lactose consumption. [Modes for carrying out the invention]

[0025] The fusion protein of the present invention constitutes a fusion of the amino acid sequences of an N-terminal domain (protein i) and a C-terminal domain (protein ii), the N-terminal and C-terminal domains being derived from two different fucosyltransferases. Wild-type proteins futL (futL from Helicobacter mustelae NCTC12198, SEQ ID NO: 5) or futC (for example, futC from H. pylori UA802, SEQ ID NO: 3) used as examples are not fusion proteins derived from two different fucosyltransferases and are therefore not included in the claims.

[0026] The term "fusion protein" means that corresponding amino acid sequences and / or coding DNA sequences have been fused in the laboratory and therefore do not occur in nature. Fusion proteins are also called hybrids or hybrid enzymes. For example, one may consist of the N-terminal domain of α-1,2-fucosyltransferase derived from the consensus sequence futC* based on the futC sequence of H. pylori UA802 and the C-terminal domain of the futL sequence of Helicobacter mustelae NCTC12198. Hybrid enzymes have high activity and specificity, efficiently transferring only fucose to lactose, for example, to the 2'-hydroxyl group of the galactose unit of the acceptor lactose, but not to the 3'-hydroxyl group of the glucose unit of lactose. Importantly, this significantly reduces the formation of the undesirable byproduct difucosyllactose. This makes the isolation of 2'-fucosyl lactose much easier and more economical and efficient, as it largely or completely eliminates the cumbersome purification process required to remove DFL.

[0027] A further advantage of using fusion proteins is that the acceptance of donors and acceptors can be altered by selecting the N-terminal and C-terminal domains, thereby enabling the more efficient production of more complex HMOs. Furthermore, higher enzymatic activity, such as that of futC, can be combined with better selectivity for acceptor substrates, such as that of futL.

[0028] To potentially combine higher futC activity with better selectivity of futL for acceptor substrates, the N-terminal domain of futC* (aa1-148 in SEQ ID NO: 7) was fused with the C-terminal domain of futL (aa142-286 in SEQ ID NO: 5), or vice versa, the N-terminal domain of futL (aa1-143 in SEQ ID NO: 5) was fused with the C-terminal domain of futC* (aa151-300 in SEQ ID NO: 7). To achieve this, a ScaI-(AGTACT) cleavage site was introduced into the linker sequence between the two enzyme domains by exchanging bases 426-427 (TA-CT) in the nucleic acid sequence of the futL gene (SEQ ID NO: 4) of the plasmid encoding futL, although this exchange did not alter the amino acid sequence. The introduction of this cleavage site allowed for the exchange of the N-terminal / C-terminal domain of futL with another suitable PCR amplification domain, such as futC*, by restriction digestion with a suitable restriction enzyme for terminal cleavage (EcoRI / XbaI) or a suitable restriction enzyme for interdomain cleavage (ScaI) using the help of this plasmid construct (see Example 2). The cds for futC* / futL (SEQ ID NO: 8) or futL / futC* (SEQ ID NO: 10) hybrids on the resulting low-copy expression plasmid were transcribed into an operon having an optimized RBS with the cds for rcsA (SEQ ID NO: 18).

[0029] The enzyme of the present invention has fucosyltransferase (FT) activity, that is, it catalyzes the transfer of fucose units from a donor such as GDP-, ADP-, CDP-, or TDP-fucose, preferably guanosine diphosphate fucose (GDP-fucose), which can be formed starting from glycerol, sucrose, glucose, or fucose, to an acceptor, the latter being an oligosaccharide, for example preferably lactose, or a glycoprotein, protein, glycolipid, or lipid.

[0030] To detect fucosyltransferase activity, the cds of a test protein with codon usage frequency optimized for E. coli are amplified by PCR using appropriate oligonucleotides and cloned by addition and cleavage sites into an expression plasmid such as pWC1, a low-copy expression plasmid downstream of a promoter, preferably an inducible promoter (see Example 2 in Figure 1).

[0031] Suitable promoters are all promoters known to those skilled in the art, such as constitutive promoters, such as the GAPDH promoter, or inductive promoters, such as the lac, tac, trc, T7, lambdaPL, ara, kmeto, or tet promoters, or sequences derived therefrom. Preferably, the promoter controlling the expression of the enzyme of the present invention is an inductive promoter, and more preferably an IPTG (isopropyl-β-D-thiogalactopyranoside)-induced promoter.

[0032] When the host cell is an E. coli cell, the cds of the E. coli endogenous transcription activator rcsA (SEQ ID NO: 18(DNA) / SEQ ID NO: 19(PRT)) for the de novo pathway of GDP-fucose, which have an optimized ribosome-binding site (RBS), are preferably polycistronically inserted into the completed expression plasmid downstream of each cds of fucosyltransferase to increase intracellular endogenous production of GDP-fucose in the de novo pathway.

[0033] Those skilled in the art can obtain strains that can deliver GDP-fucose or another activated fucose (e.g., ADP-, GDP-, CDP-, or TDP-fucose) as a donor into cells, for example, preferably a suitable E. coli strain, such as E. coli K12ΔwcaJΔlonΔsulA-lac-mod (see Example 1 and Figure 2), with an expression plasmid, thereby obtaining strains that differ only in the expressed fucosyltransferase futC (SEQ ID NO: 3), futC* (SEQ ID NO: 7), futL (SEQ ID NO: 5), futC* / futL hybrid (SEQ ID NO: 9), or futL / futC* hybrid (SEQ ID NO: 11), and therefore can be used for analysis of FT activity. For this purpose, the obtained strains that can deliver the donor into cells are cultured in the presence of a donor precursor such as glycerol, sucrose, glucose, or fucose, the strain can convert this to GDP-fucose intracellularly, and an acceptor such as lactose in the culture medium. If an inducible promoter is selected, cds are expressed constitutively or post-induction. To demonstrate the FT activity of the strain, the concentration of the fucosylated product 2'-FL will be determined by HPLC. For this purpose, at least approximately 160 cells will be used for OD. 600 A 1 ml aliquot is taken from the corresponding cell culture containing the specified material, and all solid components are then removed by centrifugation at maximum speed for 5 minutes, for example, using a bench centrifuge. The product content of the resulting supernatant is then quantified by HPLC, for example, as described in Example 4 (see also Figure 3).

[0034] The coding sequences (cds) of different fucosyltransferases used as starting sequences are known from the prior art and databases and can be optionally synthesized using codons optimized for a host organism, e.g., E. coli, or amplified from the genome of the original organism by PCR using appropriate oligonucleotides.

[0035] A coding sequence (cds) is a region of DNA or RNA located between a start codon and a stop codon that codes for the amino acid sequence of a protein.

[0036] CDS are surrounded by non-coding regions. What is called a gene is a portion of DNA that contains all the information necessary to produce biologically active RNA. Therefore, a gene includes not only the portion of DNA from which single-stranded RNA copies are produced by transcription, but also further portions of DNA that are involved in regulating this copying process.

[0037] Preferred expression signals that modulate the expression of cds for the enzyme of the present invention include at least one promoter, transcription start, translation start, ribosome binding site, and terminator. These are particularly preferably functional in the bacterial strain used, especially in E. coli. Thus, in the case of a functional promoter, the coding sequence under the regulation of this promoter may be transcribed into RNA.

[0038] Wild-type (wt) cds are a form of cds that arises naturally through evolution and is present in the wild-type genomes of organisms found in nature.

[0039] A domain or folding class refers to a region within a protein that has a stably folded, usually compact, tertiary structure. As described in detail in the prior art and as explicitly stated above, all GTs having GT-A or GT-B folds consist of an N-terminal domain and a C-terminal domain connected to each other by a linker structure / sequence, and the active site is formed from the regions of both domains. As an example, protein domains can be defined using the 3Dee database (dundee.ac.uk).

[0040] The names futL and futC refer to the corresponding wild-type fucosyltransferases, each consisting of an N-terminal and C-terminal domain, and possessing SEQ ID NO: 5 and / or SEQ ID NO: 3, respectively.

[0041] The term futC* refers to a sequence derived from futC having SEQ ID NO: 7. Similarly, it consists of two domains: an N-terminal domain and a C-terminal domain. futL, futC, and futC* are not fusion proteins.

[0042] On the other hand, the designation N / C refers to a ft-transferase (FT) that contains the N-terminal domain of one FT and the C-terminal domain of another FT. For example, futC* / futL contains the N-terminal domain of fucosyltransferase futC* (at least amino acids 1-129 or at least 80% of the same amino acid sequence as in SEQ ID NO: 7) and the C-terminal domain of fucosyltransferase futL (at least amino acids 155-286 or at least 80% of the same amino acid sequence as in SEQ ID NO: 5). Similarly, in futL / futC*, the N-terminal domain of futL and the C-terminal domain of futC* are fused.

[0043] futC* / futL(Δ8aa) and futC* / futL(Δ15aa) contain the N-terminal domain of futC* and the C-terminal domain of futL, with the C-terminal domains shortened by 8 amino acids and 15 amino acids, respectively.

[0044] Homologous amino acid sequences should be understood to mean sequences that are at least 80%, preferably at least 90%, and more preferably at least 95% identical, and each change in a homologous sequence is selected from the insertion, addition, deletion, and substitution of one or more amino acids.

[0045] The identity of amino acid sequences is determined by the "Protein blast" program, available on the publicly accessible webpage http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. The following general parameters are used as algorithm parameters for the alignment of two or more protein sequences: Maximum target sequence = 100; Short query = "Automatically adjust parameters for short input sequences"; Expected threshold = 10; Word size = 3; Maximum match in query range = 0. The default scoring parameters are Matrix = BLOSUM62; Gap cost = Presence: 11, Extension: 1; Composition adjustment = "Conditional composition score matrix adjustment". For homologous sequence identification, the above parameters were used to search the "Non-redundant protein sequences (nr)" database, but sequences from the organism Helicobacter pylori (taxid: 210) were excluded due to the high data density of homology > 80%.

[0046] α-1,2-, α-1,3 / 4-, and α-1,6-fucosyltransferases are distinguished. Preferably, the fusion protein of the present invention is an enzyme having α-1,2-fucosyltransferase activity.

[0047] Preferably, the enzyme is characterized in that the amino acid sequences of the N-terminal and C-terminal domains of the fusion protein are those of a microorganism, more preferably a Gram-negative bacterium, and particularly preferably a Helicobacter strain or a sequence homologous thereto.

[0048] In a preferred embodiment, the enzyme is characterized in that the amino acid sequences of the N-terminal and C-terminal domains of the fusion protein are sequences of the glycosyltransferase family 11 (GT-11).

[0049] More preferably, the enzyme is characterized in that the amino acid sequences of the N-terminal and C-terminal domains of the fusion protein are those of the species Helicobacter pylori or Helicobacter mustelae, or sequences homologous thereto. Particularly preferably, the fusion protein contains the C-terminal domain derived from futL of the organism Helicobacter mustelae NCTC12198 / ATCC43772 (SEQ ID NO: 5). The other N-terminal domain of the fusion protein is preferably derived from futC of the organism Helicobacter pylori UA802 (SEQ ID NO: 3).

[0050] In a preferred embodiment, the enzyme is characterized in that the N-terminal domain contains at least amino acids 1 to 129, more preferably at least amino acids 1 to 132, particularly preferably at least amino acids 1 to 148, or in each case, an amino acid sequence that is at least 80% identical thereto. In a particularly preferred embodiment, the enzyme is characterized in that the amino acid sequence of the N-terminal domain of the fusion protein is at least 80% identical to amino acids 1 to 129, more preferably amino acids 1 to 132, even more preferably amino acids 1 to 148, or an amino acid sequence that is at least 80% identical thereto.

[0051] In a preferred embodiment, the enzyme is characterized in that the C-terminal domain contains at least amino acids 155-286, more preferably at least amino acids 149-286, particularly preferably at least amino acids 142-286, or at least 80% identical to them, of SEQ ID NO: 5. Particularly preferred, the enzyme is characterized in that the amino acid sequence of the C-terminal domain of the fusion protein is at least 80% identical to amino acids 155-286, more preferably amino acids 149-286, even more preferably amino acids 142-286, or in each case, at least 80% identical to them.

[0052] The fusion protein is preferably at least 80% identical in amino acid sequence to SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or thereto. Particularly preferred is the fusion protein futC* / futL having SEQ ID NO: 9.

[0053] The 2'-FL yields after 65 hours of fermentation at 25°C from induction and total lactose input of 65 g / l (batch and continuous) showed that both futC and futC* formed 2'-FL and DFL, with the 2'-FL yield of futC being 70% higher than that of futC* (see Example 3, Table 1). As described in the literature, futL formed only 2'-FL. The 2'-FL yield of futL was 125% higher than that of futC* and 32% higher than that of futC.

[0054] Analysis of 2'-FL and DFL yields in fusion protein expression surprisingly showed, in contrast to prior art, that the fusion protein futC* / futL specifically converted 100% lactose and GDP-fucose to 2'-FL, rather than the mutant futL / futC*. Furthermore, Table 1 shows that the specific fusion protein futC* / futL increased 2'-FL yield by 56% compared to non-specific futC, 165% compared to futC*, and 18% compared to specific futL. This was unpredictable, firstly, because no usable 3D structure existed for either futC or futL to date, and secondly, because it was expected that there would be a fusion protein futL / futC* containing the N-terminal domain of lactose-specific futL, rather than futC* / futL, which specifically converts only lactose and GDP-fucose to 2'-FL, assuming a GT-B fold in the N-terminal domain responsible for acceptor substrate binding. Furthermore, the 2'-FL yield of futL / futC* was 70% lower compared to futC* / futL.

[0055] The yield of futC* (SEQ ID NO: 7), which was used for the fusion protein, was 41% lower compared to futC (SEQ ID NO: 3). Therefore, as previously described, the N-terminal domain of futC (aa1-148 of SEQ ID NO: 3) was ultimately fused with the C-terminal domain of futL (aa142-286 of SEQ ID NO: 5) to obtain futC / futL (SEQ ID NO: 12 (DNA) / SEQ ID NO: 13 (PRT)). The resulting expression plasmid was used to transform a strain suitable for 2'-FL production (E. coli K12ΔwcaJΔlonΔsulA-lac-mod) (see Examples 1 and 2). Under optimized fermentation conditions (27°C instead of 25°C, 86 g / l lactose instead of 65 g / l lactose), the 2'-FL and DFL yields of futC* / futL and futC / futL showed that DFL was not formed in this case either. However, the 2'-FL yield with the fusion protein futC / futL was 15% lower compared to the fusion protein futC* / futL (Table 1).

[0056] Nevertheless, in both cases, the fusion of the N-terminal domain of futC* and futC with the C-terminal domain of futL resulted in the selective production of 2'-FL without the formation of the very similar byproduct DFL.

[0057] Furthermore, the C-terminus of the hybrid enzymes futC* / futL was shortened by 8aa (aa1-285 in SEQ ID NO: 9) and 15aa (aa1-278 in SEQ ID NO: 9), respectively (see Example 2), and the 2'-FL yield after fermentation under optimized conditions (induction to 27°C, 86 g / l lactose) for 65 hours was similarly investigated. The 8aa shortening reduced the 2'-FL yield by 8%, but the 15aa shortening resulted in no detection of either 2'-FL or DFL (Table 1). This indicates that at least aa1-285 of the fusion protein futC* / futL are responsible for its activity.

[0058] The present invention further provides a method for producing 2'-fucosyl lactose, characterized in that lactose is present in a reaction mixture in the presence of at least one substance selected from the group consisting of glucose, glycerol, sucrose, fucose, and substances consisting of GDP-, ADP-, CDP-, and TDP-fucose, which react with the enzyme of the present invention. The substance converted to GDP-fucose in the cell is preferably glucose. The enzyme of the present invention is preferably futC* / futL having SEQ ID NO: 9. Particularly preferably, the enzyme is futC* / futL and the substance converted to GDP-fucose in the cell is glucose. In the method of the present invention, lactose can be completely converted without the formation of difucosyl lactose.

[0059] Preferably, the method for producing 2'-fucosyl lactose is characterized by the isolation of 2'-fucosyl lactose from the reaction mixture. To isolate 2'-fucosyl lactose, it is preferable that the solid components are removed from the reaction mixture in the first step by centrifugation or filtration. In subsequent steps, further impurities can be separated from the 2'-fucosyl lactose obtained, for example, by chromatography and filtration, and by evaporation.

[0060] In preferred embodiments, the method is characterized by the complete conversion of lactose without the formation of more than 5%, more preferably 2.5%, and particularly preferably 1.5% of DFL. In particularly preferred embodiments, lactose is completely converted without the formation of DFL. Thus, the method of the present invention has the major advantage that the specific formation of 2'-FL eliminates the need to remove DFL or other sugars such as lactose by crystallization, nanofiltration, or enzymatic post-treatment. Therefore, the selective production of 2'-FL significantly simplifies post-treatment.

[0061] Therefore, in a particularly preferred embodiment, the method is characterized in that 2'-fucosyl lactose is isolated without crystallization, nanofiltration and / or enzymatic post-treatment to remove other sugars such as glucose, lactose or difucosyl lactose.

[0062] In a preferred embodiment, the method for producing 2'-fucosyl lactose is characterized in that the reaction mixture is a culture of a microorganism that recombinantly expresses the enzyme of the present invention.

[0063] The cultivation of microorganisms is known in the prior art and can be carried out as described in Example 3, for example.

[0064] The microbial strain is particularly preferably the genetically modified E. coli K12 strain. Similarly, the recombinant enzyme expressed in the present invention is preferably the fusion protein futC* / futL or an amino acid sequence homologous thereto. Therefore, in a particularly preferred embodiment, the microbial strain is the genetically modified E. coli K12 strain, the recombinant enzyme expressed in the present invention is the fusion protein futC* / futL, and more preferably co-expression of rcsA.

[0065] If the reaction mixture is a microbial culture, it is preferable that 2'-fucosyl lactose be isolated from the culture supernatant. As already described above, solid components such as host cells are first separated by filtration, or more preferably by centrifugation. Further impurities can then be removed by chromatography, and the product can be obtained in crystalline form by enrichment.

[0066] As already described above, this method is preferably characterized by the complete conversion of lactose without the formation of DFL exceeding 5%, more preferably 2.5%, and particularly preferably 1.5%. In a particularly preferred embodiment, lactose is completely converted without the formation of DFL. Particularly preferably, 2'-fucosyllactose is isolated from the culture supernatant without crystallization, nanofiltration, and / or enzymatic post-treatment of the fermentation broth to remove glucose, lactose, and other sugars such as difucosyllactose from the culture supernatant.

[0067] Example 5 illustrates fermentation involving the complete conversion of lactose (see also Figure 4).

[0068] A method for producing 2'-fucosyllactose is preferably characterized by the formation of at least 4%, more preferably at least 10%, particularly preferably at least 25%, and even more preferably at least 50% more 2'-fucosyllactose with the fusion protein than with an unfused wild-type enzyme containing one domain in the fusion protein.

[0069] In a preferred embodiment, the method for producing 2'-fucosyl lactose is characterized by the formation of at least 47 g / l, preferably at least 53 g / l, and more preferably at least 60 g / l of 2'-fucosyl lactose during the reaction.

[0070] Preferably, the method for producing 2'-fucosyl lactose is characterized by the formation of less than 1 g / l, more preferably 0 g / l, of difucosyl lactose during the reaction. This means that the formation of DFL is particularly preferred, because in this case, the troublesome purification step to remove DFL can be omitted, making the isolation of 2'-fucosyl lactose much easier and therefore more economical and efficient.

[0071] In a preferred embodiment, the method for producing 2'-fucosyl lactose is characterized by the induction of enzyme expression.

[0072] In this case, the promoter that controls the expression of the enzyme of the present invention is an inducible promoter, and more preferably an IPTG (isopropyl-β-D-thiogalactopyranoside) promoter.

[0073] In this case, the method of the present invention has the advantage that the synthesis of the product does not begin until the moment of induction, which means that a high cell density is achieved first, thereby increasing the yield. [Examples]

[0074] The present invention will be described in more detail below with reference to exemplary embodiments, without being limited thereto.

[0075] All molecular biological methods used, including polymerase chain reaction (PCR), gene synthesis, DNA isolation and purification, DNA modification with restriction enzymes and ligases, and transformation, were carried out by methods known to those skilled in the art, methods described in the literature, or methods recommended by their respective manufacturers.

[0076] [Example 1] Development of strains based on E. coli K12 for the production of 2-fucosyllactose. A strain based on E. coli K12 was developed for the intracellular synthesis of fucosylated HMOs such as 2'-FL. First, the cds for undecaprenyl phosphate glucose phosphotransferase wcaJ were deleted from the genome. Next, the cds for lon protease were removed. For the lac operon, the cds for β-galactosidase (lacZ) and β-galactoside transacetylase (lacA) were deleted, while the cds for β-galactoside permease (lacY) were preserved. Finally, the cds for the cell division inhibitor sulA were deleted.

[0077] Deletion of cds for wcaJ, lon, and sulA using λ-recombinase by Datsenko and Warner (2000, Proc. Natl. Acad. Sci. USA. 97:6640-5) Due to the deletion of wcaJ from the genome of the E. coli K12 strain used, polymerase chain reaction (PCR) was initially performed using oligonucleotides wcaJ-del-fw (SEQ ID NO: 26) and wcaJ-del-rv (SEQ ID NO: 27) and a commercially available plasmid pKD3 (Coli Genetic Stock Center, CGSC: 7631) as the matrix to produce linear DNA fragments containing a chloramphenicol-resistant cassette, with approximately 50 base pairs each adjacent to the upstream and downstream regions of wcaJ cds.

[0078] Furthermore, E. coli strains were transformed with the commercially available plasmid pKD46 (CGSC:7739), and then competent cells were produced according to the details of Datsenko and Wanner. These cells were transformed with linear DNA fragments produced by PCR. Selection for integration of the chloramphenicol-resistant cassette (cat = chloramphenicol acetyltransferase) into the chromosome of E. coli K12 strain was performed using LB agar plates containing 20 mg / l chloramphenicol. Integration at the correct chromosomal position was verified by PCR using oligonucleotides wcaJ-check-fw (SEQ ID NO: 28) and wcaJ-check-rv (SEQ ID NO: 29), as well as chromosomal DNA from chloramphenicol-resistant cells as a matrix. This process yielded E. coli cells in which wcaJ cds were replaced with chloramphenicol-resistant cassettes.

[0079] Next, plasmid pKD46 was removed from the cells again according to the described procedure (Datsenko and Wanner), and the strain thus produced was named E. coli K12 wcaJ::cat.

[0080] The chloramphenicol-resistant cassette was removed from the chromosome of the E. coli K12wcaJ::cat strain using plasmid pCP 20 (CGSC:7629), which encodes FLP recombinase cds, following the Datsenko and Wanner procedure. The chloramphenicol-sensitive wcaJ deletion mutant obtained by this method was named E. coli K12ΔwcaJ.

[0081] Deletion of lon cds from E. coli K12ΔwcaJ strain was performed using the same method previously used for wcaJ cds deletion. However, for the production of linear DNA fragments using pKD3 (CGSG:7631) as the matrix, oligonucleotides lon-del-fw (SEQ ID NO: 30) and lon-del-rv (SEQ ID NO: 31) were used.

[0082] The integration of lon cds into the chromosome of the chloramphenicol-resistant E. coli K12ΔwcaJ strain was verified by the oligonucleotides lon-check-fw (SEQ ID NO: 32) and lon-check-rv (SEQ ID NO: 33), and by PCR on chromosomal DNA of chloramphenicol-resistant cells.

[0083] The chloramphenicol resistance cassette was removed from the chromosome again, as described by Datsenko and Wanner. The resulting strain, which lacked the chloramphenicol resistance cassette and was characterized by genomic deletions of wcaJ cds and lon cds, was named E. coli K12ΔwcaJΔlon.

[0084] Deletion of sulA cds from the E. coli K12ΔwcaJΔlon-lac-mod strain (constructed as described in the "Modification of the lac operon" section below) was performed using the same method previously used for wcaJ cds deletion. However, oligonucleotides sulA-del-fw (SEQ ID NO: 34) and sulA-del-rv (SEQ ID NO: 35) and pKD13 (CGSC:7633 GenBank seq.AY048744) were used as a matrix to generate linear DNA fragments containing the kanamycin resistance gene and with 50 homologous base pairs adjacent to each of the upstream and downstream regions of the sulA genomic cds.

[0085] Selection for incorporating the kanamycin-resistant cassette (kanR) into the chromosome of the E. coli K12ΔwcaJΔlon-lac-mod strain at the sulA cds position was first performed in LB agar plates containing 50 mg / l kanamycin. Subsequently, the integration was verified by PCR of oligonucleotides sulA-check-fw (SEQ ID NO: 36) and sulA-check-rv (SEQ ID NO: 37), as well as the chromosomal DNA of kanamycin-resistant cells.

[0086] Removal of the kanamycin-resistant cassette from the chromosome was performed in the same manner as for the chloramphenicol-resistant cassette, following the Datsenko and Wanner procedure. The strain obtained after removal of the kanamycin-resistant cassette was named E. coli K12ΔwcaJΔlonΔsulA-lac-mod.

[0087] To produce 2'-FL, the strain was transformed with an appropriate expression plasmid (see Example 2).

[0088] Modification of the lac operon by plasmid integration by Hamilton et al. (1989, J. Bacteriol. 171 (99:4617-4622)) For parallel deletions of lacZ and lacA from the lac operon lacZYA, which has a conserved operon structure containing a promoter, RBS, and start codon, as well as lacY cds, we used the homologous recombination method described by Hamilton et al. (1989).

[0089] This was performed by generating three linear DNA fragments (PCR1: lac-1-fw + lac-2-rv (SEQ ID NOs. 38, 39), PCR2: lac-3-fw + lac-4-rv (SEQ ID NOs. 40, 41), PCR3: lac-5-fw + lac-6-rv (SEQ ID NOs. 42, 43)) using multiple PCRs with duplicate oligonucleotides and wt E. coli K12 genomic DNA as a matrix, and then fusing them based on the overlapping region by two further polymerase chain reactions. For this purpose, the linear DNA fragments from PCR1 and PCR2 were first fused using primers lac-1-fw (SEQ ID NOs. 38) and lac-4-rv (SEQ ID NOs. 41) (PCR4), and then the resulting DNA fragment was ligated to the DNA fragment from PCR3 and oligonucleotides lac-7-fw (SEQ ID NOs. 44) and lac-8-rv (SEQ ID NOs. 45) (PCR5). The final linear DNA fragment contained a 515 bp homologous region downstream of lacA cds and lacY cds, and a 535 bp homologous region upstream of lacZ, with the fragment adjacent to BamHI cleavage sites at each end.

[0090] To clone the DNA fragments thus obtained into the temperature-sensitive vector pMAK700 (Hamilton et al., 1989, J. Bacteriol. 171 (99:4617-4622)), both the vector and the linear fragments were treated with the restriction enzyme BamHI. The vector fragments were dephosphorylated with alkaline phosphatase (rAPid Alkaline Phosphatase, Roche), purified by gel electrophoresis, then ligated, and used for transformation of competent Stellar coli (E. coli) cells (Takara, Shiga, Japan). Selection for plasmid-containing cells was based on the plasmid-encoded chloramphenicol resistance gene in LB agar containing chloramphenicol. Since the plasmid also contains a temperature-sensitive (ts) origin of replication (ori) that results in plasmid replication only possible at 30°C but not at 42°C, the cells were incubated at 30°C.

[0091] To modify the lac operon, the E. coli K12ΔwcaJΔlon strain (see above) was transformed with the vector pMAK700-lac-mod at 30°C. Chloramphenicol-resistant clones were cultured in LB medium containing chloramphenicol at 30°C, and then the cultures were plated onto LB agar containing chloramphenicol and incubated overnight at 42°C. This allowed for the selection of clones that incorporated the complete ts plasmid into the chromosome as a result of adjacent homologous regions downstream of lacA and upstream of lacZ, and thus allowed for the development of chloramphenicol resistance at high temperatures. Such clones were isolated and their correct plasmid integration was confirmed by control PCR with oligomers pMAK-fw (SEQ ID NO: 46) and lac-9-rv (SEQ ID NO: 47), or with lac-10-fw (SEQ ID NO: 48) and pMAK-rv (SEQ ID NO: 49). Since one primer on the plasmid (pMAK-fw / pMAK-rv) and the other primer on the chromosome (lac-9-rv / lac-10-fw) can undergo homologous bonding, the corresponding linear DNA fragment was formed only in the case of correct plasmid integration. The integrated strain was named E. coli K12ΔwcaJΔlon::pMAK700-lac-mod.

[0092] To remove the plasmid from the genome, a second recombination was required. Depending on how this was performed, two genomic variants of the strain were obtained. In the first case, the plasmid was recombined in the same way as the "in" recombination, resulting in the wild type again. Alternatively, the plasmid was recombined so that the modified locus remained in the genome and a plasmid with the wild-type locus was released. To degrade the plasmid from the genome, E. coli 12ΔwcaJΔlon::pMAK700-lac-mod was incubated in LB medium containing chloramphenicol at 42°C for 4 hours, then in LB medium without chloramphenicol at 30°C, and passed through multiple times. As a result of this process, some cells underwent sequential plasmid "out" recombination from the genome, and were able to lose the plasmid due to lack of selective pressure.

[0093] To isolate individual clones, diluted cultures were plated onto LB agar and incubated at 30°C. To confirm whether plasmids were lost in the clones, they were streaked onto LB agar with chloramphenicol. Finally, chloramphenicol-sensitive clones were confirmed for the desired genetic modification by PCR and sequencing using primers lac-11-fw (SEQ ID NO: 50) and lac-12-rv (SEQ ID NO: 51). The resulting strain was named E. coli K12ΔwcaJΔlon-lac-mod.

[0094] [Example 2] Cloning of fucosyltransferase cds futC, futC*, futL, hybrids, and truncated mutants for fermentation production of 2-fucosyllactose. Preparation of expression vectors: The expression vector used was pWC1, a low-copy plasmid. Based on the origin of pACYC replication, pWC1 is present in cells at approximately 10 copies per cell. A plasmid map is shown in Figure 1, showing the sequence indicated by Sequence ID No. 1, and the locations of conventional restriction enzymes (containing 6-nucleotide recognition sequences) are shown on the plasmid map.

[0095] The coding sequences (cds) of each enzyme were placed in this plasmid under the control of lactose and the IPTG-inducible promoter ptac. The vector contains restriction sites for the enzymes EcoRI and XbaI. Processing the plasmid with these enzymes formed a large fragment, particularly 4799 bp. This was isolated by agarose gel electrophoresis (QIAquick(R) Gel Extraction Kit, Quiagen) and treated with alkaline phosphatase (rAPid Alkaline Phosphatase, Roche) to avoid religation. This vector fragment was used for cloning various fucosyltransferases.

[0096] Cloning of cds for futC, futC*, and futL The cds for fucosyltransferases futC (SEQ ID NO: 2) and futC* (SEQ ID NO: 6), modified for optimal codon use of E. coli, were synthesized by GeneArt (Thermo Fisher, Regensburg), and futL (SEQ ID NO: 4) was synthesized by Genewiz (Leipzig). The cds encoding futC and futC* were PCR-amplified under standard conditions in two separate mixtures with primers futC / futC*-fw (SEQ ID NO: 20) and futC / futC*-rv (SEQ ID NO: 21), and the cds encoding futL were amplified in a third mixture with primers futL-fw (SEQ ID NO: 22) and futL-rv (SEQ ID NO: 23). These were used to introduce EcoRI or XbaI cleavage sites. Due to the substantial homology between futC cds and futC* cds, it was possible to use a single primer pair (futC / futC*-fw and futC / futC*-rv) for both of these constructs.

[0097] Subsequently, the corresponding PCR products were similarly treated with restriction enzymes EcoRI and XbaI, and then each was combined with a ligase mixture-enriched dephosphorylated vector fragment. The ligation mixtures were then transformed into competent Stellar ecoli (E. coli) cells (Takara, Shiga, Japan) using standard methods. Single colonies with successfully ligated plasmids were selected by tetracycline resistance. Several plasmids obtained from these colonies were analyzed by restriction pattern and sequencing. Finally, the correct plasmids were used for production experiments or further cloning. The obtained plasmids were pWC1-futC, pWC1-futC*, and pWC1-futL.

[0098] Introduction of ScaI-restricted transection sites to futL cds: First, the entire futL expression plasmid was amplified by PCR. The primer used here contained a novel restriction cleavage site (ScaI) in the cds of futL (SEQ ID NO: 4). The objective was to introduce a restriction cleavage site into the linker sequence between the two enzyme domains of fucosyltransferase without altering the amino acid sequence. Subsequently, the three restriction sites (EcoRI, ScaI, and XbaI) made it possible to replace the two domains with any desired alternative domain. The expression vector pWC1-futL was used as the matrix for the PCR reaction. The primers used were futL-Sca-fw (SEQ ID NO: 52) and futL-Sca-rv (SEQ ID NO: 53).

[0099] At the end of the PCR reaction, plasmid DNA was purified by chromatography, and then methylated matrix DNA was removed from the mixture by adding the restriction enzyme DpnI (10 units, NEB). The DpnI mixture was incubated at 37°C for 1 hour. Subsequently, DNA was purified by chromatography (Macherey & Nagel: NucleoSpin(R) Gel and PCR Clean-up-Kit) and transformed into competent Stellar coli (E. coli) cells (Takara, Shiga, Japan). Positive clones were selected as described above. The vector was named pWC1-futL(ScaI).

[0100] Cloning of fusion proteins futL / futC*, futC* / futL, and futC / futL cds: For the cloning of the hybrid enzyme, the plasmid pWC1-futL(ScaI) was treated with restriction enzymes ScaI and XbaI. A vector backbone fragment of approximately 5243 bp contained the N-terminal portion of futL cds (SEQ ID NO: 4). This fragment was dephosphorylated and enriched by agarose gel electrophoresis.

[0101] In parallel with this, PCR was performed using the primers C-futC*-fw and C-futC*-rv (SEQ ID NOs. 54 and 55), and the vector pWC-1-futC* as the matrix. The PCR product mainly consisted of the C-terminal domain of futC* (SEQ ID NO. 6).

[0102] At the end of the PCR reaction, the DNA was treated with restriction enzymes ScaI and XbaI, purified by chromatography, and then used in the ligation mixture along with the plasmid fragment. The ligation mixture was then transformed into competent Stellar coli (E. coli) cells (Takara, Shiga, Japan) using standard methods. Single colonies with successfully ligated plasmids were selected by tetracycline resistance. Several plasmids obtained from these colonies were analyzed by restriction pattern and sequencing. Finally, the correct plasmid was used for production experiments or further cloning.

[0103] The obtained plasmid was named pWC1-futL / futC*.

[0104] Similarly, to produce the futC* / futL hybrid (SEQ ID NO: 8 (DNA) / SEQ ID NO: 9 (PRT)), the vector pWC1-futL(ScaI) was prepared by treatment with the restriction enzymes EcoRI and ScaI. The resulting vector fragment, approximately 5240 bp in length, contained the C-terminal domain of futL cds (SEQ ID NO: 4).

[0105] Similar to the previous hybrid cloning, the N-terminal domain of futC*cds (SEQ ID NO: 6) was amplified by PCR. The vector pWC1-futC* again served as the template, and the primer pairs used were N-futC*-fw (SEQ ID NO: 56) and N-futC*-rv (SEQ ID NO: 57).

[0106] After dephosphorylation and enrichment, the vector fragment was ligated with a PCR product similarly enriched with restriction enzyme (EcoRI / ScaI), and the mixture was transformed into competent Stellar ecoli (E. coli) cells (Takara, Shiga, Japan) using standard methods. The desired hybrid plasmid was isolated as described above. The resulting plasmid was named pWC1-futC* / futL.

[0107] Similarly, the hybrid construct futC / futL (SEQ ID NO: 12(DNA) / SEQ ID NO: 13(PRT)) was cloned from the vector pWC1-futL(ScaI).

[0108] As described above, a vector fragment was generated from the C-terminal portion of futL cds, PCR products were generated as described later, and the two were further processed and ligated as described above. The matrix used for PCR was the vector pWC1-futC containing cds for futC (SEQ ID NO: 2), and the primer pairs used were N-futC*-fw (SEQ ID NO: 56) and N-futC*-rv (SEQ ID NO: 57).

[0109] The obtained plasmid was named pWC1-futC / futL.

[0110] Cloning of fucosyltransferase expression plasmids using rcsA To increase the de novo synthesis of activated fucose (GDP-fucose) in E. coli, endogenous E. coli cds were directly cloned to the C-terminus of each fucosyltransferase in an operon containing a fucosyltransferase, following an optimized Shine-Dalgarno sequence (AGGAGGU;SDS) and then RcsA (SEQ ID NO: 18). For this purpose, cds were amplified from rcsA using primers rcsA-fw (SEQ ID NO: 24) and rcsA-rv (SEQ ID NO: 25), which were used to introduce NheI or XbaI cleavage sites. Genomic DNA from E. coli K12 served as the matrix.

[0111] Similar to the cloning of fucosyltransferase expression vectors, the latter, namely pWC1-futL, pWC1-futC, pWC1-futC*, pWC1-futL / futC*, pWC1-futC* / futL, and pWC1-futC / futL, were treated with restriction enzyme XbaI, dephosphorylated, and enriched by agarose gel electrophoresis. The rcsA PCR products were treated with restriction enzymes NheI and XbaI. Subsequently, DNA was chromatographically purified (Macherey & Nagel: NucleoSpin(R) Gel and PCR Clean-up-Kit). For the ligation mixture, each enriched dephosphorylated vector fragment was combined with the enriched PCR product. Then, using standard methods, the ligation mixture was transformed into competent Stellar coli (E. coli) cells (Takara, Shiga, Japan). Single colonies with successfully ligated plasmids were selected by tetracycline resistance. Several plasmids obtained from these colonies were analyzed by restriction pattern and sequencing. Finally, the correct plasmids (pWC1-futC*-rcsA, pWC1-futC-rcsA, pWC1-futL-rcsA, pWC1-futC* / futL-rcsA, pWC1-futL / futC*-rcsA, pWC1-futC / futL-rcsA) were used for production experiments or further cloning.

[0112] Cloning of shortened futC* / futL mutants: To clone the 8aa shortened futC* / futL mutant futC* / futL (Δ8aa), in separate PCRs, the cds of a pWC1-fuc* / futL-based futC* / futL hybrid (SEQ ID NO: 8) were first amplified using primers futC*-short-fw (SEQ ID NO: 58) and futC*-short8-rv (SEQ ID NO: 59), and for rcsA (SEQ ID NO: 18), the cds based on pWC1-futC-rcsA were amplified using primers rcsA-2-fw (SEQ ID NO: 60) and rcsA-2-rv (SEQ ID NO: 61). Subsequently, the resulting linear DNA fragments were fused with primers futC*-short-fw (SEQ ID NO: 58) and rcsA-2-rv (SEQ ID NO: 61) in further PCR using terminal homologous oligonucleotides futC*-short8-rv (SEQ ID NO: 59) and rcsA-2-fw (SEQ ID NO: 60). The final linear DNA fragment contained EcoRI cleavage sites, futC* / futL cds(Δ8aa) (SEQ ID NO: 14), RBS, rcsA cds, and XbaI cleavage sites.

[0113] Linear DNA fragments of the 15aa shortened futC* / futL mutant futC* / futL (Δ15aa) were cloned using the same method, but instead of futC*-short8-rv (SEQ ID NO: 59), the oligonucleotide futC*-short15-rv (SEQ ID NO: 62) was used, and instead of rcsA-2-fw (SEQ ID NO: 60), rcsA-3-fw (SEQ ID NO: 63) was used. The final linear DNA fragments contained EcoRI cleavage sites, futC* / futL cds (Δ15aa) (SEQ ID NO: 16), RBS, rcsA cds, and XbaI cleavage sites.

[0114] Finally, both linear DNA fragments were treated with EcoRI and XbaI, and then ligated with enriched dephosphorylated vector fragments (pWC1 cleaved with EcoRI and XbaI, see above), respectively, to transform competent Stellar ecoli (E. coli) cells (Takara, Shiga, Japan). Single colonies of the ligated plasmids were selected based on the introduced tetracycline resistance. The plasmids were analyzed by restriction pattern and sequencing before use in 2'-FL production experiments to demonstrate fucosyltransferase activity. Plasmids pWC1-futC* / futL(Δ8aa)-rcsA and pWC1-futC* / futL(Δ15aa)-rcsA were obtained.

[0115] [Example 3] Effect of different fucosyl lactose transferases on the fermentation production of 2-fucosyl lactose and difucosyl lactose in a 1L fermenter. 30 mL of LB medium (3% peptone, 0.5% yeast extract, 0.5% NaCl) in a 300 mL baffled Erlenmeyer flask was inoculated using an inoculation loop with single clones of the E. coli K12ΔwcaJΔlonΔsulA-lac-mod strain produced in Example 1, which had been transformed with the respective product plasmids obtained from Example 2 (pWC1-futL-rcsA, pWC1-futC-rcsA, pWC1-futC*-rcsA, pWC1-futC* / futL-rcsA, pWC1-futC* / futL-rcsA, pWC1-futC* / futL(Δ8aa)-rcsA, pWC1-futC* / futL(Δ15aa)-rcsA), from a pre-seed, densely coated LB agar plate. After incubation in a bacterial shaker (145 rpm, 30°C) for 4.5-5 hours, OD 600 It was 1.5-3.0 (OD 600(This refers to the optical density determined spectrophotometrically at 600 nm). For fermentation in a Sartorius Biostat B-DCU research fermenter, 6–13 ml of pre-culture was transferred to the culture medium initially loaded into the fermenter in each case. The initial volume after inoculation was approximately 1 L.

[0116] The fermentation medium contained the following components: 1 g / l NaCl, 150 mg / l FeSO4·7H2O, 2 g / l trisodium citrate dihydrate, 10 g / l KH2PO4, 5 g / l (NH4)2SO4, 1.5 g / l HighExpress II (Kerry), 1.0 g / l Amisoy (Kerry), 0.5 g / l Hy-Yeast 412 (Kerry), and 10 ml / l trace element solution (the H2O solution of these components was initially charged into the fermenter and autoclaved at 121°C for 20 minutes). The trace element solution consisted of 150 mg / l Na2MoO4·2H2O, 300 mg / l H3BO3, 200 mg / l CoCl2·6H2O, 250 mg / l CuSO4·5H2O, 1.6 g / l MnCl2·4H2O, and 1.35 g / l ZnSO4·7H2O. The pH of the medium was adjusted to 6.8 by pumping in a 25% NH4OH solution. Then, 15 g / l glucose, 1.2 g / l MgSO4·7H2O, 225 mg / l CaCl2·2H2O, 5 mg / l vitamin B1, and 20 mg / l tetracycline were added from appropriate stock solutions under sterile conditions, and the inoculum was then transferred from the shaking flask to the fermenter.

[0117] During fermentation, the culture was stirred at 400-1500 rpm and a constant 2 slpm of air supplied through a sterile microbial filter was aerated. The oxygen partial pressure was maintained at 50% by adjusting the stirring speed. In the later stages of the exponential phase, the supply air of pure O2 needed to be enriched to a 32% O2 content to ensure the desired nominal value of 50% for the O2 partial pressure in the culture medium. The pH was maintained at 6.8 by automatic correction with a 25% NH4OH solution or a 20% H3PO4 solution. The temperature was initially 30°C and gradually decreased from 30°C to 25°C over 30 minutes 30 minutes before induction. The temperature was then maintained at 25°C until the end of fermentation (65 hours). Excessive foaming was prevented by the addition of an automatically controlled defoaming agent (Struktol J673, Schill & Seilnacher, 10% (v / v) in H2O).

[0118] Depending on the stage of fermentation, glucose and lactose were added via two separate (sterile) supply solutions. The glucose content was determined using a YSI glucose analyzer. In the first stage after inoculation, the glucose in the initially charged culture medium was consumed. In the second stage, which began approximately 10 hours after the start of fermentation, a 60% (w / w) glucose supply solution containing additives (660.2 g / kg of glucose monohydrate (Biesterfeld-Spezialchemie), 2.5 g / kg of vitamin B1 from a 5 g / l stock solution, 3.65 g / kg of CaCl2·2H2O from a 147 g / l stock solution, 12.31 g / kg of MgSO47·H2O from a 240 g / l stock solution, 4.04 g / kg of trace element solution (see above), and 317.3 g / kg of desalted water) was continuously supplied to the culture with a glucose concentration of 0 g / l, with the aim of supplying the culture with an unlimited supply of glucose. In the third stage, characterized by the complete consumption of a continuous glucose supply, the continuous addition of glucose supply decreased to a constant 9.2 g / L / h approximately 18.5 hours after inoculation until the end of fermentation (65 hours), and the expression of 1,2-fucosyltransferase and rcsA, respectively, was induced by the addition of 0.25 mM IPTG. In parallel, at the start of the third stage, 20 g / l lactose was added in batches from a 25% (w / w) lactose solution (263 g / kg α-D-lactose monohydrate (abcr)) dissolved in 737 g / kg desalted H2O, and then continuously supplied with a 25% (w / w) lactose solution at a rate of 4 g / l / h maintained until the end of fermentation. Thus, a total of 65 g / l of lactose was added based on a starting volume of 1 L.

[0119] In the alternative mixture (mixture 2) for 2'-FL fermentation production, the temperature was gradually lowered to 27°C over 30 minutes, 30 minutes before the start of induction. During induction, 30 g / l of lactose was added in batches, and a continuous supply of 5 g / l / h of 25% (w / w) lactose solution was maintained until the end of fermentation. This amounted to a total of 86 g / l of lactose based on an initial volume of 1 L.

[0120] The 2'-FL, 3'-FL, DFL, and lactose content of the culture medium after 65 hours was determined by chromatography from the cell-free supernatant of the sample, as described in Example 4, and summarized in Table 1 in g / l.

[0121] Table 1: Comparison of different fucosyltransferase activities in relation to the yield of 2'-FL and DFL.

[0122] [Table 1]

[0123] [Example 4] HPLC analysis of fermentation production of 2-fucosyl lactose To determine the 2'-FL, 3'-FL, DFL, and lactose content in the culture medium by chromatography, 1 ml of culture broth was fermented for 65 hours and then centrifuged at 13,000 rpm for 5 minutes in a benchtop centrifuge. The clear supernatant was diluted 1:2 to 1:5 with desalted water. Then, 300 μl of the dilution was filtered into an HPLC reservoir using a 0.2 μm syringe filter.

[0124] For analyte separation, a TSKgel amide-80 column (Tosoh Bioscience, 250 mm × 4.6 mm; particle size 5 μm) and a corresponding guard column (TSKgel Guardgel amide-80, Tosoh Bioscience, 15 mm × 3.2 mm) were used in an Agilent 1200 / 1260 HPLC system equipped with the following modules: binary pump, degasser, autosampler, thermostatic column oven, and 1260 RI detector. The column oven temperature was 30°C. The eluent used was a degassed mixture of H2O (30%) and acetonitrile (70%). After column equilibration, the prepared sample was injected in 10 μl increments from an autosampler cooled to 15°C. Elution was then performed isocratically at a flow rate of 1 ml / min for 25 minutes. For detection, a Bruker Daltonics RI detector (temperature 35°C) and a Q-TOF Impact II mass spectrometer were used.

[0125] Peaks were assigned to the analytes based on the retention times of the standard solutions (2'-FL: 15.4 min, 3'-FL: 17.3 min; DFL: 22.3 min; lactose: 12.2 min). The concentrations of the analytes in g / l were finally determined by integrating the peak areas, taking into account each dilution, and using the standard calibration curve.

[0126] [Example 5] Complete fucosylation of lactose to 2'-fucosyllactose in a 1L fermenter, without the formation of difucosyllactose.

[0127] As described in Example 3, the E. coli K12ΔwcaJΔlonΔsulA-lac-mod strain, which produces a production plasmid encoding a fusion protein homologous to mixture 2, futC* / futL, was transformed and fermented. Deviating from Example 3, the lactose supply was terminated after 65 hours, but the continuous addition of glucose was maintained. After 88 hours, fermentation was terminated, and the sugars present in the culture supernatant were determined by HPLC, as before. The formation of 67 g / l 2'-FL along with 0 g / l DFL and 0 g / l residual lactose indicates that the fusion protein can achieve complete conversion of lactose to 2'-FL without the formation of byproduct DFL, meaning that no processing steps to remove lactose and difucosyl lactose are required during subsequent post-processing.

[0128] Abbreviation aa: amino acids ΔZaa: Z amino acid deletion, Z indicates the number of deleted amino acids. OD 600 Optical density at a wavelength of 600 nm RBS: Ribosome binding site FT: Fucosyltransferase GT: Glycosyltransferase nRIU: nano-refractive index unit

[0129] HPLC analysis of the supernatant after fermentation, using enzymes homologous to FutC* / FutL, shows the production of 2'-FL and -DFL (if present), as well as residual lactose.

Claims

1. An enzyme characterized by being a fusion protein, i) The N-terminal domain contains at least amino acids 1 to 129 of SEQ ID NO: 7, or an amino acid sequence that is at least 90% identical thereto, ii) The C-terminal domain contains at least amino acids 155-286 of SEQ ID NO: 5, or an amino acid sequence that is at least 90% identical thereto, Possesses fucosyltransferase activity, The N-terminal domain and the C-terminal domain are enzymes derived from two different fucosyltransferases.

2. The enzyme according to claim 1, characterized in that the amino acid sequences of the N-terminal and C-terminal domains of the fusion protein are, respectively, the amino acid sequences of the N-terminal and C-terminal domains of a protein derived from a Gram-negative bacterium, or at least 90% identical thereto, and the protein derived from the Gram-negative bacterium has fucosyltransferase activity.

3. The enzyme according to one or more claims 1 and 2, characterized in that the amino acid sequences of the N-terminal and C-terminal domains of the fusion protein are, respectively, the amino acid sequences of the N-terminal and C-terminal domains of a protein derived from a strain of Helicobacter bacteria, or at least 90% identical thereto, and the protein derived from the strain of Helicobacter bacteria has fucosyltransferase activity.

4. The enzyme according to one or more of claims 1 to 3, characterized in that the amino acid sequence of the N-terminal domain of the fusion protein is at least 90% identical to amino acids 1 to 148 of SEQ ID NO:

7.

5. The enzyme according to one or more of claims 1 to 4, characterized in that the amino acid sequence of the C-terminal domain of the fusion protein is at least 90% identical to amino acids 142 to 286 of SEQ ID NO:

5.

6. The enzyme according to one or more of claims 1 to 5, characterized in that the fusion protein has an amino acid sequence that is at least 90% identical to SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, or thereto.

7. A method for producing 2'-fucosyl lactose, characterized by reacting at least one substance selected from the group consisting of glucose, glycerol, sucrose, fucose, and substances comprising GDP-, ADP-, CDP-, and TDP-fucose with at least one enzyme described in one or more of claims 1 to 6, in the presence of lactose.

8. The method according to claim 7, characterized in that lactose is completely converted without the formation of more than 5% difucosyl lactose.

9. The method according to one or more claims 7 and 8, characterized in that the reaction is carried out in a culture medium containing a culture of a microorganism that recombinantly expresses the enzyme.

10. The method according to claim 9, characterized in that 2'-fucosyl lactose is isolated from the culture supernatant.

11. The method according to one or more claims 7 to 10, characterized in that 2'-fucosyl lactose is formed by the enzyme according to one or more claims 1 to 6, in an amount at least 4% greater than that formed by the unfused wild-type enzyme, which contains either the N-terminal domain or the C-terminal domain contained in the enzyme according to one or more claims 1 to 6 and has fucosyltransferase activity.

12. The method according to one or more of claims 7 to 11, characterized in that at least 47 g / l of 2'-fucosyl lactose is formed in the reaction.

13. The method according to one or more of claims 7 to 12, characterized in that difucosyl lactose of less than 1 g / l is formed in the above reaction.

14. The method according to claim 13, characterized in that 0 g / l difucosyl lactose is formed in the above reaction.

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