IDENTIFICATION OF AN alpha-1,2-FUCOSYLTRANSFERASE FOR THE IN VIVO PRODUCTION OF PURE LNFP-I

Abstract

The present disclosure discloses the identification and introduction of a specific heterologous gene (denoted as smob), which encodes an -1,2-fucosyltransferase, into an LNT production strain to produce LNFP-I in particular.

The smob gene originates from the organism Sulfuriflexus mobilis (https://www.dsmz.de/collection/catalogue/details/culture/DSM-102939), which is a sulfur-oxidizing bacterium isolated from a brackish lake sediment.

FIG. 1 should accompany the abstract.

Claims

1. A method for producing one or more fucosylated human milk oligosaccharides (HMOs), the method comprising the steps of, a. providing a genetically engineered cell capable of producing LNFP-I, wherein said cell comprises a recombinant nucleic acid encoding an -1,2-fucosyl-transferase protein as shown in SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to SEQ ID NO: 1, wherein said -1,2-fucosyl-transferase is capable of transferring of a fucosyl moiety from a donor molecule onto a galactose moiety of LNT through an -1,2 coupling, b. culturing the cell according to (a) in a suitable cell culture medium to express said nucleic acid; and c. harvesting the one or more HMOs produced in step (b).

2. The method according to claim 1, wherein the one or more fucosylated HMOs are selected from the group consisting of 2-FL, LNFP-I, DFL and LNDFH-I.

3. The method according to claim 1, wherein the method predominantly produces LNFP-1.

4. The method according to claim 1, wherein the one or more fucosylated HMOs comprise LNFP-I and 2-FL, and wherein the method produces a molar ratio of LNFP-I:2-FL in the harvested HMOs in step c) in the range of 20:1-2:1.

5. The method according to claim 1, wherein the one or more fucosylated HMOs comprise LNFP-I, and wherein the method produces a molar ratio of LNFP-I:LNT in the harvested HMOs in step c), in the range of 1000:1 to 10:1.

6. The method according to claim 1, wherein said genetically engineered cell comprises a heterologous nucleotide sequence encoding a heterologous polypeptide capable of hydrolyzing sucrose into fructose and glucose which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell, and wherein the polypeptide capable of hydrolyzing sucrose into fructose and glucose is SacC_AagI or Bff (GenBank accession IDs: WP_103853210.1 and BAD18121.1) or a functional homologue thereof having a amino acid sequence which is at least 80% identical to any one of SEQ ID NOs: 13 or 14.

7. The method according to claim 1, wherein the genetically engineered cell is cultured in sucrose as sole carbon and energy source.

8. The method according to claim 1, wherein the genetically engineered cell comprises a heterologous nucleotide sequence encoding a MFS transporter protein selected from Nec and YberC (GenBank accession ID WP_092672081.1 and GenBank accession ID EEQ08298.1) or a functional homologue thereof having an amino acid sequence which is at least 80% identical to any one of SEQ ID NOs: 4 or 5.

9. A genetically engineered cell capable of producing LNFP-I comprising a recombinant nucleic acid sequence encoding an -1,2-fucosyltransferase protein as shown in SEQ ID NO: 1, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to SEQ ID NO: 1, wherein said -1,2-fucosyl-transferase is capable of transferring of a fucosyl moiety from a donor molecule onto a galactose moiety of LNT through an -1,2 coupling.

10. The genetically engineered cell according to claim 9, wherein the predominant fucosylated HMO produced by the cell is LNFP-I.

11. The genetically engineered cell according to claim 9, wherein the cell further comprises a nucleic acid sequence encoding a -1,3-N-acetyl-glucosaminyltransferase protein and a -1,3-galactosyltransferase protein.

12. The genetically engineered cell according to claim 9, further comprising a recombinant nucleic acid sequence encoding the MFS transporter protein Nec or YberC (GenBank accession ID WP_092672081.1. and GenBank accession ID EEQ08298.1.) or a functional homologue thereof having an amino acid sequence which is at least 80% identical to any one of SEQ ID NOs: 4 or 5.

13.-15. (canceled)

16. The genetically engineered cell according to claim 9, wherein the cell is selected from the group consisting of E. coli, C. glutamicum, L. lactis, B. subtilis, S. lividans, P. pastoris, and S. cerevisiae.

17. The genetically engineered cell according to claim 9, wherein the expression of the recombinant nucleic acid is regulated by a promoter sequence selected from the group consisting of SEQ ID NO: 16-38 and 39.

18. (canceled)

19. The genetically engineered cell according to claim 17, wherein said promoter sequence is PglpF.

20. The genetically engineered cell according to claim 15, wherein said genetically engineered cell is capable of utilizing sucrose as sole carbon and energy source.

21. The genetically engineered cell according to claim 20, wherein the sucrose utilization system is a polypeptide capable of hydrolyzing sucrose into fructose and glucose selected from the group consisting of the GenBank accession IDs: WP_103853210.1 and BAD18121.1, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to any one of SEQ ID NOs: 13 or 14.

22. A nucleic acid construct comprising a recombinant nucleic acid sequence which is at least 80% identical to SEQ ID NO: 2.

23. The nucleic acid construct according to claim 22, further comprising one or more recombinant nucleic acid sequence(s) comprising a regulatory element with a promoter sequence selected from the group consisting SEQ ID NO: 16-38 and 39.

24.-27. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0332] FIG. 1 The choice of the -1,2-fucosyltransferase that is introduced in an E. coli DH1 K12 strain producing LNT has a large impact on the HMO content of the final HMO blend. % change in the final LNFP-I titer of fucT54- and smob-expressing cells relative to cells expressing futC.

[0333] FIG. 2 LNFP-I to 2-FL molar ratios for strains expressing the FutC, FucT54 or Smob enzymes.

[0334] FIG. 3 Molar % fraction of each HMO in the final HMO blend acquired by strain MP1 expressing the futC gene.

[0335] FIG. 4 Molar % fraction of each HMO in the final HMO blend acquired by strain MP2 expressing the fucT54 gene.

[0336] FIG. 5 Molar % fraction of each HMO in the final HMO blend acquired by strain MP3 expressing the smob gene.

[0337] FIG. 6 The final HMO titers for smob-expressing strains that bear a genomic copy of the nec (strains MP5 and MP7) or yberC (strain MP6) genes, shown relative to the final HMO titers of smob-expressing cells that do not express an MFS transporter (strain MP4), as revealed by the analysis of total samples. The reference level (given as 100%) is shown for strain MP4.

[0338] FIG. 7 Fraction of LNFP-I (in molar %) in the final HMO blend for smob-expressing cells that do not express a MFS transporter (strain MP4) and cells that bear a genomic copy of the nec (strains MP5 and MP7) or yberC (strain MP6) genes, as revealed by the analysis of total samples.

[0339] FIG. 8 The final HMO titers for smob-expressing strains that bear a genomic copy of the nec (strains MP5 and MP7) or yberC (strain MP6) genes, shown relative to the final HMO titers of smob-expressing cells that do not express an MFS transporter (strain MP4), as revealed by the analysis of the supernatant fraction of the corresponding cultures. The reference level (given as 100%) is shown for strain MP4.

[0340] FIG. 9 Fraction of LNFP-I detected in the supernatant (in % of total LNFP-I) in cultures of smob-expressing cells that do not express a MFS transporter (strain MP4) and smob-expressing cells that bear a genomic copy of the nec (strains MP5 and MP7) or yberC (strain MP6) genes.

[0341] FIG. 10 Pathways for producing LNFP-I and 2-FL respectively from lactose. 2FL is produced in a single step from lactose in the presence of the enzyme -1,2-fucosyltransferase (-1,2-ft) adding fucose to the lactose. Production of LNFP-I is a 3 step process where a -1,3-N-acetyl-glucosaminyltransferase (-1,3-GlcNacT) adds N-acetylglucosamine to lactose to form LNT-II to which a -1,3-galactosyltransferase (-1,3-GalT) adds galactose forming LNT on which an -1,2-fucosyltransferase (-1,2-ft) adds a fucose to form LNFP-I. As illustrated in example 1 different -1,2-fucosyltransferase may have different substrate specificities, i.e. FutC seem to have higher specificity for lactose whereas smob seems to have higher specificity for LNT as substrate.

[0342] FIG. 11 Batch growth profile on sucrose for E. coli cells that do or do not express protein(s) that enable the utilization of sucrose as the main and/or the sole, carbon source and/or energy source. The strain MP5 is not capable of utilizing sucrose, while the strains MP8 and MP9 express the extracellular sucrose hydrolase SacC_Agal from one or two PglpF-driven genomic copies, respectively

EXAMPLES

Example 1The LNFP-I Content in Neutral HMO Blends can be Modulated by the Choice of the Expressed -1,2-Fucosyltransferase

Description of the Genotype of Strains MP1, MP2 and MP3 Tested in Deep Well Assays

[0343] The strains (genetically engineered cells) constructed in the present application were based on Escherichia coli K-12 DH1 with the genotype: F.sup., .sup., gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the platform strain MDO with the following modifications: lacZ: deletion of 1.5 kbp, lacA: deletion of 0.5 kbp, nanKETA: deletion of 3.3 kbp, melA: deletion of 0.9 kbp, wcaJ: deletion of 0.5 kbp, mdoH: deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.

[0344] Methods of inserting or deleting gene(s) of interest into the genome of E. coli are well known to the person skilled in the art. Insertion of genetic cassettes into the E. coli chromosome can be done using gene gorging (see e.g., Herring and Blattner 2004 J. Bacteriol. 186:2673-81 and Warming et al 2005 Nucleic Acids Res. 33(4):e36) with specific selection marker genes and screening methods.

[0345] Based on the platform strain (MDO), the modifications summarised in Table 2, were made to obtain the fully chromosomal strains MP1, MP3 and MP2. The strains can produce the pentasaccharide HMO, LNFP-I. The glycosyltransferase enzymes LgtA (a -1,3-N-acetyloglucosamine transferase) from N. meningitidis and GalTK (a -1,3-galactosyltransferase) from H. pylori are present in all three strains.

[0346] In addition, each of these strains bears a single genomic copy of a gene encoding an -1,2-fucosyltransferase as indicated in table 2, whose expression is driven by the synthetic inducible promoter PglpF.

[0347] Specifically, the strain MP1 expresses a single PglpF-driven copy of the futC gene (Helicobacter pylori 26695, Wang et al, Mol. Microbiol., 1999, 31, 1265-1274, GenBank ID: WP_080473865.1), the strain MP3 expresses a single PglpF-driven copy of the fucT54 gene (Sideroxydans lithotrophicus ES-11,WO2019008133A1, GenBank ID: WP_013031010.1) and the strain MP3 expresses a single PglpF-driven copy of the smob gene (Sulfuriflexus mobilis, SEQ ID NO: 1 of the present disclosure).

[0348] The present Example describes for the first time an -1,2-fucosyltransferase that is found in nature, Smob, and shows an unpreceded high specificity for LNT and simultaneously a very low specificity for lactose. Contrary to previously tested -1,2-fucosyltransferases, cells that concomitantly express two glycosyltransferases (SEQ ID NO: 40 and 41) required for LNT synthesis and the Smob enzyme produce almost exclusively LNFP-I. In this manner, the present disclosure demonstrates how the simple strain engineering approach of introducing a single heterologous gene, smob, into the genome of an E. coli DH1 K12 strain that already produces LNT can be advantageously employed to either increase the LNFP-I content of neutral HMO blends or establish an in vivo production process that results in an almost pure LNFP-I HMO product.

[0349] See: Table 2. Genotypes of the strains MP1, MP3 and MP2

Description of the Applied Deep Well Assay Protocol for Strain Characterization

[0350] The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1, fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 C. and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7.5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20% glucose solution (50 ul per 100 mL) and a bolus of 10% lactose solution (5 ml per 100 ml). Moreover, 50% sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 C. and 1000 rpm shaking.

[0351] For the analysis of total broth, the 96-well plates were boiled at 100 C., subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.

Results of the Deep Well Assays Since the FutC enzyme is known to have a higher specificity for lactose than for LNT, the formation of 2-FL is favoured in futC-expressing cells (Wang et al, Mol. Microbiol., 1999, 31, 1265-1274). To promote LNFP-I synthesis in vivo, it is therefore desirable to identify -1,2-fucosyltransferases other than FutC that show higher specificity for LNT than for lactose. This has been attempted before in patent WO2019008133A1, where several -1,2-fucosyltransferases were associated with a high specificity for LNT, including the FucT54 enzyme.

[0352] In our experiments, strains that express the FutC, FucT54 or Smob enzymes were constructed and characterized in deep well assays, and samples were collected from the total broth of the cultures. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs in each sample was used to calculate the relative differences in the HMO content of the strains tested, i.e., the % HMO content of fucT54- and smob-expressing cells relative to the HMO content of futC-expressing cells. LNFP-I to 2-FL ratios were also calculated based on the HMO concentrations determined by HPLC analysis. Finally, the molar % fraction of each HMO in the final blend acquired by each strain was calculated to report the overall HMO profile of each strain.

[0353] As revealed by the analysis of the total samples in deep-well cultures and the calculations mentioned above, the enzyme identified in the present disclosure, namely Smob, appears to be superior to the FutC and FucT54 enzymes in terms of producing LNFP-I due to its inherent high specificity for LNT. As shown in FIG. 1, final LNFP-I titers of the strains expressing the fucT54 gene (strain MP2) and the smob gene (strain MP3) are, respectively, 40% and 70% higher than the LNFP-I titer reached with the strain expressing the futC gene (strain MP1). Likewise, the 1:1 LNFP-1/2-FL ratio in futC-expressing cells is changed to 2:1 in fucT54- and 11:1 in smob-expressing cells (FIG. 2). As shown in FIG. 3-5, the HMO content in the blends acquired by cells expressing the futC, fucT54 or smob genes differs markedly from strain to strain. Specifically, the almost 50%:50% LNFP-I:2FL content in the HMO blend delivered by the strain MP1 (FutC) is turned to almost 70%:30% in the strain MP2 (FucT54) or even 90%:10% in the strain MP3 (Smob).

[0354] These results highlight the unique advantage offered by the Smob enzyme to generate neutral HMO blends enriched in LNFP-I, or cells producing almost exclusively LNFP-I, which is a highly desired strain engineering goal. Also, the fact that the Smob enzyme meets the above expectations to a higher degree than the previously identified FucT54 enzyme (WO2019008133A1) is unexpected and thereby strengthens the impact of the present disclosure on the HMO field.

Example 2The Concomitant Expression of the Smob Enzyme and Either of the Heterologous MFS Transporters Nec or YberC is the Key for an Efficient LNFP-I Cell Factory

Description of the Genotype of Strains MP4, MP5, MP6 and MP7 Tested in Deep Well Assays

[0355] Based on the platform strain (MDO) described in example 1, the modifications summarised in Table 3, were made to obtain the fully chromosomal strains MP4, MP5, MP6 and MP7. The strains can produce the pentasaccharide HMO LNFP-I. The glycosyltransferase enzymes LgtA (a -1,3-N-acetyloglucosamine transferase, SEQ ID NO: 40) from N. meningitidis, GalTK (a -1,3-galactosyltransferase, SEQ ID NO: 41) from H. pylori and Smob (-1,2-fucosyltransferase (SEQ ID NO: 1) from S. mobilis are present in all four strains. Moreover, the strain MP6 expresses the heterologous transporter of the Major Facilitator Superfamily (MFS) YberC (SEQ ID NO: 5) from Yersinia bercovieri, while the strains MP5 and MP7 express the heterologous MFS transporter Nec (SEQ ID NO: 4) from Rosenbergiella nectarea. The only difference between the latter two strains lies in the strength of the promoter that drives the expression of the nec gene, i.e. a PglpF-driven nec copy is present in the strain MP5, while the strain MP7 expresses the nec gene under the control of the Plac promoter.

[0356] The present Example describes an optimized strain engineering approach to construct a highly efficient LNFP-I cell factory that produces LNFP-I at high titers, with a significant fraction of the product being found in the supernatant of the culture. Following the approach described here, HMOs other than LNFP-I constitute only a minor fraction of the total HMO blend delivered by the engineered cell. In the framework of the present Example, introducing the heterologous genes, smob and nec or yberC, ito the genome of an E. coli DH1 K12 strain that already produces LNT can be advantageously employed with a high copy number for the IgtA gene to deliver an efficient LNFP-I cell factory with the beneficial traits described above.

[0357] See: Table 3. Genotypes of the strains MP4, MP5, MP6 and MP7

Description of the Applied Deep Well Assay Protocol for Strain Characterization

[0358] The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1, fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 C. and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7.5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20% glucose solution (50 ul per 100 mL) and a bolus of 20% lactose solution (10 ml per 175 ml). Moreover, 50% sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 C. and 1000 rpm shaking.

[0359] For the analysis of total broth, the 96-well plates were boiled at 100 C., subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.

Results of the Deep Well Assays

[0360] The expression of many host genes as well as the heterologous genes encoding enzymes involved in the in vivo synthesis of a HMO of interest needs to be fine-tuned to achieve an optimal fermentation output, where the desired HMO product is formed at high titers while molecules other than the main product (i.e., precursor or heavily decorated sugars) are formed in minimal amounts. Moreover, the export of the newly formed HMO of interest needs to be exported in the cell exterior to alleviate the cell from the HMO-imposed osmotic stress. The identification of sugar exporters and the fine balancing of their expression can be a key for the success of such production systems. This task can though be challenging, since only the HMO of interest, and not the precursor or elongated versions thereof, should be bound and exported by the chosen sugar exporter.

[0361] After following some strain engineering rounds to balance the expression of the enzymes involved in LNFP-I synthesis, sugar transporters that were proven to be able to export the LNFP-I product out of the cell, namely Nec and YberC (FIG. 9), were also introduced in the LNFP-I production system. Relevant strains were constructed and characterized in deep well assays as described in the previous sections. Samples were collected from the total broth as well as the supernatant and pellet fractions of the cultures. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs in each sample was used to calculate the % quantitative differences in the HMO content of the strains tested, i.e., the % HMO content of nec- and yberC-expressing cells relative to the HMO content of cells that do not express a heterologous transporter.

[0362] As revealed by the analysis of the total samples in deep-well cultures, minor gains in LNFP-I titers can be achieved when a transporter is expressed in a LNFP-I system with balanced expression of glycosyltransferases, i.e. up to 10% higher LNFP-I titers are observed for then strain MP7 (Nec) than the strain MP4 (no transporter) (FIG. 6). Moreover, the introduction of a sugar exporter in LNFP-I production strains induces drastic changes in the abundance of the other HMOs in the final blend. Specifically, LNT II is absent from the total broth of all transporter-expressing cells, with the latter reaching approximately 20% lower 2-FL and up to 40% higher LNT titers (strains MP6 and MP5) compared to the strain that does not express a sugar transporter (strain MP4) (FIG. 6). It should be noted that the relatively large increase in LNT titers (up to 40%) observed for the transporter-expressing cells can be attributed to the low values of LNT concentrations reported for all four strains.

[0363] The minor gains in the relative final LNFP-I titers and the drastic changes in the relative abundance of other HMOs observed for MFS-expressing cells was expectedly reflected in the absolute LNFP-I fraction (%) that was measured in the final HMO blend. Specifically, as revealed by the analysis of the total samples, the absolute fraction of LNFP-I in the final HMO blend reached approximately 88% for cells expressing the Nec transporter (strains MP5 and MP7), while this fraction corresponded to approximately 81% for cells that do not express a MFS transporter (FIG. 7).

[0364] The analysis of the supernatant and total samples of strains tested in deep-well cultures revealed similar trends regarding the observed relative abundance changes in LNT II, LNT and 2-FL for transporter-expressing cells (FIGS. 6 and 8). However, the analysis of supernatant samples revealed a striking fact about LNFP-I. In detail, although only a minor relative overall gain in LNFP-I titer can be achieved by the strain MP7, which expresses the Nec transporter under the control of the Plac promoter (FIG. 6), the extracellular LNFP-I fraction of this strain is much higher (up to 30%) than the strain MP4, which does not express a sugar transporter (FIG. 8). The same trend is observed for the strains expressing the YberC transporter (strain MP6) or the Nec transporter from a PglpF-driven genomic copy (strain MP5) (FIG. 8).

[0365] The marked increase in the supernatant fraction of LNFP-I for MFS-relative to no MFS-expressing cells was expectedly reflected in the absolute LNFP-I fraction (%) that was detected in the supernatant of the corresponding cultures. In detail, only 24% of the total LNFP-I was detected in the supernatant for cells that do not express an MFS transporter (strain MP4), while approximately 38% of the synthesized LNFP-I was detected in the supernatant of cultures for cells expressing the Nec transporter (FIG. 9).

[0366] In conclusion, the balanced expression of the -1,3-N-acetyloglucosamine transferase LgtA, the -1,3-galactosyltransferase GalTK, the -1,2-fucosyltransferase Smob and either of the MFS transporters Nec or YberC constitute an effective strain engineering strategy for the generation of a highly productive LNFP-I cell factories. Such microbial systems produce LNFP-I at high titers, with a significant fraction of the product being found in the supernatant of the culture, and HMOs other than LNFP-I representing only a minor fraction of the total HMO blend delivered by the engineered cell.

Example 3The SacC_Agal Sucrose Utilization Technology can be Successfully Applied to Engineer E. coli Cells Producing the Complex Pentasaccharide LNFP-I

Description of the Genotype of Strains MP5, MP8 and MP9

[0367] Based on the platform strain (MDO, MP1) described in example 1, the modifications summarised in Table 6 were made to obtain the fully chromosomal strains MP5, MP8 and MP9.

[0368] The strains can produce the pentasaccharide HMO LNFP-I, the tetrasaccharide HMO LNT and the trisaccharide HMO 2-FL. The glycosyltransferase enzymes LgtA (a -1,3-N-acetyloglucosamine transferase) from N. meningitidis, GalTK (a -1,3-galactosyltransferase) from H. pylori, Smob (-1,2-fucosyltransferase) from S. mobilis and the heterologous MFS transporter Nec from Rosenbergiella nectarea.are present in all three strains. Contrary to the strain MP5, the strains MP8 and MP11 can utilize sucrose as the carbon and energy source since the gene sacC_Agal from Avibacterium gallinarum is integrated on their genome in one or two loci, respectively.

[0369] This invention demonstrates how the introduction of an extracellular invertase such as SacC_Agal can be advantageously used to confer an engineered E. coli that produces the complex pentasaccharide LNFP-I the ability to utilize sucrose as carbon and/or energy source. The only difference between the strains MP5 and MP8 or MP9, as shown in the table below, is the absence of the SacC_Agal enzyme from the former and its presence in the latter two strains. Although the strain MP8 bears a single PglpF-driven copy of the sacC_Agal gene, the strain MP9 bears two such copies.

[0370] In the present Example, it is demonstrated that sacC_Agal-expressing cells not only grow robustly in batch cultures containing sucrose, but they also produce LNFP-I at high titers in fed-batch fermentation processes.

Description of the Protocol Applied During Growth Monitoring Assays

[0371] The strains disclosed in the present example were screened in 96 well microtiter plates using a 2,5-day protocol. During the first 24 hours, cells were grown to high densities while in the next 36 hours cells were transferred to a medium containing sucrose as the main carbon and energy source. Specifically, during day 1, fresh inoculums were prepared using a Luria-Bertani broth containing 20% glucose. After 24 hours of incubation of the prepared cultures at 34 C., cells were transferred to a basal minimal medium (200 uL) supplemented with magnesium sulphate and thiamine to which an initial bolus of 20% glucose solution and 15 g/L sucrose solution as carbon source was provided to the cells. After inoculation of the new medium, cells were shaken at 1200 rpm at 28 C. for 72 hours. The cells were grown in a batch mode of cultivation in microtiter plates that were compatible with the Varioskan LUX Multimode Microplate Reader from ThermoFisher Scientific.

Fermentation Protocol

[0372] The E. coli strains were cultivated in 250 mL fermenters (Ambr250 HT Bioreactor system, Sartorius) starting with 100 mL of mineral culture medium consisting of 30 g/L glucose or sucrose (AL-X16 and AL-X17 respectively) and a mineral medium comprised of NH.sub.4H.sub.2PO.sub.4, KH.sub.2PO.sub.4, MgSO.sub.47H.sub.2O, NaOH, citric acid, trace element solution, antifoam and thiamine. The dissolved oxygen level was kept at 20% by a cascade of first agitation and then airflow starting at 700 rpm (up to max 4500 rpm) and 1 VVM (up to max 3 VVM). The pH was kept at 6.8 by titration with 8.5% NH.sub.4OH solution. The cultivations were started with 2% (v/v) inoculums from pre-cultures comprised of 10 g/L glucose (AL-X16) or sucrose (AL-X17), (NH.sub.4).sub.2HPO.sub.4, KH.sub.2PO.sub.4, MgSO.sub.47H.sub.2O, KOH, NaOH, citric acid, trace element solution, antifoam and thiamine. After depletion of the glucose or sucrose contained in the basal minimal medium, a glucose (AL-X16) or sucrose-(AL-X17) containing feed solution was continuously added to the fermenter at a rate that maintained carbon-limiting conditions. The temperature was initially at 33 C. but was dropped to 30 C. after 3 hours of feeding. Lactose was added as a bolus addition of 25% lactose monohydrate solution 36 hours after feed start and then every 19 hours to keep lactose from being a rate limiting factor. The growth, metabolic activity and metabolic state of the cells was followed by on-line measurements of reflectance and CO.sub.2 evolution rate. Throughout the fermentations, samples were taken to determine the concentration of HMO products, lactose and other minor by-products using HPLC.

Results of the Growth Monitoring in Assays

[0373] Strains were tested in growth monitoring assays using the 60-hour protocol described above, with the cultures being operated at the batch mode in the presence of sucrose. To evaluate the ability of different strains to grow on sucrose as a function of their genetic makeup (i.e., expression or not of the SacC_Agal enzyme that is directly associated with sucrose utilization), the raw data on culture absorbance (in 600 nm), reported by the Varioskan LUX system, was used to inspect the growth curves on sucrose for the strains tested, namely MP5, MP8 and MP9. The data analysis software Skanlt was used to extract all growth curves and execute various calculations.

[0374] As shown in FIG. 11, the strain MP5, which does not bear the sacC_Agal on its genome, cannot grow on the sucrose that is provided in the medium being present in the prepared batch cultures. After a little growth (due to the provided low levels of glucose and the potentially partially degraded sucrose that could be present in the medium), the strain MP5 has a flat growth profile (FIG. 11). On the contrary, the strain MP8 and MP9, which bear a single or two PglpF-driven copies of the sacC_Agal gene, respectively, grow nicely on sucrose over time and reach much higher optical density values than the strain MP5 (FIG. 10). It is also noteworthy that the strains MP8 and MP9 have an almost identical growth profile, which indicates that a single PglpF-driven copy of the sacC_Agal gene should be sufficient to support robust growth on sucrose (FIG. 11).

[0375] In this manner, the present disclosure indicates an efficient strain engineering tool for producing flexi-fuel strains (capable of growing on more than one carbon source) with a normal cell physiology, which could indicate a presumably low metabolic burden in sacC_Agal-expressing cells compared to other multi-gene sucrose utilization technologies that are known in the art (e.g., scrBRYA).

[0376] In the present example the SacC_Agal sucrose invertase was introduced into the LNFP-I expressing host cell, identified in table 4 as MP5. In detail, the sacC_Agal gene was placed under control of the PglpF promoter and integrated in the chromosome in a single (strain MP8) or two copies (strain MP9). Also, it is hereby demonstrated that sacC_Agal-expressing cells not only grow robustly in batch cultures containing sucrose, but they also produce LNFP-I at high titers in fed-batch fermentation processes as shown in the fermentation results below.

Fermentation Results

[0377] The production of LNFP-I, LNT, 2FL and LNT-II is shown as the fraction % of the total HMO produced. A single fermentation was run with the strain MP5, while the fermentations of the strain MP9 were done in duplicate. The fermentation end-point data is presented in Table 5. In general, in the selected fermentation processes, both strains MP5 and MP9 were producing LNFP-I at high levels and similar titers.

[0378] Specifically, the strain that cannot grow on sucrose, namely MP5, provided an HMO profile that consisted of 3 HMOs when a glucose-based process (AL-X16) was implemented. In detail, the HMO profile of the strain MP5 contains approximately 95% LNFP-I, 1% LNT and 4% 2-FL (Table 5). The strain expressing sacC_Agal under the control of the PglpF promoter (strain MP9) produced a bit higher amount of 2-FL compared to the strain MP5 when a sucrose-based process (AL-X17) was implemented. In particular, the HMO profile of the strain MP9 contains approximately 90% LNFP-I, and 10% 2-FL, but no LNT (Table 5).

TABLE-US-00007 TABLE 5 HMO blend composition in total broth sample at fermentation timepoint 89 h. The strain MP5 grows on glucose (process AL-X16) while the strain MP9 (two independent runs) expresses the SacC_Agal enzyme and can thus utilize sucrose as the carbon and/or energy source (process AL-X17). Fermentation LNFP-I/ 2-FL/ LNT/ LNT-II/ Batch ID Process HMO HMO HMO HMO GDF22xxx Strain ID (%) (%) (%) (%) 240 MP5 AL-X16 94.9 4.2 0.9 0.0 243 MP9 AL-X17 90.5 9.5 0.0 0.0 244 MP9 AL-X17 90.3 9.7 0.0 0.0

[0379] In conclusion, the SacC_Agal sucrose utilization technology enables the high-level LNFP-I production using an accordingly engineered cell, which provides an HMO profile that is highly similar to the one obtained using glucose as the carbon source.

Sequences

[0380] The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference as are the sequences listed in the corrected sequence list in the priority application DK PA 2021 70250. Below is a summary of the sequences which are not presented in Table 4. [0381] SEQ ID NO: 1 [smob protein] [0382] SEQ ID NO: 2 [smob gene] [0383] SEQ ID NO: 3 [bad] [0384] SEQ ID NO: 4 [nec] [0385] SEQ ID NO: 5 [YberC] [0386] SEQ ID NO: 6 [Fred] [0387] SEQ ID NO: 7 [Vag] [0388] SEQ ID NO: 8 [Marc] [0389] SEQ ID NO: 9 [scrY] [0390] SEQ ID NO: 10 [scrA] [0391] SEQ ID NO: 11 [scrB] [0392] SEQ ID NO: 12 [scrR] [0393] SEQ ID NO: 13 [SacC_Agal protein] [0394] SEQ ID NO: 14 [Bff protein] [0395] SEQ ID NO: 40 [IgtA] [0396] SEQ ID NO: 41 [GalTK] [0397] SEQ ID NO: 42 [GlpR]