ENHANCING FORMATION OF HUMAN MILK OLIGOSACCHARIDES (HMOS) BY MODIFYING LACTOSE IMPORT IN THE CELL

Abstract

This invention relates to a method of producing one or more human milk oligosaccharides (HMOs), in particular LNT and/or LNnT, in a genetically engineered cell comprising an enhanced oligosaccharide transport capability. The genetically modified cell comprises a series of genetic modification which enable the production of one or more HMO(s), and a series of genetic modification that enhances the transport of lactose and produced HMO(s).

Claims

1.-19. (canceled)

20. A method for producing one or more human milk oligosaccharides (HMOs) comprising: a) providing a genetically engineered cell which i. overexpresses one or more lactose permeases, ii. expresses a heterologous MFS transporter protein selected from the group consisting of I. Vag with the amino acid sequence according to SEQ ID NO: 9, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 9, II. Nec with the amino acid sequence according to SEQ ID NO: 6, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 6 III. Fred with the amino acid sequence according to SEQ ID NO: 8, or a functional homologue thereof having an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 8, IV. Marc with the amino acid sequence according to SEQ ID NO: 10, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 10, and V. Bad with the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 5, and iii. expresses two or more glycosyltransferases selected from the group consisting of ?-1,3-GlcNAc-transferases, ?-1,3-Gal-transferases and ?-1,4-gal-transferases, b) culturing said cell in a suitable media with added lactose, and c) harvesting the one or more HMOs, wherein the one or more HMOs are LNT, LNnT, or both.

21. The method for producing one or more HMOs according to claim 20, wherein the genetically engineered cell expresses a sucrose utilisation system.

22. The method for producing one or more HMOs according to claim 21, wherein the sucrose utilization system is a polypeptide capable of hydrolysing sucrose into glucose and fructose, selected from the group consisting of SEQ ID NOs: 11 and 12, or a functional homologue of any one of SEQ ID NOs: 11 and 12, having an amino acid sequence which is at least 80% identical, to any one of SEQ ID NOs: 11 or 12.

23. The method according to claim 20, wherein the amino acid sequence of the one or more lactose permeases is selected from the group consisting of GenBank IDs NP_414877.1, WP_042094275.1, WP_000291549.1, WP_089607162.1 and WP_152280604.1, EGT4952364.1, WP_134216118.1, ED11749185.1, WP_084912833.1, WP_103826752.1, WP_021804673.1, WP 084984472.1, WP 199428647.1, WP 046596210.1, XP 452193.1 and an amino acid sequence which is at least 70% identical to any one of the GenBank IDs NP_414877.1, WP_042094275.1, WP_000291549.1, WP_089607162.1, WP_152280604.1, EGT4952364.1, WP_134216118.1, EDI1749185.1, WP_084912833.1, WP_103826752.1, WP_021804673.1, WP 084984472.1, WP 199428647.1, WP 046596210.1 or XP_452193.1 and which encodes a functional homologue.

24. The method according to claim 20, wherein the MFS transporter is Nec or Vag.

25. The method according to claim 20, wherein d) the ?-1,3-Gal-transferase is selected from the group consisting of CvB3galT and GalTK, or a functional homologue of CvB3galT or GalTK, having an amino acid sequence which is at least 80% identical to any one of the amino acid sequences of GenBank IDs WP_080969100.1 (CvB3galT), SEQ ID NO: 1 (GalTK), or e) the ?-1,4-gal-transferase is GalT, or a functional homologue of GalT, having an amino acid sequence which is at least 80% identical to WP_001262061.1 (GalT).

26. The method according to claim 20, wherein the ?-1,3-GlcNAc-transferase is selected from the group consisting of LgtA, PmnagT, HD0466 and a functional homologue of any one of LgtA, PmnagT or HD0466, having an amino acid sequence which is at least 80% identical to any one of GenBank IDs WP_033911473.1 (LgtA), WP_014390683.1 (PmnagT) or WP_010944479.1 (HD0466).

27. The method according to claim 20, wherein said genetically engineered cell comprises one or more heterologous nucleic acid sequence encoding one or more heterologous polypeptides, which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell.

28. The method according to claim 20, wherein the genetically engineered cell expresses one or more polypeptides involved in the biosynthesis of activated sugar nucleotides selected from the group consisting of Pgm, GalU, GalE, GlmM, GlmU and GlmS, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to any one of GenBank IDs NP_415214.1 (Pgm), WP_001396326.1 (Pgm), NP_415752.1 (GalU), WP_000718995.1 (GalU), NP 415280.3 (GalE), WP_001265438.1 (GalE), NP_417643.1 (GlmM), WP_000933736.1 (GlmM), NP 418186.1 (GlmU), WP_000071134.1 (GlmM), NP_418185.1 (GlmS), or WP_000334099.1 (GlmS).

29. The method according to claim 20, wherein the genetically engineered cell comprises more than one nucleic acid sequence encoding one or more lactose permeases according to claim 23.

30. The method according to claim 20, wherein the genetically engineered cell comprises at least one nucleic acid sequence encoding one or more heterologous polypeptides involved in the biosynthesis of activated sugars according to claim 29.

31. The method according to claim 29, wherein at least one of the nucleic acid sequences encoding lactose permease is regulated by one or more promoter sequences selected from the group consisting of Plac, PgatY_70UTR, PgIpF, PgIpF_SDI, PgIpF_SD10, PgIpF_SD2, PgIpF_SD3, PgIpF_SD4, PgIpF_SD5, PgIpF_SD6, PgIpF_SD7, PgIpF_SD8, PgIpF_SD9, Plac 16UTR, PmgIB_70UTR, PmgIB_70UTR_SD4, CP6 and PosmY.

32. The method according to claim 20, wherein at least one nucleic acid sequence encoding the one or more lactose permeases is integrated into the genome of the genetically engineered cell.

33. The method according to claim 20, wherein at least one nucleic acid sequence encoding the heterologous MFS transporter is integrated into the genome of the genetically engineered cell.

34. The method according to claim 20, wherein at least one nucleic acid sequence encoding the two or more glycosyltransferases is integrated into the genome of the genetically engineered cell.

35. The method according to claim 29, wherein the at least one nucleic acid sequence encoding one or more heterologous polypeptides involved in the biosynthesis of activated sugars is integrated into the genome of the genetically engineered cell.

36. The method according to claim 20 wherein the genetically engineered cell is Escherichia coli.

37. The method according to claim 20, wherein the genetically engineered cell is derived from Escherichia coli K-12.

38. A genetically engineered cell capable of producing LNT, LNnT, or LNT and LNnT and wherein the cell f) overexpresses one or more lactose permease genes, g) expresses a heterologous MFS transporter protein selected from the group consisting of: i. Vag with the amino acid sequence according to SEQ ID NO: 9, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 9, ii. Nec with the amino acid sequence according to SEQ ID NO: 6, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO 6: iii. Fred, with the amino acid sequence according to SEQ ID NO: 8, or a functional homologue thereof having an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 8, iv. Marc with the amino acid sequence according to SEQ ID NO: 10, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 10 and v. Bad with the amino acid sequence according to SEQ ID NO: 5, or a functional homologue thereof having an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO: 5, and h) expresses two or more glycosyltransferases selected from the group consisting of ?-1,3-GlcNAc-transferases, ?-1,3-Gal-transferases and ?-1,4-gal-transferases, and optionally, i) expresses a sucrose utilisation system and/or j) expresses one or more polypeptides involved in the biosynthesis of activated sugars.

39. The genetically engineered cell according to claim 38, wherein the cell comprises, i. one or more lactose permeases selected from the group consisting of GenBank IDs NP_414877.1, WP_042094275.1, WP_000291549.1, WP_089607162.1, WP_152280604.1, EGT4952364.1, WP_134216118.1, ED11749185.1, WP_084912833.1, WP 103826752.1, WP_021804673.1, WP_084984472.1, WP_199428647.1, WP 046596210.1, XP_452193.1, and a functional homologue thereof, having an amino acid sequence which is at least 80% identical to any one of GenBank IDs NP_414877.1, WP_042094275.1, WP_000291549.1, WP_089607162.1, WP_152280604.1, EGT4952364.1, WP_134216118.1, ED11749185.1, WP_084912833.1, WP_103826752.1, WP_021804673.1, WP_084984472.1, WP_199428647.1, WP_046596210.1 or XP_452193.1, ii. two or more glycosyltransferases selected from the group consisting of CvB3galT, GalTK, GalT, LgtA, PmnagT, HD0466, and a functional homologue thereof, having an amino acid sequence which is at least 80% identical to any one of GenBank IDs WP_080969100.1 (CvB3galT), SEQ ID NO: 1 (GalTK), WP_001262061.1 (GalT), WP_033911473.1 (LgtA), WP_014390683.1 (PmnagT), or WP_010944479.1 (HD0466), and optionally, ii. one or more polypeptides involved in the biosynthesis of activated sugars selected from the group consisting of GenBank IDs NP_415214.1 (Pgm), WP_001396326.1 (Pgm), NP_415752.1 (GalU), WP_000718995.1 (GalU), NP 415280.3 (GalE), WP_001265438.1 (GalE), NP_417643.1 (GlmM), WP_000933736.1 (GlmM), NP_418186.1 (GlmU), WP_000071134.1 (GlmM), NP 418185.1 (GlmS) and WP_000334099.1 (GlmS).

Description

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

[0299] The effect of lacY over-expression on the relative titers of LNT-II, LNT, pLNH2 and the total HMO content for (a) the yberC-expressing strains MP1 and MP2 and (b) the nec-expressing strains MP3 and MP4, as revealed by the analysis of total samples.

FIG. 2

[0300] The effect of lacY over-expression on (a) the LNT fraction in % of the final HMO blend and (b) the final optical density reached by the yberC-expressing strains MP1 and MP2 and the nec-expressing strains MP3 and MP4.

FIG. 3

[0301] The effect of lacY over-expression on the fraction of LNT-II, LNT and pLNH2 detected in the supernatant of cultures of (a) the yberC-expressing strains MP1 and MP2 and (b) the nec-expressing strains MP3 and MP4, as revealed by the analysis of supernatant and cell pellet samples.

FIG. 4

[0302] The effect of lacY over-expression on the relative titers of LNT-II, LNT, pLNnH and the total HMO content for the vag-expressing strains MP5 and MP6, as revealed by the analysis of total samples.

FIG. 5

[0303] The effect of lacY over-expression on (a) the LNnT fraction in % of the final HMO blend and (b) the final optical density reached by the vag-expressing strains MP5 and MP6.

FIG. 6

[0304] Performance of LNT producing strains MP7 and MP8 in fermentation runs using a high lactose process. Time course profiles shown for: (a) lactose concentration, (b) biomass concentration, (c) relative accumulated LNT yields on sucrose, (d) ratio of LNT-11 to LNT, and (e) ratio of pLNH2 to LNT. FIG. 7

[0305] Performance of LNnT producing strains MP5 and MP6 in fermentation runs using a high lactose process. Time course profiles shown for: (a) lactose concentration, (b) biomass concentration, (c) relative accumulated LNnT yields on sucrose, (d) ratio of LNT-II to LNnT, and (e) ratio of pLNnH to LNnT.

FIG. 8

[0306] 3SL production as % of the respective MFS transporter strain set to 100%. Strains containing the MFS transporter nec are presented as the dotted bars, yberC strains are cross stiped and fred strains are horizontally striped

FIG. 9

[0307] 2FL production as % of the control nec transporter strain (MP18) set to 100%. The 2FL titers of strains containing the MFS transporter nec in combination with an additional copy of lacY expressed from either PgIpF_SD7 (MP19) or PgIpF (MP20) are shown relative to the nec transporter strain.

SEQUENCE ID'S

[0308] The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference. Table 9 provides a summary of the sequences in the present application.

TABLE-US-00004 TABLE 9 sequences Protein/gene/promoter Abbreviation Function Public reference SEQ ID NO: LacY Lactose permease NP_414877.1 1 WP_042094275.1 3 WP_000291549.1 4 WP_089607162.1 5 WP_152280604.1 6 EGT4952364.1 7 WP_134216118.1 8 EDI1749185.1 9 WP_084912833.1 10 WP_103826752.1 11 WP_021804673.1 12 WP_084984472.1 13 WP_199428647.1 14 WP_046596210.1 15 XP_452193.1 16 lacY E. Coli K-12 lactose NC_000913.3 2 permease gene CvB3galT ?-1,3-Gal-transferase WP_080969100.1 17 GalTK ?-1,3-Gal-transferase homologous to BD182026.1 18 GalT ?-1,4-gal-transferase WP_001262061.1 19 LgtA ?-1,3-GlcNAc- WP_033911473.1 20 transferase PmnagT ?-1,3-GlcNAc- WP_014390683.1 21 transferase HD0466 ?-1,3-GlcNAc- WP_010944479.1 22 transferase FutC ?-1,2-fucosyl-transferase WP_080473865.1 (with two 23 additional amino acids (LG) at the C-terminus) Smob ?-1,2-fucosyl-transferase WP_126455392.1 24 Mtun ?-1,2-fucosyl-transferase WP_031437198.1 25 FutA ?-1,3-fucosyl-transferase NP_207177.1 26 FucT ?-1,3-fucosyl-transferase AAB81031.1 27 FucTIII ?-1,3-fucosyl-transferase AAR88243.1 28 Pd2 ?-2,3-sialyl-transferase 29 Nst ?-2,3-sialyl-transferase 30 Gmd GDP-mannose 4,6- NP_416557.1 31 dehydratas WP_000048190.1 32 WcaG GDP-L-fucose synthase NP_416556.1 33 WP_000043654.1 34 WcaH GDP-mannose NP_416555.2 35 mannosyl hydrolase WP_001393539.1 36 Wcal colanic acid biosynthesis NP_416554.1 37 fucosyltransferase Wcal WP_000699693.1 38 ManC mannose-1-phosphate NP_416553.1 39 guanylyltransferase WP_000079274.1 40 ManB Phosphomannomutase NP_416552.1 41 WP_001350528.1 42 Pgm Phosphoglucomutase NP_415214.1 43 WP_001396326.1 44 GalU UTP-glucose-1- NP_415752.1 45 phosphate WP_000718995.1 46 uridylyltransferase GalE UDP-glucose 4- NP_415280.3 47 epimerase WP_001265438.1 48 GlmM Phosphoglucosamine NP_417643.1 49 mutase WP_000071134.1 50 GlmU N-acetylglucosamine-1- NP_418186.1 51 phosphate WP_000933736.1 52 uridyltransferase and glucosamine-1- phosphate acetyltransferase GlmS L-glutamine-D-fructose- NP_418185.1 53 6-phosphate WP_000334099.1 54 aminotransferase NeuA CMP-Neu5Ac AAK91728.1 55 synthetase WP_006881452.1 56 NeuB Neu5 Ac synthase AAK91726.1 57 WP_023580510.1 58 NeuC UDP-GlcNAc 2- AAK91727.1 59 epimerase WP_000723250.1 60 CAR04561.1 61 Vag MFS transporter WP_048785139.1 62 Fred MFS transporter WP_087817556.1 63 Marc MFS transporter WP_060448169.1 64 Bad MFS transporter WP_017489914.1 65 Nec MFS transporter WP_092672081.1 66 YberC MFS transporter EEQ08298.1 67 Plac promoter 68 PgatY_70UTR promoter WO2020255054A1 69 PglpF promoter WO2019123324A1 70 PglpF_SD1 promoter WO2019123324A1 71 PglpF_SD10 promoter WO2019123324A1 72 PglpF_SD2 promoter WO2019123324A1 73 PglpF_SD3 promoter WO2019123324A1 74 PglpF_SD4 promoter WO2019123324A1 75 PglpF_SD5 promoter WO2019123324A1 76 PglpF_SD6 promoter WO2019123324A1 77 PglpF_SD7 promoter WO2019123324A1 78 PglpF_SD8 promoter WO2019123324A1 79 PglpF_SD9 promoter WO2019123324A1 80 Plac_16UTR promoter WO2020255054A1 81 PmglB_70UTR promoter WO2020255054A1 82 PmglB_70UTR_SD4 promoter WO2020255054A1 83 CP6 promoter 84 PosmY promoter 85 SacC_Agal glycoside hydrolase WP_103853210.1 86 family 32 protein Bff beta-fructofuranosidase BAD18121.1 87 protein scrY Sucrose porin WO2015197082 88 scrA PTS system sucrose- WO2015197082 89 specific EIIBC component scrB Sucrose-6-phosphate WO2015197082 90 hydrolase scrR Sucrose operon WO2015197082 91 repressor glpR Regulator of the Glp 92 Operon PmglB_70UTR_SD8 promoter WO2020255054 93 PmglB_70UTR_SD10 promoter WO2020255054 94 PmglB_54UTR promoter WO2020255054 95 Plac_70UTR promoter WO2019123324 96 PmglB_70UTR_SD9 promoter WO2020255054 97 PmglB_70UTR_SD5 promoter WO2020255054 98 PmglB_70UTR_SD7 promoter WO2020255054 99 PglpA_70UTR promoter WO2019123324 100 PglpT_70UTR promoter WO2019123324 101 PmglB_16UTR promoter WO2020255054 102 PglpA_16UTR promoter WO2019123324 103

EXAMPLES

Example 1Improvement of LNT Production Systems by Increasing the Expression of the lacY Gene Encoding Lactose Permease in Cells Expressing a Heterologous MFS Transporter

Genotype of Strains MP1, MP2, MP3 and MP4

[0309] 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, me/A: 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.

[0310] 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.

[0311] Based on the platform strain MDO (e.g., also reported in WO2020255054A1 or WO2019123324A1), the modifications summarised in the table below, were made to obtain the fully chromosomal strains MP1, MP2, MP3 and MP4. The strains can produce the tetrasaccharide HMO LNT. The glycosyltransferase enzymes LgtA (a beta-1,3-N-acetyloglucosamine transferase) from N. meningitidis and GalTK (a beta-1,3-galactosyltransferase) from H. pylon are present in all four strains. Moreover, MP1 and MP2 express the heterologous transporter YberC from Yersinia bercovieri, while the strains MP3 and MP4 express the heterologous transporter Nec from Rosenbergiella nectarea. Moreover, the strains MP2 and MP4 over-express the lacY gene from an additional PgIpF-driven genomic copy, while the strains MP1 and MP3 do not.

[0312] In the present example, it is demonstrated how the over-expression of the lacY gene coding lactose permease is used as a genetic tool to enhance LNT production in strains that already express the heterologous transporter YberC or Nec. This invention also demonstrates how the over-expression of the lacY gene can be advantageously used to increase the total HMO content of the broth, and simultaneously reduce the formation of other HMOs, such as LNT-II, and increase the LNT content in the final HMO blend. As shown in table 2, the only difference among each strain of the two strain pairs, namely MP1-MP2 and MP3-MP4, is the presence of an additional lacY expression cassette at a genomic locus that is different from the native lacY locus.

TABLE-US-00005 TABLE 2 Genotypes of the strains MP1, MP2, MP3 and MP4 Strain ID Genotype Plasmid-free MP1 MDO x3 GlcNACT* x2 GalTK**, Plac-yberC LNT MP2 MDO x3 GlcNACT* x2 GalTK**, LNT Plac-yberC, PglpF-lacY MP3 MDO x3 GlcNACT* x2 GalTK**, PglpF-nec LNT MP4 MDO x3 GlcNACT* x2 GalTK**, LNT PglpF-nec, PglpF-lacY *GlcNAcT: beta-1,3-N-acetyloglucosamine transferase **GalTK: beta-1,3-galactosyltransferase

Deep Well Assay

[0313] 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 (BMM) 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 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 (0.5 ?L per mL) and a bolus of 20% lactose solution (0.1 ?L per ?L).

[0314] Moreover, a 20% stock solution of a specific polysaccharide was provided as carbon source, accompanied by the addition of a specific hydrolytic enzyme, so that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed-batch fermentation process. The main cultures were incubated for 72 hours at 28? C. and 1000 rpm shaking. For the analysis of total broth, the 96 well plates were boiled at 100? C., subsequently centrifuged, and finally the supernatants were analyzed by HPLC.

Results

[0315] Strains were characterized in deep well assays and samples were collected from the total broth, the supernatant, and the cell pellet. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above.

[0316] The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the HMO content of the strains tested, i.e., the % differences in the HMO concentrations of lacY-expressing cells relative to the ones expressing lacY at physiological levels. Moreover, the absolute fraction (%) of LNT in the final HMO blend was calculated by considering the HMO concentrations detected by HPLC, i.e., LNT-II and LNT concentrations. The final optical density at 600 nm was also measured for all strains at the end of the experiment, i.e., after 72 hours in the production phase. Finally, the HPLC measurements for the supernatant and pellet samples were used to calculate the absolute sugar ratio (%) of the supernatant (S) fraction to the sum of the supernatant and pellet fractions (total, T).

[0317] As revealed by the analysis of total samples in deep-well cultures, some gains in LNT and total HMO titers can be obtained by over-expressing the lacY gene both in nec- and yberC-expressing cells. Specifically, the strain expressing the Nec transporter and over-expressing the lacY gene, MP2, produced approximately 15% more LNT and provided approximately 10% more total HMO content than the nec-expressing strain MP1 that has wild-type expression levels of the lacY gene (FIG. 1a). Similarly, gains up to 15% both in LNT titers and the total HMO content can be obtained by over-expressing the lacY gene in the yberC-expressing strain MP3 to generate the strain MP4748 (FIG. 1b). Moreover, the increase in the intracellular lactose concentration mediated by lacY over-expression in both nec- and yberC-expressing cells (strains MP2 and MP4) results in markedly lower by-product (LNT-II) formation compared to cells that express lacY at physiological levels (strains MP1 and MP3) (FIG. 1a-1b).

[0318] The facts mentioned above are directly reflected to the LNT content (%) in the final HMO blend of both nec- and yberC-expressing cells that over-express the lacY gene. Specifically, the LNT fraction of the final HMO blend generated by cells over-expressing the lacY and nec genes (strain MP3) or the lacY and yberC genes (strain MP2) is approximately 10% higher than for cells that express the nec (strain MP4) or yberC (strain MP1) gene alone (FIG. 2a). Interestingly, the effect of lacY over-expression on biomass formation for heterologous MFS-expressing strains can vary depending on the heterologous MFS being expressed. In detail, the strain MP4 that over-expresses both the lacY and nec genes reaches significantly lower optical density values after 72 hours of cultivation compared to the similar strain MP3 that does not over-express the lacY gene (FIG. 2b). The opposite is true for the strain MP2 that over-expresses both the lacY and yberC genes, i.e., MP2 forms higher biomass than the strain MP1 that expresses the yberC gene alone (FIG. 2b) In conclusion, the over-expression of the lacY gene in transporter expressing LNT production strains seems to change the sugar transport dynamics across the cell membrane in such a manner that the final LNT and total HMO titer in these strains is much higher when the lacY gene is over-expressed (strains MP2 and MP4) than when it is expressed at physiological levels (strains MP1 and MP3).

[0319] As it is apparent from the analysis of the supernatant and pellet fractions of the broth, the % fraction of LNT and LNT-II in the supernatant is not affected by the over-expression of the lacY gene in yberC-expressing cells (FIG. 3a). Approximately 25% less pLNH2 is, however, detected in the culture medium when yberC-expressing cells over-express lacY (strain MP2) rather than when lacY is solely expressed from the Plac promoter of the lac operon of E. coli (strain MP1) (FIG. 3a).

[0320] As for yberC-expressing cells, the fraction of LNT and LNT-II detected in the supernatant is not affected by the over-expression of the lacY gene in nec-expressing cells (FIG. 3b). Contrary to yberC-expressing cells (strain MP3), however, the over-expression of the lacY gene in nec-expressing cells (strain MP4) results in higher pLNH2 in the culture medium (FIG. 3b).

Example 2Improvement of LNnT Production Systems by Increasing the Expression of the lacY Gene Encoding Lactose Permease in Cells Expressing a Heterologous MFS Transporter

Genotype of Strains MP5 and MP6

[0321] Based on the platform strain MDO (e.g., see example 1 and also reported in WO2020255054A1 or WO2019123324A1), the modifications summarised in the table below, were made to obtain the fully chromosomal strains MP5 and MP6. The strains can produce the tetrasaccharide HMO LNnT. The glycosyltransferase enzymes LgtA (a beta-1,3-N-acetyloglucosamine transferase) from N. meningitidis and GalT (a beta-1,4-galactosyltransferase) from H. pylon are present in both strains. Moreover, MP5 and MP6 express the heterologous transporter Vag from Pantoea vagans, and the strain MP6, but not MP5, over-expresses the lacY gene from an additional genomic copy under the control of the PgIpF promoter.

[0322] In the present Example, it is demonstrated how the over-expression of the lacY gene coding lactose permease is used as a genetic tool to enhance LNnT production in strains that already express the heterologous transporter Vag. This invention also demonstrates how the over-expression of the lacY gene can be advantageously used to increase the pLNnH and total HMO content of the broth. As shown in table 3 below, the only difference between the two vag-expressing strains, MP5 and MP6, is the presence of an additional lacY expression cassette at a genomic locus that is different from the native lacY locus.

TABLE-US-00006 TABLE 3 Genotypes of the strains MP5 and MP6 Strain ID Genotype Product MP5 MDO x2 GlcNACT* x1 GalT**, LNnT PglpF-vag, scrBRYA*** MP6 MDO x2 GlcNACT* x1 GalT**, PglpF-vag, LNnT PglpF-lacY, scrBRYA*** *GlcNACT: beta-1,3-N-acetyloglucosamine transferase **GalT: beta-1,4-galactosyltransferase ***scrBRYA: sucrose utilization genes

Deep Well Assay

[0323] 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 (BMM) 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 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 (0.5 ?L per mL) and a bolus of 20% lactose solution (0.1 ?L per ?L).

[0324] Moreover, a 20% stock solution of a specific polysaccharide was provided as carbon source, accompanied by the addition of a specific hydrolytic enzyme, so that glucose was released at a rate suitable for carbon-limited growth and similar to that of a typical fed-batch fermentation process. The main cultures were incubated for 72 hours at 28? C. and 1000 rpm shaking. 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.

Results

[0325] Strains were characterized in deep well assays and samples were collected from the total broth and analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the HMO content of the strains tested, i.e., the % differences in the HMO concentrations of lacY-expressing cells (strain MP6) relative to the ones expressing lacY at physiological levels (strain MP5). Moreover, the absolute fraction (%) of LNnT in the final HMO blend was calculated by considering the HMO concentrations detected by HPLC, i.e., LNT-II, LNnT and pLNnH concentrations. The final optical density at 600 nm was also measured for all strains at the end of the experiment, i.e., after 72 hours in the production phase.

[0326] As revealed by the analysis of total samples in deep-well cultures, marked gains in LNnT, pLNnH and total HMO titers can be obtained by over-expressing the lacY gene in vag-expressing cells. Specifically, the strain expressing the Vag transporter and over-expressing the lacY gene, MP6, produced approximately 20% more LNnT, 20% more pLNnH and had 20% more total HMO content than the vag-expressing strain MP5 that expresses the lacY gene at wild-type levels (FIG. 4). No significant differences in the LNT-II content among the two strains could be detected (FIG. 4).

[0327] The facts mentioned above are directly reflected to the LNnT content (%) in the final HMO blend of vag-expressing cells that over-express the lacY gene. Specifically, the LNnT fraction of the final HMO blend generated by the strain MP6 that over-expresses the lacY gene and the vag gene is approximately 10% higher than for the strain MP5 that expresses the vag gene alone (FIG. 5a). Contrary to the marked impact of lacY over-expression on biomass formation for the LNT production strains presented in example 1, the impact of lacY over-expression on biomass formation for the heterologous MFS-expressing LNnT strain MP6 is negligible. In detail, the final optical density of cells over-expressing both the lacY and vag genes (strain MP6) is very similar to the one of cells expressing the vag gene alone (strain MP5) (FIG. 5b).

Example 3Fermentation Performance of LNT and LNnT Strains Over-Expressing the lacY Gene and Expressing a Gene Encoding a Heterologous Sugar Exporter

Genotype of Strains MP7, MP8, MP5 and MP6

[0328] Based platform strain MDO (e.g., see Example 1 and also reported in WO2020255054A1 or WO2019123324A1), the modifications summarised in table 4, were made to obtain the fully chromosomal strains MP7, MP8, MP5 and MP6. The strains can produce the tetrasaccharide HMO LNT (MP7 and MP8) or LNnT (MP5 and MP6). All four strains can grow on sucrose. The glycosyltransferase enzymes LgtA (a beta-1,3-N-acetyloglucosamine transferase) from N. meningitidis is present in all four strains, while the GalTK (a beta-1,3-galactosyltransferase) or the GalT (a beta-1,4-galactosyltransferase) from H. pylon is introduced in strains MP7-MP8 and MP5-MP6, respectively. Moreover, the strains MP7 and MP8 express the heterologous transporter YberC from Yersinia bercovieri, while the strains MP5 and MP6 express the heterologous transporter Vag from Pantoea vagans. Moreover, the strains MP8 and MP6 over-express the lacY gene from an additional PgIpF-driven genomic copy, while the strains MP7 and MP5 do not.

[0329] In the present Example, it is demonstrated how the over-expression of the lacY gene coding lactose permease is used as a strain engineering tool to enhance LNT or LNnT production in fed-batch fermentations using strains that already express the heterologous transporter YberC or Vag, respectively. This invention also demonstrates how the over-expression of the lacY gene can be advantageously used to obtain higher total HMO content in the fermentation broth, and simultaneously modulate the formation of HMOs other than LNT and LNnT, e.g., LNT-II, pLNnH and pLNH2. As shown in table 4, the only difference between the two pairs of strains, MP7-MP8 and MP5-MP6, is the presence of an additional lacY expression cassette at a genomic locus that is different from the native lacY locus.

TABLE-US-00007 TABLE 4 Genotypes of the strains MP7, MP8, MP5 and MP6 Strain ID Genotype Product MP7 MDO x3 GlcNACT* x2 GalTK**, LNT Plac-yberC, scrBRYA**** MP8 MDO x3 GlcNACT* x2 GalTK**, Plac-yberC, LNT PglpF-lacY, scrBRYA**** MP5 MDO x2 GlcNACT* x1 GalT***, LNnT PglpF-vag, scrBRYA**** MP6 MDO x2 GlcNAcT* x1 GalT***, PglpF-vag, LNnT PglpF-lacY, scrBRYA**** *GlcNAcT: beta-1,3-N-acetyloglucosamine transferase **GalTK: beta-1,3-galactosyltransferase ***GalT: beta-1,4-galactosyltransferase ****scrBRYA: sucrose utilization genes

Fermentation Processes

[0330] The fermentations were carried out in 200 mL DasBox bioreactors (Eppendorf, Germany), starting with 100 mL of defined mineral culture medium, consisting of a suitable concentration of a carbon source (sucrose or glucose), MgSO4?7H2O, KOH, NaOH, NH4H2PO4, KH2PO4, trace metal solution, citric acid, antifoam and thiamine. The trace metal solution (TMS) contained Mn, Cu, Fe, Zn as sulfate salts and citric acid. Fermentations were started by inoculation with 2% (v/v) of pre-cultures grown in a defined minimal medium. After depletion of the carbon source present in the batch medium, a sterile feed solution containing sucrose (or glucose), NH.sub.4SO.sub.4, and TMS was fed continuously in a carbon-limited manner using a predetermined, linear profile.

[0331] Lactose addition was done by a high lactose process (LNT98 and LNT108 for LNT fermentations, and L232 for LNnT fermentations), where lactose monohydrate solution was added by two bolus additions, the first one at approx. 10 hours after feed start, the second one at approx. 70 hours EFT. For LNT fermentations, the processes LNT98 and LNT108 differ only in the fact that the second lactose pulse was performed approx. 20 h earlier for LNT108 than for LNT98. In this manner, lactose was ensured not to be the limiting factor for HMO formation at least until 90 hours EFT, as shown in FIGS. 6a and 7a. The process L232 is identical with LNT98.

[0332] The pH throughout fermentation was controlled at 6.8 by titration with 14% NH4OH solution. Aeration was controlled at 1 vvm using air, and dissolved oxygen was kept above 23% of air saturation, controlled by the stirrer rate. At 15 min after sucrose feed start, the fermentation temperature setpoint was lowered from 33? C. to 28? C. This temperature drop was conducted without a ramp. The total duration of the fermentations was 4-5 days.

[0333] Throughout the fermentations, samples were taken to determine the concentration of LNT or LNnT, LNT-II, lactose, pLNnH or pLNH2 and other minor by-products using HPLC. Total broth samples were diluted three-fold in deionized water and boiled for 20 minutes. This was followed by centrifugation at 17000g for 3 minutes, where after the resulting supernatant was analysed by HPLC. The above measurements were used along with data on carbon source utilization to accurately calculate product yields on sucrose as well as the ratios of LNT-II and hexasaccharide (pLNnH or pLNH2) relative to the main product (respectively, LNnT or LNT).

Results

[0334] All four fermentations ran in a stable manner for at least 4 days (FIGS. 6 and 7).

[0335] As shown in FIG. 6c, the performance of the LNT strain that bears an extra copy of the lacY gene (strain MP8) is superior to the one observed by the strain that expresses lacY at physiological levels (strain MP7), with the former showing an approximately 50% higher LNT yield on sucrose (FIG. 6c), while retaining the biomass formation at a comparable level (FIG. 6B). Moreover, as revealed by the calculations of ratios of by-products to LNT, the strains MP8 and MP7 show similar LNT-II levels (FIG. 6d), while the over-expression of the lacY gene in strain MP8 results in increased pLNH2 formation (FIG. 6e).

[0336] As shown in FIG. 7c, the performance of the LNnT strain that bears an extra copy of the lacY gene (strain MP6) is superior to the one observed by the strain that expresses lacY at physiological levels (strain MP5), with the former showing an approximately 25% higher LNnT yield on sucrose (FIG. 7c), while retaining the biomass formation at a comparable level (FIG. 7b). As mentioned above for the LNT strains, the strains MP8 and MP7 show similar LNT-II levels (FIG. 7d), and the over-expression of the lacY gene in strain MP6 results in higher pLNnH formation than in strain MP5 (FIG. 7e).

Example 4Increased Expression of lacY in Combination with an MFS Transporter does not Improve 3SL Expression

[0337] In Examples 1 to 3 it has been illustrated that LacY overexpression in combination with certain MFS transporters has a positive effect on LNT and LNnT production.

[0338] In the following example the MFS transporters nec, yberC or fred (1 copy genomically integrated) have been tested in a 3-SL producing strain without (MP9, MP12 and MP15) or with overexpression of LacY, either as an extra copy from the chromosome (MP10, MP13 and MP16) or from a medium copy nr plasmid (pSU2719-lacY-chr) (MP11, MP14 and MP17).

[0339] All strains were based on the platform strain MDO (e.g., see example 1 and also reported in WO2020255054A1 or WO2019123324A1) and contained a truncated version (29 aa n-terminal deletion) of the ?-2,3-sialyltransferase from Neisseria meningitidis, nst (GenBank assession nr. AAC44541.1), heterologous CMP-Neu5Ac synthetase, neuA (GenBank assession nr. AAK91728.1), heterologous sialic acid synthase, neuB (GenBank assession nr. AAK91726.1) and heterologous GlcNAc-6-phosphate 2 epimerase, neuC (GenBank assession nr. AAK91727.1) incorporated with a single copy at different loci in the genome of the MDO strain. The genotypes of the tested strains are given in table 5.

TABLE-US-00008 TABLE 5 Genotypes of the strains 3-SL producing strains with different MFS transporters Strain ID Genotype Transporter MP9 MDO x1 PglpF-nst, PglpF-nec, 1xPglpF-neuB, 1xPglpF- nec neuC, 1xPglpF-neuA MP10 MDO x1 PglpF-nst, PglpF-nec, 1xPglpF-neuB, 1xPglpF- nec neuC, 1xPglpF-neuA, x1 PglpF-LacY MP11 MDO x1 PglpF-nst, PglpF-nec, 1xPglpF-neuB, 1xPglpF- nec neuC, 1xPglpF-neuA, pSU2719-lacY-chr MP12 MDO x1 PglpF-nst, PglpF-yberC, 1xPglpF-neuB, 1xPglpF- yberC neuC, 1xPglpF-neuA MP13 MDO x1 PglpF-nst, PglpF-yberC, 1xPglpF-neuB, 1xPglpF- yberC neuC, 1xPglpF-neuA, x1 PglpF-LacY MP14 MDO x1 PglpF-nst, PglpF-yberC, 1xPglpF-neuB, 1xPglpF- yberC neuC, 1xPglpF-neuA, pSU2719-lacY-chr MP15 MDO x1 PglpF-nst, PglpF-fred, 1xPglpF-neuB, 1xPglpF- fred neuC, 1xPglpF-neuA, x1 PglpF-LacY MP16 MDO x1 PglpF-nst, PglpF-fred, 1xPglpF-neuB, 1xPglpF- fred neuC, 1xPglpF-neuA, MP17 MDO x1 PglpF-nst, PglpF-fred, 1xPglpF-neuB, 1xPglpF- fred neuC, 1xPglpF-neuA, pSU2719-lacY-chr

Deep-Well Assay

[0340] The strains disclosed in the present example were screened for 3SL production in a 96-deep well plate assay. Three replicates per strain were tested. More specifically, during day 1, fresh precultures were prepared using a basal minimal medium (BMM) supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated at 34? C. with 1000 rpm for 24 h and then transferred to a new 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 (0.5 ?L/mL) and a bolus of 20% lactose solution (0.1 ?L/?L).

[0341] To the main culture 50% sucrose solution as carbon source (52.5 ?l/ml) was provided to the cells accompanied by the addition of sucrose hydrolase (invertase 0.1 g/L) and 50 mg/ml IPTG, so that glucose was released at a rate suitable for C-limited growth allowing for gene expression and 3SL production.

[0342] Antibiotic (chloramphenicol 20 mg/ml) was added in wells when required for plasmid maintenance. The main cultures were incubated at 34? C. with 1000 rpm for 96 h. 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.

Results

[0343] The strains of the present example only produce 3SL. The results from deep-well assays are shown in FIG. 8. The 3SL production for the strain expressing the MFS transporter was set to 100 and the corresponding strains with either one extra copy of LacY on the chromosome or LacY expressed from a multicopy plasmid was normalized to the respective MFS transporter strain.

[0344] From FIG. 8 it can be seen that in a 3SL producing strain, overexpression of LacY does not increase the total 3SL production. In fact, the production seems to be reduced with increased overexpression of lacY as indicated by the strains expressing lacY from a medium copy nr plasmid (MP11, MP14 and MP17, respectively) which have further reduced 3SL levels compared to strain with a single additional copy of LacY (MP10, MP13 and MP16, respectively).

Example 5Increased Expression of lacY in Combination with an MFS Transporter does not Improve 2FL Expression

[0345] In Examples 1 to 3 it has been illustrated that lacY overexpression in combination with certain MFS transports has a positive effect on LNT and LNnT production.

[0346] In the following example the MFS transporter Nec (1 copy genomically integrated) was tested in 2FL producing strains with (MP19 and MP20) or without (MP18) overexpression of lacY. In the strains overexpressing lacy the additional genomic copy was placed under control of a weak promoter (MP19) or a strong promoter (MP20) to assess if the lacy expression level affected the 2FL production.

[0347] All strains were based on the platform strain MDO (e.g., see example 1 and also reported in WO2020255054A1 or WO2019123324A1) and contained the ?-1,2-fucolyltransferase from Helicobacter pylori, futC (GenBank assession nr. CP003904), incorporated in a single copy at two different loci in the genome of the MDO strain. The genotypes of the tested strains are given in table 6.

TABLE-US-00009 TABLE 6 Genotypes of the 2FL producing strains with different expression levels of lacY Strain ID Genotype Transporter MP18 MDO x2 PglpF-futC, PglpF-nec, 1xCA* nec MP19 MDO x2 PglpF-futC, PglpF-nec, nec PglpF_SD7-lacY, 1xCA* MP20 MDO x2 PglpF-futC, PglpF-nec, nec PglpF-lacY, 1xCA* *CA: extra colanic acid gene cluster (gmd-wcaG-wcaH-wcal-manC-manB) under the control of a PglpF promoter at a locus that is different than the native locus.

Deep-Well Assay

[0348] The strains disclosed in the present example were screened for 2FL production in a 96-deep well plate assay. Three replicates per strain were tested. More specifically, during day 1, fresh precultures were prepared using a basal minimal medium (BMM) supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated at 34? C. with 700 rpm for 24 h and then transferred to a new 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 (0.5 ?L/mL) and a bolus of 10% lactose solution (0.1 ?L/?L).

[0349] To the main culture 50% (52.5 ?l/ml) was provided to the cells accompanied by the addition of sucrose hydrolase (invertase 0.1 g/L), so that glucose was released at a rate suitable for C-limited growth allowing for gene expression and 2FL production. The main cultures were incubated at 28? C. with 700 rpm for 48 h. 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.

Results

[0350] The strains of the present example produce 2FL and very low levels of DFL. The results from deep-well assays are shown in FIG. 9. The 2FL production for the strain expressing the MFS transporter nec was set to 100 and the corresponding strains with one additional copy of lacY on the chromosome expressed from either PgIpF or PgIpF SD7 promoter was normalized to the respective nec transporter strain.

[0351] From FIG. 9 it can be seen that in a 2FL producing strain, overexpression of lacY does not increase the total 2FL production. In fact, the production seems to be reduced with increased overexpression of lacY as indicated by the strains expressing lacY from a weak promoter (PgIpF_SD7 in strain MP19) and from a strong promoter, (PgIpF in strain MP20).