Enhanced HMO formation by modifying intracellular lactose import

Abstract

The present invention relates to a method for 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 modifications capable of producing one or more HMOs, and a series of genetic modifications that enhance the transport of lactose and the produced HMOs.

Description

Technical Field

[0001] The present invention relates to methods for producing one or more human milk oligosaccharides (HMOs), particularly LNT and / or LNnT, in genetically engineered cells comprising enhanced oligosaccharide transport capabilities. The genetically modified cells comprise a series of genetic modifications that enable the production of one or more HMOs, as well as a series of genetic modifications that enhance the transport of lactose and the produced HMOs. Background Art

[0002] Human milk oligosaccharides (HMOs) have garnered significant interest over the past decade due to the discovery of their important functions in human development. Beyond their prebiotic properties, HMOs possess additional positive effects that have expanded their applications. The health benefits of HMOs have led to their approval for use in foods (such as infant formula and food) as well as consumer healthcare products.

[0003] To date, the structures of at least 115 HMOs have been determined, and many more are likely to be present in human milk.

[0004] Due to the limited availability of HMOs, efficient commercialization, i.e., large-scale production, is highly desirable. Currently, chemical synthesis of HMOs, at the scale and high quality required for their food and medical applications, is not readily available. Furthermore, the chemical synthesis pathways for HMOs involve multiple toxic chemicals, which pose a contamination risk to the final product.

[0005] To overcome the shortcomings associated with the chemical synthesis of HMOs, several enzymatic and fermentation methods have been developed. Fermentation-based processes have been developed for a variety of HMOs, such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, 3'-sialyllactose, and 6'-sialyllactose. Fermentation-based processes typically utilize genetically modified bacterial strains, such as recombinant Escherichia coli (E. coli) or yeast, such as Saccharomyces cerevisiae (S. cerevisiae).

[0006] Biosynthetic production of HMOs in engineered bacterial strains is a valuable, cost-effective, and scalable solution for HMO manufacturing. It relies on genetically engineered bacteria with modified metabolic engineering (e.g., modified sugar nucleotide synthesis pathways) and constructed to express the glycosyltransferases required to synthesize the desired oligosaccharides, and typically utilizes the bacterial nucleotide sugar pool as HMO precursors.

[0007] Recent developments in biotechnological production of HMOs have made it possible to overcome some of the inherent limitations of bacterial expression systems. For example, HMO-producing bacterial cells can be genetically modified to increase the limited intracellular nucleotide sugar pool (WO2012112777), to enhance the activity of enzymes involved in HMO production (WO2016 / 040531), or to promote the import of HMO precursors and the secretion of synthesized HMOs into the extracellular medium (WO2010142305, WO2017042382). Furthermore, the expression of a gene of interest in recombinant cells can be modulated through the use of specific promoter sequences or other gene expression regulators, such as those recently described in WO2019123324.

[0008] One approach to optimizing HMO production is to modify sugar transport, particularly the import of the HMO precursor lactose, which is the starting point of HMO biosynthesis, which can have a favorable effect on HMO production. This was achieved by overexpressing the E. coli lactose permease LacY, which represents one of the most thoroughly characterized solute transporters (Guan and Kaback, Annu Rev Biophys Biomol Struct. 2006; 35: 67-91).

[0009] WO2014018596 proposes to enhance the production of 2'-FL, LDFT, LNFP-I or LNDFH-1 by modified lactose catabolism and overexpression of the Escherichia coli lactose permease LacY, wherein the expression of LacY is driven by the lacIq promoter, an inducible synthetic promoter whose transcription level is much higher than that of the wild-type promoter (Penumetcha et al., BIOS, 81(1):7-15 (2010)). This enhances the intracellular pool of lactose and nucleotide phosphates, resulting in enhanced HMO production.

[0010] WO2017042382 combines inactivation of the endogenous β-galactosidase gene with overexpression or deletion of native or heterologous sugar efflux transporters and a functional lactose permease protein to produce one or more HMOs. Specific examples show that deletion of an oligosaccharide exporter can increase LNT formation.

[0011] EP2927316 discloses the production of 2'FL from a host with a native LacY gene under the control of the Ptet promoter and a YberC transporter expressed from a plasmid. It also discloses that overexpression of LacY in the presence of lactose can cause lactose-induced cell stress, and therefore the cells have been genetically modified to be able to fully ferment the desired oligosaccharide without the need for exogenous addition of lactose.

[0012] Disclosed herein for the first time are methods for producing one or more HMOs comprising enhancing expression of a lactose permease gene (e.g., an endogenous lactose permease gene) in combination with expression of a heterologous non-LacY sugar transporter, which surprisingly promotes an optimized transport balance, resulting in effectively enhanced production of one or more HMOs, while also reducing the amount of by-product formation. Summary of the Invention

[0013] The present disclosure relates to genetically engineered cells and methods for producing one or more HMOs (particularly LNT and / or LNnT), wherein the methods comprise genetically engineered cells in which upregulation of the lactose uptake system is combined with expression of a second sugar transporter, thereby promoting the import / export of lactose and / or oligosaccharides in the genetically engineered cells to promote HMO production. The identification of key steps in the cellular pathway that regulates HMO biosynthesis enables modification of the pathway to enhance cellular HMO production by modulating the cellular balance between metabolites and products. Specifically, maintenance of a favorable lactose / HMO balance has been achieved by overexpressing the lactose permease LacY in combination with another sugar transporter from the MFS transporter family, which enhances overall LNT and / or LNnT production by the cells and transport of specific HMOs into the fermentation medium. Overall, the present invention facilitates a more sustainable manufacturing process in which conversion of carbon sources to HMO products during fermentation occurs with higher overall yields.

[0014] Thus, in its broadest sense, the present disclosure relates to a method for producing one or more HMOs, particularly LNT and / or LNnT, comprising providing a genetically engineered cell, wherein the cell overexpresses one or more lactose permeases, expresses a heterologous MFS transporter selected from the group consisting of Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, wherein the amino acid sequence of the functional homolog has 80% identity to any one of Vag, Nec, Fred, Marc, YberC, or Bad, and wherein the cell further expresses one or more glycosyltransferases selected from the group consisting of β-1,3-GlcNAc-transferase, β-1,3-Gal-transferase, and β-1,4-gal-transferase, and optionally a sucrose utilization system. The method comprises culturing the cell in a suitable medium containing lactose and harvesting the one or more HMOs produced.

[0015] In a preferred embodiment, the genetically engineered cells of the present invention overexpress one or more native lactose permeases.

[0016] The amino acid sequence of the one or more lactose permeases is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 [LacY variants] and amino acid sequences that are functional homologs and have at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 [LacY variants].

[0017] In a currently preferred embodiment, the one or more overexpressed lactose permeases are identical to or have at least 70% identity to SEQ ID NO: 1 [LacY K-12]. The genetically engineered cell may contain at least one nucleic acid sequence encoding a lactose permease, or it may contain more than one nucleic acid sequence encoding one or more lactose permeases. The nucleic acid sequence encoding the lactose permease is preferably a native gene of the genetically engineered cell, but may be encoded by a recombinant nucleic acid sequence.

[0018] The heterologous MFS transporter is selected from the group consisting of SEQ ID NO: 62 [Vag], 63 [Fred], 64 [Marc], 65 [Bad], 66 [Nec], 67 [YberC], and a functional homolog of any one of SEQ ID NO: 62 [Vag], 63 [Fred], 64 [Marc], 65 [Bad], 66 [Nec], 67 [YberC], having an amino acid sequence at least 70% identical to any one of SEQ ID NO: 62 [Vag], 63 [Fred], 64 [Marc], 65 [Bad], 66 [Nec], 67 [YberC]. In one or more exemplary embodiments, the MFS transporter is Nec, YberC, and / or Vag. According to the present invention, the genetically engineered cell may comprise at least one nucleic acid sequence encoding one or more heterologous MFS transporters. It may comprise more than one nucleic acid sequence encoding one or more heterologous MFS transporters. The nucleic acid sequence encoding the heterologous MFS transporter may be encoded by a synthetic or recombinant nucleic acid sequence.

[0019] The genetically engineered cell may comprise a nucleic acid sequence encoding one or more glycosyltransferases selected from the group consisting of β-1,3-GlcNAc-transferase, β-1,3-Gal-transferase, and β-1,4-gal-transferase according to the present invention. It may comprise more than one nucleic acid sequence encoding one or more glycosyltransferases. The nucleic acid sequence encoding one or more glycosyltransferases may be encoded by a synthetic or recombinant nucleic acid sequence.

[0020] In one or more exemplary embodiments of the present disclosure, the β-1,3-Gal-transferase or β-1,4-gal-transferase is selected from CvB3galT and GalTK or GalT, and a functional homolog of any one of CvB3galT, GalTK or GalT, respectively, whose amino acid sequence is at least 70% identical to any one of SEQ ID NO: 17 [CvB3galT], 18 [GalTK] and 19 [GalT].

[0021] In one or more additional exemplary embodiments of the present disclosure, the β-1,3-GlcNAc-transferase is selected from LgtA, PmnagT, HD0466, and a functional homolog of any one of LgtA, PmnagT, or HD0466, whose amino acid sequence is at least 70% identical to any one of SEQ ID NO: 20 [LgtA], 21 [PmnagT], or 22 [HD0466].

[0022] In one or more exemplary embodiments, the genetically engineered cell comprises one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides that, when expressed, constitute a sucrose utilization system capable of utilizing sucrose as the sole carbon and energy source for the genetically engineered cell. In one or more preferred exemplary embodiments, the genetically engineered cell comprises a PTS-dependent sucrose utilization system and further comprises the scrYA and scrBR operons. In another preferred embodiment, the genetically engineered cell expresses a polypeptide, preferably a single polypeptide, capable of hydrolyzing sucrose into glucose and fructose.

[0023] One method for enhancing HMO production is also to modify the biosynthesis of activated sugar nucleotides used for HMO biosynthesis. Thus, the genetically engineered cells can contain at least one nucleic acid sequence encoding one or more heterologous polypeptides involved in the biosynthesis of activated sugars according to the present disclosure. In one or more exemplary embodiments, the genetically engineered cells express one or more polypeptides involved in the biosynthesis of activated sugars, such as one or more polypeptides selected from Pgm [SEQ ID NO: 43 or 44], GalU [SEQ ID NO: 45 or 46], GalE [SEQ ID NO: 47 or 48], GlmM [SEQ ID NO: 49 or 50], GlmU [SEQ ID NO: 51 or 52], GlmS [SEQ ID NO: 33 or 54], and functional homologs thereof, whose amino acid sequences are at least 70% identical to any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54.

[0024] According to the gene expression of protein in the genetically engineered cell of the present invention, it is regulated by regulatory elements.Therefore, any one or more nucleotide sequences according to the present invention can be regulated by one or more regulatory elements.In one or more exemplary embodiments, regulatory elements comprise one or more promoter sequences, and nucleotide sequence is as shown in Table 8, preferably selected from the promoter sequence of SEQ ID NO:70 (PglpF) or SEQ ID NO:68 (Plac) or SEQ ID NO:69 (PgatY_70UTR), SEQ ID NO:82 (PmglB_UTR70) or SEQ ID NO:100 (PglpA_70UTR) or SEQ ID NO:101 (PglpT_70UTR) or cp6 (SEQ ID NO:84) or Posmy (SEQ ID NO:85) or its variant.

[0025] In the presently exemplified embodiment, in the genetically engineered cells of the present invention, one or more lactose permeases are regulated by the promoter sequence PglpF.

[0026] In another presently exemplified embodiment, in the genetically engineered cells of the present invention, one or more heterologous MFS transporters are regulated by the promoter sequences Plac and / or PglpF.

[0027] Thus, in a currently preferred embodiment, in the genetically engineered cells of the present invention, the one or more lactose permeases are native and regulated by the promoter sequence PglpF, and the one or more heterologous MFS transporters are selected from Nec, YberC and / or Vag and are regulated by the promoter sequences Plac and / or PglpF.

[0028] Regulatory elements may also comprise endogenous, heterologous, synthetic and / or optimized Shine-Dalgarno sequences.

[0029] The nucleic acid sequences comprising the elements of the present disclosure may be integrated into the genome of the genetically engineered cell. Alternatively, one or more nucleic acid sequences may be expressed from a plasmid, such as one or more low, medium and / or high copy number plasmids.

[0030] In one or more exemplary embodiments, the genetically engineered cell is a bacterial or yeast cell, such as an E. coli cell, eg, preferably a strain derived from E. coli K-12.

[0031] The present invention also relates to methods and / or the use of genetically engineered cells for producing and / or manufacturing one or more HMOs (eg, LNT and / or LNnT). DETAILED DESCRIPTION

[0032] The present disclosure relates to a genetically engineered cell and a method for producing one or more HMOs, wherein upregulation of the cell's lactose uptake system is combined with expression of a second sugar transporter, thereby promoting import / export of oligosaccharides in the genetically engineered cell to promote HMO production, particularly of LNT and LNnT.

[0033] In the present disclosure, cellular production of LNT and / or LNnT is enhanced by regulating the cellular balance between metabolites and products. Specifically, maintenance of a favorable lactose / HMO balance has been achieved, wherein overexpression of the lactose permease LacY in combination with another sugar transporter from the MFS transporter family (particularly selected from Vag, Nec, Fred, Marc, YberC, and Bad) enhances the overall HMO levels produced by the cells and the levels of HMOs transported into the fermentation medium, resulting in a higher overall yield of HMOs, particularly LNT and / or LNnT, produced by the engineered cells.

[0034] Modulation of HMO biosynthesis can modify cellular pathways, which can enhance cellular production of HMOs by regulating the cellular balance between metabolites and products. The term "biosynthesis" refers to the synthesis of one or more HMOs by and / or in genetically engineered cells. The cellular balance between metabolites and products is an important feature for maintaining cell viability, where accumulation of metabolites (e.g., phosphate) and / or product precursors (e.g., lactose) can lead to cellular stress, thereby reducing the efficiency of HMO production. On the other hand, cellular accumulation of products (e.g., one or more HMOs) can also lead to cellular stress responses, which can also hinder the efficiency of HMO production. Therefore, engineering of cellular pathways to customize each point of HMO biosynthesis combined with customized input / output systems to reduce cellular accumulation of metabolites and / or HMOs while enhancing the output of produced HMOs is highly advantageous in the large-scale production of HMOs.

[0035] In the present disclosure, maintenance of a favorable cellular balance between metabolites and products has been achieved by overexpression of the lactose permease LacY in combination with another sugar transporter (such as, but not limited to, the MFS transporters YberC, Nec, or Vag), which enhances the overall levels of LNT or LNnT produced and in some cases affects biomass formation, as described in Examples 1-3.

[0036] Specifically, the present disclosure relates to a genetically engineered cell that overexpresses one or more lactose permeases in combination with a heterologous MFS transporter selected from the group consisting of Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, whose amino acid sequence is 70% identical to the amino acid sequence of any one of Vag, Nec, Fred, Marc, YberC, or Bad, and further expresses one or more glycosyltransferases. In a preferred embodiment, the genetically engineered cell expresses two or more glycosyltransferases, wherein the glycosyltransferases are β-1,3-GlcNAc-transferase and β-1,3-Gal-transferase, or β-1,3-GlcNAc-transferase and β-1,4-Gal-transferase, or β-1,3-GlcNAc-transferase and β-1,3-Gal-transferase and β-1,4-Gal-transferase.

[0037] In a preferred embodiment, the present disclosure relates to a genetically engineered cell that overexpresses one or more native lactose permeases in combination with a heterologous MFS transporter selected from the group consisting of Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, whose amino acid sequence has 70% identity to the amino acid sequence of any one of Vag, Nec, Fred, Marc, YberC, or Bad, and further expresses one or more glycosyltransferases. In a preferred embodiment, the genetically engineered cell expresses two or more glycosyltransferases, wherein the glycosyltransferases are β-1,3-GlcNAc-transferase and β-1,3-Gal-transferase, or β-1,3-GlcNAc-transferase and β-1,4-Gal-transferase, or β-1,3-GlcNAc-transferase and β-1,3-Gal-transferase and β-1,4-Gal-transferase.

[0038] A method for producing one or more human milk oligosaccharides

[0039] The present disclosure provides a method for producing one or more human milk oligosaccharides (HMOs), particularly LNT and / or LNnT, comprising enhancing expression of a lactose permease gene in combination with expression of a heterologous non-LacY sugar transporter, which surprisingly facilitates enhanced production of one or more HMOs while also reducing the amount of by-product formation. Preferably, the lactose permease gene is endogenous to the host cell.

[0040] This is demonstrated, for example, in Examples 1 and 2, where additional copies of LacY were inserted into strains already expressing the heterologous transporters YberC, Nec, or Vag, and enhanced the production of LNT in Example 1 and LNnT and pLNnH in Example 2, while simultaneously reducing byproduct formation, such as LNT-II and pLNH2 production. As also evident from Example 3, when LacY was overexpressed, HMO titers in fermentation reached higher levels compared to normal LacY expression levels. Lower byproduct formation and higher titers are essential features in large-scale production environments, where lower byproduct formation can lead to simpler post-fermentation purification, and higher titers can lead to higher production yields, thereby reducing HMO production costs.

[0041] Human milk oligosaccharides (HMOs)

[0042] In the context of the present disclosure, the term "oligosaccharide" refers to a sugar polymer containing multiple monosaccharide units. In some embodiments, preferred oligosaccharides are sugar polymers composed of three, four, five or six monosaccharide units, i.e., trisaccharides, tetrasaccharides, pentasaccharides or hexasaccharides. Preferred oligosaccharides of the present disclosure are human milk oligosaccharides (HMOs).

[0043] The term "human milk oligosaccharide" or "HMO" in this context refers to complex carbohydrates found in human milk. HMOs have a core structure comprising a lactose unit at the reducing end, which may be elongated by one or more β-N-acetyl-lactosamine and / or one or more β-lacto-N-biosyl units, and which may be substituted with α-L-fucopyranyl and / or α-N-acetyl-neuraminic acid (sialic acid) moieties.

[0044] In the context of this disclosure, lactose is not considered an HMO species.

[0045] Non-acidic (or neutral) HMOs do not contain sialic acid residues, and acidic HMOs have at least one sialic acid residue in their structure. The methods of the present disclosure are specifically designed to increase the production and overall yield of neutral and acidic HMOs.

[0046] Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose II (LNT-II), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), p-lacto-N-neohexaose (pLNnH), p-lacto-N-hexaose (pLNH), and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3'-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-II), and lacto-N-difucohexaose III (LNDFH-III). III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-p-lacto-N-hexaose I (FpLNH-I), fucosyl-p-lacto-N-neohexaose II (F-pLNnHII) and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), 3'-O-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6'-O-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6'-O-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3'-O-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d). d), sialic acid-lacto-N-hexaose (SLNH), sialic acid-lacto-N-neohexaose I (SLNH-I), sialic acid-lacto-N-neohexaose II (SLNH-II), and disialyl-lacto-N-tetraose (DSLNT).

[0047] In one or more preferred embodiments, the one or more HMOs produced are selected from the group consisting of LNT-II, pLNnH, LNT and LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, 2'-FL, DFL, 3-FL, LST-a, 3'-SL, 6'-SL, LST-b, LST-c, FSL, FLST-a, DSLNT, LNnH, and LNH.

[0048] Preferably, the HMO produced by the methods and / or genetically engineered cells disclosed herein is an HMO comprising an LNT-II core, such as LNT and / or LNnT, pLNnH and / or LNFP-I.

[0049] Even more preferably, the HMO produced is LNT and / or LNnT.

[0050] In one or more exemplary embodiments, the HMO produced is LNT. In one or more additional exemplary embodiments, the HMO produced is LNnT.

[0051] LNT, LNnT, and LNFP-I contain an LNT-II core and can be synthesized in appropriately genetically engineered cells by selecting a series of glycosyltransferases that have the ability to attach specific sugars such as galactose (Gal) or N-acetylglucosamine (GlcNAc) to the lactose backbone.

[0052] Genetically engineered cells

[0053] The genetically engineered cells of the present invention are host cells that have been genetically engineered for HMO production.

[0054] Specifically, the present disclosure relates to an engineered cell that overexpresses one or more lactose permeases in combination with a heterologous MFS transporter selected from Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, whose amino acid sequence has 70% identity to the amino acid sequence of any one of Vag, Nec, Fred, Marc, YberC, or Bad, and further expresses one or more glycosyltransferases. The one or more lactose permeases can be native to the host cell.

[0055] Genetically engineered cells can typically contain one or more genes that are not present in the natural (non-genetically engineered) form of the cell. Techniques for introducing exogenous nucleic acid molecules / sequences and / or inserting exogenous nucleic acid molecules / sequences (recombinant, heterologous) into the genetic information of the cell to insert, delete or alter the nucleic acid sequence of the genetic information of the cell are known to those skilled in the art.

[0056] The genetically engineered cells according to the present disclosure may contain one or more genes that are naturally present in the cell, wherein the genes are modified by artificial means and reintroduced into the microbial cell.

[0057] The term "genetically engineered cell" also encompasses cells containing nucleic acid molecules endogenous to the cell that have been modified but not removed from the cell. Such modifications include those obtained by gene replacement, site-specific mutagenesis, and related techniques.

[0058] Genetically engineered cells of the present invention can be provided using standard methods in the art, such as those described in the manuals of Sambrook et al., Wilson & Walker, Maniatis et al., and Ausubel et al.

[0059] The genetically engineered cells can be any cell useful for HMO production, including mammalian cell lines. Preferably, the host cell is a unicellular microorganism of eukaryotic or prokaryotic origin. Suitable microbial cells that can serve as host cells include yeast cells, bacterial cells, archaeal cells, algae cells, and fungal cells.

[0060] In one or more exemplary embodiments, the genetically engineered cell is a prokaryotic cell. In one or more preferred exemplary embodiments, the genetically engineered cell is a bacterial cell.

[0061] The genetically engineered cells of the present disclosure can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow genetic manipulation to insert the gene of interest and can be cultured on a manufacturing scale. Preferably, the host cell has properties that allow it to be cultured to high cell densities.

[0062] In an embodiment of the present invention, the genetically engineered cell is a bacterial or yeast cell. In a preferred embodiment, the genetically engineered cell is preferably a prokaryotic cell, such as a bacterial cell.

[0063] Non-limiting examples of bacterial cells suitable for use in the present disclosure are selected from recipient organisms that do not naturally contain any of the mentioned MFS transporters Vag, Nec, Fred, Marc, YberC and Bad. These include, but are not limited to, E. coli K-12 strains (e.g., EMG2, MG1655, W3110, W3350, C600 and DH5α) (this is the strain background for most genetic engineering work performed in E. coli), ATCC 8739 (E. coli C), ATCC 11303 (E. coli B), BL21 (see New England Biolabs catalog, 2007-2008), and derivatives of these strains.

[0064] In a currently preferred embodiment, the genetically engineered cells of the present invention are E. coli cells. In a particularly preferred embodiment, the genetically engineered cells of the present invention are E. coli cells derived from E. coli K-12 strain or DE3 strain.

[0065] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli, Corynebacterium glutamicum (C. glutamicum), Lactococcus lactis (L. lactis), Bacillus subtilis (B. subtilis), and Streptomyces lividans (S. lividans).

[0066] In one or more exemplary embodiments, the genetically engineered cell is selected from Bacillus subtilis and Escherichia coli.

[0067] In one or more exemplary embodiments, the genetically engineered cell is selected from Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, Bacillus subtilis, and Streptomyces lividans.

[0068] In one or more exemplary embodiments, the genetically engineered cell is Bacillus subtilis.

[0069] In one or more exemplary embodiments, the genetically engineered cell is Corynebacterium glutamicum.

[0070] In one or more exemplary embodiments, the genetically engineered cell is Escherichia coli.

[0071] Non-limiting examples of fungal host cells suitable for the recombinant industrial production of HMO products can be yeast cells, such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae, or filamentous fungi such as Aspargillus sp., Fusarium sp., or Thricoderma sp., exemplary species being A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum, and T. reesei.

[0072] In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.

[0073] In one or more exemplary embodiments, the genetically engineered cell is Saccharomyces cerevisiae or Pichia pastoris.

[0074] In one or more exemplary embodiments, the genetically engineered cell is Pichia pastoris.

[0075] Expression in genetically engineered cells

[0076] In connection with the present invention, the term "expression" may refer to the expression of a gene or nucleic acid sequence resulting in the production of a protein, or it may relate to the expression of a protein, i.e., it may be directly involved in the production of a protein. In particular, the term "expression" may refer to the expression of a lactose permease gene resulting in the expression of a lactose permease protein, or the expression of a lactose permease protein and the expression of an MFS transporter gene or an MFS transporter protein, respectively.

[0077] The term "enhanced expression" is used interchangeably in this context with the term "overexpression" of, for example, a lactose permease protein, and refers to an increase in the level of expressed protein compared to the normal protein expression level of the native protein in the cell. Enhanced expression of a protein or gene can be achieved in a variety of ways and can include modification of the gene copy number, controlling the expression of any copy of the gene at the transcriptional or translational level, for example by replacing the native promoter with a strong promoter, deleting regulatory elements that inhibit gene expression, or introducing episomal elements, such as a plasmid that carries and expresses the coding sequence of the gene of interest.

[0078] It may be advantageous to increase gene copy number and / or expression of one or more genes encoding enzymes and / or transporters directly or indirectly involved in the HMO biosynthetic pathway, as described herein and exemplified in Examples 1-3.

[0079] In one or more exemplary embodiments, expression is controlled by increasing the copy number of a gene or nucleic acid sequence encoding a protein of interest.

[0080] In one or more exemplary embodiments, expression of a gene or nucleic acid sequence encoding a protein of interest is enhanced by controlling expression using a strong promoter sequence.

[0081] Overexpression of one or more lactose permeases

[0082] The present disclosure relates to a genetically engineered cell and a method comprising the genetically engineered cell, the cell overexpressing one or more lactose permeases.

[0083] In a preferred embodiment, the present disclosure relates to a genetically engineered cell and a method comprising the genetically engineered cell that overexpresses one or more native lactose permeases.

[0084] Overexpression of one or more lactose permeases according to the present disclosure results in increased production of one or more HMOs, particularly LNT and / or LNnT, while in some cases also resulting in reduced amounts of by-products in production, as described in Examples 1-3.

[0085] "Overexpression" in this context refers to expression levels of a protein that are higher than the levels naturally obtained by the cells of the present invention. Therefore, the introduction and expression of a heterologous gene is not considered "overexpression" in relation to the present invention. Overexpression can be determined by transcriptional or translational analysis, such as quantitative measurement of mRNA levels or protein levels by any method known to those skilled in the art, such as, but not limited to, quantitative PCR or mass spectrometry. In the present disclosure, overexpression is preferably determined by experimental observation of effects that can be associated with enhanced expression of one or more lactose permeases, such as those described in Examples 1-3 and Figure 1-5 The experimental data provided in .

[0086] Lactose permease gene

[0087] In one or more embodiments of the present invention, the genetically engineered cells comprise one or more overexpressed lactose permease genes. The lactose permease gene may be a lacY gene.

[0088] The genetically engineered cells according to the present invention may comprise at least one, such as at least two, three, four, five, six, seven, eight, nine or ten nucleic acid sequences encoding a lactose permease.

[0089] In one or more preferred exemplary embodiments, the genetically engineered cell comprises more than one nucleic acid sequence encoding one or more lactose permeases.

[0090] In one or more exemplary embodiments, the genetically engineered cells comprise one or more additional lactose permeases, such as one or more heterologous lactose permeases.

[0091] In one or more additional exemplary embodiments, the one or more lactose permeases are encoded by heterologous and / or recombinant nucleic acid sequences.

[0092] In one or more preferred exemplary embodiments, the nucleic acid sequence encoding one or more lactose permeases is a native gene of the genetically engineered cell.

[0093] native lactose permease gene

[0094] In one or more exemplary embodiments, the genetically engineered cells of the present disclosure comprise an overexpressed host cell native lactose permease gene.

[0095] The native lactose permease disclosed herein is a gene found endogenously in the host cells of the present invention. For example, the native lactose permease gene can be selected from the lacY gene of E. coli K-12MG1655 of SEQ ID NO: 2 encoding the lactose permease LacY of SEQ ID NO: 1 in an E. coli K-12MG1655 derivative strain. Thus, assuming that the gene encoding E. coli BL-21(DE3) LacY is not identical to the gene encoding E. coli K-12MG1655 LacY, the native lacY gene of E. coli BL-21(DE3) overexpressed in the E. coli K-12MG1655 derivative strain will not be native to the E. coli K-12MG1655 derivative strain.

[0096] LacY

[0097] In one embodiment, the one or more lactose permeases may be any one or more amino acid sequences selected from SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 [LacY variants] and functional homologues thereof, the amino acid sequences of the functional homologues having at least 70% identity, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identity to any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 [LacY variants].

[0098] In one or more preferred exemplary embodiments, the one or more lactose permeases are identical to SEQ ID NO:1.

[0099] In one or more embodiments, the lactose permease is a native lactose permease.

[0100] MFS transporter

[0101] The genetically engineered cells of the present disclosure comprise at least one nucleic acid sequence encoding one or more heterologous MFS transporters. Thus, the genetically engineered cells of the present disclosure express heterologous MFS transporters.

[0102] Transporters of the major facilitator superfamily (MFS) facilitate the transport of molecules such as, but not limited to, oligosaccharides across cell membranes.

[0103] The term "major facilitator superfamily (MFS)" refers to a large and unusually diverse family of secondary active transporters, whose members are responsible for the transport of a range of different substrates, including sugars, drugs, hydrophobic molecules, peptides, organic ions, etc.

[0104] The term "MFS transporter" in this context refers to a protein that facilitates the transport of oligosaccharides (preferably HMOs), preferably HMOs / oligosaccharides synthesized by the genetically engineered cells described herein, across or across the cell membrane from the cell cytosol to the cell culture medium. Additionally or alternatively, MFS transporters can also facilitate the efflux of molecules not considered to be HMOs or oligosaccharides, such as lactose, glucose, cellular metabolites, and / or toxins. In preferred embodiments, MFS transporters are capable of exposing LNT and / or LNnT from the cell cytosol to the cell culture medium.

[0105] In the context of the present invention, lactose permease is not considered to be a heterologous MFS transporter.

[0106] In Examples 1-3, it is shown how the introduction of selected heterologous genes encoding sugar efflux transporters into the genetic background of LacY expressing or LacY overexpressing strains can reverse the order of abundance of HMO precursors and / or byproducts of cellular HMO production and thus facilitate increased HMO production in genetically modified cells.

[0107] In one or more exemplary embodiments, the MFS transporter is selected from the group consisting of Bad, Nec, YberC, Fred, Vag, and Marc.

[0108] Thus, in one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a heterologous MFS transporter selected from Vag, Nec, Fred, Marc, YberC, Bad, and a functional homolog of any one of Vag, Nec, Fred, Marc, YberC, or Bad, the amino acid sequence of which has 70% identity to the amino acid sequence of any one of SEQ ID NO: 62 [Vag], 63 [Fred], 64 [Marc], 65 [Bad], 66 [Nec], or 67 [YberC].

[0109] Bad

[0110] The amino acid sequence of the MFS transporter, identified interchangeably herein as "Bad protein" or "Bad transporter" or "Bad," is SEQ ID NO: 65; the amino acid sequence identified herein as SEQ ID NO: 65 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession ID WP_017489914.1.

[0111] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Bad. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is Bad.

[0112] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein bad or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of bad [SEQ ID NO: 65].

[0113] In one or more embodiments, expression of bad is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0114] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter Bad of SEQ ID NO: 65 or a functional homolog thereof having an amino acid sequence that is 70% identical to the amino acid sequence of SEQ ID NO: 65, and further express one or more glycosyltransferases.

[0115] Nec

[0116] The amino acid sequence of the MFS transporter, identified interchangeably herein as "Nec protein" or "Nec transporter" or "Nec," is SEQ ID NO: 66; the amino acid sequence identified herein as SEQ ID NO: 66 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession ID WP_092672081.1.

[0117] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Nec. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is Nec.

[0118] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein Nec or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of Nec [SEQ ID NO: 66].

[0119] In one or more embodiments, expression of Nec is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0120] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter nec of SEQ ID NO: 66 or a functional homolog thereof having an amino acid sequence that is 70% identical to the amino acid sequence of SEQ ID NO: 66, and further express one or more glycosyltransferases.

[0121] YberC

[0122] The amino acid sequence of the MFS transporter, identified interchangeably herein as "YberC protein" or "YberC transporter" or "YberC," is SEQ ID NO:67; the amino acid sequence identified herein as SEQ ID NO:67 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession ID EEQ08298.1.

[0123] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is YberC. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is YberC.

[0124] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein YberC or a functional homolog thereof, whose amino acid sequence is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of YberC [SEQ ID NO: 67].

[0125] In one or more embodiments, expression of YberC is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0126] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter YberC of SEQ ID NO: 67 or a functional homolog thereof having an amino acid sequence that is 70% identical to the amino acid sequence of SEQ ID NO: 67, and further express one or more glycosyltransferases.

[0127] Fred

[0128] The amino acid sequence of the MFS transporter, identified interchangeably herein as "Fred protein" or "Fred transporter" or "Fred," is SEQ ID NO:63; the amino acid sequence identified herein as SEQ ID NO:63 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession ID WP_087817556.1.

[0129] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Fred. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is Fred.

[0130] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein fred or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of fred [SEQ ID NO: 63].

[0131] In one or more embodiments, expression of fred is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0132] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter Fred of SEQ ID NO: 63 or a functional homolog thereof having an amino acid sequence with 70% identity to the amino acid sequence of SEQ ID NO: 63, and further express one or more glycosyltransferases.

[0133] Vag

[0134] The amino acid sequence of the MFS transporter, identified interchangeably herein as "Vag protein" or "Vag transporter" or "Vag," is SEQ ID NO: 62; the amino acid sequence identified herein as SEQ ID NO: 62 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession ID WP_048785139.1.

[0135] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Vag. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is Vag.

[0136] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter protein vag or a functional homolog thereof, the amino acid sequence of which is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of vag [SEQ ID NO: 62].

[0137] In one or more embodiments, expression of vag is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0138] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter vag of SEQ ID NO: 62 or a functional homolog thereof having an amino acid sequence that is 70% identical to the amino acid sequence of SEQ ID NO: 62, and further express one or more glycosyltransferases.

[0139] Marc

[0140] The amino acid sequence of the MFS transporter, identified interchangeably herein as "Marc protein" or "Marc transporter" or "Marc," is SEQ ID NO: 64; the amino acid sequence identified herein as SEQ ID NO: 64 is an amino acid sequence that is 100% identical to the amino acid sequence of GenBank Accession No. WP_060448169.1.

[0141] In one or more exemplary embodiments, the MFS transporter expressed according to the present disclosure is Marc. Thus, in one or more exemplary embodiments, the heterologous MFS transporter expressed by the genetically engineered cells of the present disclosure is Marc.

[0142] In one or more embodiments of the invention, the genetically engineered cells express a heterologous MFS transporter marc or a functional homolog thereof, whose amino acid sequence is at least 80%, such as at least 90%, such as at least 95%, such as at least 99% identical to the amino acid sequence of marc [SEQ ID NO: 64].

[0143] In one or more embodiments, expression of marc is regulated by a regulatory element comprising the Plac promoter element of SEQ ID NO:68.

[0144] In one embodiment of the present invention, the genetically engineered cells overexpress one or more native lactose permeases in combination with the heterologous MFS transporter marc of SEQ ID NO: 64 or a functional homolog thereof having an amino acid sequence that is 70% identical to the amino acid sequence of SEQ ID NO: 64, and further express one or more glycosyltransferases.

[0145] Thus, in one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a heterologous MFS transporter that is Vag, Nec, Fred, Marc, YberC, or Bad. In one or more additional exemplary embodiments, the genetically engineered cells of the present disclosure express more than one heterologous MFS transporter selected from the group consisting of SEQ ID NO: 62 [Vag], 63 [Fred], 64 [Marc], 65 [Bad], 66 [Nec], and 67 [YberC].

[0146] In one or more exemplary embodiments, the genetically engineered cells of the present disclosure express a functional homolog of Vag, Nec, Fred, Marc, YberC and / or Bad, whose amino acid sequence is at least 70%, 80%, 85%, 90%, 95% or at least 99% identical to any one of SEQ ID NO: 62, 63, 64, 65, 66 or 67.

[0147] In a presently preferred embodiment, the MFS transporter expressed is Nec, Vag and / or YberC.

[0148] In a particularly preferred embodiment, the expressed MFS transporter is YberC.

[0149] In a particularly preferred embodiment, the expressed MFS transporter is Nec.

[0150] In a particularly preferred embodiment, the expressed MFS transporter is Vag.

[0151] Heterologous expression

[0152] In this disclosure, the term heterologous refers to a protein that is experimentally placed into a cell that does not normally produce (i.e., express) the protein. Thus, heterologous means that the transferred protein is typically originally cloned or derived from a different cell type or a different species than the recipient. The protein itself does not necessarily have to be transferred, but rather "correctly edited" genetic material (complementary DNA or cDNA) encoding the protein can be added to the recipient cell. The transferred genetic material typically must be in a format that encourages the recipient cell to express the cDNA as protein (i.e., it is placed into an expression vector). Methods for transferring exogenous genetic material into recipient cells include transfection and transduction. The choice of recipient cell type is typically based on the experimental needs for detailed examination of protein function, and the most common receptors (called heterologous expression systems) are typically chosen because they are easy to transfer DNA into, or because they can more simply assess the function of the protein.

[0153] Glycosyltransferase

[0154] The genetically engineered cells of the present disclosure used in accordance with the present disclosure also express one or more glycosyltransferases. The nucleic acid sequence encoding the one or more expressed glycosyltransferases can be integrated into the genome of the genetically engineered cells (via chromosomal integration), or alternatively, it can be contained in a plasmid and expressed as plasmid-borne, as described in the present disclosure.

[0155] If two or more glycosyltransferases are required for the genetically engineered cell to be suitable for producing an HMO, such as LNT or LNnT, two or more heterologous nucleic acids encoding different enzymes having glycosyltransferase activity can be incorporated into the nucleic acid construct, or they can be provided as separate nucleic acid sequences that can be integrated into the genome and / or expressed from a plasmid.

[0156] In one exemplary embodiment, a β-1,3-N-acetylglucosaminyltransferase (a first heterologous nucleic acid sequence encoding a first glycosyltransferase) is introduced into a host cell in combination with a β-1,3-galactosyltransferase (a second heterologous nucleic acid sequence encoding a second glycosyltransferase) for LNT production, wherein the first heterologous nucleic acid and the second heterologous nucleic acid can be independently integrated into the chromosome or expressed from a plasmid, or they can be combined into a nucleic acid construct, optionally containing additional features of the present disclosure. Thus, in one or more exemplary embodiments, the genetically engineered cells of the present invention contain at least one nucleic acid sequence encoding one or more heterologous glycosyltransferases.

[0157] In another exemplary embodiment, a β-1,3-N-acetylglucosaminyltransferase (a first heterologous nucleic acid sequence encoding a first glycosyltransferase) is introduced into a host cell in combination with a β-1,4-galactosyltransferase (a second heterologous nucleic acid sequence encoding a second glycosyltransferase) to produce LNnT, wherein the first and second heterologous nucleic acids can be independently integrated into the chromosome or expressed from a plasmid, or they can be combined into a nucleic acid construct, optionally containing additional features of the present disclosure. Thus, in one or more exemplary embodiments, the genetically engineered cells of the present invention contain at least one nucleic acid sequence encoding one or more heterologous glycosyltransferases.

[0158] In one or more exemplary embodiments, the first and second heterologous nucleic acids encoding one or more glycosyltransferases are both stably integrated into the chromosome of the genetically engineered cell; in another preferred embodiment, according to the present invention, the first and second heterologous nucleic acids encoding one or more glycosyltransferases are integrated independently of the nucleic acid sequence encoding the native lactose permease and / or MFS transporter. In one or more additional exemplary embodiments, the first and second heterologous nucleic acids encoding one or more glycosyltransferases are integrated into a nucleic acid construct. In one or more additional exemplary embodiments, at least one heterologous nucleic acid sequence encoding a glycosyltransferase is plasmid-borne.

[0159] The glycosyltransferase of the present invention is selected from the group consisting of enzymes having the following activities: α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3 / 4-fucosyltransferase, α-1,4-fucosyltransferase, α-2,3-sialyltransferase, α-2,6-sialyltransferase, β-1,3-N-acetylglucosaminyltransferase, β-1,6-N-acetylglucosaminyltransferase, β-1,3-galactosyltransferase, and β-1,4-galactosyltransferase.

[0160] In a preferred embodiment, the glycosyltransferase is selected from the group consisting of β-1,3-GlcNAc-transferase, β-1,3-Gal-transferase and β-1,4-gal-transferase.

[0161] Table 1 shows some non-limiting embodiments of proteins having glycosyltransferase activity that can be encoded by a heterologous gene contained in the production cell.

[0162] Table 1

[0163]

[0164]

[0165]

[0166]

[0167] The enzymes are capable of biosynthesizing the HMO core structure as well as fucosylated and / or sialylated HMOs and their glycoside derivatives.

[0168] The production of neutral N-acetylglucosamine-containing HMOs in modified bacteria is known in the art (see, for example, Gebus C et al. (2012) Carbohydrate Research 36:383-90).

[0169] In one or more exemplary embodiments, the β-1,3-Gal-transferase or β-1,4-gal-transferase is selected from CvB3galT and GalTK or GalT, respectively, and functional homologs thereof, whose amino acid sequence is at least 70% identical to any one of SEQ ID NOs: 17, 18 or 19, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical.

[0170] In one or more preferred exemplary embodiments, the β-1,3-Gal-transferase is CvB3galT having an amino acid sequence of SEQ ID NO:17.

[0171] In one or more preferred exemplary embodiments, the β-1,3-Gal-transferase is GalTK having an amino acid sequence of SEQ ID NO:18.

[0172] In another currently preferred embodiment, the β-1,4-gal-transferase is GalT having an amino acid sequence of SEQ ID NO:19.

[0173] In one or more additional exemplary embodiments, the β-1,3-GlcNAc-transferase is selected from LgtA, PmnagT, HD0466, and functional homologs thereof, whose amino acid sequence is at least 70% identical to any one of SEQ ID NO: 20, 21 or 22, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.9% identical.

[0174] In one or more additional exemplary embodiments, the β-1,3-GlcNAc-transferase is LgtA having an amino acid sequence of SEQ ID NO:20.

[0175] In another one or more additional exemplary disclosures, the α-1,2-fucosyltransferase is selected from FutC, Smob, Mtun and functional homologs thereof, and its amino acid sequence is at least 70% identical to any one of SEQ ID NO: 23, 24 or 25, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical.

[0176] In one or more additional exemplary disclosures, the α-1,3-fucosyltransferase is selected from FutA, FucT, FucTIII and functional homologs thereof, and its amino acid sequence is at least 70% identical to any one of SEQ ID NO: 26, 27 or 28, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical.

[0177] In one or more further exemplary disclosures, the α-2,3-sialyltransferase is Pd2 and / or Nst and / or a functional homolog thereof, whose amino acid sequence is at least 70% identical to any one of SEQ ID NO: 29 or 30, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identical.

[0178] Sucrose fermentation

[0179] In the production of one or more HMOs, the starting material significantly impacts the quality, purity, and quantity of the resulting product. In some cases, the utilization of glucose as a carbon and energy source can be problematic due to difficulties in handling and sterilizing glucose, which often results in glucose degradation, derivative formation, and caramelization. Therefore, the ability to utilize alternative sugars (such as, but not limited to, sucrose) with simpler processing parameters is highly advantageous in the production of one or more HMOs.

[0180] In large-scale manufacturing, the cost price of raw materials and purity are most important for the feasibility of production. Therefore, using the more flexible sucrose comprising glucose and fructose part is very advantageous in large-scale manufacturing. However, many manufactures and / or production bacterial strains can not utilize sucrose as a carbon source owing to the lack of an enzyme that can hydrolyze sucrose. In some cases, this has been overcome by adding sucrose and sucrose hydrolase (for example invertase) to fermentation medium, thereby allowing sucrose to be hydrolyzed into glucose and fructose.

[0181] In one or more preferred exemplary embodiments, the genetically engineered cell expresses a sucrose utilization system. Such a system may be endogenous to the cell, but it may also be heterologous if the cell is unable to utilize sucrose.

[0182] In one or more preferred exemplary embodiments, the genetically engineered cell comprises one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides, enabling it to utilize sucrose as the sole carbon and energy source of the genetically engineered cell.

[0183] In one or more preferred exemplary embodiments, the genetically engineered cell expresses a polypeptide capable of hydrolyzing sucrose into glucose and fructose. Preferably, the polypeptide capable of hydrolyzing sucrose into glucose and fructose is a single heterologous enzyme.

[0184] Thus, in one or more exemplary embodiments, the heterologous polypeptide capable of hydrolyzing sucrose to fructose and glucose expressed by the genetically modified cells of the present disclosure is selected from SEQ ID NO: 86 [SacC_Agal, glycoside hydrolase family 32 protein, WP_103853210.1] and SEQ ID NO: 87 [Bff, β-fructofuranosidase protein, BAD18121.1], and a functional homolog of any one of SEQ ID NO: 86 or 87, whose amino acid sequence has at least 70% identity to any one of SEQ ID NO: 86 or 87, such as at least 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.9% identity.

[0185] Another approach is to express a heterologous sucrose utilization system, such as, but not limited to, a PTS-dependent system or expression of an invertase, enabling the cell to utilize sucrose as a carbon and energy source, as described in Examples 2 and 3 of this disclosure.

[0186] A heterologous PTS-dependent sucrose utilization and transport system comprising a sucrose-specific porin, a sucrose transporter, and a sucrose 6-phosphate hydrolase is described, for example, in WO2015197082. The oxidation of glucose-6-phosphate and fructose provides bioenergy through the organism's own metabolic system. In addition, glucose-6-phosphate and fructose can be used as carbon sources for producing sugar nucleotides in the cell's natural biosynthetic pathway. The sugar nucleotides thus produced are donors for glycosylated carbohydrate acceptors (e.g., lactose) and are internalized by specific permeases of the cell to produce the target oligosaccharides. Glycosylation is mediated by one or more glycosyltransferases directly produced by expressing heterologous genes. The organism lacks any enzyme that degrades the acceptor or oligosaccharide products in the cell.

[0187] Thus, in one or more exemplary embodiments, the genetically engineered cells of the present disclosure comprise one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides that enable the utilization of sucrose as the sole carbon and energy source for the genetically engineered cells. In one or more preferred exemplary embodiments, the genetically engineered cells comprise a PTS-dependent sucrose utilization system further comprising scrYA and scrBR operons.

[0188] Thus, in one or more exemplary embodiments, a genetically engineered cell according to the present disclosure comprises a PTS-dependent sucrose utilization transport system and / or a recombinant nucleic acid sequence encoding a heterologous polypeptide capable of hydrolyzing sucrose into fructose and glucose.

[0189] In one or more exemplary embodiments, the polypeptide encoded by the scrYA operon is a polypeptide having an amino acid sequence according to SEQ ID NOs: 88 and 89 [scrY and scrA] or a functional homolog of any one of SEQ ID NOs: 88 and 89 [scrY and scrA], the amino acid sequence of the functional homolog having at least 80% identity, such as at least 90% identity, such as at least 95% identity, to any one of SEQ ID NOs: 88 and 89 [scrY and scrA].

[0190] In one or more exemplary embodiments, the polypeptide encoded by the scrBR operon is a polypeptide having an amino acid sequence according to SEQ ID NOs: 90 and 91 [scrB and scrR], or a functional homolog of any one of SEQ ID NOs: 90 and 91 [scrB and scrR], the amino acid sequence of the functional homolog having at least 80% identity, such as at least 90% identity, such as at least 95% identity, to any one of SEQ ID NOs: 90 and 91 [scrB and scrR].

[0191] Such cells as described above are capable of utilizing sucrose as a carbon and energy source.

[0192] Additionally, the culturing step of the methods disclosed herein can include a two-step sucrose feed, wherein in the second feeding stage, less sucrose is continuously added to the culture than in the first feeding stage to slow cell growth and increase product content produced in high cell density cultures.

[0193] The feed rate of sucrose continuously added to the cell culture during the second feeding phase can be about 30-40% lower than the feed rate of sucrose continuously added during the first feeding phase.

[0194] During both feed phases, lactose can be added continuously, preferably together with sucrose in the same feed solution, or sequentially.

[0195] Optionally, when a large amount of unused receptor remains in the extracellular fraction after the second phase, the culture further comprises a third feeding phase.

[0196] The addition of sucrose is then continued without the addition of acceptor, preferably at approximately the same feed rate as set for the second feed phase until the acceptor is exhausted.

[0197] Sugar nucleotide synthesis

[0198] The biosynthesis of activated sugar nucleotides can affect the production of one or more HMOs in a variety of ways. First, increased production of specific sugar nucleotides, which serve as substrates in HMO synthesis, can increase overall HMO production or reduce byproduct formation. Second, decreased production of specific sugar nucleotides can reduce the amount of byproduct production while maintaining the level of target HMO production. Therefore, in one or more exemplary embodiments of the present disclosure, the genetically engineered cells contain at least one nucleic acid sequence encoding one or more heterologous polypeptides involved in the biosynthesis of activated sugars.

[0199] The terms "activated sugar" or "activated sugar nucleotide" or "sugar nucleotide" are used interchangeably and, in this context, refer to modified sugars, such as nucleotide sugars (e.g., UDP-galactose), that serve as precursors in HMO synthesis. Typically, monosaccharides are activated for glycosyltransferase-mediated reactions to synthesize HMOs by conjugation to nucleotides or phosphorylation. Therefore, modifications in sugar nucleotide synthesis can be highly beneficial for the production of one or more HMOs.

[0200] Activated sugar nucleotides generally have a phosphorylated glycosyl residue connected to a nucleoside. Specific glycosyltransferases only accept specific activated sugar nucleotides. Therefore, preferably, the following activated sugar nucleotides participate in glycosyl transfer: glucose-UDP-GlcNAc, UDP-galactose, UDP-glucose, UDP-N-acetyl glucosamine, UDP-N-acetyl galactosamine (GlcNAc) and CMP-N-acetyl neuraminic acid. Genetically modified cells according to the present disclosure can comprise one or more pathways that produce nucleotide activated sugars selected from glucose-UDP-GlcNAc, GDP-fucose, UDP-galactose, UDP-glucose, UDP-N-acetyl glucosamine, UDP-N-acetyl galactosamine and CMP-N-acetyl neuraminic acid (CMP-Neu5Ac). Table 7 below is a non-limiting example of glycosyl donors and the HMO products they can be used for production, and this list may not be exhaustive.

[0201] Table 7. Glycosyl donor HMO product list

[0202]

[0203] One or more exemplary embodiments of the present disclosure relate to a genetically modified cell having modified sugar nucleotide synthesis. Specifically, increased sugar nucleotide synthesis of UDP-GlcNAc and / or UDP-Gal can facilitate the production of LNT and / or LNnT.

[0204] In one or more exemplary disclosures, the genetically engineered cells express one or more polypeptides involved in activated sugar nucleotide biosynthesis. The one or more polypeptides involved in activated sugar nucleotide biosynthesis can be selected from Gmd, WcaG, WcaH, WcaI, ManC, ManB, Pgm, GalU, GalE, GlmM, GlmU, GlmS, NeuA, NeuB, NeuC, and functional homologs thereof, and have an amino acid sequence at least 70% identical to any one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45.

[0205] In one or more exemplary embodiments of the present disclosure, the genetically engineered cells express one or more polypeptides involved in activated sugar nucleotide biosynthesis, wherein the one or more polypeptides involved in activated sugar nucleotide biosynthesis are selected from Pgm [SEQ ID NO: 43 or 44], GalU [SEQ ID NO: 45 or 46], GalE [SEQ ID NO: 47 or 48], GlmM [SEQ ID NO: 49 or 50], GlmU [SEQ ID NO: 51 or 52], GlmS [SEQ ID NO: 33 or 54], and functional homologs thereof, and their amino acid sequences have at least 70% identity with any one of SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or 54.

[0206] Colanic acid gene cluster

[0207] The colanic acid gene cluster of Escherichia coli K-12 is responsible for the production of the extracellular polysaccharide colanic acid, the major oligosaccharide of the bacterial cell wall.

[0208] Since the colanic acid gene cluster is typically introduced into genetically engineered cells in the form of a PglpF-driven expression cassette, as described in the Examples, deletion of the glpR gene (which encodes the DNA-binding transcriptional repressor GlpR) eliminates the repression exerted by GlpR on transcription from all PglpF promoters in the cell and in this way enhances gene expression of all PglpF-based cassettes (containing the colanic acid gene cluster).

[0209] In one or more exemplary disclosures, the colanic acid gene cluster can be expressed from its natural genomic site. Expression can be actively regulated. This expression can be regulated by exchanging a natural promoter with a target promoter and / or by expressing a gene cluster from another genomic site to increase the copy number of the protein encoding the colanic acid gene or by additional expression of the colanic acid gene cluster or its specific gene.

[0210] Thus, in one or more exemplary disclosures, the expression can be regulated by exchanging the native promoter with a desired promoter, and / or by increasing the copy number of the colanic acid gene encoding the protein by expressing a gene cluster from another genomic locus, or by episomal expression of the colanic acid gene cluster.

[0211] With respect to the present disclosure, the term "natural genomic site" in relation to the colanic acid gene cluster relates to the original and natural location of the gene cluster in the genome of a genetically engineered cell.

[0212] Gmd

[0213] The gmd gene encodes the protein GDP-mannose-4,6-dehydratase, which catalyzes the conversion of GDP-D-mannose to GDP-4-dehydro-6-deoxy-D-mannose. This protein participates in the synthesis of GDP-L-fucose from GDP-α-D-mannose. In one or more exemplary disclosures, the gmd gene is overexpressed.

[0214] wcaG

[0215] The wcaG gene, also known as fcl, encodes the protein GDP-L-fucose synthase (EC 1.1.1.271), which catalyzes the two-step NADP-dependent conversion of GDP-4-dehydro-6-deoxy-D-mannose to GDP-fucose, involving epimerase and reductase reactions. In one or more exemplary disclosures, the wcaG gene is overexpressed.

[0216] wxya

[0217] The wcaH gene encodes the protein GDP-mannose mannosyl hydrolase (EC 3.6.1.-), which hydrolyzes GDP-mannose and GDP-glucose. In one or more exemplary disclosures, the wcaH gene is overexpressed.

[0218] wxya

[0219] The wcal gene encodes the colanic acid biosynthesis glycosyltransferase Wcal, which catalyzes the transfer of unmodified fucose to UPP-Glc (α-D-glucopyranosyl-diphosphoundecyl-glucose). In one or more exemplary disclosures, the wcal gene is overexpressed.

[0220] manB

[0221] The manB gene encodes a protein phosphomannose mutase (EC 5.4.2.8), which participates in the biosynthesis of GDP-mannose by catalyzing the conversion of α-D-mannose-1-phosphate to D-mannose-6-phosphate. Thus, the expression level of manB regulates the formation of GDP-mannose. In one or more exemplary disclosures, the manB gene is overexpressed.

[0222] manC

[0223] The manC gene encodes the protein mannose-1-phosphate guanylyltransferase (EC 2.7.7.13), which is involved in the biosynthesis of GDP-mannose by synthesizing GDP-mannose from GTP and α-D-mannose-1-phosphate. In one or more exemplary disclosures, the manC gene is overexpressed.

[0224] Sialic acid catabolism pathway

[0225] Additionally, genetically engineered cells may also include sialic acid synthesis capabilities. For example, the cells are transformed with exogenous UDP-GlcNAc 2-epimerase (e.g., neuC from Campylobacter jejuni (GenBank AAK91727.1) or equivalent (e.g., (GenBank CAR04561.1), Neu5Ac synthase (e.g., neuB from Campylobacter jejuni (GenBank AAK91726.1) or equivalent (Flavobacterium limnosediminis sialic acid synthase, NCBI Reference Sequence WP_023580510.1), and / or CMP-Neu5Ac synthase (e.g., NeuA from Campylobacter jejuni (GenBank AAK91728.1) or equivalent (e.g., Vibrio brasiliensis CMP sialic acid synthase, NCBI Reference Sequence WP_006881452.1).

[0226] In one or more exemplary disclosures, genetically engineered cells contain defective sialic acid catabolism pathways. A "sialic acid catabolism pathway" refers to a series of reactions typically controlled and catalyzed by enzymes that result in the degradation of sialic acid. The exemplary sialic acid catabolism pathway described herein is an E. coli pathway. In this pathway, sialic acid (Neu5Ac; N-acetylneuraminic acid) is degraded by the enzymes NanA (N-acetylneuraminic acid lyase), NanK (N-acetylmannosamine kinase), and NanE (N-acetylmannosamine-6-phosphate epimerase), all encoded by the nanATEK-yhcH operon and inhibited by NanR (http: / / ecocyc.org / ECOLI). A defective sialic acid catabolic pathway is generated in an E. coli host by introducing mutations in the endogenous nanA (N-acetylneuraminic acid lyase) (e.g., GenBank Accession No. D00067.1 (GL216588)) and / or nanK (N-acetylmannosamine kinase) genes (e.g., GenBank Reference (amino acids) BAE77265.1 (GL85676015)) and / or nanE (N-acetylmannosamine-6-phosphate epimerase, Gene ID: 947745, incorporated herein by reference). Optionally, the nanT (N-acetylneuraminic acid transporter) gene is also inactivated or mutated. Other intermediates of sialic acid metabolism include: (ManNAc-6-P) N-acetylmannosamine-6-phosphate; (GlcNAc-6-P) N-acetylglucosamine-6-phosphate; (GlcN-6-P) glucosamine-6-phosphate and (Fruc-6-P) fructose-6-phosphate.

[0227] In one or more preferred exemplary disclosures, nanA is mutated. In other preferred embodiments, nanA and nanK are mutated, and nanE remains functional. In another preferred embodiment, nanA and nanE are mutated, and nanK is not mutated, inactivated or missing. Mutation in this context refers to one or more changes in the nucleotide sequence of the gene product encoding nanA, nanK, nanE and / or nanT. For example, mutation can be 1, 2, at most 5, at most 10, at most 25, at most 50 or at most 100 changes in the nucleotide sequence. For example, nanA, nanK, nanE and / or nanT genes mutate by invalid (null) mutation. Null mutation as herein described encompasses amino acid replacement, addition, deletion or insertion, which causes enzyme function loss (i.e., activity reduction or inactivity) or enzyme loss (i.e., no gene product). "deletion" refers to that the coding region is removed in whole or in part so as not to produce (functional) gene product. Inactivation refers to a coding sequence that has been altered such that the resulting gene product is functionally inactive or encodes a gene product that has less than 100%, e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the activity of the native, naturally occurring, endogenous gene product. A "non-mutated" gene or protein does not differ from the native, naturally occurring or endogenous coding sequence or the corresponding encoded amino acid sequence by 1, 2, at most 5, at most 10, at most 20, at most 50, at most 100, at most 200 or at most 500 or more codons.

[0228] repressor

[0229] In one or more exemplary embodiments, the genetically engineered cells disclosed herein comprise a non-functional or absent gene product that normally binds a regulatory element and inhibits expression of any protein of the disclosure that is regulated by the regulatory element.

[0230] In this context, the term non-functional (or absent) gene product which generally binds and inhibits expression driven by regulatory elements relates to a DNA binding site upstream of the coding sequence of the gene of interest and specifically in the promoter region of said gene.

[0231] In one or more exemplary embodiments, the cells can have a non-functional (or absent) gene product that normally binds to and inhibits expression of the lacY gene and / or any protein with an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or an upstream region of a regulatory element for controlling expression of the lacY gene and / or any protein with an amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0232] In one or more exemplary embodiments, the gene product is the DNA-binding transcriptional repressor GlpR.

[0233] GlpR

[0234] GlpR belongs to the DeoR family of transcriptional regulators and acts as a repressor of the glycerol-3-phosphate regulon, which is composed of different operons. This regulator is part of the glpEGR operon, but it can also be constitutively expressed as an independent (glpR) transcription unit. In addition, when E. coli is grown in the presence of inducers, glycerol or glycerol-3-phosphate (G3P) and in the absence of glucose, the regulated operon is induced. In the absence of inducers, the repressor binds in tandem to an inverted repeat sequence consisting of a 20-nucleotide-long DNA target site.

[0235] The term "non-functional or absent" with respect to the glpR gene refers to inactivation of the glpR gene by complete or partial deletion of the corresponding nucleic acid sequence (e.g., SEQ ID NO: 92 or a variant thereof encoding glpR capable of downregulating a glpF-derived promoter) from the bacterial genome. The glpR gene encodes the DNA-binding transcriptional repressor GlpR. In this manner, due to the deletion of the repressor gene, promoter sequences of the PglpF family are upregulated in the genetically engineered cells, while conversely, the PglpF promoter is downregulated.

[0236] In one or more exemplary embodiments, the glpR gene is deleted.

[0237] activator

[0238] In one or more exemplary embodiments, the genetically engineered cells disclosed herein comprise an overexpressed gene product that enhances the expression of any gene and / or protein of the disclosure that is regulated by the regulatory elements.

[0239] In one or more exemplary embodiments, the cells of the present disclosure may comprise an overexpressed gene product that enhances the expression of a gene encoding the lactose permease LacY and / or any protein having an amino acid sequence of SEQ ID NOs: 1-16.

[0240] In one or more exemplary embodiments, the gene product is cAMP DNA-binding dual regulator of transcription CRP.

[0241] CRP

[0242] CRP belongs to the CRP-FNR superfamily of transcription factors. CRP regulates the expression of several Escherichia coli genes, many of which are involved in the catabolism of secondary carbon sources. After activation by cyclic adenosine monophosphate (cAMP), CRP directly binds to a specific promoter sequence, and this binding recruits RNA polymerase through direct interaction, thereby activating the transcription of the nucleic acid sequence following the promoter sequence, resulting in the expression of the target gene. Therefore, overexpression of CRP can lead to enhanced expression of the target gene / nucleic acid sequence. Among other functions, CRP exerts its function on the PglpF promoter, and contrary to the repressor GlpR, it activates the promoter sequence of the PglpF family. In this way, overexpression of CRP in the genetically engineered cells of the present disclosure promotes the expression of genes regulated by the PglpF family promoter.

[0243] Thus, in one or more exemplary embodiments, the crp gene is overexpressed.

[0244] As suggested in this disclosure, genetic engineering of GlpR and / or CRP in 2'-FL producing strains facilitates the overall production of 2'-FL by these strains.

[0245] Regulating elements

[0246] Genetically engineered cells according to the present disclosure may comprise regulatory elements capable of controlling the overexpression of endogenous, heterologous and / or synthetic nucleic acid sequences.

[0247] The term "regulatory element" encompasses promoter sequences, signal sequences, and / or transcription factor binding site arrays that influence the transcription and / or translation of a nucleic acid sequence to which the regulatory element is operably linked.

[0248] Regulatory elements are found at both the transcriptional and post-transcriptional levels, further enabling molecular networks at these levels. For example, at the post-transcriptional level, biochemical signals that control mRNA stability, translation, and subcellular localization are processed by regulatory elements. RNA-binding proteins are another type of post-transcriptional regulatory element and are further categorized as either sequence elements or structural elements. Specific sequence motifs that function as regulatory elements are also associated with mRNA modification. A variety of DNA regulatory elements participate in the regulation of gene expression and rely on biochemical interactions between DNA, cellular proteins that constitute chromatin, and transcription factors.

[0249] Generally, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.

[0250] Promoters and enhancers are the main genomic regulatory elements of gene expression. Promoters are regions of DNA within 1-2 kilobases (kb) of the transcription start site (TSS) of a gene; they contain short regulatory elements (DNA motifs) necessary for the assembly of the RNA polymerase transcription machinery. However, transcription is usually minimal without the contribution of DNA regulatory elements located distal to the TSS. These regions, often referred to as enhancers, are position-independent DNA regulatory elements that interact with site-specific transcription factors to establish cell type recognition and regulate gene expression. Enhancers can function independently of their sequence context and at distances of several to hundreds of kb from their target genes through a process called looping. Due to these characteristics, it is difficult to identify appropriate enhancers and link them to their target genes based solely on DNA sequence.

[0251] A promoter, along with other transcriptional and translational regulatory nucleic acid sequences (also called "control sequences"), is necessary for the expression of a given gene or group of genes (operon).

[0252] Identifying appropriate promoter sequences that promote expression of a particular gene of interest is a tedious task that in many cases requires laborious work.With respect to the present disclosure, a regulatory element may or may not be a post-translational regulatory factor, or it may or may not be a translational regulatory factor.

[0253] In one or more exemplary embodiments of the present invention, expression of any one or more nucleic acid sequences and / or genes is regulated by one or more regulatory elements.

[0254] In one or more exemplary embodiments, the regulatory element comprises one or more elements capable of enhancing expression, ie, overexpression, of any one or more nucleic acid sequences according to the present disclosure.

[0255] In this respect, regulatory elements can be endogenous or heterologous, and / or recombinant and / or synthetic nucleic acid sequences. In this article, the term "heterologous regulatory element" is understood to be a regulatory element that is not endogenous to the original genetically engineered cell as described herein. A heterologous regulatory element can also be a recombinant regulatory element, in which two or more non-operably linked natural regulatory elements are recombined into heterologous and / or synthetic regulatory elements. Methods well known to those skilled in the art can be used to introduce heterologous regulatory elements into genetically engineered cells.

[0256] The regulatory element may be a native regulatory element, such as the Plac promoter, which promotes expression of the native lac operon of E. coli.

[0257] Promoter sequence

[0258] The present disclosure relates to genetically engineered cells and / or methods comprising providing such genetically engineered cells, wherein the expression of any one or more of the nucleic acid sequences is regulated by one or more regulatory elements. The regulatory elements may comprise one or more promoter sequences.

[0259] Regulatory elements that regulate the expression of genes and / or nucleic acid sequences may comprise one or more promoter sequences, wherein the promoter sequence is operably linked to the nucleic acid sequence of the gene of interest, in such a way that the expression of the nucleic acid sequence of the gene of interest is regulated.

[0260] Generally, the promoter can comprise a natural, heterologous and / or synthetic nucleic acid sequence, and can be a recombinant nucleic acid sequence, recombining two or more nucleic acid sequences or the same or different sources as described above, thereby producing a homologous, heterologous or synthetic nucleic acid promoter sequence, and / or a homologous, heterologous or synthetic nucleic acid regulatory element. Promoter sequences derived from a wide range of selections of PglpF, PlgpA, PlgpT, PgatY, PmglB and Plac promoter systems are described in detail in WO2019123324A1 and WO2020255054A1.

[0261] In one or more exemplary embodiments, the regulatory elements of the genes and / or heterologous nucleic acid sequences of the genetically engineered cells comprise more than one native or heterologous promoter sequence.

[0262] In one or more exemplary embodiments, the regulatory elements of the genes and / or heterologous nucleic acid sequences of the genetically engineered cells comprise two or more regulatory elements having the same promoter sequence.

[0263] In one or more exemplary embodiments, the regulatory elements of the genes and / or heterologous nucleic acid sequences of the genetically engineered cells comprise two or more regulatory elements having non-identical promoter sequences.

[0264] The regulatory architecture, i.e., the gene-by-gene distribution of transcription factor binding sites and the identity of the transcription factors that bind to these sites, can be used for a variety of different growth conditions, and more than 100 genes from the E. coli genome can serve as regulatory elements. Thus, any promoter sequence that is capable of transcribing and / or regulating the level of transcription of one or more heterologous or native nucleic acid sequences encoding one or more proteins described herein may be suitable.

[0265] One approach to increasing product production could be to modulate the production of a required enzyme activity for the production of the product or precursor / substrate input, such as a glycosyltransferase or MFS transporter or lactose permease.

[0266] Increasing the strength of the promoter driving expression of the desired enzyme may be one way to achieve this. The strength of the promoter can be assessed using the lacZ enzyme assay, in which β-galactosidase activity is determined as previously described (see, for example, Miller J.H. Experiments in molecular genetics, Cold Spring Harbor Laboratory Press, NY, 1972). Briefly, cells are diluted in Z buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The lacZ assay is performed at 30°C. The sample is preheated, the assay is initiated by adding 200 μl of o-nitrophenyl-β-galactosidase (4 mg / ml), and the assay is stopped by adding 500 μl of 1 M Na2CO3 when the sample turns slightly yellow. The release of o-nitrophenol is then determined as the change in optical density at 420 nm. Specific activity is reported in Miller units (MU) [A420 / (min*ml*A600)]. Regulatory elements with an activity greater than 10,000 MU are considered strong regulatory elements, while regulatory elements with an activity less than 3,000 MU are considered weak regulatory elements, with regulatory elements in between having intermediate strengths. An example of a strong regulatory element is the PglpF promoter, which has an activity of approximately 14,000 MU, while an example of a weak promoter is Plac, which has an activity of approximately 2,300 MU when induced with IPTG.

[0267] Alternatively, if it is necessary to balance the expression levels of one or more proteins to optimize production, it may be beneficial to use a promoter with a desired strength (e.g., medium or low strength). Table 8 below lists a range of wild-type and recombinant promoters based on their strength relative to the PglpF promoter.

[0268] Table 8 - Promoter sequences according to strength

[0269]

[0270]

[0271] na=Not visited

[0272] *Promoter activity was assessed in the LacZ assay described below, and the PglpF promoter was run as a positive reference in the same assay. For comparison between assays, activity was calculated relative to the PglpF promoter, and ranges indicate results from multiple assays.

[0273] The promoter may be of heterologous origin, native to the genetically modified cell, or it may be a recombinant promoter combining heterologous and / or native elements.

[0274] In one or more exemplary embodiments, the regulatory element comprises a promoter sequence selected from the group consisting of PBAD, Pxyl, Plac, PsacB, PxylA, PrpR, PnitA, PT7, Ptac, PL, PR, PnisA, Pb, PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, Plac_16UTR, PmglB_70UTR, PmglB_70UTR_SD4, CP6, PosmY, Pspc, Pbla, Prrn1, and Prrn2.

[0275] In a currently preferred embodiment, the promoter sequence is selected from PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, Plac_16UTR and Plac.

[0276] In one or more exemplary embodiments, the regulatory element is a promoter selected from the group consisting of PglpF (SEQ ID NO: 70), PglpT_70UTR (SEQ ID NO: 101), SEQ ID NO: 69 (PgatY_70UTR), Plac (SEQ ID NO: 68, PmglB_70UTR (SEQ ID NO: 82), PglpA_70UTR (SEQ ID NO: 100), Cp6 (SEQ ID NO: 84), Posmy (SEQ ID NO: 85), or variants thereof, and variants thereof. In particular, the variants disclosed in Table 8 are preferred.

[0277] In one or more exemplary embodiments, the regulatory element is a promoter with high or medium strength, for example, a promoter sequence selected from the group consisting of: PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_7 0UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR.

[0278] In a preferred embodiment, the promoter is a strong promoter selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, and PmglB_16UTR. This may be particularly advantageous for expression of heterologous glycosyltransferases and / or MFS transporters.

[0279] In another embodiment, the promoter is selected from promoters of medium strength, such as PglpF_SD9, PglpF_SD7, PglpF_SD6, and PglpA_16UTR. This may be particularly advantageous for the expression of heterologous MFS transporters if balanced expression with expression of lactose permease is desired.

[0280] In another embodiment, the promoter is selected from low strength promoters such as Plac, PglpF_SD3 and PglpF_SD1. This may be particularly advantageous for the expression of heterologous MFS transporters if balanced expression with expression of lactose permease is desired.

[0281] In a currently preferred embodiment, the promoter sequence is PglpF as shown in SEQ ID NO: 70. In a preferred embodiment of the present invention, the regulatory elements for regulating the heterologous MFS transporter include a medium or low strength promoter element, such as the Plac promoter (SEQ ID NO: 68), and the regulatory elements for regulating LacY include a high strength promoter, such as PglpF (SEQ ID NO: 70).

[0282] In a currently preferred embodiment, the Plac promoter element of SEQ ID NO: 68 is contained within the regulatory elements regulating YberC, and the PglpF shown in SEQ ID NO: 70 is contained within the regulatory elements regulating LacY.

[0283] Another example of a regulatory element is the Shine-Dalgarno sequence, a ribosome binding site in bacterial and archaeal messenger RNAs, typically located approximately eight bases upstream of the AUG start codon. It helps recruit ribosomes to the mRNA and initiate protein synthesis. Therefore, modification of the Shine-Dalgarno sequence upstream of a coding nucleic acid sequence can alter the expression level of any one or more genes and / or nucleic acid sequences and / or polypeptides of the present invention.

[0284] Thus, in one or more exemplary embodiments, a genetically engineered cell according to the present disclosure comprises one or more endogenous, heterologous, synthetic and / or optimized Shine-Dalgarno sequences.

[0285] Sequence identity

[0286] In the context of two or more nucleic acid or amino acid sequences, the term "% sequence identity" refers to the two or more sequences having a given percentage of common nucleotides or amino acid residues (i.e., sequences having at least 90% (%) identity) when compared and aligned for maximum correspondence over a comparison window or a specified nucleic acid or amino acid sequence. The percent identity of nucleic acid or amino acid sequences can be measured using the BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see, for example, http: / / www.ncbi.nlm.nih.gov / BLAST / ). This definition also applies to the complement of the test sequence and sequences with deletions and / or additions, as well as sequences with substitutions. An example of an algorithm suitable for determining percent identity, sequence similarity, and alignment is the BLAST 2.2.20+ algorithm, which is described in Altschul et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ is used to determine the percent sequence identity of nucleic acids and proteins disclosed herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). Examples of commonly used sequence alignment algorithms are:

[0287] CLUSTAL Omega(http: / / www.ebi.ac.uk / Tools / msa / clustalo / ),

[0288] EMBOSS Needle(http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ), MAFFT(http: / / mafft.cbrc.jp / alignment / server / ), or

[0289] MUSCLE (http: / / www.ebi.ac.uk / Tools / msa / muscle / ).

[0290] For purposes of the present invention, the sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mo / .Biol. 48: 443-453) implemented in the Needle program of EMBOSS package (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher (accessible https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5, and EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix (substitution matrix). The Needle output labeled "longest identity" (obtained using the -nobrief option) was used as the percent identity, calculated as follows: (identical residues x 100) / (length of alignment - total number of gaps in the alignment).

[0291] For purposes of the present invention, the sequence identity between two nucleotide sequences is preferably used as at the EMBOSS bag, 10 preferred versions 5.0.0 or higher versions (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277) in the Needle program implemented in the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970 as above) determine.The parameter used is gap open penalty (gap open penalty) 10, gap extension penalty (gap extension penalty) 0.5 and DNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix.The Needle output (using-nobrief option to obtain) labeled as " longest identity " is used as identity percentage, is calculated as follows: (identical deoxyribonucleotide × 100) / (total number of gaps in comparison length-comparison).

[0292] Functional homologs

[0293] Functional homologues of the proteins / nucleotides described herein are proteins / nucleotides with altered genetic codes that retain their original function. Functional homologues can be obtained by mutagenesis. Compared to the functionality of the protein / nucleotide, a functional homologue should have at least 50%, for example 60%, 70%, 80%, 90% or 100% residual functionality.

[0294] Functional homologs of any of the disclosed amino acid sequences may also have increased functionality. Functional homologs of any of the polypeptides disclosed herein should ideally be capable of participating in HMO production in terms of HMO yield, purity, reduction in biomass formation, viability of genetically engineered cells, robustness of genetically engineered cells according to the disclosure, or reduction in consumables.

[0295] Integrated into the genome

[0296] The nucleic acid sequence expressed in the genetically engineered cell according to the present invention may be integrated into the genome of the genetically engineered cell.

[0297] Integration of the heterologous nucleic acid of interest, which may be contained in a construct (expression cassette), into the genome can be achieved by conventional methods, for example, by using a linear cassette containing flanking sequences homologous to a specific site on the chromosome, as described for the attTn7 site (Waddell CS and Craig NL, Genes Dev. (1988) Feb; 2(2): 137-49.); methods for genomic integration of nucleic acid sequences, wherein recombination is mediated by the Red recombinase function of bacteriophage lambda or the RecE / RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998); 180(8): 2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128; Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red / ET recombination (Wenzel et al., Chem. Biol. Am. Am. (1996) 18(1 ... (1996) 18(1): 239-243); methods based on Red / ET recombination (Wenzel et al., Chem. Biol. Am. (1996) 18(1): 239-243); methods based on Red / ET recombination (Wenzel et al. Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005); 71(4):1829-35); or positive clones can be selected, ie clones carrying the expression cassette, for example by selection of a marker gene, or loss or gain of gene function.

[0298] The expression of natural or heterologous gene and / or overexpression can be obtained from the episomal integration of said gene, that is, the nucleic acid encoding heterologous polypeptide can be inserted in the nucleic acid construct, then included in the expression vector of plasmid or another chromosome independence, wherein expression vector can be integrated in the chromosome in some cases, yet do not need to do so to obtain the expression of the heterologous nucleic acid encoding heterologous polypeptide of the present invention.The example of episome is for example transposon and insertion sequence.

[0299] In the context of the disclosed invention, the term "genomic integration" refers to the integration of one or more native or heterologous nucleic acid sequences into a chromosome or endogenous plasmid and thereby into the genome of the genetically engineered cell of the present invention.

[0300] Thus, in one or more exemplary preferred embodiments, the present invention relates to a genetically engineered cell as described above, comprising one or more heterologous nucleic acid sequences encoding one or more episomal and / or genomically integrated copies of, for example, one or more lactose permease and / or heterologous MFS transporter genes and / or one or more glycosyltransferases as described above.

[0301] In one or more exemplary embodiments, one or more native and / or heterologous nucleic acid sequences encoding, for example, one or more lactose permeases and / or heterologous MFS transporters and / or one or more glycosyltransferases according to the present invention are stably integrated into the genome of the genetically engineered cell.

[0302] In one or more further exemplary embodiments, one or more native and / or heterologous nucleic acid sequences encoding, for example, one or more lactose permeases and / or heterologous MFS transporters and / or one or more glycosyltransferases according to the present invention are transiently integrated into the genome of the genetically engineered cell.

[0303] Expression from plasmids

[0304] In one or more additional exemplary embodiments, the one or more nucleic acid sequences are expressed from a plasmid.

[0305] In the present disclosure, one or more nucleic acid sequences encoding, for example, one or more lactose permeases and / or heterologous MFS transporters and / or one or more glycosyltransferases according to the present invention may be transiently introduced into genetically engineered cells.

[0306] Thus, in one or more exemplary embodiments, one or more heterologous nucleic acid sequences, e.g., encoding one or more lactose permeases and / or heterologous MFS transporter genes and / or one or more glycosyltransferases, are plasmid-borne, such as, but not limited to, low, medium, or high copy number plasmids.

[0307] In the present disclosure, low / medium / high copy number plasmids refer to the number of plasmid copies per bacterial cell. In one or more exemplary embodiments of the present disclosure, a low copy number plasmid is one in which cells contain an average of 1-20 copies of the plasmid, such as an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 copies per cell. Thus, in one or more exemplary embodiments of the present disclosure, a medium copy number plasmid is one in which cells contain an average of 21-100 copies of the plasmid, such as an average of 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 98, 99, or 100 copies of a plasmid. In addition, in one or more exemplary embodiments of the present disclosure, a high copy number plasmid is one in which cells contain an average of at least 101 copies of the plasmid, such as an average of at least 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies per cell.

[0308] Cell culture medium

[0309] In this context, a growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells or plantlets. The culture medium contains an appropriate energy source and may contain compounds that regulate the cell cycle. The culture medium may be semi-defined, i.e., containing complex culture medium compounds (e.g., yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined without any complex compounds. Exemplary suitable culture media are provided in the Experimental Examples.

[0310] In one or more exemplary embodiments, the culture medium is a minimal culture medium.

[0311] In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from the group consisting of glycerol, sucrose, glucose, and fructose.

[0312] In one or more exemplary embodiments, the culture medium is supplemented with one or more energy and carbon sources selected from the group consisting of glycerol, sucrose, and glucose.

[0313] In one or more exemplary embodiments, the culture medium is supplemented with glycerol, sucrose and / or glucose.

[0314] In one or more exemplary embodiments, the culture medium is supplemented with glycerol and / or glucose.

[0315] In one or more exemplary embodiments, the culture medium is supplemented with sucrose and / or glucose.

[0316] In one or more exemplary embodiments, the culture medium is supplemented with glycerol and / or sucrose.

[0317] In one or more exemplary embodiments, the culture medium is supplemented with sucrose only.

[0318] In one or more exemplary embodiments, the culture medium contains sucrose as the sole carbon and energy source.

[0319] In one or more exemplary embodiments, the culture medium is supplemented with lactose as a suitable acceptor for the glycosyltransferases of the present invention.

[0320] The cell culture

[0321] The present disclosure relates to a method for producing HMOs in a biosynthetic process, comprising culturing a genetically engineered cell according to the present disclosure in a suitable culture medium and harvesting one or more HMOs. Preferably, the culture medium contains lactose.

[0322] According to the present invention, the term "culturing" (or "cultivating" or "cultivation", also called "fermentation") relates to the propagation of bacterial expression cells in controlled bioreactors according to methods known in the industry.

[0323] According to the method, the genetically engineered cell, preferably a bacterium, more preferably E. coli, is a strain optimized for industrially profitable transformations such as HMO production. Such optimization may include the steps described in the present disclosure.

[0324] To produce one or more HMOs, the HMO-producing microorganisms described herein are cultured according to procedures known in the art, and the produced HMOs are harvested from the culture medium and biomass formed during the culture process. Thereafter, the HMOs are purified according to procedures known in the art, such as those described in WO2015188834, WO2017182965, or WO2017152918, and the purified HMOs are used as nutraceuticals, pharmaceuticals, or for any other purpose, such as for research.

[0325] During the culture of the genetically engineered cells disclosed herein, the oligosaccharide-producing cells are supplied with a carbon and energy source, such as, but not limited to, sucrose, which provides energy for growth, reproduction, and maintenance of their structure through glycolysis. Furthermore, as described above, the energy and carbon source absorbed by the cells provide or serve as precursors for the synthesis of activated sugar nucleotides required for the glycosylation process occurring in the cells. Furthermore, a lactose solution is added to the culture medium.

[0326] Lactose can be provided as a bolus of a 20% lactose solution (0.1 ml) into a basal minimal medium supplemented with magnesium sulfate and thiamine as demonstrated in Example 1. Alternatively or additionally, lactose can be added continuously, preferably together with sucrose in the same feed solution, or sequentially.

[0327] Lactose levels during culture

[0328] In one or more exemplary embodiments, the lactose level in the culture medium is adjusted.

[0329] In one or more exemplary embodiments, lactose levels are adjusted from low to high during the genetically engineered cell culture. In this context, this results in the following lactose concentration ranges: 30-80 g / L for high lactose processes and 0-15 g / L, e.g., 0.5-15 g / L, for low lactose processes.

[0330] In one or more exemplary embodiments, a high level of lactose levels relates to 30-80 g / L, such as, but not limited to, 30-40 g / L, 30-50 g / L, 30-60 g / L, 30-70 g / L, 40-50 g / L, 40-60 g / L, 40-70 g / L, 40-80 g / L, 50-60 g / L, 50-70 g / L, 50- 80g / L, 60-70g / L, 60-80g / L, 35-50g / L, 35-60g / L, 35-70g / L, 35-75g / L, 35-80g / L, 45-55g / L, 45-75g / L, 55-65g / L, 55-75g / L, 55-80g / L, 65-75g / L, or 65-80g / L.

[0331] In one or more exemplary embodiments, low levels of lactose levels relate to 0-15 g / L, such as, but not limited to, 0-5 g / L, 0-7.5 g / L, 0-10 g / L, 0-12.5 g / L, 0.5-5 g / L, 0.5-7.5 g / L, 0.5-10 g / L, 0.5-12.5, 0.5-15 g / L g / L, 2.5-5g / L, 2.5-7.5g / L, 2.5-10g / L, 2.5-12.5g / L, 2.5-15g / L, 5-7.5g / L, 5-10g / L, 5-12 .5g / L, 5-15g / L, 7.5-10g / L, 7.5-12.5g / L, 7.5-15g / L, 10-12.5g / L, 10-15g / L, or 12.5-15g / L.

[0332] Internalized HMO backbone precursors, such as lactose, participate in glycosyltransferase-induced glycosylation reactions in which the glycosyl residue of the activated nucleotide donor produced by the cell is transferred, resulting in glycosylation of the acceptor. Optionally, when the cell expresses more than one glycosyltransferase, additional glycosylation reactions can occur, leading to the formation of target oligosaccharides and / or byproducts. The cell preferably lacks any enzymatic activity that would degrade oligosaccharide derivatives produced in the cell.

[0333] Harvest

[0334] The term "harvesting" in the context of the present invention relates to the collection of produced HMOs after fermentation has terminated. In various embodiments, this may include collecting HMOs contained in both the biomass (i.e., genetically engineered cells) and the culture medium, i.e., before / after separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and the fermentation broth, i.e., after / after separation of the biomass from the culture medium (i.e., the fermentation broth).

[0335] Separation of the cells from the culture medium can be performed by any method known to those skilled in the art, such as any suitable type of centrifugation or filtration. Separation of the cells from the culture medium can be performed immediately after harvesting the fermentation broth, or at a later stage after storage of the fermentation broth under appropriate conditions. Recovery of the produced HMOs from the remaining biomass (or total fermentation) includes extraction of the HMOs from the biomass (producer cells).

[0336] It can be performed by any suitable method in the art, for example by sonication, boiling, homogenization, enzymatic lysis using lysozyme, or freezing and grinding.

[0337] After recovery from fermentation, HMOs are available for further processing and purification.

[0338] Use of the method or genetically engineered cell for production and / or manufacturing

[0339] The present disclosure also relates to any commercial use of the methods or genetically engineered cells described herein.

[0340] Thus, in one or more exemplary embodiments, the methods or genetically engineered cells according to the present invention are used to produce one or more HMOs. The one or more HMOs can be selected from the group consisting of LNT-II, pLNnH, LNT, LNnT, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, 2'-FL, DFL, 3-FL, LST-a, 3'-SL, 6'-SL, LST-b, LST-c, FSL, FLST-a, DSLNT, LNnH, and LNH. In a currently preferred embodiment, the one or more HMOs have an LNT-II core, for example, the one or more HMOs are selected from the group consisting of LNT, LNnT, and LNFP-I.

[0341] In one or more exemplary embodiments, the methods and / or genetically engineered cells are used to produce more than one HMO, wherein the one or more HMOs are selected from LNT, LNnT, and LNFP-I.

[0342] In one or more additional exemplary embodiments, methods and / or genetically engineered cells according to the present disclosure are used to produce more than one HMO, wherein the HMO is LNT and / or LNnT.

[0343] Manufacturing of HMOs

[0344] To produce one or more HMOs, the genetically engineered cells described herein are cultured in the presence of a suitable carbon and energy source (e.g., glucose, glycerol, or sucrose) and a suitable acceptor (e.g., lactose or any HMO) according to procedures known in the art, and the resulting HMO blend is harvested from the culture medium and microbial biomass formed during the culture process. Thereafter, the HMOs are purified according to procedures known in the art, such as those described in WO2016095924, WO2015188834, WO2017152918, WO2017182965, and US20190119314, and the purified HMOs are used as nutraceuticals, pharmaceuticals, or for any other purpose, such as for research.

[0345] HMO production is typically accomplished through large-volume cultivation. The terms "manufacturing" and "manufacturing-scale," as used herein, define fermentation with a minimum volume of 5 L of culture broth. Typically, a "manufacturing-scale" process is one that can process large volumes of a preparation containing the desired product and produce a sufficient amount of the desired HMO product, for example, in the case of a therapeutic compound or composition, to meet the needs of clinical trials and market supply. In addition to their large size, manufacturing-scale processes, compared to simpler laboratory-scale methods such as shake flask cultivation, are characterized by the use of bioreactor (fermenter) technology systems equipped with agitation, aeration, nutrient feed, and monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, backpressure, etc.). To a large extent, the behavior of expression systems in laboratory-scale methods, such as the shake flask, benchtop bioreactor, or deep-well formats described in the examples of this disclosure, allows for prediction of how such systems will behave in the complex environment of a bioreactor.

[0346] manufactured products

[0347] The term "manufactured product" in terms of use of a genetically engineered cell or nucleic acid construct refers to one or more HMOs defined as one or more product HMOs. Various products are described above.

[0348] Advantageously, the methods disclosed herein provide a reduced ratio of by-products to products and an increased overall yield of products (and / or total HMOs). Reduced by-product formation associated with product formation facilitates increased product yields and increases the efficiency of the production and product recovery processes, thereby providing a superior HMO manufacturing process.

[0349] The manufactured product may be a powder, composition, suspension or gel comprising one or more HMOs.

[0350] General

[0351] The terms lacto-N-triose, LNT-II, LNT II, ​​LNT2 and LNT 2 are used interchangeably. Sequence Listing <110> DSM IP ASSETS BV <120> Enhanced HMO formation by modifying intracellular lactose import <130> 340340-WO-PCT <150> DKPA202170249 <151> 2021-05-17 <160> 103 <170> BiSSAP 1.3.6 <210> 1 <211> 417 <212> PRT <213> Escherichia coli <220> <223> Lactose permease LacY GenBank ID: NP_414877.1 <400> 1 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 That <210> 2 <211> 1254 <212> Ms <213> Escherichia coli K-12 <220> <223> Nucleic acid sequence encoding LacY, GenBank ID: NC_000913.3 <400> 2 atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60 tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120 agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180 ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240 accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300 tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360 ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420 ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480 atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540 gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600 gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660 aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720 caacgtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780 tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840 ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900 gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960 ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020 tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttct taagcaactg 1080 gcgatgattt ttgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140 gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200 agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc ttaa 1254 <210> 3 <211> 433 <212> PRT <213> Escherichia coli <220> <223> Lactose permease, GenBank ID: WP_042094275.1 <400> 3 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 Ala Tyr Gly Cys Gly Ala Ser Ala Leu Ser Asp Gln His Ile Arg Ala 420 425 430 Glu <210> 4 <211> 417 <212> PRT <213> Proteobacteria <220> <223> Lactose permease, GenBank ID: WP_000291549.1 <400> 4 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Island Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Island Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 To the <210> 5 <211> 417 <212> PRT <213> Campylobacter jejuni <220> <223> Lactose permease GenBank ID: WP_089607162.1 <400> 5 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Cys Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 Ala <210> 6 <211> 417 <212> PRT <213> Klebsiella pneumoniae <220> <223> Lactose permease, GenBank ID: WP_152280604.1 <400> 6 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Leu Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Ile Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Lys Val Asn Glu Val 405 410 415 Ala <210> 7 <211> 417 <212> PRT <213> Cronobacter sakazakii <220> <223> Lactose permease, GenBank ID: EGT4952364.1 <400> 7 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Tyr Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Phe Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Leu Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Ile Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 Ala <210> 8 <211> 417 <212> PRT <213> Citrobacter freundii <220> <223> Lactose permease, GenBank ID NO: WP_134216118.1 <400> 8 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Phe Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Glu Val Asn His Ile Ser Lys Gly Asp Thr Gly Ile Ile 35 40 45 Phe Ala Cys Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Ile Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys His Leu Leu Trp Val Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Tyr Val Phe Gly 85 90 95 Pro Leu Leu Gln Val Asn Ile Leu Leu Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Ile Tyr Asn Ala Gly Ala Pro Ala Ile Glu Ala Tyr 115 120 125 Ile Glu Lys Ala Ser Arg Arg Ser Asn Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Ala Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Val Ile Leu Ala Leu Leu Leu Leu Phe Ser Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Lys Val Ala Asp Ala Val Gly Ala Asn Asn Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Lys Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Val Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Leu Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Val Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Thr Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Ile Pro Phe Leu Ile Val Gly Cys Phe Lys Tyr 325 330 335 Island Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Island Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Lys Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Ile Ile Ala Leu Ser Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Phe Ser Leu Leu Arg Arg Arg Glu Ser Val Ala 405 410 415 Leu <210> 9 <211> 418 <212> PRT <213> Citrobacter freundii <220> <223> Lactose permease, GenBank ID: EDI1749185.1 <400> 9 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Leu Gly Phe Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Gly Asp Thr Gly Ile Ile 35 40 45 Phe Ala Cys Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Val Phe Gly 50 55 60 Leu Met Ser Asp Lys Leu Gly Leu Arg Lys Asn Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Tyr Val Phe Gly 85 90 95 Pro Leu Leu His Phe Asn Ile Leu Leu Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Ile Tyr Asn Ala Gly Ala Pro Ala Ile Glu Ala Tyr 115 120 125 Ile Glu Lys Ala Ser Arg Arg Ser His Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Val 145 150 155 160 Met Phe Thr Ile Asn Asn Glu Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Val Ile Leu Ala Leu Leu Leu Phe Phe Ser Arg Thr Asp Ser Gly 180 185 190 Ser Ser Ala Val Val Ala Asp Ala Val Gly Ala Asn Ser Ser Pro Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Lys Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Val Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Ser Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Cys Ile Met Phe Phe Ala Pro Phe Ile Val Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Met Ala Gly Ile Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Thr Ala Gly Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Ile Pro Phe Leu Ile Val Gly Cys Phe Lys Tyr 325 330 335 Island Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Island Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Val Leu 355 360 365 Ala Gly Asn Met Tyr Glu Lys Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Ile Ala Leu Val Phe Thr Leu Ile Ser Val Ala Thr Leu 385 390 395 400 Ser Gly Ala Gly Pro Leu Ala Val Met Arg Val Ser Gln Gln Lys Ser 405 410 415 Ala Ser <210> 10 <211> 423 <212> PRT <213> Rouxiella badensis <220> <223> Lactose permease, GenBank ID: WP_084912833.1 <400> 10 Met Tyr Tyr Ile Lys Asn Thr Asn Phe Trp Thr Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Asn Gln Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ser Ile Ser Phe Phe Ser Leu Val Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Lys Lys His Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Tyr Val Phe Gly 85 90 95 Pro Leu Leu Lys Thr Asn Ile Leu Leu Gly Ser Ile Val Gly Gly Val 100 105 110 Tyr Leu Gly Phe Ile Tyr Asn Gly Gly Ala Pro Ala Ile Glu Ala Tyr 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Ser Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Ile Cys Ala Ser Val Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Ser Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Val Ile Leu Ala Val Leu Leu Leu Val Ala Lys Pro Ala Val Gly 180 185 190 Ala Thr Ala Lys Val Ala Asn Glu Leu Gly Ala Asn Ser Lys Pro Phe 195 200 205 Ser Leu Arg Leu Ala Ala Glu Leu Leu Lys Asp Lys Lys Leu Trp Phe 210 215 220 Leu Gly Leu Tyr Val Val Gly Val Ser Cys Thr Tyr Glu Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ser Ser Ala Asp Glu 245 250 255 Gly Thr Arg Val Phe Gly Tyr Ile Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Leu Val Met Phe Phe Ala Pro Leu Ile Val Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Ile Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Ser Thr Pro Val Glu Val Val Val Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Ile Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Val Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Ile Ala Met Ile Phe Met Ser Val Phe 355 360 365 Ala Gly Asp Met Tyr Gly Lys Ile Gly Phe His Gly Thr Tyr Leu Ile 370 375 380 Leu Gly Leu Ile Ala Leu Ala Phe Thr Leu Leu Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Arg Gly Pro Leu Gln Ala Phe Lys Pro Val Ala Arg Thr Val 405 410 415 Glu Lys Pro Val Ser Asn Val 420 <210> 11 <211> 417 <212> PRT <213> Serratia marcescens <220> <223> Lactose permease, GenBank ID: WP_103826752.1 <400> 11 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Phe Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ala Ile Ser Leu Phe Ser Leu Leu Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys Tyr Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Met Phe Ala Pro Phe Phe Ile Phe Ile Phe Gly 85 90 95 Pro Leu Leu Gln Tyr Asn Ile Leu Val Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Cys Phe Asn Ala Gly Ala Pro Ala Val Glu Ala Phe 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Asn Phe Glu Tyr Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Ile Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Phe Ile Leu Ala Val Leu Leu Phe Phe Ala Lys Thr Asp Ala Pro 180 185 190 Ser Ser Ala Thr Val Ala Asn Ala Val Gly Ala Asn His Ser Ala Phe 195 200 205 Ser Leu Lys Leu Ala Leu Glu Leu Phe Arg Gln Pro Lys Leu Trp Phe 210 215 220 Leu Ser Leu Tyr Val Ile Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Gly Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Ile Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Thr Ser Ala Leu Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Val Pro Phe Leu Leu Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Gln Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Met Ile Phe Met Ser Ile Leu 355 360 365 Ala Gly Asn Met Tyr Glu Ser Ile Gly Phe Gln Gly Ala Tyr Leu Val 370 375 380 Leu Gly Leu Val Ala Leu Gly Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Pro Gly Pro Leu Ser Leu Leu Arg Arg Gln Val Asn Glu Val 405 410 415 Ala <210> 12 <211> 420 <212> PRT <213> Serratia fonticola <220> <223> Lactose permease, GenBank ID: WP_021804673.1 <400> 12 Met Tyr Tyr Leu Lys Asn Thr Asn Phe Trp Met Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn Gln Val Ser Lys Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Ser Ile Ser Phe Phe Ser Leu Leu Phe Gln Pro Ile Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Arg Lys His Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Ile Leu Phe Ala Pro Phe Phe Ile Tyr Val Phe Gly 85 90 95 Pro Leu Leu Arg Thr Asn Ile Val Leu Gly Ser Ile Ala Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Ile Tyr Asn Gly Gly Ala Pro Ala Ile Glu Ala Tyr 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Ser Phe Glu Phe Gly Arg Ala Arg 130 135 140 Leu Phe Gly Cys Val Gly Trp Ala Leu Cys Ala Ser Val Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Glu Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Val Ile Leu Ala Ile Leu Leu Leu Ile Ala Lys Pro Glu Ala Gly 180 185 190 Ser Thr Ala Gln Val Ala Asp Lys Leu Gly Ala Asn Ser Lys Pro Phe 195 200 205 Asn Leu Arg Leu Ala Phe Glu Leu Leu Lys Asp Ser Lys Leu Trp Phe 210 215 220 Leu Ala Leu Tyr Val Val Gly Val Ser Cys Thr Tyr Asp Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ser Ser Ser Glu Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Val Thr Thr Met Gly Glu Leu Leu Asn 260 265 270 Ala Ser Ile Met Phe Phe Ala Pro Leu Ile Val Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Leu Ile Met Ser Ile Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Ser Thr Pro Val Glu Val Val Ile Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Ile Pro Phe Leu Ile Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Val Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Leu Ala Ile Met Phe Met Ser Val Phe 355 360 365 Ala Gly Asn Met Tyr Glu Lys Val Gly Phe His Gly Thr Tyr Leu Ile 370 375 380 Leu Gly Leu Ile Ala Leu Ser Phe Thr Leu Ile Ser Val Phe Thr Leu 385 390 395 400 Ser Gly Arg Gly Pro Leu Ser Ala Phe Lys Pro Glu Pro Glu Ala Val 405 410 415 Arg Asn Met Ala 420 <210> 13 <211> 423 <212> PRT <213> Rouxiella silvae <220> <223> Lactose permease, GenBank ID: WP_084984472.1 <400> 13 Met Asn Tyr Met Arg Asn Thr Asn Phe Trp Thr Phe Gly Leu Phe Phe 1 5 10 15 Phe Phe Tyr Phe Phe Ile Met Gly Ala Tyr Phe Pro Phe Phe Pro Ile 20 25 30 Trp Leu His Asp Ile Asn His Ile Asn Gln Ser Asp Thr Gly Ile Ile 35 40 45 Phe Ala Cys Ile Ser Phe Phe Ser Leu Val Phe Gln Pro Leu Phe Gly 50 55 60 Leu Leu Ser Asp Lys Leu Gly Leu Lys Lys His Leu Leu Trp Ile Ile 65 70 75 80 Thr Gly Met Leu Val Leu Phe Ala Pro Phe Phe Ile Tyr Val Phe Gly 85 90 95 Pro Leu Leu Gln Thr Asn Ile Leu Leu Gly Ser Ile Val Gly Gly Ile 100 105 110 Tyr Leu Gly Phe Ile Tyr Asn Gly Gly Ala Pro Ala Ile Glu Ala Tyr 115 120 125 Ile Glu Lys Val Ser Arg Arg Ser Ser Phe Glu Phe Gly Arg Ala Arg 130 135 140 Met Phe Gly Cys Val Gly Trp Ala Ile Cys Ala Ser Val Val Gly Ile 145 150 155 160 Met Phe Thr Ile Asn Asn Gln Phe Val Phe Trp Leu Gly Ser Gly Cys 165 170 175 Ala Val Ile Leu Ala Ile Leu Leu Leu Ile Ala Lys Pro Ala Val Gly 180 185 190 Ala Ser Ala Lys Val Ala Asn Glu Leu Gly Ala Asn Ser Lys Pro Phe 195 200 205 Asn Leu Arg Leu Ala Ala Glu Leu Leu Lys Asp Lys Lys Leu Trp Phe 210 215 220 Leu Gly Leu Tyr Val Val Gly Val Ser Cys Thr Tyr Glu Val Phe Asp 225 230 235 240 Gln Gln Phe Ala Asn Phe Phe Thr Ser Phe Phe Ser Ser Ala Asp Gln 245 250 255 Gly Thr Arg Val Phe Gly Tyr Ile Thr Thr Leu Gly Glu Leu Leu Asn 260 265 270 Ala Phe Ile Met Phe Phe Ala Pro Ala Ile Val Asn Arg Ile Gly Gly 275 280 285 Lys Asn Ala Leu Leu Leu Ala Gly Ala Ile Met Ser Val Arg Ile Ile 290 295 300 Gly Ser Ser Phe Ala Ser Thr Pro Val Glu Val Val Leu Leu Lys Thr 305 310 315 320 Leu His Met Phe Glu Ile Pro Phe Leu Ile Val Gly Cys Phe Lys Tyr 325 330 335 Ile Thr Ser Val Phe Glu Val Arg Phe Ser Ala Thr Ile Tyr Leu Val 340 345 350 Cys Phe Cys Phe Phe Lys Gln Ile Ala Met Ile Phe Met Ser Ile Phe 355 360 365 Ala Gly Glu Met Tyr Gly Lys Val Gly Phe His Gly Thr Tyr Leu Ile 370 375 380 Leu Gly Leu Ile Ala Leu Ala Phe Thr Leu Val Ser Ile Phe Thr Leu 385 390 395 400 Thr Gly Arg Gly Pro Leu Gln Ala Phe Lys Pro Ser Gln Arg Val Ser 405 410 415 Glu Thr Arg Pro Val Ser Pro 420 <210> 14 <211> 412 <212> PRT <213> Aeromonas <220> <223> Lactose permease, GenBank ID: WP_199428647.1 <400> 14 Met Phe Gly Ala Ile Phe Phe Leu Tyr Phe Phe Ile Met Gly Ala Tyr 1 5 10 15 Phe Pro Phe Phe Pro Ile Trp Leu His Asp Val Asn Gly Ile Ser Lys 20 25 30 Ala Glu Thr Gly Ile Val Phe Gly Thr Ile Ser Leu Phe Ala Leu Leu 35 40 45 Phe Gln Pro Val Phe Gly Leu Val Ser Asp Lys Leu Gly Leu Lys Lys 50 55 60 His Leu Leu Trp Ile Ile Ile Gly Leu Leu Val Phe Phe Ala Pro Phe 65 70 75 80 Phe Leu Tyr Val Leu Ala Pro Leu Leu Lys Leu Asn Ile Tyr Leu Gly 85 90 95 Ser Ile Val Gly Gly Met Tyr Ile Gly Phe Val Phe Ala Gly Gly Ala 100 105 110 Pro Ala Ile Glu Ala Tyr Val Glu Lys Val Ser Arg Ser Ser Gln Phe 115 120 125 Glu Phe Gly Arg Ala Arg Met Phe Gly Ala Ile Gly Trp Ala Leu Cys 130 135 140 Ala Ser Ile Val Gly Ile Met Phe Thr Ile Asn Asn Glu Phe Val Phe 145 150 155 160 Trp Met Gly Ser Ala Phe Ala Val Val Met Ala Val Leu Phe Tyr Phe 165 170 175 Ile Asn Pro Gly Arg Gly Ser Thr Ser Glu Val Leu Asp Ser Ile Gly 180 185 190 Ala Asn Gln Lys Gly Phe Ser Ile Lys Leu Ala Met Arg Leu Leu Lys 195 200 205 Gln Ala Lys Phe Trp Phe Phe Thr Leu Tyr Val Ile Gly Val Ala Cys 210 215 220 Thr Tyr Asp Val Phe Asp Gln Gln Phe Ala Asn Phe Phe Thr Ala Phe 225 230 235 240 Phe Asp Thr Lys Glu Glu Gly Thr Arg Tyr Phe Gly Tyr Val Thr Thr 245 250 255 Met Gly Glu Leu Leu Asn Ala Ser Ile Met Phe Phe Ala Pro Leu Ile 260 265 270 Val Asn Arg Ile Gly Gly Lys Asn Ala Leu Leu Ile Ala Gly Thr Ile 275 280 285 Met Ser Ile Arg Ile Ile Gly Ser Ser Phe Ala Glu Thr Val Ser His 290 295 300 Val Ile Leu Leu Lys Thr Leu His Met Leu Glu Val Pro Phe Leu Leu 305 310 315 320 Val Gly Thr Phe Lys Tyr Ile Thr Ser Glu Phe Asp Val Arg Phe Ser 325 330 335 Ala Thr Ile Trp Leu Ile Gly Tyr Gln Phe Phe Lys Gln Leu Ala Thr 340 345 350 Met Phe Met Ser Val Trp Ala Gly Ser Met Tyr Glu Val Met Gly Phe 355 360 365 Arg Glu Thr Tyr Leu Val Leu Gly Ile Ile Ala Ala Thr Phe Thr Phe 370 375 380 Ile Ser Ile Phe Thr Leu Ser Gly Met Gly Pro Leu Gly Leu Leu Lys 385 390 395 400 Gly Arg Thr Met Val Ser Lys Lys Thr Ala Ser Tyr 405 410 <210> 15 <211> 425 <212> PRT <213> Yersinia pestis <220> <223> Lactose permease, GenBank ID: WP_046596210.1 <400> 15 Met Lys Ile Ser Lys Thr Gly Asn Tyr Thr Ile Leu Phe Thr Val Ser 1 5 10 15 Phe Phe Ile Met Ala Ser Tyr Tyr Pro Phe Phe Pro Ile Trp Leu His 20 25 30 Asp Ile Asn Asn Leu Ser Lys Thr Asp Thr Gly Ile Val Phe Gly Ser 35 40 45 Ile Ser Leu Phe Ala Leu Ala Phe Gln Pro Ile Met Gly Pro Leu Ser 50 55 60 Asp Lys Leu Gly Leu Arg Lys Thr Leu Met Trp Ile Ile Val Gly Leu 65 70 75 80 Leu Val Leu Phe Ala Pro Phe Phe Ile Tyr Val Phe Ser Pro Leu Leu 85 90 95 Lys Tyr Asn Ile Phe Ile Gly Ala Ile Val Gly Gly Cys Tyr Leu Gly 100 105 110 Phe Val Phe Thr Gly Gly Ser His Ala Ile Glu Ala Tyr Ile Glu Lys 115 120 125 Val Ser Arg His Ser Asn Phe Glu Tyr Gly Arg Val Arg Met Phe Gly 130 135 140 Cys Ile Gly Trp Ala Leu Cys Ala Thr Val Val Gly Ile Leu Tyr Thr 145 150 155 160 Val Asn Asn Gln Leu Ile Phe Trp Met Ala Ser Gly Cys Ala Leu Ile 165 170 175 Leu Ala Val Leu Leu Phe Phe Ala Arg Pro Asp Arg Gln Ser Thr Ala 180 185 190 Phe Val Val Asp Thr Leu Gly Ala Asn Lys Ala Val Phe Asn Leu Lys 195 200 205 Asn Ala Leu Ala Leu Leu Arg Lys Arg Glu Leu Trp Phe Phe Val Met 210 215 220 Tyr Ile Val Gly Val Ala Cys Ile Tyr Asp Val Phe Asp Gln Gln Phe 225 230 235 240 Ala Asn Phe Phe Thr Ser Phe Phe Ala Thr Lys Gln Gln Gly Thr Glu 245 250 255 Ile Phe Gly Phe Val Thr Thr Gly Gly Glu Ile Leu Asn Ala Thr Val 260 265 270 Met Phe Phe Ala Pro Val Ile Ile Ala Arg Ile Gly Ser Lys Asn Ala 275 280 285 Leu Leu Leu Ala Gly Thr Ile Met Ser Val Arg Ile Leu Gly Ser Ala 290 295 300 Phe Ala Thr Thr Ala Thr Gln Val Val Phe Leu Lys Met Leu His Met 305 310 315 320 Phe Glu Val Pro Phe Leu Leu Val Gly Ser Phe Lys Tyr Ile Thr Gln 325 330 335 Val Phe Glu Val Arg Phe Ser Ala Thr Val Tyr Leu Ile Gly Phe Cys 340 345 350 Phe Ser Lys Gln Leu Ser Met Met Phe Met Ser Val Phe Ala Gly Arg 355 360 365 Met Tyr Gly Ser Met Gly Tyr Gln Asp Thr Tyr Met Val Leu Gly Val 370 375 380 Ile Val Leu Ser Phe Thr Leu Ile Ser Ala Phe Thr Leu Ser Gly Arg 385 390 395 400 Ser Ala Val Ala Asn Leu Ala Ser Arg Leu Lys Glu Asp Pro Ser Val 405 410 415 Thr Pro Pro Val Ser Gln Pro Gln Ser 420 425 <210> 16 <211> 587 <212> PRT <213> Kluyveromyces lactis <220> <223> Lactose permease, GenBank ID: XP_452193.1 <400> 16 Met Ala Asp His Ser Ser Ser Ser Ser Ser Leu Gln Lys Lys Pro Ile 1 5 10 15 Asn Thr Ile Glu His Lys Asp Thr Leu Gly Asn Asp Arg Asp His Lys 20 25 30 Glu Ala Leu Asn Ser Asp Asn Asp Asn Thr Ser Gly Leu Lys Ile Asn 35 40 45 Gly Val Pro Ile Glu Asp Ala Arg Glu Glu Val Leu Leu Pro Gly Tyr 50 55 60 Leu Ser Lys Gln Tyr Tyr Lys Leu Tyr Gly Leu Cys Phe Ile Thr Tyr 65 70 75 80 Leu Cys Ala Thr Met Gln Gly Tyr Asp Gly Ala Leu Met Gly Ser Ile 85 90 95 Tyr Thr Glu Asp Ala Tyr Leu Lys Tyr Tyr His Leu Asp Ile Asn Ser 100 105 110 Ser Ser Gly Thr Gly Leu Val Phe Ser Ile Phe Asn Val Gly Gln Ile 115 120 125 Cys Gly Ala Phe Phe Val Pro Leu Met Asp Trp Lys Gly Arg Lys Pro 130 135 140 Ala Ile Leu Ile Gly Cys Leu Gly Val Val Ile Gly Ala Ile Ile Ser 145 150 155 160 Ser Leu Thr Thr Thr Lys Ser Ala Leu Ile Gly Gly Arg Trp Phe Val 165 170 175 Ala Phe Phe Ala Thr Ile Ala Asn Ala Ala Ala Pro Thr Tyr Cys Ala 180 185 190 Glu Val Ala Pro Ala His Leu Arg Gly Lys Val Ala Gly Leu Tyr Asn 195 200 205 Thr Leu Trp Ser Val Gly Ser Ile Val Ala Ala Phe Ser Thr Tyr Gly 210 215 220 Thr Asn Lys Asn Phe Pro Asn Ser Ser Lys Ala Phe Lys Ile Pro Leu 225 230 235 240 Tyr Leu Gln Met Met Phe Pro Gly Leu Val Cys Ile Phe Gly Trp Leu 245 250 255 Ile Pro Glu Ser Pro Arg Trp Leu Val Gly Val Gly Arg Glu Glu Glu 260 265 270 Ala Arg Glu Phe Ile Ile Lys Tyr His Leu Asn Gly Asp Arg Thr His 275 280 285 Pro Leu Leu Asp Met Glu Met Ala Glu Ile Ile Glu Ser Phe His Gly 290 295 300 Thr Asp Leu Ser Asn Pro Leu Glu Met Leu Asp Val Arg Ser Leu Phe 305 310 315 320 Arg Thr Arg Ser Asp Arg Tyr Arg Ala Met Leu Val Ile Leu Met Ala 325 330 335 Trp Phe Gly Gln Phe Ser Gly Asn Asn Val Cys Ser Tyr Tyr Leu Pro 340 345 350 Thr Met Leu Arg Asn Val Gly Met Lys Ser Val Ser Leu Asn Val Leu 355 360 365 Met Asn Gly Val Tyr Ser Ile Val Thr Trp Ile Ser Ser Ile Cys Gly 370 375 380 Ala Phe Phe Ile Asp Lys Ile Gly Arg Arg Glu Gly Phe Leu Gly Ser 385 390 395 400 Ile Ser Gly Ala Ala Leu Ala Leu Thr Gly Leu Ser Ile Cys Thr Ala 405 410 415 Arg Tyr Glu Lys Thr Lys Lys Lys Ser Ala Ser Asn Gly Ala Leu Val 420 425 430 Phe Ile Tyr Leu Phe Gly Gly Ile Phe Ser Phe Ala Phe Thr Pro Met 435 440 445 Gln Ser Met Tyr Serving Thr Glu Val Serving Thr Asn Leu Thr Arg Serving Lys 450 455 460 Ala Gln Leu Leu Asn Phe Val Ser Gly Val Ala Gln Phe Val Asn 465 470 475 480 Gln Phe Ala Thr Pro Lys Ala Met Lys Asn Ile Lys Tyr Trp Phe Tyr 485,490,495 Val Phe Tyr Val Phe Phe Asp Ile Phe Glu Phe Ile Val Ile Tyr Phe 500 505 510 Phe Phe Val Glu Thr Lys Gly Arg Ser Leu Glu Glu Leu Glu Val Val 515,520,525 Phe Glu Ala Pro Asn Pro Arg Lys Ala Ser Val Asp Gln Ala Phe Leu 530 535 540 Gln Val Arg Val Ala Thr Leu Val Gln Arg Asn Asp Val Arg Val Ala 545 550 555 560 Asn Ala Gln Asn Leu Lys Glu Gln Glu Pro Leu Lys Ser Ser Asp Ala Asp 565,570,575 His Val Glu Lys Leu Ser Glu Ala Glu Ser Val 580,585 <210> 17 <211> 262 <212> PRT <213> Chromobacterium violaceum <220> <223> cvb3galT, Î2-1,3-galactosyltransferase, GeneBank ID: WP_080969100.1 <400> 17 Met Asp Thr Ile Met Ile Lys Arg Pro Leu Val Ser Val Ile Leu Pro 1 5 10 15 Val Asn Lys Asn Pro His Leu Glu Glu Ala Ile Gln Ser Ile Lys 20 25 30 Gln Gln Thr Tyr Lys Glue Lead Glue Induced and Alternatively Asn Asn Cys 35 40 45 Glu Asp Asn Phe Ser Tyr Leu Leu Lys Tyr Gln Asp Gln Lys Thr 50 55 60 Lys Ile Arg Thr Ser Ile Lys Tyr Leu Pro Phe Ser Leu Asn Leu 65 70 75 80 Gly Val His Leu Ser Gln Gly Glu Tyr Ile Ala Arg With Asp Ser Asp 85 90 95 Asp Is Containing Val Leu Asp Arg Is Glued To Lys Gln Val Lys Arg Phe Leu 100 105 110 Asn Thr Pro Glu Leu Ser Ile Leu Gly Ser Asn Val Glu Tyr Ile Asn 115 120 125 Glu Ala Ser Glu Ser Ile Gly Tyr Ser Asn Tyr Pro Leu Asp His Ser 130 135 140 Ser Ile Val Asn Ser Phe Pro Phe Arg Cys Asn Leu Ala His Pro Thr 145 150 155 160 Ile Met Val Lys Lys Glu Val Ile Thr Thr Leu Gly Gly Tyr Met Tyr 165 170 175 Gly Ser Leu Ser Glu Asp Tyr Asp Leu Trp Ile Arg Ala Ser Arg His 180 185 190 Gly Asn Phe Lys Phe Ser Asn Ile Asp Glu Pro Leu Leu Lys Tyr Arg 195 200 205 Ile His Lys Gly Gln Ala Thr Asn Lys Ser Asn Ala Tyr Asn Ile Phe 210 215 220 Ala Phe Asp Ser Ser Leu Lys Ile Arg Glu Phe Leu Leu Asn Gly Asn 225 230 235 240 Val Gln Tyr Leu Leu Gly Ala Ala Arg Gly Phe Phe Ala Phe Leu Tyr 245 250 255 Val Arg Phe Ile Lys Lys 260 <210> 18 <211> 439 <212> PRT <213> Artificial Sequence <220> <223> galTK, β-1,3-galactosyltransferase, homologous to GeneBank ID: BD182026.1 <400> 18 Met Ile Ser Val Tyr Ile Ile Ser Leu Lys Glu Ser Gln Arg Arg Leu 1 5 10 15 Asp Thr Glu Lys Leu Val Leu Glu Ser Asn Glu Lys Phe Lys Gly Arg 20 25 30 Cys Val Phe Gln Ile Phe Asp Ala Ile Ser Pro Lys His Glu Asp Phe 35 40 45 Glu Lys Phe Val Gln Glu Leu Tyr Asp Ser Ser Ser Leu Leu Lys Ser 50 55 60 Asp Trp Phe His Ser Asp Tyr Cys Tyr Gln Glu Leu Leu Pro Gln Glu 65 70 75 80 Phe Gly Cys Tyr Leu Ser His Tyr Leu Leu Trp Lys Glu Cys Val Lys 85 90 95 Leu Asn Gln Pro Val Val Ile Leu Glu Asp Asp Val Ala Leu Glu Ser 100 105 110 Asn Phe Met Gln Ala Leu Glu Asp Cys Leu Lys Ser Pro Phe Asp Phe 115 120 125 Val Arg Leu Tyr Gly His Tyr Trp Gly Gly His Lys Thr Asn Leu Cys 130 135 140 Ala Leu Pro Val Tyr Thr Glu Thr Glu Glu Ala Glu Ala Ser Ile Glu 145 150 155 160 Lys Thr Pro Ile Glu Asn Tyr Glu Val Thr Ser Pro Pro Pro Pro Asn 165 170 175 Pro Thr Arg Asp Thr Gln Gln Asp Phe Ile Thr Glu Thr Gln Gln Asp 180 185 190 Pro Lys Glu Leu Ser Glu Pro Cys Lys Ile Ala Pro Gln Lys Ile Ser 195 200 205 Phe Asn Gln Val Val Phe Lys Lys Ile Lys Arg Lys Leu Asn Arg Phe 210 215 220 Ile Gly Ser Ile Leu Ala Arg Thr Glu Val Tyr Lys Asn Ile Val Ala 225 230 235 240 Lys Tyr Asp Asp Leu Thr Thr Lys Tyr Asp Asp Leu Thr Thr Lys Tyr 245 250 255 Asp Asp Leu Thr Thr Lys Tyr Asp Asp Leu Thr Thr Lys Tyr Asp Asp 260 265 270 Leu Asn Lys Asn Ile Ala Glu Lys Tyr Asp Glu Leu Met Gly Lys Tyr 275 280 285 Glu Ser Leu Leu Ala Lys Glu Val Asn Ile Lys Glu Thr Phe Trp Glu 290,295,300 Ser Arg Ala Asp Ser Glu Lys Glu Ala Leu Phe Leu Asp His Phe Tyr 305 310 315 320 Leu Thr Ser Val Tyr Val Ala Thr Thr Ala Gly Tyr Tyr Leu Thr Pro 325 330 335 Lys Gly Ala Lys Thr Phe Ile Glu Ala Thr Glu Arg Phe Lys Ile Ile 340 345 350 Glu Pro Val Asp Met Phe Ile Asn Asn Pro Thr Tyr His Asp Ile Ala 355 360 365 Asn Phe Thr Tyr Val Pro Cys Pro Val Ser Leu Asn Lys His Ala Phe 370 375 380 The Three Asns With Gln Asn With Lys Pro Asp With Leu Lys Pro 385 390 395 400 Pro Lys Lys Ser Tyr Phe Asp Asn Leu Phe Tyr His Lys Phe Asn Ala 405 410 415 Arg Lys Cys Leu Lys Ala Phe Asn Lys Tyr Ser Lys Gln Tyr Ala Pro 420 425 430 Read Lys Thr Pro Lys Glu Val 435 <210> 19 <211> 273 <212> PRT <213> Helicobacter pylori <220> <223> β-1,4-gal-transferase, GalT, GenBank ID: WP_001262061.1 <400> 19 Met Arg Val Phe Ala Ile Ser Leu Asn Gln Lys Val Cys Asp Thr Phe 1 5 10 15 Gly Leu Val Phe Arg Asp Thr Thr Thr Leu Leu Asn Ser Ile Asn Ala 20 25 30 Thr His His Gln Ala Gln Ile Phe Asp Ala Ile Tyr Ser Lys Thr Phe 35 40 45 Glu Gly Gly Leu His Pro Leu Val Lys Lys His Leu His Pro Tyr Phe 50 55 60 Ile Thr Gln Asn Ile Lys Asp Met Gly Ile Thr Thr Asn Leu Ile Ser 65 70 75 80 Glu Val Ser Lys Phe Tyr Tyr Ala Leu Lys Tyr His Ala Lys Phe Met 85 90 95 Ser Leu Gly Glu Leu Gly Cys Tyr Ala Ser His Tyr Ser Leu Trp Glu 100 105 110 Lys Cys Ile Glu Leu Asn Glu Ala Ile Cys Ile Leu Glu Asp Asp Ile 115 120 125 Thr Leu Lys Glu Asp Phe Lys Glu Gly Leu Asp Phe Leu Glu Lys His 130 135 140 Ile Gln Glu Leu Gly Tyr Ile Arg Leu Met His Leu Leu Tyr Asp Ala 145 150 155 160 Ser Val Lys Ser Glu Pro Leu Ser His Lys Asn His Glu Ile Gln Glu 165 170 175 Arg Val Gly Ile Ile Lys Ala Tyr Ser Glu Gly Val Gly Thr Gln Gly 180 185 190 Tyr Val Ile Thr Pro Lys Ile Ala Lys Val Phe Leu Lys Cys Ser Arg 195 200 205 Lys Trp Val Val Pro Val Asp Thr Ile Met Asp Ala Thr Phe Ile His 210 215 220 Gly Val Lys Asn Leu Val Leu Gln Pro Phe Val Ile Ala Asp Asp Glu 225 230 235 240 Gln Ile Ser Thr Ile Ala Arg Lys Glu Glu Pro Tyr Ser Pro Lys Ile 245 250 255 Ala Leu Met Arg Glu Leu His Phe Lys Tyr Leu Lys Tyr Trp Gln Phe 260 265 270 Val <210> 20 <211> 332 <212> PRT <213> Neisseria meningitidis <220> <223> lgtA, β-1,3-N-acetyl-glucosaminyltransferase, GeneBank ID: WP_033911473.1 <400> 20 Met Gln Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val Glu Lys 1 5 10 15 Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Asn Gln Thr Trp Arg Asn 20 25 30 Leu Glu Ile Leu Ile Val Asp Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser Arg Ile Lys Ile Leu Ala 50 55 60 Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Leu Asn Ile Gly Leu Asp 65 70 75 80 Glu Leu Ala Lys Ser Gly Met Gly Glu Tyr Ile Ala Arg Thr Asp Ala 85 90 95 Asp Asp Ile Ala Ala Pro Asp Trp Ile Glu Lys Ile Val Gly Glu Met 100 105 110 Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Ala Trp Leu Glu Val Leu 115 120 125 Ser Glu Glu Lys Asp Gly Asn Arg Leu Ala Arg His His Arg His Gly 130 135 140 Lys Ile Trp Lys Lys Pro Thr Arg Pro Glu Asp Ile Ala Asp Phe Phe 145 150 155 160 Pro Phe Gly Asn Pro Ile His Asn Asn Thr Met Ile Met Arg Arg Ser 165 170 175 Val Ile Asp Gly Gly Leu Arg Tyr Asn Thr Glu Arg Asp Trp Ala Glu 180 185 190 Asp Tyr Gln Phe Trp Tyr Asp Val Ser Lys Leu Gly Arg Leu Ala Tyr 195 200 205 Tyr Pro Glu Ala Leu Val Lys Tyr Arg Leu His Ala Asn Gln Val Ser 210 215 220 Ser Lys Tyr Ser Ile Arg Gln His Glu Ile Ala Gln Gly Ile Gln Lys 225 230 235 240 Thr Ala Arg Asn Asp Phe Leu Gln Ser Met Gly Phe Lys Thr Arg Phe 245 250 255 Asp Ser Leu Glu Tyr Arg Gln Ile Lys Ala Val Ala Tyr Glu Leu Leu 260 265 270 Glu Lys His Leu Pro Glu Glu Asp Phe Glu Arg Ala Arg Arg Phe Leu 275 280 285 Tyr Gln Cys Phe Lys Arg Thr Asp Thr Leu Pro Ala Gly Ala Trp Leu 290,295,300 Asp Phe Ala Ala Asp Gly Arg Met Arg Leu Phe Thr Leu Arg Gln 305 310 315 320 Tyr Phe Gly with His Arg and with Lys Asn Arg 325 330 <210> 21 <211> 334 <212> PRT <213> Pasteurella multocida <220> <223> pmnagT, β-1,3-N-acetylglucosaminyl-transferase, GeneBankID: WP_014390683.1 <400> 21 Met Glu Asn Lys Pro Leu Val Ser Val Leu Ile Cys Ala Tyr Asn Val 1 5 10 15 Glue to Lys Tyr Glue to Cys Asn Ala Val Asn to Gln Thr Tyr 20 25 30 Lys Asn Leu Glue Ile Ile Val Asn Asp Gly Ser Ser Asp Asn Thr 35 40 45 Tyr Phe Leu Lys Lys Leu Glu Lys Asp Asn Arg Ile Lys Ile 50 55 60 Leu Asn Phe Asn Asn His Ile Gly Ile Ser Ala Leu Asn Glu Gly 65 70 75 80 Leu Lys Glu Ile Ala Gly Glu Tyr Ile Ala Arg Thr Asp Ser Asp Asp 85 90 95 Ile Thr Lys Pro Asp Trp Ile Glu Lys Ile Leu Thr Cys Met Gln Asn 100 105 110 Asp Pro Lys Ile Ile Ala Met Gly Ser Tyr Leu Thr Val Leu Ser Glu 115 120 125 Glu Asn Asn Gly Ser Val Leu Ala Asn His His Lys Asn Lys Val Glu 130 135 140 Trp Lys Asn Pro Leu Glu His Lys Asp Ile Val Glu Lys Met Leu Phe 145 150 155 160 Gly Asn Pro Ile His Asn Asn Ser Met Val Met Arg Ser Glu Ile Tyr 165 170 175 Thr Lys Tyr His Leu Ile Tyr Asp Pro Asp Tyr His Tyr Ala Glu Asp 180 185 190 Tyr Lys Phe Trp Leu Glu Val Ser Arg Ile Gly Lys Leu Ala Asn Tyr 195 200 205 Pro Glu Ser Leu Val Tyr Tyr Arg Leu His Arg Asn Gln Thr Ser Ser 210 215 220 Ile His Asn Ser Gln Gln Glu Ile Asn Gly Lys Lys Leu Arg Leu Gln 225 230 235 240 Only Lew Donkey Tyr Tyr Lys Asp Lew Gly And Asp Tyr Gln Leu Pro 245 250 255 Glu Lys Phe Leu Phe Lys Asp Ile Ala Leu Leu Gln Glu Phe Tyr 260 265 270 Glu Arg Gly Met Phe Arg Glu Asn With Arg Arg With Tyr Glu 275 280 285 Cys Tyr Ser Leu Gly Glu Tyr Asn Tyr Lys To Tyr Phe 290,295,300 Leu Ile Asn Lys Asn Asn Phe Leu Ser Ile Lys Asp Lys Phe Lys Ile 305 310 315 320 Ile Lys Tyr Leu Arg Pro Asp Lys Tyr Ser Ser Thr Tyr 325 330 <210> 22 <211> 330 <212> PRT <213> Haemophilus ducrei <220> <223> HD0466, glycosyltransferase family 2 protein, GeneBank ID: WP_010944479.1 <400> 22 Met Thr Thr Leu Will Ser Will Leu Ile Cys Ala Tyr Asn Will Glu Lys 1 5 10 15 Tyr Ile Asp Glu Cys Leu Asn Ala Val Ile Ala Gln Thr Tyr Lys Asn 20 25 30 Leu Glu Ile Ile Val Val Asn Asp Gly Ser Thr Asp Gly Thr Leu Ala 35 40 45 Lys Leu Arg Gln Phe Glu Ala Lys Asp Pro Arg Val Lys Ile Ile Asp 50 55 60 Asn Ile Val Asn Gln Gly Thr Ser Lys Ser Leu Asn Ile Gly Ile Gln 65 70 75 80 Tyr Cys Gln Gly Glu Ile Ile Ala Arg Thr Asp Ser Asp Asp Ile Val 85 90 95 Asp Ile His Trp Ile Glu Thr Leu Met Arg Glu Leu Asp Asn Ser Pro 100 105 110 Glu Thr Ile Ala Ile Ser Ala Tyr Leu Glu Phe Leu Ala Glu Lys Gly 115 120 125 Asn Gly Ser Lys Leu Ser Arg Ser Arg Lys His Gly Lys Asn Ala Glu 130 135 140 Asn Pro Ile Ser Ser Glu Ala Ile Ser Gln Arg Met Leu Phe Gly Asn 145 150 155 160 Pro Val His Asn Asn Val Ala Leu Val Arg Arg Lys Val Phe Ser Glu 165 170 175 Tyr Gly Leu Arg Phe Asp Pro Asp Tyr Ile His Ala Glu Asp Tyr Lys 180 185 190 Phe Trp Phe Glu Val Ser Lys Leu Gly Lys Met Arg Thr Tyr Pro Lys 195 200 205 Only Leu Will Lys Tyr Arg Leu His Only Thr Gln Will Be Only Tyr 210 215 220 Asn Gln Lys Gln Arg Ser Ile Ala Lys Lys Ile Lys Arg Glu Ala Ile 225 230 235 240 Ser His Tyr Leu Gln Gln Tyr Gly Ile Leu Pro Glu Lys Thr 245 250 255 I His Asp Leu Phe Ser Ile Phe Ser Pro Gln Ile Glu Leu Ser Leu 260 265 270 Thr Val Only Asn Lys Gln Glu Leu Phe Trp Ser Leu Only Thr Ser Leu 275 280 285 Ser Glu Tyr His Phe Arg Asp Leu Leu Lys Ile Tyr Ser Leu Asp Ile 290,295,300 Phe His Gln Leu Ser Phe Lys Tyr Lys Lys Arg And Phe Arg Lys Phe 305 310 315 320 Leu Leu Pro Asn Arg Tyr Pro Ser Val Ile 325 330 <210> 23 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> FutC (α-1,2-fucosyl-transferase) <400> 23 Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met 1 5 10 15 Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Ser Asn Thr Pro 20 25 30 Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asp Arg Lys Met Gln 35 40 45 Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Ala Lys Glu Ile 50 55 60 Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Ala Leu 65 70 75 80 Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr 85 90 95 Glu Pro Lys Leu Lys Pro Ser Arg Leu Thr Tyr Phe Phe Gly Tyr 100 105 110 Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln 115 120 125 Thr Phe Thr Leu Pro Pro Pro Pro Glu Asn Asn Lys Asn Asn Asn Lys 130 135 140 Lys Glu Glu Glu Tyr Gln Cys Lys Leu Ser Leu Ile Leu Ala Ala Lys 145 150 155 160 Asn Ser Val Phe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly 165 170 175 Cys Gln Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala 180 185 190 Lys Arg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Glu 195 200 205 Phe Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr 210 215 220 Arg Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser 225 230 235 240 Cys Gln His Gly Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala 245 250 255 Tyr Leu Ile Glu Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp 260 265 270 Leu Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu 275 280 285 Ser His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala Leu Gly 290 295 300 <210> 24 <211> 292 <212> PRT <213> Sulfuriflexus mobilis <220> <223> Smob, α-1,2-fucosyltransferase, GeneBank ID: WP_126455392.1 <400> 24 Met Ile Ile Ser Gln Ile Ile Gly Gly Leu Gly Asn Gln Met Phe Gln 1 5 10 15 Tyr Ala Ala Gly Arg Ala Leu Ser Leu Val Arg Gly Gln Pro Leu Leu 20 25 30 Leu Asp Val Thr Gly Phe Ala Gly Tyr Gly Leu His Gln Gly Phe Glu 35 40 45 Leu Gln Arg Val Phe Asp Cys Pro Ile Gly Ile Ala Thr Glu Glu Asp 50 55 60 Val Arg Gly Ile Leu Gly Trp Gln Phe Ser Ala Gly Ile Arg Arg Ile 65 70 75 80 Val Ala Arg Pro Gly Met Ala Ala Phe Arg Arg Lys Gly Phe Ile Val 85 90 95 Glu Pro His Phe His Tyr Trp Pro Glu Ile Lys Asn Val Pro Arg Asp 100 105 110 Cys Tyr Leu Leu Gly Tyr Trp Gln Ser Glu Arg Tyr Phe Arg Ala Ala 115 120 125 Thr Ala Asp Ile Arg Ala Asp Phe Ser Phe Lys Ser Pro Leu Val Asn 130 135 140 Arg Asn Ala Glu Thr Ala Ala Gln Ile Asp Gln Val Asn Ala Ile Ser 145 150 155 160 Leu His Met Arg Arg Gly Asp Tyr Val Asn Asn Pro Lys Thr Ser Ala 165 170 175 Thr His Gly Leu Cys Ser Leu Asp Tyr Tyr Gln Ala Ala Ile Lys Phe 180 185 190 Val Ser Glu Arg Val Glu Glu Pro Phe Phe Phe Ile Phe Ser Asp Asp 195 200 205 Ile Ala Trp Val Lys Ala Asn Leu Lys Leu Asp Phe Pro Cys Gln Tyr 210 215 220 Val Asp His Asn His Gly Ala Glu Ser Phe Asn Asp Met His Leu Met 225 230 235 240 Ser Leu Cys Gln His His Ile Ile Ala Asn Ser Ser Phe Ser Trp Trp 245 250 255 Gly Ala Trp Leu Asn Ser Asp Pro Lys Lys Ile Val Leu Ala Pro Lys 260 265 270 Lys Trp Phe Ala Asn Lys Asn Asn Ile Lys Asp Leu Phe Pro Pro Gly 275 280 285 Trp Val Ser Leu 290 <210> 25 <211> 292 <212> PRT <213> Methylobacter tundripaludum <220> <223> Mtun, α-1,2-fucosyltransferase, GeneBank ID: WP_031437198.1 <400> 25 Met Val Ile Thr His Leu Ile Gly Gly Leu Gly Asn Gln Met Phe Gln 1 5 10 15 Tyr Ala Ala Gly Arg Ala Val Ser Leu Glu Arg Gly Val Ser Leu Ser 20 25 30 Leu Asp Ile Ser Gly Phe Ala Asn Tyr Gly Leu His Gln Gly Phe Glu 35 40 45 Leu Gln Arg Ile Phe Asn Cys Thr Ala Glu Ile Ala Asn Glu Ala Asp 50 55 60 Val Arg Gly Ile Leu Gly Trp Gln Ser Ser Pro Arg Ile Arg Gln Leu 65 70 75 80 Leu Ser Arg Gln Asn Met Ala Ile Phe Arg Arg Glu Gly Phe Val Val 85 90 95 Glu Pro His Phe His Tyr Trp Gln Gly Ile Lys Ser Val Pro Arg Asp 100 105 110 Cys Tyr Leu Thr Gly Tyr Trp Gln Ser Glu Gln Tyr Phe Leu Glu Ala 115 120 125 Ala Ala Gln Ile Arg Ala Asp Phe Thr Phe Lys Leu Pro Leu Asp Asn 130 135 140 Gln Asn Ile Glu Leu Ala Lys Gln Ile Asn Ala Val Asn Ala Val Ser 145 150 155 160 Leu His Val Arg Arg Gly Asp Tyr Ala Asn Thr Pro Glu Thr Thr Ala 165 170 175 Thr His Gly Leu Cys Ser Leu Asp Tyr Tyr Arg Val Ala Ile Arg His 180 185 190 Ile Ala Glu Gln Val Gln Gln Pro His Phe Phe Val Phe Ser Asp Asp 195 200 205 Ile Ala Trp Val Lys Asn Asn Leu Ser Ile Asp Phe Pro Cys Gln Tyr 210 215 220 Val Asp His Asn Gln Gly Ala Glu Ser Tyr Asn Asp Met Arg Leu Met 225 230 235 240 Ser Met Cys Arg His His Ile Ile Ala Asn Ser Ser Phe Ser Trp Trp 245 250 255 Gly Ala Trp Leu Asn Pro Asn Val Asn Lys Ile Val Val Ala Pro Ser 260 265 270 Arg Trp Phe Ala Lys Gln Thr Asp Val Arg Asp Leu Leu Pro Gln Gly 275 280 285 Trp Ile Lys Gln 290 <210> 26 <211> 425 <212> PRT <213> Helicobacter pylori <220> <223> FutA, α-1,3-fucosyl-transferase, GenBank ID: NP_207177.1 <400> 26 Met Phe Gln Pro Leu Leu Asp Ala Phe Ile Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro Pro Pro Leu Lys Ile Ala Val Ala 20 25 30 Asn Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Lys Ser Val Leu 35 40 45 Tyr Phe Ile Leu Ser Gln Arg Tyr Ala Ile Thr Leu His Gln Asn Pro 50 55 60 Asn Glu Phe Ser Asp Leu Val Phe Ser Asn Pro Leu Gly Ala Ala Arg 65 70 75 80 Lys Ile Leu Ser Tyr Gln Asn Thr Lys Arg Val Phe Tyr Thr Gly Glu 85 90 95 Asn Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp 100 105 110 Glu Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala 115 120 125 His Leu His Tyr Lys Ala Glu Leu Val Asn Asp Thr Thr Ala Pro Tyr 130 135 140 Lys Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His 145 150 155 160 Phe Lys Glu Asn His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser 165 170 175 Asp Leu Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Ala Asn 180 185 190 Ala Pro Met Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro 195 200 205 Val Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Lys Val Gly 210 215 220 Asn Lys Ser Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu 225 230 235 240 Asn Ser Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr 245 250 255 Phe Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys 260 265 270 Asp Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Asn Asn Phe 275 280 285 Asp Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Pro Asn Ala 290 295 300 Tyr Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys 305 310 315 320 Ala Tyr Phe Tyr Gln Asp Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe 325 330 335 Lys Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Lys Phe Ser Thr Ser 340 345 350 Phe Met Trp Glu Tyr Asp Leu His Lys Pro Leu Val Ser Ile Asp Asp 355 360 365 Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Arg Leu Leu 370 375 380 Gln Asn Ala Ser Pro Leu Leu Glu Leu Ser Gln Asn Thr Thr Phe Lys 385 390 395 400 Tyr Arg Lys Wing Tyr Gln Lys Serving Leu Pro Leu Arg Wing Val 405 410 415 Arg Lys Leu Val Lys Leu Gly Leu 420 425 <210> 27 <211> 478 <212> PRT <213> Helicobacter pylori NCTC 11639 <220> <223> FucT, alpha1,3-fucosyltransferase, GenBank ID NO: AAB81031.1 <400> 27 Met Phe Gln Pro Leu Leu Asp Ala Tyr Val Glu Ser Ala Ser Ile Glu 1 5 10 15 Lys Met Ala Ser Lys Ser Pro Pro Leu Lys Ile Ala Val Ala Asn 20 25 30 Trp Trp Gly Asp Glu Glu Ile Lys Glu Phe Lys Asn Ser Val Leu Tyr 35 40 45 He Has Gln Arg Tyr Thr Thr Has His Gln Asn Pro Asn 50 55 60 Glu Phe Ser Asp Leu Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys 65 70 75 80 Tyr Gln Asn Is Contained With Lys Arg Val Phe Tyr Gly Glu Asn 85 90 95 Glu Ser Pro Asn Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu 100 105 110 Leu Asp Phe Asn Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Asp Arg 115 120 125 Leu His His Lys Ala Glu Ser Val Asn Asp Thr Thr Ala Pro Tyr Lys 130 135 140 Leu Lys Asp Asn Ser Leu Tyr Ala Leu Lys Lys Pro Ser His Cys Phe 145 150 155 160 Lys Glu Lys His Pro Asn Leu Cys Ala Val Val Asn Asp Glu Ser Asp 165 170 175 Pro Leu Lys Arg Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala 180 185 190 Pro Ile Arg Asn Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val 195 200 205 Thr Gly Gly Gly Ser Val Arg Asn Thr Leu Gly Tyr Asn Val Lys Asn 210 215 220 Lys Asn Glu Phe Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn 225 230 235 240 Thr Gln Gly Tyr Gly Tyr Val Thr Glu Lys Ile Ile Asp Ala Tyr Phe 245 250 255 Ser His Thr Ile Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp 260 265 270 Phe Asn Pro Lys Ser Phe Val Asn Val His Asp Phe Lys Asn Phe Asp 275 280 285 Glu Ala Ile Asp Tyr Ile Lys Tyr Leu His Thr His Lys Asn Ala Tyr 290 295 300 Leu Asp Met Leu Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala 305 310 315 320 Tyr Phe Tyr Gln Asn Leu Ser Phe Lys Lys Ile Leu Ala Phe Phe Lys 325 330 335 Thr Ile Leu Glu Asn Asp Thr Ile Tyr His Asp Asn Pro Phe Ile Phe 340 345 350 Cys Arg Asp Leu Asn Glu Pro Leu Val Thr Ile Asp Asp Leu Arg Val 355 360 365 Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr 370 375 380 Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp 385 390 395 400 Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg 405 410 415 Ile Asn Tyr Asp Asp Leu Arg Val Asn Tyr Asp Asp Leu Arg Val Asn 420 425 430 Tyr Glu Arg Leu Leu Ser Lys Ala Thr Pro Leu Leu Glu Leu Ser Gln 435 440 445 Asn Thr Thr Ser Lys Ile Tyr Arg Lys Ala Tyr Gln Lys Ser Leu Pro 450 455 460 Leu Leu Arg Ala Ile Arg Arg Trp Val Lys Lys Leu Gly Leu 465 470 475 <210> 28 <211> 432 <212> PRT <213> Helicobacter pylori <220> <223> FucTIII, alpha-1,4 fucosyltransferase, GenBank ID: AAR88243.1 <400> 28 Met Phe Gln Pro Leu Leu Asp Ala Tyr Ile Asp Ser Thr Arg Leu Asp 1 5 10 15 Glu Thr Asp Tyr Lys Pro Pro Leu Lys Ile Ala Val Ala Asn Trp Trp 20 25 30 Gly Gly Val Glu Glu Phe Lys Lys Ser Thr Leu Tyr Phe Ile Leu Ser 35 40 45 Gln Arg Tyr Thr Ile Thr Leu His Arg Asn Pro Asp Lys Pro Ala Asp 50 55 60 Ile Val Phe Gly Asn Pro Leu Gly Ser Ala Arg Lys Ile Leu Ser Tyr 65 70 75 80 Gln Asn Ala Lys Arg Val Phe Tyr Thr Gly Glu Asn Glu Val Pro Asn 85 90 95 Phe Asn Leu Phe Asp Tyr Ala Ile Gly Phe Asp Glu Leu Asp Phe Asn 100 105 110 Asp Arg Tyr Leu Arg Met Pro Leu Tyr Tyr Ala His Leu His Tyr Glu 115 120 125 Ala Glu Leu Val Asn Asp Thr Thr Ser Pro Tyr Lys Ile Lys Asp Asn 130 135 140 Ser Leu Tyr Ala Leu Lys Lys Pro Ser His His Phe Lys Glu Asn His 145 150 155 160 Pro Asn Leu Cys Ala Val Val Asn Asn Glu Ser Asp Pro Leu Lys Arg 165 170 175 Gly Phe Ala Ser Phe Val Ala Ser Asn Pro Asn Ala Pro Lys Arg Asn 180 185 190 Ala Phe Tyr Asp Ala Leu Asn Ser Ile Glu Pro Val Thr Gly Gly Gly 195 200 205 Ser Val Lys Asn Thr Leu Gly Tyr Asn Val Lys Asn Lys Asn Glu Phe 210 215 220 Leu Ser Gln Tyr Lys Phe Asn Leu Cys Phe Glu Asn Ser Gln Gly Tyr 225 230 235 240 Gly Tyr Val Thr Glu Lys Ile Leu Asp Ala Tyr Phe Ser His Thr Ile 245 250 255 Pro Ile Tyr Trp Gly Ser Pro Ser Val Ala Lys Asp Phe Asn Pro Lys 260 265 270 Ser Phe Val Asn Val His Asp Phe Asn Asn Phe Asp Glu Ala Ile Asp 275 280 285 His Val Arg Tyr Leu His Thr His Pro Asn Ala Tyr Leu Asp Met Leu 290 295 300 Tyr Glu Asn Pro Leu Asn Thr Leu Asp Gly Lys Ala Tyr Phe Tyr Gln 305 310 315 320 Asn Leu Ser Phe Lys Lys Ile Leu Asp Phe Phe Lys Thr Ile Leu Glu 325 330 335 Asn Asp Thr Ile Tyr His Cys Asp Ala His Asn Tyr Ser Ala Leu His 340 345 350 Arg Asp Leu Asn Glu Pro Leu Val Ser Ile Asp Asp Leu Arg Ile Asn 355 360 365 Tyr Asp Asp Leu Arg Ile Asn Tyr Asp Asp Leu Arg Ile Asn Tyr Asp 370 375 380 Asp Leu Arg Ile Asn Tyr Glu Arg Leu Leu Gln Asn Ala Ser Pro Leu 385 390 395 400 Leu Glu Leu Ser Gln Asn Thr Ser Phe Lys Ile Tyr Arg Lys Ala Tyr 405 410 415 Gln Lys Ser Leu Pro Leu Leu Arg Ala Ile Arg Arg Trp Val Lys Lys 420 425 430 <210> 29 <211> 483 <212> PRT <213> Artificial Sequence <220> <223> Pd2 (α-1,3-sialyl-transferase) <400> 29 Met Cys Asn Ser Asp Asn Thr Ser Leu Lys Glu Thr Val Ser Ser Asn 1 5 10 15 Ser Ala Asp Val Val Glu Thr Glu Thr Tyr Gln Leu Thr Pro Ile Asp 20 25 30 Ala Pro Ser Ser Phe Leu Ser His Ser Trp Glu Gln Thr Cys Gly Thr 35 40 45 Pro Ile Leu Asn Glu Ser Asp Lys Gln Ala Ile Ser Phe Asp Phe Val 50 55 60 Ala Pro Glu Leu Lys Gln Asp Glu Lys Tyr Cys Phe Thr Phe Lys Gly 65 70 75 80 Ile Thr Gly Asp His Arg Tyr Ile Thr Asn Thr Thr Leu Thr Val Val 85 90 95 Ala Pro Thr Leu Glu Val Tyr Ile Asp His Ala Ser Leu Pro Ser Leu 100 105 110 Gln Gln Leu Ile His Ile Ile Gln Ala Lys Asp Glu Tyr Pro Ser Asn 115 120 125 Gln Arg Phe Val Ser Trp Lys Arg Val Thr Val Asp Ala Asp Asn Ala 130 135 140 Asn Lys Leu Asn Ile His Thr Tyr Pro Leu Lys Gly Asn Asn Thr Ser 145 150 155 160 Pro Glu Met Val Ala Ala Ile Asp Glu Tyr Ala Gln Ser Lys Asn Arg 165 170 175 Leu Asn Ile Glu Phe Tyr Thr Asn Thr Ala His Val Phe Asn Asn Leu 180 185 190 Pro Pro Ile Ile Gln Pro Leu Tyr Asn Asn Glu Lys Val Lys Ile Ser 195 200 205 His Ile Ser Leu Tyr Asp Asp Gly Ser Ser Glu Tyr Val Ser Leu Tyr 210 215 220 Gln Trp Lys Asp Thr Pro Asn Lys Ile Glu Thr Leu Glu Gly Glu Val 225 230 235 240 Ser Leu Leu Ala Asn Tyr Leu Ala Gly Thr Ser Pro Asp Ala Pro Lys 245 250 255 Gly Met Gly Asn Arg Tyr Asn Trp His Lys Leu Tyr Asp Thr Asp Tyr 260 265 270 Tyr Phe Leu Arg Glu Asp Tyr Leu Asp Val Glu Ala Asn Leu His Asp 275 280 285 Leu Arg Asp Tyr Leu Gly Ser Ser Ala Lys Gln Met Pro Trp Asp Glu 290 295 300 Phe Ala Lys Leu Ser Asp Ser Gln Gln Thr Leu Phe Leu Asp Ile Val 305 310 315 320 Gly Phe Asp Lys Glu Gln Leu Gln Gln Gln Tyr Ser Gln Ser Pro Leu 325 330 335 Pro Asn Phe Ile Phe Thr Gly Thr Thr Thr Trp Ala Gly Gly Glu Thr 340 345 350 Lys Glu Tyr Tyr Ala Gln Gln Gln Val Asn Val Ile Asn Asn Ala Ile 355 360 365 Asn Glu Thr Ser Pro Tyr Tyr Leu Gly Lys Asp Tyr Asp Leu Phe Phe 370 375 380 Lys Gly His Pro Ala Gly Gly Val Ile Asn Asp Ile Ile Leu Gly Ser 385 390 395 400 Phe Pro Asp Met Ile Asn Ile Pro Ala Lys Ile Ser Phe Glu Val Leu 405 410 415 Met Met Thr Asp Met Leu Pro Asp Thr Val Ala Gly Ile Ala Ser Ser 420 425 430 Leu Tyr Phe Thr Ile Pro Ala Asp Lys Val Asn Phe Ile Val Phe Thr 435 440 445 Ser Ser Asp Thr Ile Thr Asp Arg Glu Glu Ala Leu Lys Ser Pro Leu 450 455 460 Val Gln Val Met Leu Thr Leu Gly Ile Val Lys Glu Lys Asp Val Leu 465 470 475 480 Phe Trp Ala <210> 30 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> Nst (α-1,3-sialyl-transferase) <400> 30 Met Glu Arg Asn Ala Val Ser Leu Leu Lys Glu Lys Leu Phe Asn Glu 1 5 10 15 Glu Gly Glu Pro Val Asn Leu Ile Phe Cys Tyr Thr Ile Leu Gln Met 20 25 30 Lys Val Ala Glu Arg Ile Met Ala Gln His Pro Gly Glu Arg Phe Tyr 35 40 45 Val Val May Also Be Glu Asn Arg Asn Glu Lys Tyr Asp Tyr Phe 50 55 60 Asn Gln Ile Lys Asp Lys Ala Glu Arg Ala Tyr Phe His Leu Pro 65 70 75 80 Tyr Gly Leu Asn Lys Ser Phe Asn Phe Ile Pro Thr Met Ala Glu Leu 85 90 95 Lys Val Lys Ser Met Lion Pro Lys Val Lys Arg Ile Tyr Lion Wing 100 105 110 Glu Lys Val Thr Tyr Pro Asp 115 120 125 Only Glu with Lys Thr Phe Asp Asp Gly Thr Gly Asn with Gln Ser 130 135 140 Ser Ser Tyr Leu Gly Asp Glu Phe Ser Val Asn Gly Thr Ile Lys Arg 145 150 155 160 Asn Phe Ala Arg Met Met Ile Gly Asp Trp Ser Ile Ala Lys Thr Arg 165 170 175 Asn Ala Ser Asp Glu His Tyr Thr Ile Phe Lys Gly Leu Lys Asn Ile 180 185 190 Met Asp Asp Gly Arg Arg Lys Met Thr Tyr Leu Pro Leu Phe Asp Ala 195 200 205 Ser Glu Leu Lys Thr Gly Asp Glu Thr Gly Gly Thr Val Arg Ile Leu 210 215 220 Leu Gly Ser Pro Asp Lys Glu Met Lys Glu Ile Ser Glu Lys Ala Ala 225 230 235 240 Lys Asn Phe Lys Ile Gln Tyr Val Ala Pro His Pro Arg Gln Thr Tyr 245 250 255 Gly Leu Ser Gly Val Thr Thr Leu Asn Ser Pro Tyr Val Ile Glu Asp 260 265 270 Tyr Ile Leu Arg Glu Ile Lys Lys Asn Pro His Thr Arg Tyr Glu Ile 275 280 285 Tyr Thr Phe Phe Ser Gly Ala Ala Leu Thr Met Lys Asp Phe Pro Asn 290 295 300 Val His Val Tyr Ala Leu Lys Pro Ala Ser Leu Pro Glu Asp Tyr Trp 305 310 315 320 Leu Lys Pro Val Tyr Ala Leu Phe Thr Gln Ser Gly Ile Pro Ile Leu 325 330 335 Thr Phe Asp Asp Lys Asn 340 <210> 31 <211> 373 <212> PRT <213> Escherichia coli K-12 <220> <223> gmd, GDP-mannose 4,6-dehydratas, GenBank ID: NP_416557.1 <400> 31 Met Ser Lys Val Ala Leu Ile Thr Gly Val Thr Gly Gln Asp Gly Ser 1 5 10 15 Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr Glu Val His Gly Ile 20 25 30 Lys Arg Arg Ala Ser Ser Phe Asn Thr Glu Arg Val Asp His Ile Tyr 35 40 45 Gln Asp Pro His Thr Cys Asn Pro Lys Phe His Leu His Tyr Gly Asp 50 55 60 Leu Ser Asp Thr Ser Asn Leu Thr Arg Ile Leu Arg Glu Val Gln Pro 65 70 75 80 Asp Glu Val Tyr Asn Leu Gly Ala Met Ser His Val Ala Val Ser Phe 85 90 95 Glu Ser Pro Glu Tyr Thr Ala Asp Val Asp Ala Met Gly Thr Leu Arg 100 105 110 Leu Leu Glu Ala Ile Arg Phe Leu Gly Leu Glu Lys Lys Thr Arg Phe 115 120 125 Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly Leu Val Gln Glu Ile Pro 130 135 140 Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Ala Val Ala 145 150 155 160 Lys Leu Tyr Ala Tyr Trp Ile Thr Val Asn Tyr Arg Glu Ser Tyr Gly 165 170 175 Met Tyr Ala Cys Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg 180 185 190 Gly Glu Thr Phe Val Thr Arg Lys Ile Thr Arg Ala Ile Ala Asn Ile 195 200 205 Ala Gln Gly Leu Glu Ser Cys Leu Tyr Leu Gly Asn Met Asp Ser Leu 210 215 220 Arg Asp Trp Gly His Ala Lys Asp Tyr Val Lys Met Gln Trp Met Met 225 230 235 240 Leu Gln Gln Glu Gln Pro Glu Asp Phe Val Ile Ala Thr Gly Val Gln 245 250 255 Tyr Ser Val Arg Gln Phe Val Glu Met Ala Ala Ala Gln Leu Gly Ile 260 265 270 Lys Leu Arg Phe Glu Gly Thr Gly Val Glu Glu Lys Gly Ile Val Val 275 280 285 Ser Val Thr Gly His Asp Ala Pro Gly Val Lys Pro Gly Asp Val Ile 290 295 300 Ile Ala Val Asp Pro Arg Tyr Phe Arg Pro Ala Glu Val Glu Thr Leu 305 310 315 320 Leu Gly Asp Pro Thr Lys Ala His Glu Lys Leu Gly Trp Lys Pro Glu 325 330 335 Ile Thr Leu Arg Glu Met Val Ser Glu Met Val Ala Asn Asp Leu Glu 340 345 350 Ala Ala Lys Lys His Ser Leu Leu Lys Ser His Gly Tyr Asp Val Ala 355 360 365 Ile Ala Leu Glu Ser 370 <210> 32 <211> 373 <212> PRT <213> Enterobacteriaceae <220> <223> gmd, GDP-mannose 4,6-dehydratas, GenBank ID: WP_000048190.1 <400> 32 Met Ser Lys Val Ala Leu Ile Thr Gly Val Thr Gly Gln Asp Gly Ser 1 5 10 15 Tyr Leu Ala Glu Phe Leu Leu Glu Lys Gly Tyr Glu Val His Gly Ile 20 25 30 Lys Arg Arg Ala Ser Ser Phe Asn Thr Glu Arg Val Asp His Ile Tyr 35 40 45 Gln Asp Pro His Thr Cys Asn Pro Lys Phe His Leu His Tyr Gly Asp 50 55 60 Leu Ser Asp Thr Ser Asn Leu Thr Arg Ile Leu Arg Glu Val Gln Pro 65 70 75 80 Asp Glu Val Tyr Asn Leu Gly Ala Met Ser His Val Ala Val Ser Phe 85 90 95 Glu Ser Pro Glu Tyr Thr Ala Asp Val Asp Ala Met Gly Thr Leu Arg 100 105 110 Leu Leu Glu Ala Ile Arg Phe Leu Gly Leu Glu Lys Lys Thr Arg Phe 115 120 125 Tyr Gln Ala Ser Thr Ser Glu Leu Tyr Gly Leu Val Gln Glu Ile Pro 130 135 140 Gln Lys Glu Thr Thr Pro Phe Tyr Pro Arg Ser Pro Tyr Ala Val Ala 145 150 155 160 Lys Leu Tyr Ala Tyr Trp Ile Thr Val Asn Tyr Arg Glu Ser Tyr Gly 165 170 175 Met Tyr Ala Cys Asn Gly Ile Leu Phe Asn His Glu Ser Pro Arg Arg 180 185 190 Gly Glu Thr Phe Val Thr Arg Lys Ile Thr Arg Ala Ile Ala Asn Ile 195 200 205 Ala Gln Gly Leu Glu Ser Cys Leu Tyr Leu Gly Asn Met Asp Ser Leu 210 215 220 Arg Asp Trp Gly His Ala Lys Asp Tyr Val Lys Met Gln Trp Met Met 225 230 235 240 Leu Gln Gln Glu Gln Pro Glu Asp Phe Val Ile Ala Thr Gly Val Gln 245 250 255 Tyr Ser Val Arg Gln Phe Val Glu Met Ala Ala Ala Gln Leu Gly Ile 260 265 270 Lys Leu Arg Phe Glu Gly Thr Gly Val Glu Glu Lys Gly Ile Val Val 275 280 285 Ser Val Thr Gly His Asp Ala Pro Gly Val Lys Pro Gly Asp Val Ile 290 295 300 Ile Ala Val Asp Pro Arg Tyr Phe Arg Pro Ala Glu Val Glu Thr Leu 305 310 315 320 Leu Gly Asp Pro Thr Lys Ala His Glu Lys Leu Gly Trp Lys Pro Glu 325 330 335 Ile Thr Leu Arg Glu Met Val Ser Glu Met Val Ala Asn Asp Leu Glu 340 345 350 Ala Ala Lys Lys His Ser Leu Leu Lys Ser His Gly Tyr Asp Val Ala 355 360 365 Ile Ala Leu Glu Ser 370 <210> 33 <211> 321 <212> PRT <213> Escherichia coli K-12 <220> <223> WcaG, GDP-L-fucose synthase <400> 33 Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly 1 5 10 15 Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val 20 25 30 Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His 35 40 45 Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala 50 55 60 Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile 65 70 75 80 Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln 85 90 95 Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro 100 105 110 Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr 115 120 125 Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile 130 135 140 Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser 145 150 155 160 Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser 165 170 175 Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr 180 185 190 Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met 195 200 205 Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val 210 215 220 Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu 225 230 235 240 Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Glu Leu 245 250 255 Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe 260 265 270 Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr 275 280 285 Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly 290 295 300 Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg 305 310 315 320 Gly <210> 34 <211> 321 <212> PRT <213> Enterobacteriaceae <220> <223> WcaG, GDP-L-fucose synthase, GenBank ID: WP_000043654.1 <400> 34 Met Ser Lys Gln Arg Val Phe Ile Ala Gly His Arg Gly Met Val Gly 1 5 10 15 Ser Ala Ile Arg Arg Gln Leu Glu Gln Arg Gly Asp Val Glu Leu Val 20 25 30 Leu Arg Thr Arg Asp Glu Leu Asn Leu Leu Asp Ser Arg Ala Val His 35 40 45 Asp Phe Phe Ala Ser Glu Arg Ile Asp Gln Val Tyr Leu Ala Ala Ala 50 55 60 Lys Val Gly Gly Ile Val Ala Asn Asn Thr Tyr Pro Ala Asp Phe Ile 65 70 75 80 Tyr Gln Asn Met Met Ile Glu Ser Asn Ile Ile His Ala Ala His Gln 85 90 95 Asn Asp Val Asn Lys Leu Leu Phe Leu Gly Ser Ser Cys Ile Tyr Pro 100 105 110 Lys Leu Ala Lys Gln Pro Met Ala Glu Ser Glu Leu Leu Gln Gly Thr 115 120 125 Leu Glu Pro Thr Asn Glu Pro Tyr Ala Ile Ala Lys Ile Ala Gly Ile 130 135 140 Lys Leu Cys Glu Ser Tyr Asn Arg Gln Tyr Gly Arg Asp Tyr Arg Ser 145 150 155 160 Val Met Pro Thr Asn Leu Tyr Gly Pro His Asp Asn Phe His Pro Ser 165 170 175 Asn Ser His Val Ile Pro Ala Leu Leu Arg Arg Phe His Glu Ala Thr 180 185 190 Ala Gln Asn Ala Pro Asp Val Val Val Trp Gly Ser Gly Thr Pro Met 195 200 205 Arg Glu Phe Leu His Val Asp Asp Met Ala Ala Ala Ser Ile His Val 210 215 220 Met Glu Leu Ala His Glu Val Trp Leu Glu Asn Thr Gln Pro Met Leu 225 230 235 240 Ser His Ile Asn Val Gly Thr Gly Val Asp Cys Thr Ile Arg Glu Leu 245 250 255 Ala Gln Thr Ile Ala Lys Val Val Gly Tyr Lys Gly Arg Val Val Phe 260 265 270 Asp Ala Ser Lys Pro Asp Gly Thr Pro Arg Lys Leu Leu Asp Val Thr 275 280 285 Arg Leu His Gln Leu Gly Trp Tyr His Glu Ile Ser Leu Glu Ala Gly 290 295 300 Leu Ala Ser Thr Tyr Gln Trp Phe Leu Glu Asn Gln Asp Arg Phe Arg 305 310 315 320 Gly <210> 35 <211> 159 <212> PRT <213> Escherichia coli <220> <223> WcaH, GDP-mannose mannosyl hydrolase, GenBank ID: NP_416555.2 <400> 35 Met Phe Leu Arg Gln Glu Asp Phe Ala Thr Val Val Arg Ser Thr Pro 1 5 10 15 Leu Val Ser Leu Asp Phe Ile Val Glu Asn Ser Arg Gly Glu Phe Leu 20 25 30 Leu Gly Lys Arg Thr Asn Arg Pro Ala Gln Gly Tyr Trp Phe Val Pro 35 40 45 Gly Gly Arg Val Gln Lys Asp Glu Thr Leu Glu Ala Ala Phe Glu Arg 50 55 60 Leu Thr Met Ala Glu Leu Gly Leu Arg Leu Pro Ile Thr Ala Gly Gln 65 70 75 80 Phe Tyr Gly Val Trp Gln His Phe Tyr Asp Asp Asn Phe Ser Gly Thr 85 90 95 Asp Phe Thr Thr His Tyr Val Val Leu Gly Phe Arg Phe Arg Val Ser 100 105 110 Glu Glu Glu Leu Leu Leu Pro Asp Glu Gln His Asp Asp Tyr Arg Trp 115 120 125 Leu Thr Ser Asp Ala Leu Leu Ala Ser Asp Asn Val His Ala Asn Ser 130 135 140 Arg Ala Tyr Phe Leu Ala Glu Lys Arg Thr Gly Val Pro Gly Leu 145 150 155 <210> 36 <211> 159 <212> PRT <213> Escherichia <220> <223> WcaH, GDP-mannose mannosyl hydrolase, GenBank ID: WP_001393539.1 <400> 36 Met Phe Leu Arg Gln Glu Asp Phe Ala Thr Val Val Arg Ser Thr Pro 1 5 10 15 Leu Val Ser Leu Asp Phe Ile Val Glu Asn Ser Arg Gly Glu Phe Leu 20 25 30 Leu Gly Lys Arg Thr Asn Arg Pro Ala Gln Gly Tyr Trp Phe Val Pro 35 40 45 Gly Gly Arg Val Gln Lys Asp Glu Thr Leu Glu Ala Ala Phe Glu Arg 50 55 60 Leu Thr Met Ala Glu Leu Gly Leu Arg Leu Pro Ile Thr Ala Gly Gln 65 70 75 80 Phe Tyr Gly Val Trp Gln His Phe Tyr Asp Asp Asn Phe Ser Gly Thr 85 90 95 Asp Phe Thr Thr His Tyr Val Val Leu Gly Phe Arg Phe Arg Val Ser 100 105 110 Glu Glu Glu Leu Leu Leu Pro Asp Glu Gln His Asp Asp Tyr Arg Trp 115 120 125 Leu Thr Ser Asp Ala Leu Leu Ala Ser Asp Asn Val His Ala Asn Ser 130 135 140 Arg Ala Tyr Phe Leu Ala Glu Lys Arg Thr Gly Val Pro Gly Leu 145 150 155 <210> 37 <211> 407 <212> PRT <213> Escherichia coli <220> <223> WcaI, colanic acid biosynthesis fucosyltransferase, GenBank ID: NP_416554.1 <400> 37 Met Lys Ile Leu Val Tyr Gly Ile Asn Tyr Ser Pro Glu Leu Thr Gly 1 5 10 15 Ile Gly Lys Tyr Thr Gly Glu Met Val Glu Trp Leu Ala Ala Gln Gly 20 25 30 His Glu Val Arg Val Ile Thr Ala Pro Pro Tyr Tyr Pro Gln Trp Gln 35 40 45 Val Gly Glu Asn Tyr Ser Ala Trp Arg Tyr Lys Arg Glu Glu Gly Ala 50 55 60 Ala Thr Val Trp Arg Cys Pro Leu Tyr Val Pro Lys Gln Pro Ser Thr 65 70 75 80 Leu Lys Arg Leu Leu His Leu Gly Ser Phe Ala Val Ser Ser Phe Phe 85 90 95 Pro Leu Met Ala Gln Arg Arg Trp Lys Pro Asp Arg Ile Ile Gly Val 100 105 110 Val Pro Thr Leu Phe Cys Ala Pro Gly Met Arg Leu Leu Ala Lys Leu 115 120 125 Ser Gly Ala Arg Thr Val Leu His Ile Gln Asp Tyr Glu Val Asp Ala 130 135 140 Met Leu Gly Leu Gly Leu Ala Gly Lys Gly Lys Gly Gly Lys Val Ala 145 150 155 160 Gln Leu Ala Thr Ala Phe Glu Arg Ser Gly Leu His Asn Val Asp Asn 165 170 175 Val Ser Thr Ile Ser Arg Ser Met Met Asn Lys Ala Ile Glu Lys Gly 180 185 190 Val Ala Ala Glu Asn Val Ile Phe Phe Pro Asn Trp Ser Glu Ile Ala 195 200 205 Arg Phe Gln His Val Ala Asp Ala Asp Val Asp Ala Leu Arg Asn Gln 210 215 220 Leu Asp Leu Pro Asp Asn Lys Lys Ile Ile Leu Tyr Ser Gly Asn Ile 225 230 235 240 Gly Glu Lys Gln Gly Leu Glu Asn Val Ile Glu Ala Ala Asp Arg Leu 245 250 255 Arg Asp Glu Pro Leu Ile Phe Ala Ile Val Gly Gln Gly Gly Gly Lys 260 265 270 Ala Arg Leu Glu Lys Met Ala Gln Gln Arg Gly Leu Arg Asn Met Gln 275 280 285 Phe Phe Pro Leu Gln Ser Tyr Asp Ala Leu Pro Ala Leu Leu Lys Met 290 295 300 Gly Asp Cys His Leu Val Val Gln Lys Arg Gly Ala Ala Asp Ala Val 305 310 315 320 Leu Pro Ser Lys Leu Thr Asn Ile Leu Ala Val Gly Gly Asn Ala Val 325 330 335 Ile Thr Ala Glu Ala Tyr Thr Glu Leu Gly Gln Leu Cys Glu Thr Phe 340 345 350 Pro Gly Ile Ala Val Cys Val Glu Pro Glu Ser Val Glu Ala Leu Val 355 360 365 Ala Gly Ile Arg Gln Ala Leu Leu Leu Pro Lys His Asn Thr Val Ala 370 375 380 Arg Glu Tyr Ala Glu Arg Thr Leu Asp Lys Glu Asn Val Leu Arg Gln 385 390 395 400 Phe Ile Asn Asp Ile Arg Gly 405 <210> 38 <211> 407 <212> PRT <213> Escherichia <220> <223> WcaI, colanic acid biosynthesis fucosyltransferase , GenBank ID: WP_000699693.1 <400> 38 Met Lys Ile Leu Val Tyr Gly Ile Asn Tyr Ser Pro Glu Leu Thr Gly 1 5 10 15 Ile Gly Lys Tyr Thr Gly Glu Met Val Glu Trp Leu Ala Ala Gln Gly 20 25 30 His Glu Val Arg Val Ile Thr Ala Pro Pro Tyr Tyr Pro Gln Trp Gln 35 40 45 Val Gly Glu Asn Tyr Ser Ala Trp Arg Tyr Lys Arg Glu Glu Gly Ala 50 55 60 Ala Thr Val Trp Arg Cys Pro Leu Tyr Val Pro Lys Gln Pro Ser Thr 65 70 75 80 Leu Lys Arg Leu Leu His Leu Gly Ser Phe Ala Val Ser Ser Phe Phe 85 90 95 Pro Leu Met Ala Gln Arg Arg Trp Lys Pro Asp Arg Ile Ile Gly Val 100 105 110 Val Pro Thr Leu Phe Cys Ala Pro Gly Met Arg Leu Leu Ala Lys Leu 115 120 125 Ser Gly Ala Arg Thr Val Leu His Ile Gln Asp Tyr Glu Val Asp Ala 130 135 140 Met Leu Gly Leu Gly Leu Ala Gly Lys Gly Lys Gly Gly Lys Val Ala 145 150 155 160 Gln Leu Ala Thr Ala Phe Glu Arg Ser Gly Leu His Asn Val Asp Asn 165 170 175 Val Ser Thr Ile Ser Arg Ser Met Met Asn Lys Ala Ile Glu Lys Gly 180 185 190 Val Ala Ala Glu Asn Val Ile Phe Phe Pro Asn Trp Ser Glu Ile Ala 195 200 205 Arg Phe Gln His Val Ala Asp Ala Asp Val Asp Ala Leu Arg Asn Gln 210 215 220 Leu Asp Leu Pro Asp Asn Lys Lys Ile Ile Leu Tyr Ser Gly Asn Ile 225 230 235 240 Gly Glu Lys Gln Gly Leu Glu Asn Val Ile Glu Ala Ala Asp Arg Leu 245 250 255 Arg Asp Glu Pro Leu Ile Phe Ala Ile Val Gly Gln Gly Gly Gly Lys 260 265 270 Ala Arg Leu Glu Lys Met Ala Gln Gln Arg Gly Leu Arg Asn Met Gln 275 280 285 Phe Phe Pro Leu Gln Ser Tyr Asp Ala Leu Pro Ala Leu Leu Lys Met 290 295 300 Gly Asp Cys His Leu Val Val Gln Lys Arg Gly Ala Ala Asp Ala Val 305 310 315 320 Leu Pro Ser Lys Leu Thr Asn Ile Leu Ala Val Gly Gly Asn Ala Val 325 330 335 Ile Thr Ala Glu Ala Tyr Thr Glu Leu Gly Gln Leu Cys Glu Thr Phe 340 345 350 Pro Gly Ile Ala Val Cys Val Glu Pro Glu Ser Val Glu Ala Leu Val 355 360 365 Ala Gly Ile Arg Gln Ala Leu Leu Leu Pro Lys His Asn Thr Val Ala 370 375 380 Arg Glu Tyr Ala Glu Arg Thr Leu Asp Lys Glu Asn Val Leu Arg Gln 385 390 395 400 Phe Ile Asn Asp Ile Arg Gly 405 <210> 39 <211> 478 <212> PRT <213> Escherichia coli K-12 <220> <223> ManC, mannose-1-phosphate guanylyltransferase, GenBank ID: NP_416553.1 <400> 39 Met Ala Gln Ser Lys Leu Tyr Pro Val Val Met Ala Gly Gly Ser Gly 1 5 10 15 Ser Arg Leu Trp Pro Leu Ser Arg Val Leu Tyr Pro Lys Gln Phe Leu 20 25 30 Cys Leu Lys Gly Asp Leu Thr Met Leu Gln Thr Thr Ile Cys Arg Leu 35 40 45 Asn Gly Val Glu Cys Glu Ser Pro Val Val Ile Cys Asn Glu Gln His 50 55 60 Arg Phe Ile Val Ala Glu Gln Leu Arg Gln Leu Asn Lys Leu Thr Glu 65 70 75 80 Asn Ile Ile Leu Glu Pro Ala Gly Arg Asn Thr Ala Pro Ala Ile Ala 85 90 95 Leu Ala Ala Leu Ala Ala Lys Arg His Ser Pro Glu Ser Asp Pro Leu 100 105 110 Met Leu Val Leu Ala Ala Asp His Val Ile Ala Asp Glu Asp Ala Phe 115 120 125 Arg Ala Ala Val Arg Asn Ala Met Pro Tyr Ala Glu Ala Gly Lys Leu 130 135 140 Val Thr Phe Gly Ile Val Pro Asp Leu Pro Glu Thr Gly Tyr Gly Tyr 145 150 155 160 Ile Arg Arg Gly Glu Val Ser Ala Gly Glu Gln Asp Met Val Ala Phe 165 170 175 Glu Val Ala Gln Phe Val Glu Lys Pro Asn Leu Glu Thr Ala Gln Ala 180 185 190 Tyr Val Ala Ser Gly Glu Tyr Tyr Trp Asn Ser Gly Met Phe Leu Phe 195 200 205 Arg Ala Gly Arg Tyr Leu Glu Glu Leu Lys Lys Tyr Arg Pro Asp Ile 210 215 220 Leu Asp Ala Cys Glu Lys Ala Met Ser Ala Val Asp Pro Asp Leu Asn 225 230 235 240 Phe Ile Arg Val Asp Glu Glu Ala Phe Leu Ala Cys Pro Glu Glu Ser 245 250 255 Val Asp Tyr Ala Val Met Glu Arg Thr Ala Asp Ala Val Val Val Pro 260 265 270 Met Asp Ala Gly Trp Ser Asp Val Gly Ser Trp Ser Ser Leu Trp Glu 275 280 285 Ile Ser Ala His Thr Ala Glu Gly Asn Val Cys His Gly Asp Val Ile 290 295 300 Asn His Lys Thr Glu Asn Ser Tyr Val Tyr Ala Glu Ser Gly Leu Val 305 310 315 320 Thr Thr Val Gly Val Lys Asp Leu Val Val Val Gln Thr Lys Asp Ala 325 330 335 Val Leu Ile Ala Asp Arg Asn Ala Val Gln Asp Val Lys Lys Val Val 340 345 350 Glu Gln Ile Lys Ala Asp Gly Arg His Glu His Arg Val His Arg Glu 355 360 365 Val Tyr Arg Pro Trp Gly Lys Tyr Asp Ser Ile Asp Ala Gly Asp Arg 370 375 380 Tyr Gln Val Lys Arg Ile Thr Val Lys Pro Gly Glu Gly Leu Ser Val 385 390 395 400 Gln Met His His His Arg Ala Glu His Trp Val Val Val Ala Gly Thr 405 410 415 Ala Lys Val Thr Ile Asp Gly Asp Ile Lys Leu Leu Gly Glu Asn Glu 420 425 430 Ser Ile Tyr Ile Pro Leu Gly Ala Thr His Cys Leu Glu Asn Pro Gly 435 440 445 Lys Ile Pro Leu Asp Leu Ile Glu Val Arg Ser Gly Ser Tyr Leu Glu 450 455 460 Glu Asp Asp Val Val Arg Phe Ala Asp Arg Tyr Gly Arg Val 465 470 475 <210> 40 <211> 478 <212> PRT <213> Escherichia <220> <223> ManC, mannose-1-phosphate guanyltransferase, GenBank ID: WP_000079274.1 <400> 40 Met Ala Gln Ser Lys Leu Tyr Pro Val Val Met Ala Gly Gly Ser Gly 1 5 10 15 Ser Arg Leu Trp Pro Leu Ser Arg Val Leu Tyr Pro Lys Gln Phe Leu 20 25 30 Cys Leu Lys Gly Asp Leu Thr Met Leu Gln Thr Thr Ile Cys Arg Leu 35 40 45 Asn Gly Val Glu Cys Glu Ser Pro Val Val Ile Cys Asn Glu Gln His 50 55 60 Arg Phe Ile Val Ala Glu Gln Leu Arg Gln Leu Asn Lys Leu Thr Glu 65 70 75 80 Asn Ile Ile Leu Glu Pro Ala Gly Arg Asn Thr Ala Pro Ala Ile Ala 85 90 95 Leu Ala Ala Leu Ala Ala Lys Arg His Ser Pro Glu Ser Asp Pro Leu 100 105 110 Met Leu Val Leu Ala Ala Asp His Val Ile Ala Asp Glu Asp Ala Phe 115 120 125 Arg Ala Ala Val Arg Asn Ala Met Pro Tyr Ala Glu Ala Gly Lys Leu 130 135 140 Val Thr Phe Gly Ile Val Pro Asp Leu Pro Glu Thr Gly Tyr Gly Tyr 145 150 155 160 Ile Arg Arg Gly Glu Val Ser Ala Gly Glu Gln Asp Met Val Ala Phe 165 170 175 Glu Val Ala Gln Phe Val Glu Lys Pro Asn Leu Glu Thr Ala Gln Ala 180 185 190 Tyr Val Ala Ser Gly Glu Tyr Tyr Trp Asn Ser Gly Met Phe Leu Phe 195 200 205 Arg Ala Gly Arg Tyr Leu Glu Glu Leu Lys Lys Tyr Arg Pro Asp Ile 210 215 220 Leu Asp Ala Cys Glu Lys Ala Met Ser Ala Val Asp Pro Asp Leu Asn 225 230 235 240 Phe Ile Arg Val Asp Glu Glu Ala Phe Leu Ala Cys Pro Glu Glu Ser 245 250 255 Val Asp Tyr Ala Val Met Glu Arg Thr Ala Asp Ala Val Val Val Pro 260 265 270 Met Asp Ala Gly Trp Ser Asp Val Gly Ser Trp Ser Ser Leu Trp Glu 275 280 285 Ile Ser Ala His Thr Ala Glu Gly Asn Val Cys His Gly Asp Val Ile 290 295 300 Asn His Lys Thr Glu Asn Ser Tyr Val Tyr Ala Glu Ser Gly Leu Val 305 310 315 320 Thr Thr Val Gly Val Lys Asp Leu Val Val Val Gln Thr Lys Asp Ala 325 330 335 Val Leu Ile Ala Asp Arg Asn Ala Val Gln Asp Val Lys Lys Val Val 340 345 350 Glu Gln Ile Lys Ala Asp Gly Arg His Glu His Arg Val His Arg Glu 355 360 365 Val Tyr Arg Pro Trp Gly Lys Tyr Asp Ser Ile Asp Ala Gly Asp Arg 370 375 380 Tyr Gln Val Lys Arg Ile Thr Val Lys Pro Gly Glu Gly Leu Ser Val 385 390 395 400 Gln Met His His His Arg Ala Glu His Trp Val Val Val Ala Gly Thr 405 410 415 Ala Lys Val Thr Ile Asp Gly Asp Ile Lys Leu Leu Gly Glu Asn Glu 420 425 430 Ser Ile Tyr Ile Pro Leu Gly Ala Thr His Cys Leu Glu Asn Pro Gly 435 440 445 Lys Ile Pro Leu Asp Leu Ile Glu Val Arg Ser Gly Ser Tyr Leu Glu 450 455 460 Glu Asp Asp Val Val Arg Phe Ala Asp Arg Tyr Gly Arg Val 465 470 475 <210> 41 <211> 456 <212> PRT <213> Escherichia coli K-12 <220> <223> ManB, phosphomannomutase, GenBank ID: NP_416552.1 <400> 41 Met Lys Lys Leu Thr Cys Phe Lys Ala Tyr Asp Ile Arg Gly Lys Leu 1 5 10 15 Gly Glu Glu Leu Asn Glu Asp Ile Ala Trp Arg Ile Gly Arg Ala Tyr 20 25 30 Gly Glu Phe Leu Lys Pro Lys Thr Ile Val Leu Gly Gly Asp Val Arg 35 40 45 Leu Thr Ser Glu Thr Leu Lys Leu Ala Leu Ala Lys Gly Leu Gln Asp 50 55 60 Ala Gly Val Asp Val Leu Asp Ile Gly Met Ser Gly Thr Glu Glu Ile 65 70 75 80 Tyr Phe Ala Thr Phe His Leu Gly Val Asp Gly Gly Ile Glu Val Thr 85 90 95 Ala Ser His Asn Pro Met Asp Tyr Asn Gly Met Lys Leu Val Arg Glu 100 105 110 Gly Ala Arg Pro Ile Ser Gly Asp Thr Gly Leu Arg Asp Val Gln Arg 115 120 125 Leu Ala Glu Ala Asn Asp Phe Pro Pro Val Asp Glu Thr Lys Arg Gly 130 135 140 Arg Tyr Gln Gln Ile Asn Leu Arg Asp Ala Tyr Val Asp His Leu Phe 145 150 155 160 Gly Tyr Ile Asn Val Lys Asn Leu Thr Pro Leu Lys Leu Val Ile Asn 165 170 175 Ser Gly Asn Gly Ala Ala Gly Pro Val Val Asp Ala Ile Glu Ala Arg 180 185 190 Phe Lys Ala Leu Gly Ala Pro Val Glu Leu Ile Lys Val His Asn Thr 195 200 205 Pro Asp Gly Asn Phe Pro Asn Gly Ile Pro Asn Pro Leu Leu Pro Glu 210 215 220 Cys Arg Asp Asp Thr Arg Asn Ala Val Ile Lys His Gly Ala Asp Met 225 230 235 240 Gly Ile Ala Phe Asp Gly Asp Phe Asp Arg Cys Phe Leu Phe Asp Glu 245 250 255 Lys Gly Gln Phe Ile Glu Gly Tyr Tyr Ile Val Gly Leu Leu Ala Glu 260 265 270 Ala Phe Leu Glu Lys Asn Pro Gly Ala Lys Ile Ile His Asp Pro Arg 275 280 285 Leu Ser Trp Asn Thr Val Asp Val Val Thr Ala Ala Gly Gly Thr Pro 290 295 300 Val Met Ser Lys Thr Gly His Ala Phe Ile Lys Glu Arg Met Arg Lys 305 310 315 320 Glu Asp Ala Ile Tyr Gly Gly Glu Met Ser Ala His His Tyr Phe Arg 325 330 335 Asp Phe Ala Tyr Cys Asp Ser Gly Met Ile Pro Trp Leu Leu Val Ala 340 345 350 Glu Leu Val Cys Leu Lys Asp Lys Thr Leu Gly Glu Leu Val Arg Asp 355 360 365 Arg Met Ala Ala Phe Pro Ala Ser Gly Glu Ile Asn Ser Lys Leu Ala 370 375 380 Gln Pro Val Glu Ala Ile Asn Arg Val Glu Gln His Phe Ser Arg Glu 385 390 395 400 Ala Leu Ala Val Asp Arg Thr Asp Gly Ile Ser Met Thr Phe Ala Asp 405 410 415 Trp Arg Phe Asn Leu Arg Thr Ser Asn Thr Glu Pro Val Val Arg Leu 420 425 430 Asn Val Glu Ser Arg Gly Asp Val Pro Leu Met Glu Ala Arg Thr Arg 435 440 445 Thr Leu Leu Thr Leu Leu Asn Glu 450 455 <210> 42 <211> 456 <212> PRT <213> Escherichia <220> <223> ManB, phosphomannomutase, GenBank ID: WP_001350528.1 <400> 42 Met Lys Lys Leu Thr Cys Phe Lys Ala Tyr Asp Ile Arg Gly Lys Leu 1 5 10 15 Gly Glu Glu Leu Asn Glu Asp Ile Ala Trp Arg Ile Gly Arg Ala Tyr 20 25 30 Gly Glu Phe Leu Lys Pro Lys Thr Ile Val Leu Gly Gly Asp Val Arg 35 40 45 Leu Thr Ser Glu Thr Leu Lys Leu Ala Leu Ala Lys Gly Leu Gln Asp 50 55 60 Ala Gly Val Asp Val Leu Asp Ile Gly Met Ser Gly Thr Glu Glu Ile 65 70 75 80 Tyr Phe Ala Thr Phe His Leu Gly Val Asp Gly Gly Ile Glu Val Thr 85 90 95 Ala Ser His Asn Pro Met Asp Tyr Asn Gly Met Lys Leu Val Arg Glu 100 105 110 Gly Ala Arg Pro Ile Ser Gly Asp Thr Gly Leu Arg Asp Val Gln Arg 115 120 125 Leu Ala Glu Ala Asn Asp Phe Pro Pro Val Asp Glu Thr Lys Arg Gly 130 135 140 Arg Tyr Gln Gln Ile Asn Leu Arg Asp Ala Tyr Val Asp His Leu Phe 145 150 155 160 Gly Tyr Ile Asn Val Lys Asn Leu Thr Pro Leu Lys Leu Val Ile Asn 165 170 175 Ser Gly Asn Gly Ala Ala Gly Pro Val Val Asp Ala Ile Glu Ala Arg 180 185 190 Phe Lys Ala Leu Gly Ala Pro Val Glu Leu Ile Lys Val His Asn Thr 195 200 205 Pro Asp Gly Asn Phe Pro Asn Gly Ile Pro Asn Pro Leu Leu Pro Glu 210 215 220 Cys Arg Asp Asp Thr Arg Asn Ala Val Ile Lys His Gly Ala Asp Met 225 230 235 240 Gly Ile Ala Phe Asp Gly Asp Phe Asp Arg Cys Phe Leu Phe Asp Glu 245 250 255 Lys Gly Gln Phe Ile Glu Gly Tyr Tyr Ile Val Gly Leu Leu Ala Glu 260 265 270 Ala Phe Leu Glu Lys Asn Pro Gly Ala Lys Ile Ile His Asp Pro Arg 275 280 285 Leu Ser Trp Asn Thr Val Asp Val Val Thr Ala Ala Gly Gly Thr Pro 290 295 300 Val Met Ser Lys Thr Gly His Ala Phe Ile Lys Glu Arg Met Arg Lys 305 310 315 320 Glu Asp Ala Ile Tyr Gly Gly Glu Met Ser Ala His His Tyr Phe Arg 325 330 335 Asp Phe Ala Tyr Cys Asp Ser Gly Met Ile Pro Trp Leu Leu Val Ala 340 345 350 Glu Leu Val Cys Leu Lys Asp Lys Thr Leu Gly Glu Leu Val Arg Asp 355 360 365 Arg Met Ala Ala Phe Pro Ala Ser Gly Glu Ile Asn Ser Lys Leu Ala 370 375 380 Gln Pro Val Glu Ala Ile Asn Arg Val Glu Gln His Phe Ser Arg Glu 385 390 395 400 Ala Leu Ala Val Asp Arg Thr Asp Gly Ile Ser Met Thr Phe Ala Asp 405 410 415 Trp Arg Phe Asn Leu Arg Thr Ser Asn Thr Glu Pro Val Val Arg Leu 420 425 430 Asn Val Glu Ser Arg Gly Asp Val Pro Leu Met Glu Ala Arg Thr Arg 435 440 445 Thr Leu Leu Thr Leu Leu Asn Glu 450 455 <210> 43 <211> 546 <212> PRT <213> Escherichia coli K-12 <220> <223> pgm, Phosphoglucomutase, GenBank ID: NP_415214.1 <400> 43 Met Ala Ile His Asn Arg Ala Gly Gln Pro Ala Gln Gln Ser Asp Leu 1 5 10 15 Ile Asn Val Ala Gln Leu Thr Ala Gln Tyr Tyr Val Leu Lys Pro Glu 20 25 30 Ala Gly Asn Ala Glu His Ala Val Lys Phe Gly Thr Ser Gly His Arg 35 40 45 Gly Ser Ala Ala Arg His Ser Phe Asn Glu Pro His Ile Leu Ala Ile 50 55 60 Ala Gln Ala Ile Ala Glu Glu Arg Ala Lys Asn Gly Ile Thr Gly Pro 65 70 75 80 Cys Tyr Val Gly Lys Asp Thr His Ala Leu Ser Glu Pro Ala Phe Ile 85 90 95 Ser Val Leu Glu Val Leu Ala Ala Asn Gly Val Asp Val Ile Val Gln 100 105 110 Glu Asn Asn Gly Phe Thr Pro Thr Pro Ala Val Ser Asn Ala Ile Leu 115 120 125 Val His Asn Lys Lys Gly Gly Pro Leu Ala Asp Gly Ile Val Ile Thr 130 135 140 Pro Ser His Asn Pro Pro Glu Asp Gly Gly Ile Lys Tyr Asn Pro Pro 145 150 155 160 Asn Gly Gly Pro Ala Asp Thr Asn Val Thr Lys Val Val Glu Asp Arg 165 170 175 Ala Asn Ala Leu Leu Ala Asp Gly Leu Lys Gly Val Lys Arg Ile Ser 180 185 190 Leu Asp Glu Ala Met Ala Ser Gly His Val Lys Glu Gln Asp Leu Val 195 200 205 Gln Pro Phe Val Glu Gly Leu Ala Asp Ile Val Asp Met Ala Ala Ile 210 215 220 Gln Lys Ala Gly Leu Thr Leu Gly Val Asp Pro Leu Gly Gly Ser Gly 225 230 235 240 Ile Glu Tyr Trp Lys Arg Ile Gly Glu Tyr Tyr Asn Leu Asn Leu Thr 245 250 255 Ile Val Asn Asp Gln Val Asp Gln Thr Phe Arg Phe Met His Leu Asp 260 265 270 Lys Asp Gly Ala Ile Arg Met Asp Cys Ser Ser Glu Cys Ala Met Ala 275 280 285 Gly Leu Leu Ala Leu Arg Asp Lys Phe Asp Leu Ala Phe Ala Asn Asp 290 295 300 Pro Asp Tyr Asp Arg His Gly Ile Val Thr Pro Ala Gly Leu Met Asn 305 310 315 320 Pro Asn His Tyr Leu Ala Val Ala Ile Asn Tyr Leu Phe Gln His Arg 325 330 335 Pro Gln Trp Gly Lys Asp Val Ala Val Gly Lys Thr Leu Val Ser Ser 340 345 350 Ala Met Ile Asp Arg Val Val Asn Asp Leu Gly Arg Lys Leu Val Glu 355 360 365 Val Pro Val Gly Phe Lys Trp Phe Val Asp Gly Leu Phe Asp Gly Ser 370 375 380 Phe Gly Phe Gly Gly Glu Glu Ser Ala Gly Ala Ser Phe Leu Arg Phe 385 390 395 400 Asp Gly Thr Pro Trp Ser Thr Asp Lys Asp Gly Ile Ile Met Cys Leu 405 410 415 Leu Ala Ala Glu Ile Thr Ala Val Thr Gly Lys Asn Pro Gln Glu His 420 425 430 Tyr Asn Glu Leu Ala Lys Arg Phe Gly Ala Pro Ser Tyr Asn Arg Leu 435 440 445 Gln Ala Ala Ala Thr Ser Ala Gln Lys Ala Ala Leu Ser Lys Leu Ser 450 455 460 Pro Glu Met Val Ser Ala Ser Thr Leu Ala Gly Asp Pro Ile Thr Ala 465 470 475 480 Arg Leu Thr Ala Ala Pro Gly Asn Gly Ala Ser Ile Gly Gly Leu Lys 485 490 495 Val Met Thr Asp Asn Gly Trp Phe Ala Ala Arg Pro Ser Gly Thr Glu 500 505 510 Asp Ala Tyr Lys Ile Tyr Cys Glu Ser Phe Leu Gly Glu Glu His Arg 515 520 525 Lys Gln Ile Glu Lys Glu Ala Val Glu Ile Val Ser Glu Val Leu Lys 530 535 540 Asn Ala 545 <210> 44 <211> 553 <212> PRT <213> Escherichia coli <220> <223> pgm, phosphoglucomutase, GenBank ID: WP_001396326.1 <400> 44 Met Gln Thr Lys Asp Lys Ala Met Ala Ile His Asn Arg Ala Gly Gln 1 5 10 15 Pro Ala Gln Gln Ser Asp Leu Ile Asn Val Ala Gln Leu Thr Ala Gln 20 25 30 Tyr Tyr Val Leu Lys Pro Glu Ala Gly Asn Ala Glu His Ala Val Lys 35 40 45 Phe Gly Thr Ser Gly His Arg Gly Ser Ala Ala Arg His Ser Phe Asn 50 55 60 Glu Pro His Ile Leu Ala Ile Ala Gln Ala Ile Ala Glu Glu Arg Ala 65 70 75 80 Lys Asn Gly Ile Thr Gly Pro Cys Tyr Val Gly Lys Asp Thr His Ala 85 90 95 Leu Ser Glu Pro Ala Phe Ile Ser Val Leu Glu Val Leu Ala Ala Asn 100 105 110 Gly Val Asp Val Ile Val Gln Glu Asn Asn Gly Phe Thr Pro Thr Pro 115 120 125 Ala Val Ser Asn Ala Ile Leu Val His Asn Lys Lys Gly Gly Pro Leu 130 135 140 Ala Asp Gly Ile Val Ile Thr Pro Ser His Asn Pro Pro Glu Asp Gly 145 150 155 160 Gly Ile Lys Tyr Asn Pro Pro Asn Gly Gly Pro Ala Asp Thr Asn Val 165 170 175 Thr Lys Val Val Glu Asp Arg Ala Asn Ala Leu Leu Ala Asp Gly Leu 180 185 190 Lys Gly Val Lys Arg Ile Ser Leu Asp Glu Ala Met Ala Ser Gly His 195 200 205 Val Lys Glu Gln Asp Leu Val Gln Pro Phe Val Glu Gly Leu Ala Asp 210 215 220 Ile Val Asp Met Ala Ala Ile Gln Lys Ala Gly Leu Thr Leu Gly Val 225 230 235 240 Asp Pro Leu Gly Gly Ser Gly Ile Glu Tyr Trp Lys Arg Ile Gly Glu 245 250 255 Tyr Tyr Asn Leu Asn Leu Thr Ile Val Asn Asp Gln Val Asp Gln Thr 260 265 270 Phe Arg Phe Met His Leu Asp Lys Asp Gly Ala Ile Arg Met Asp Cys 275 280 285 Ser Ser Glu Cys Ala Met Ala Gly Leu Leu Ala Leu Arg Asp Lys Phe 290 295 300 Asp Leu Ala Phe Ala Asn Asp Pro Asp Tyr Asp Arg His Gly Ile Val 305 310 315 320 Thr Pro Ala Gly Leu Met Asn Pro Asn His Tyr Leu Ala Val Ala Ile 325 330 335 Asn Tyr Leu Phe Gln His Arg Pro Gln Trp Gly Lys Asp Val Ala Val 340 345 350 Gly Lys Thr Leu Val Ser Ser Ala Met Ile Asp Arg Val Val Asn Asp 355 360 365 Leu Gly Arg Lys Leu Val Glu Val Pro Val Gly Phe Lys Trp Phe Val 370 375 380 Asp Gly Leu Phe Asp Gly Ser Phe Gly Phe Gly Gly Glu Glu Ser Ala 385 390 395 400 Gly Ala Ser Phe Leu Arg Phe Asp Gly Thr Pro Trp Ser Thr Asp Lys 405 410 415 Asp Gly Ile Ile Met Cys Leu Leu Ala Ala Glu Ile Thr Ala Val Thr 420 425 430 Gly Lys Asn Pro Gln Glu His Tyr Asn Glu Leu Ala Lys Arg Phe Gly 435 440 445 Ala Pro Ser Tyr Asn Arg Leu Gln Ala Ala Ala Thr Ser Ala Gln Lys 450 455 460 Ala Ala Leu Ser Lys Leu Ser Pro Glu Met Val Ser Ala Ser Thr Leu 465 470 475 480 Ala Gly Asp Pro Ile Thr Ala Arg Leu Thr Ala Ala Pro Gly Asn Gly 485 490 495 Ala Ser Ile Gly Gly Leu Lys Val Met Thr Asp Asn Gly Trp Phe Ala 500 505 510 Ala Arg Pro Ser Gly Thr Glu Asp Ala Tyr Lys Ile Tyr Cys Glu Ser 515 520 525 Phe Leu Gly Glu Glu His Arg Lys Gln Ile Glu Lys Glu Ala Val Glu 530 535 540 Ile Val Ser Glu Val Leu Lys Asn Ala 545 550 <210> 45 <211> 302 <212> PRT <213> Escherichia coli K-12 <220> <223> GalU, UTP-glucose-1-phosphate uridylyltransferase, GenBank: NP_415752.1 <400> 45 Met Ala Ala Ile Asn Thr Lys Val Lys Lys Ala Val Ile Pro Val Ala 1 5 10 15 Gly Leu Gly Thr Arg Met Leu Pro Ala Thr Lys Ala Ile Pro Lys Glu 20 25 30 Met Leu Pro Leu Val Asp Lys Pro Leu Ile Gln Tyr Val Val Asn Glu 35 40 45 Cys Ile Ala Ala Gly Ile Thr Glu Ile Val Leu Val Thr His Ser Ser 50 55 60 Lys Asn Ser Ile Glu Asn His Phe Asp Thr Ser Phe Glu Leu Glu Ala 65 70 75 80 Met Leu Glu Lys Arg Val Lys Arg Gln Leu Leu Asp Glu Val Gln Ser 85 90 95 Ile Cys Pro Pro His Val Thr Ile Met Gln Val Arg Gln Gly Leu Ala 100 105 110 Lys Gly Leu Gly His Ala Val Leu Cys Ala His Pro Val Val Gly Asp 115 120 125 Glu Pro Val Ala Val Ile Leu Pro Asp Val Ile Leu Asp Glu Tyr Glu 130 135 140 Ser Asp Leu Ser Gln Asp Asn Leu Ala Glu Met Ile Arg Arg Phe Asp 145 150 155 160 Glu Thr Gly His Ser Gln Ile Met Val Glu Pro Val Ala Asp Val Thr 165 170 175 Ala Tyr Gly Val Val Asp Cys Lys Gly Val Glu Leu Ala Pro Gly Glu 180 185 190 Ser Val Pro Met Val Gly Val Val Glu Lys Pro Lys Ala Asp Val Ala 195 200 205 Pro Ser Asn Leu Ala Ile Val Gly Arg Tyr Val Leu Ser Ala Asp Ile 210 215 220 Trp Pro Leu Leu Ala Lys Thr Pro Pro Gly Ala Gly Asp Glu Ile Gln 225 230 235 240 Leu Thr Asp Ala Ile Asp Met Leu Ile Glu Lys Glu Thr Val Glu Ala 245 250 255 Tyr His Met Lys Gly Lys Ser His Asp Cys Gly Asn Lys Leu Gly Tyr 260 265 270 Met Gln Ala Phe Val Glu Tyr Gly Ile Arg His Asn Thr Leu Gly Thr 275 280 285 Glu Phe Lys Ala Trp Leu Glu Glu Glu Met Gly Ile Lys Lys 290 295 300 <210> 46 <211> 302 <212> PRT <213> Enterobacteriaceae <220> <223> GalU, UTP-glucose-1-phosphate uridylyltransferase, WP_000718995.1 <400> 46 Met Ala Ala Ile Asn Thr Lys Val Lys Lys Ala Val Ile Pro Val Ala 1 5 10 15 Gly Leu Gly Thr Arg Met Leu Pro Ala Thr Lys Ala Ile Pro Lys Glu 20 25 30 Met Leu Pro Leu Val Asp Lys Pro Leu Ile Gln Tyr Val Val Asn Glu 35 40 45 Cys Ile Ala Ala Gly Ile Thr Glu Ile Val Leu Val Thr His Ser Ser 50 55 60 Lys Asn Ser Ile Glu Asn His Phe Asp Thr Ser Phe Glu Leu Glu Ala 65 70 75 80 Met Leu Glu Lys Arg Val Lys Arg Gln Leu Leu Asp Glu Val Gln Ser 85 90 95 Ile Cys Pro Pro His Val Thr Ile Met Gln Val Arg Gln Gly Leu Ala 100 105 110 Lys Gly Leu Gly His Ala Val Leu Cys Ala His Pro Val Val Gly Asp 115 120 125 Glu Pro Val Ala Val Ile Leu Pro Asp Val Ile Leu Asp Glu Tyr Glu 130 135 140 Ser Asp Leu Ser Gln Asp Asn Leu Ala Glu Met Ile Arg Arg Phe Asp 145 150 155 160 Glu Thr Gly His Ser Gln Ile Met Val Glu Pro Val Ala Asp Val Thr 165 170 175 Ala Tyr Gly Val Val Asp Cys Lys Gly Val Glu Leu Ala Pro Gly Glu 180 185 190 Ser Val Pro Met Val Gly Val Val Glu Lys Pro Lys Ala Asp Val Ala 195 200 205 Pro Ser Asn Leu Ala Ile Val Gly Arg Tyr Val Leu Ser Ala Asp Ile 210 215 220 Trp Pro Leu Leu Ala Lys Thr Pro Pro Gly Ala Gly Asp Glu Ile Gln 225 230 235 240 Leu Thr Asp Ala Ile Asp Met Leu Ile Glu Lys Glu Thr Val Glu Ala 245 250 255 Tyr His Met Lys Gly Lys Ser His Asp Cys Gly Asn Lys Leu Gly Tyr 260 265 270 Met Gln Ala Phe Val Glu Tyr Gly Ile Arg His Asn Thr Leu Gly Thr 275 280 285 Glu Phe Lys Ala Trp Leu Glu Glu Glu Met Gly Ile Lys Lys 290 295 300 <210> 47 <211> 338 <212> PRT <213> Escherichia coli K-12 <220> <223> GalE, UDP-glucose 4-epimerase, GenBank ID: NP_415280.3 <400> 47 Met Arg Val Leu Val Thr Gly Gly Ser Gly Tyr Ile Gly Ser His Thr 1 5 10 15 Cys Val Gln Leu Leu Gln Asn Gly His Asp Val Ile Ile Leu Asp Asn 20 25 30 Leu Cys Asn Ser Lys Arg Ser Val Leu Pro Val Ile Glu Arg Leu Gly 35 40 45 Gly Lys His Pro Thr Phe Val Glu Gly Asp Ile Arg Asn Glu Ala Leu 50 55 60 Met Thr Glu Ile Leu His Asp His Ala Ile Asp Thr Val Ile His Phe 65 70 75 80 Ala Gly Leu Lys Ala Val Gly Glu Ser Val Gln Lys Pro Leu Glu Tyr 85 90 95 Tyr Asp Asn Asn Val Asn Gly Thr Leu Arg Leu Ile Ser Ala Met Arg 100 105 110 Ala Ala Asn Val Lys Asn Phe Ile Phe Ser Ser Ser Ala Thr Val Tyr 115 120 125 Gly Asp Gln Pro Lys Ile Pro Tyr Val Glu Ser Phe Pro Thr Gly Thr 130 135 140 Pro Gln Ser Pro Tyr Gly Lys Ser Lys Leu Met Val Glu Gln Ile Leu 145 150 155 160 Thr Asp Leu Gln Lys Ala Gln Pro Asp Trp Ser Ile Ala Leu Leu Arg 165 170 175 Tyr Phe Asn Pro Val Gly Ala His Pro Ser Gly Asp Met Gly Glu Asp 180 185 190 Pro Gln Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Ala Gln Val Ala 195 200 205 Val Gly Arg Arg Asp Ser Leu Ala Ile Phe Gly Asn Asp Tyr Pro Thr 210 215 220 Glu Asp Gly Thr Gly Val Arg Asp Tyr Ile His Val Met Asp Leu Ala 225 230 235 240 Asp Gly His Val Val Ala Met Glu Lys Leu Ala Asn Lys Pro Gly Val 245 250 255 His Ile Tyr Asn Leu Gly Ala Gly Val Gly Asn Ser Val Leu Asp Val 260 265 270 Val Asn Ala Phe Ser Lys Ala Cys Gly Lys Pro Val Asn Tyr His Phe 275 280 285 Ala Pro Arg Arg Glu Gly Asp Leu Pro Ala Tyr Trp Ala Asp Ala Ser 290 295 300 Lys Ala Asp Arg Glu Leu Asn Trp Arg Val Thr Arg Thr Leu Asp Glu 305 310 315 320 Met Ala Gln Asp Thr Trp His Trp Gln Ser Arg His Pro Gln Gly Tyr 325 330 335 Pro Asp <210> 48 <211> 338 <212> PRT <213> Enterobacteriaceae <220> <223> GalE, UDP-glucose 4-epimerase, GenBank ID: WP_001265438.1 <400> 48 Met Arg Val Leu Val Thr Gly Gly Ser Gly Tyr Ile Gly Ser His Thr 1 5 10 15 Cys Val Gln Leu Leu Gln Asn Gly His Asp Val Ile Ile Leu Asp Asn 20 25 30 Leu Cys Asn Ser Lys Arg Ser Val Leu Pro Val Ile Glu Arg Leu Gly 35 40 45 Gly Lys His Pro Thr Phe Val Glu Gly Asp Ile Arg Asn Glu Ala Leu 50 55 60 Met Thr Glu Ile Leu His Asp His Ala Ile Asp Thr Val Ile His Phe 65 70 75 80 Ala Gly Leu Lys Ala Val Gly Glu Ser Val Gln Lys Pro Leu Glu Tyr 85 90 95 Tyr Asp Asn Asn Val Asn Gly Thr Leu Arg Leu Ile Ser Ala Met Arg 100 105 110 Ala Ala Asn Val Lys Asn Phe Ile Phe Ser Ser Ser Ala Thr Val Tyr 115 120 125 Gly Asp Gln Pro Lys Ile Pro Tyr Val Glu Ser Phe Pro Thr Gly Thr 130 135 140 Pro Gln Ser Pro Tyr Gly Lys Ser Lys Leu Met Val Glu Gln Ile Leu 145 150 155 160 Thr Asp Leu Gln Lys Ala Gln Pro Asp Trp Ser Ile Ala Leu Leu Arg 165 170 175 Tyr Phe Asn Pro Val Gly Ala His Pro Ser Gly Asp Met Gly Glu Asp 180 185 190 Pro Gln Gly Ile Pro Asn Asn Leu Met Pro Tyr Ile Ala Gln Val Ala 195 200 205 Val Gly Arg Arg Asp Ser Leu Ala Ile Phe Gly Asn Asp Tyr Pro Thr 210 215 220 Glu Asp Gly Thr Gly Val Arg Asp Tyr Ile His Val Met Asp Leu Ala 225 230 235 240 Asp Gly His Val Val Ala Met Glu Lys Leu Ala Asn Lys Pro Gly Val 245 250 255 His Ile Tyr Asn Leu Gly Ala Gly Val Gly Asn Ser Val Leu Asp Val 260 265 270 Val Asn Ala Phe Ser Lys Ala Cys Gly Lys Pro Val Asn Tyr His Phe 275 280 285 Ala Pro Arg Arg Glu Gly Asp Leu Pro Ala Tyr Trp Ala Asp Ala Ser 290 295 300 Lys Ala Asp Arg Glu Leu Asn Trp Arg Val Thr Arg Thr Leu Asp Glu 305 310 315 320 Met Ala Gln Asp Thr Trp His Trp Gln Ser Arg His Pro Gln Gly Tyr 325 330 335 Pro Asp <210> 49 <211> 445 <212> PRT <213> Escherichia coli K-12 <220> <223> GlmM, Phosphoglucosamine mutase, GenBank ID: NP_417643.1 <400> 49 Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30 Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 <210> 50 <211> 445 <212> PRT <213> Enterobacteriaceae <220> <223> GlmM, Phosphoglucosamine mutase, GenBank ID: WP_000071134.1 <400> 50 Met Ser Asn Arg Lys Tyr Phe Gly Thr Asp Gly Ile Arg Gly Arg Val 1 5 10 15 Gly Asp Ala Pro Ile Thr Pro Asp Phe Val Leu Lys Leu Gly Trp Ala 20 25 30 Ala Gly Lys Val Leu Ala Arg His Gly Ser Arg Lys Ile Ile Ile Gly 35 40 45 Lys Asp Thr Arg Ile Ser Gly Tyr Met Leu Glu Ser Ala Leu Glu Ala 50 55 60 Gly Leu Ala Ala Ala Gly Leu Ser Ala Leu Phe Thr Gly Pro Met Pro 65 70 75 80 Thr Pro Ala Val Ala Tyr Leu Thr Arg Thr Phe Arg Ala Glu Ala Gly 85 90 95 Ile Val Ile Ser Ala Ser His Asn Pro Phe Tyr Asp Asn Gly Ile Lys 100 105 110 Phe Phe Ser Ile Asp Gly Thr Lys Leu Pro Asp Ala Val Glu Glu Ala 115 120 125 Ile Glu Ala Glu Met Glu Lys Glu Ile Ser Cys Val Asp Ser Ala Glu 130 135 140 Leu Gly Lys Ala Ser Arg Ile Val Asp Ala Ala Gly Arg Tyr Ile Glu 145 150 155 160 Phe Cys Lys Ala Thr Phe Pro Asn Glu Leu Ser Leu Ser Glu Leu Lys 165 170 175 Ile Val Val Asp Cys Ala Asn Gly Ala Thr Tyr His Ile Ala Pro Asn 180 185 190 Val Leu Arg Glu Leu Gly Ala Asn Val Ile Ala Ile Gly Cys Glu Pro 195 200 205 Asn Gly Val Asn Ile Asn Ala Glu Val Gly Ala Thr Asp Val Arg Ala 210 215 220 Leu Gln Ala Arg Val Leu Ala Glu Lys Ala Asp Leu Gly Ile Ala Phe 225 230 235 240 Asp Gly Asp Gly Asp Arg Val Ile Met Val Asp His Glu Gly Asn Lys 245 250 255 Val Asp Gly Asp Gln Ile Met Tyr Ile Ile Ala Arg Glu Gly Leu Arg 260 265 270 Gln Gly Gln Leu Arg Gly Gly Ala Val Gly Thr Leu Met Ser Asn Met 275 280 285 Gly Leu Glu Leu Ala Leu Lys Gln Leu Gly Ile Pro Phe Ala Arg Ala 290 295 300 Lys Val Gly Asp Arg Tyr Val Leu Glu Lys Met Gln Glu Lys Gly Trp 305 310 315 320 Arg Ile Gly Ala Glu Asn Ser Gly His Val Ile Leu Leu Asp Lys Thr 325 330 335 Thr Thr Gly Asp Gly Ile Val Ala Gly Leu Gln Val Leu Ala Ala Met 340 345 350 Ala Arg Asn His Met Ser Leu His Asp Leu Cys Ser Gly Met Lys Met 355 360 365 Phe Pro Gln Ile Leu Val Asn Val Arg Tyr Thr Ala Gly Ser Gly Asp 370 375 380 Pro Leu Glu His Glu Ser Val Lys Ala Val Thr Ala Glu Val Glu Ala 385 390 395 400 Ala Leu Gly Asn Arg Gly Arg Val Leu Leu Arg Lys Ser Gly Thr Glu 405 410 415 Pro Leu Ile Arg Val Met Val Glu Gly Glu Asp Glu Ala Gln Val Thr 420 425 430 Glu Phe Ala His Arg Ile Ala Asp Ala Val Lys Ala Val 435 440 445 <210> 51 <211> 456 <212> PRT <213> Escherichia coli K-12 <220> <223> GlmU, N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase, GenBank ID: NP_418186.1 <400> 51 Met Leu Asn Asn Ala Met Ser Val Val Ile Leu Ala Ala Gly Lys Gly 1 5 10 15 Thr Arg Met Tyr Ser Asp Leu Pro Lys Val Leu His Thr Leu Ala Gly 20 25 30 Lys Ala Met Val Gln His Val Ile Asp Ala Ala Asn Glu Leu Gly Ala 35 40 45 Ala His Val His Leu Val Tyr Gly His Gly Gly Asp Leu Leu Lys Gln 50 55 60 Ala Leu Lys Asp Asp Asn Leu Asn Trp Val Leu Gln Ala Glu Gln Leu 65 70 75 80 Gly Thr Gly His Ala Met Gln Gln Ala Ala Pro Phe Phe Ala Asp Asp 85 90 95 Glu Asp Ile Leu Met Leu Tyr Gly Asp Val Pro Leu Ile Ser Val Glu 100 105 110 Thr Leu Gln Arg Leu Arg Asp Ala Lys Pro Gln Gly Gly Ile Gly Leu 115 120 125 Leu Thr Val Lys Leu Asp Asp Pro Thr Gly Tyr Gly Arg Ile Thr Arg 130 135 140 Glu Asn Gly Lys Val Thr Gly Ile Val Glu His Lys Asp Ala Thr Asp 145 150 155 160 Glu Gln Arg Gln Ile Gln Glu Ile Asn Thr Gly Ile Leu Ile Ala Asn 165 170 175 Gly Ala Asp Met Lys Arg Trp Leu Ala Lys Leu Thr Asn Asn Asn Ala 180 185 190 Gln Gly Glu Tyr Tyr Ile Thr Asp Ile Ile Ala Leu Ala Tyr Gln Glu 195 200 205 Gly Arg Glu Ile Val Ala Val His Pro Gln Arg Leu Ser Glu Val Glu 210 215 220 Gly Val Asn Asn Arg Leu Gln Leu Ser Arg Leu Glu Arg Val Tyr Gln 225 230 235 240 Ser Glu Gln Ala Glu Lys Leu Leu Leu Ala Gly Val Met Leu Arg Asp 245 250 255 Pro Ala Arg Phe Asp Leu Arg Gly Thr Leu Thr His Gly Arg Asp Val 260 265 270 Glu Ile Asp Thr Asn Val Ile Ile Glu Gly Asn Val Thr Leu Gly His 275 280 285 Arg Val Lys Ile Gly Thr Gly Cys Val Ile Lys Asn Ser Val Ile Gly 290 295 300 Asp Asp Cys Glu Ile Ser Pro Tyr Thr Val Val Glu Asp Ala Asn Leu 305 310 315 320 Ala Ala Ala Cys Thr Ile Gly Pro Phe Ala Arg Leu Arg Pro Gly Ala 325 330 335 Glu Leu Leu Glu Gly Ala His Val Gly Asn Phe Val Glu Met Lys Lys 340 345 350 Ala Arg Leu Gly Lys Gly Ser Lys Ala Gly His Leu Thr Tyr Leu Gly 355 360 365 Asp Ala Glu Ile Gly Asp Asn Val Asn Ile Gly Ala Gly Thr Ile Thr 370 375 380 Cys Asn Tyr Asp Gly Ala Asn Lys Phe Lys Thr Ile Ile Gly Asp Asp 385 390 395 400 Val Phe Val Gly Ser Asp Thr Gln Leu Val Ala Pro Val Thr Val Gly 405 410 415 Lys Gly Ala Thr Ile Ala Ala Gly Thr Thr Val Thr Arg Asn Val Gly 420 425 430 Glu Asn Ala Leu Ala Ile Ser Arg Val Pro Gln Thr Gln Lys Glu Gly 435 440 445 Trp Arg Arg Pro Val Lys Lys Lys 450 455 <210> 52 <211> 456 <212> PRT <213> Enterobacteriaceae <220> <223> GlmU, N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase, GenBank ID: WP_000933736.1 <400> 52 Met Leu Asn Asn Ala Met Ser Val Val Ile Leu Ala Ala Gly Lys Gly 1 5 10 15 Thr Arg Met Tyr Ser Asp Leu Pro Lys Val Leu His Thr Leu Ala Gly 20 25 30 Lys Ala Met Val Gln His Val Ile Asp Ala Ala Asn Glu Leu Gly Ala 35 40 45 Ala His Val His Leu Val Tyr Gly His Gly Gly Asp Leu Leu Lys Gln 50 55 60 Ala Leu Lys Asp Asp Asn Leu Asn Trp Val Leu Gln Ala Glu Gln Leu 65 70 75 80 Gly Thr Gly His Ala Met Gln Gln Ala Ala Pro Phe Phe Ala Asp Asp 85 90 95 Glu Asp Ile Leu Met Leu Tyr Gly Asp Val Pro Leu Ile Ser Val Glu 100 105 110 Thr Leu Gln Arg Leu Arg Asp Ala Lys Pro Gln Gly Gly Ile Gly Leu 115 120 125 Leu Thr Val Lys Leu Asp Asp Pro Thr Gly Tyr Gly Arg Ile Thr Arg 130 135 140 Glu Asn Gly Lys Val Thr Gly Ile Val Glu His Lys Asp Ala Thr Asp 145 150 155 160 Glu Gln Arg Gln Ile Gln Glu Ile Asn Thr Gly Ile Leu Ile Ala Asn 165 170 175 Gly Ala Asp Met Lys Arg Trp Leu Ala Lys Leu Thr Asn Asn Asn Ala 180 185 190 Gln Gly Glu Tyr Tyr Ile Thr Asp Ile Ile Ala Leu Ala Tyr Gln Glu 195 200 205 Gly Arg Glu Ile Val Ala Val His Pro Gln Arg Leu Ser Glu Val Glu 210 215 220 Gly Val Asn Asn Arg Leu Gln Leu Ser Arg Leu Glu Arg Val Tyr Gln 225 230 235 240 Ser Glu Gln Ala Glu Lys Leu Leu Leu Ala Gly Val Met Leu Arg Asp 245 250 255 Pro Ala Arg Phe Asp Leu Arg Gly Thr Leu Thr His Gly Arg Asp Val 260 265 270 Glu Ile Asp Thr Asn Val Ile Ile Glu Gly Asn Val Thr Leu Gly His 275 280 285 Arg Val Lys Ile Gly Thr Gly Cys Val Ile Lys Asn Ser Val Ile Gly 290 295 300 Asp Asp Cys Glu Ile Ser Pro Tyr Thr Val Val Glu Asp Ala Asn Leu 305 310 315 320 Ala Ala Ala Cys Thr Ile Gly Pro Phe Ala Arg Leu Arg Pro Gly Ala 325 330 335 Glu Leu Leu Glu Gly Ala His Val Gly Asn Phe Val Glu Met Lys Lys 340 345 350 Ala Arg Leu Gly Lys Gly Ser Lys Ala Gly His Leu Thr Tyr Leu Gly 355 360 365 Asp Ala Glu Ile Gly Asp Asn Val Asn Ile Gly Ala Gly Thr Ile Thr 370 375 380 Cys Asn Tyr Asp Gly Ala Asn Lys Phe Lys Thr Ile Ile Gly Asp Asp 385 390 395 400 Val Phe Val Gly Ser Asp Thr Gln Leu Val Ala Pro Val Thr Val Gly 405 410 415 Lys Gly Ala Thr Ile Ala Ala Gly Thr Thr Val Thr Arg Asn Val Gly 420 425 430 Glu Asn Ala Leu Ala Ile Ser Arg Val Pro Gln Thr Gln Lys Glu Gly 435 440 445 Trp Arg Arg Pro Val Lys Lys Lys 450 455 <210> 53 <211> 609 <212> PRT <213> Escherichia coli K-12 <220> <223> GlmS, L-glutamine--D-fructose-6-phosphate aminotransferase, GenBank ID: NP_418185.1 <400> 53 Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95 Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115 120 125 Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175 Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205 Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val 385 390 395 400 Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430 Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510 Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 Glu <210> 54 <211> 609 <212> PRT <213> Enterobacteriaceae <220> <223> GlmS, L-glutamine--D-fructose-6-phosphate aminotransferase, GenBank ID: WP_000334099.1 <400> 54 Met Cys Gly Ile Val Gly Ala Ile Ala Gln Arg Asp Val Ala Glu Ile 1 5 10 15 Leu Leu Glu Gly Leu Arg Arg Leu Glu Tyr Arg Gly Tyr Asp Ser Ala 20 25 30 Gly Leu Ala Val Val Asp Ala Glu Gly His Met Thr Arg Leu Arg Arg 35 40 45 Leu Gly Lys Val Gln Met Leu Ala Gln Ala Ala Glu Glu His Pro Leu 50 55 60 His Gly Gly Thr Gly Ile Ala His Thr Arg Trp Ala Thr His Gly Glu 65 70 75 80 Pro Ser Glu Val Asn Ala His Pro His Val Ser Glu His Ile Val Val 85 90 95 Val His Asn Gly Ile Ile Glu Asn His Glu Pro Leu Arg Glu Glu Leu 100 105 110 Lys Ala Arg Gly Tyr Thr Phe Val Ser Glu Thr Asp Thr Glu Val Ile 115 120 125 Ala His Leu Val Asn Trp Glu Leu Lys Gln Gly Gly Thr Leu Arg Glu 130 135 140 Ala Val Leu Arg Ala Ile Pro Gln Leu Arg Gly Ala Tyr Gly Thr Val 145 150 155 160 Ile Met Asp Ser Arg His Pro Asp Thr Leu Leu Ala Ala Arg Ser Gly 165 170 175 Ser Pro Leu Val Ile Gly Leu Gly Met Gly Glu Asn Phe Ile Ala Ser 180 185 190 Asp Gln Leu Ala Leu Leu Pro Val Thr Arg Arg Phe Ile Phe Leu Glu 195 200 205 Glu Gly Asp Ile Ala Glu Ile Thr Arg Arg Ser Val Asn Ile Phe Asp 210 215 220 Lys Thr Gly Ala Glu Val Lys Arg Gln Asp Ile Glu Ser Asn Leu Gln 225 230 235 240 Tyr Asp Ala Gly Asp Lys Gly Ile Tyr Arg His Tyr Met Gln Lys Glu 245 250 255 Ile Tyr Glu Gln Pro Asn Ala Ile Lys Asn Thr Leu Thr Gly Arg Ile 260 265 270 Ser His Gly Gln Val Asp Leu Ser Glu Leu Gly Pro Asn Ala Asp Glu 275 280 285 Leu Leu Ser Lys Val Glu His Ile Gln Ile Leu Ala Cys Gly Thr Ser 290 295 300 Tyr Asn Ser Gly Met Val Ser Arg Tyr Trp Phe Glu Ser Leu Ala Gly 305 310 315 320 Ile Pro Cys Asp Val Glu Ile Ala Ser Glu Phe Arg Tyr Arg Lys Ser 325 330 335 Ala Val Arg Arg Asn Ser Leu Met Ile Thr Leu Ser Gln Ser Gly Glu 340 345 350 Thr Ala Asp Thr Leu Ala Gly Leu Arg Leu Ser Lys Glu Leu Gly Tyr 355 360 365 Leu Gly Ser Leu Ala Ile Cys Asn Val Pro Gly Ser Ser Leu Val Arg 370 375 380 Glu Ser Asp Leu Ala Leu Met Thr Asn Ala Gly Thr Glu Ile Gly Val 385 390 395 400 Ala Ser Thr Lys Ala Phe Thr Thr Gln Leu Thr Val Leu Leu Met Leu 405 410 415 Val Ala Lys Leu Ser Arg Leu Lys Gly Leu Asp Ala Ser Ile Glu His 420 425 430 Asp Ile Val His Gly Leu Gln Ala Leu Pro Ser Arg Ile Glu Gln Met 435 440 445 Leu Ser Gln Asp Lys Arg Ile Glu Ala Leu Ala Glu Asp Phe Ser Asp 450 455 460 Lys His His Ala Leu Phe Leu Gly Arg Gly Asp Gln Tyr Pro Ile Ala 465 470 475 480 Leu Glu Gly Ala Leu Lys Leu Lys Glu Ile Ser Tyr Ile His Ala Glu 485 490 495 Ala Tyr Ala Ala Gly Glu Leu Lys His Gly Pro Leu Ala Leu Ile Asp 500 505 510 Ala Asp Met Pro Val Ile Val Val Ala Pro Asn Asn Glu Leu Leu Glu 515 520 525 Lys Leu Lys Ser Asn Ile Glu Glu Val Arg Ala Arg Gly Gly Gln Leu 530 535 540 Tyr Val Phe Ala Asp Gln Asp Ala Gly Phe Val Ser Ser Asp Asn Met 545 550 555 560 His Ile Ile Glu Met Pro His Val Glu Glu Val Ile Ala Pro Ile Phe 565 570 575 Tyr Thr Val Pro Leu Gln Leu Leu Ala Tyr His Val Ala Leu Ile Lys 580 585 590 Gly Thr Asp Val Asp Gln Pro Arg Asn Leu Ala Lys Ser Val Thr Val 595 600 605 Glu <210> 55 <211> 221 <212> PRT <213> Campylobacter jejuni <220> <223> NeuA, CMP-Neu5Ac synthetase, GenBank ID: AAK91728.1 <400> 55 Met Ser Leu Ala Ile Ile Pro Ala Arg Gly Gly Ser Lys Gly Ile Lys 1 5 10 15 Asn Lys Asn Leu Val Leu Leu Asn Asn Lys Pro Leu Ile Tyr Tyr Thr 20 25 30 Ile Lys Ala Ala Leo Asn Ala Lys Ser Ile Ser Lys Val Val Val Ser 35 40 45 Ser Asp Ser Asp Glu Ile Leu Asn Tyr Ala Lys Ser Gln Asn Val Asp 50 55 60 Ile Leu Lys Arg Pro Ile Ser Leu Ala Gln Asp Asp Thr Thr Ser Asp 65 70 75 80 Lys Val Leu Leu His Ala Leu Lys Phe Tyr Lys Asp Tyr Glu Asp Val 85 90 95 Val Phe Leu Gln Pro Thr Ser Pro Leu Arg Thr Asn Ile His Ile Asn 100 105 110 Glu Ala Phe Asn Leu Tyr Lys Asn Ser Asn Ala Asn Ala Leu Ile Ser 115 120 125 Val Ser Glu Cys Asp Asn Lys Ile Leu Lys Ala Phe Val Cys Asn Asp 130 135 140 Cys Gly Asp Leu Ala Gly Ile Cys Asn Asp Glu Tyr Pro Phe Met Pro 145 150 155 160 Arg Gln Lys Leu Pro Lys Thr Tyr Met Ser Asn Gly Ala Ile Tyr Ile 165 170 175 Leu Lys Ile Lys Glu Phe Leu Asn Asn Pro Ser Phe Leu Gln Ser Lys 180 185 190 Thr Lys His Phe Leu Met Asp Glu Ser Ser Ser Leu Asp Ile Asp Cys 195 200 205 Leu Glu Asp Leu Lys Lys Val Glu Gln Ile Trp Lys Lys 210 215 220 <210> 56 <211> 231 <212> PRT <213> Vibrio brasiliensis <220> <223> NeuA, CMP-Neu5Ac synthetase, GenBank ID: WP_006881452.1 <400> 56 Met Arg Val Ala Leu Ile Thr Ala Arg Gly Gly Ser Lys Gly Leu Pro 1 5 10 15 Arg Lys Asn Val Leu Asn Val Asn Gly Leu Pro Leu Ile Gly Trp Thr 20 25 30 Ile Asn Ala Ala Arg Glu Cys Ser Tyr Ile Asp Arg Val Phe Val Ser 35 40 45 Thr Glu Asp Glu Glu Ile Ala Lys Ile Ser Leu Gly Met Gly Ala Glu 50 55 60 Leu Ile Glu Arg Pro Cys Glu Leu Ala Gln Asp Asn Ser Thr Ser Asp 65 70 75 80 Glu Val Ile Arg His Ala Ile Asp Trp Leu Asp Lys His Ser Ile Leu 85 90 95 Ser Asp Leu Val Val Leu Leu Gln Pro Thr Ser Pro Leu Arg Thr Ser 100 105 110 Ala His Ile Ser Glu Ala Ile Asp Leu Tyr Asp Ser Leu Asn Asn Pro 115 120 125 Asn Ala Thr Ile Ile Ser Val Tyr Glu Pro Glu His Ser Pro Leu Lys 130 135 140 Ala Phe Val Gln Leu Glu Asn Gly Thr Leu Lys Gly Ser Tyr Asn Glu 145 150 155 160 Asp Ala Pro Tyr Thr Arg Arg Gln Glu Leu Pro Lys Ser Tyr Leu Ala 165 170 175 Asn Gly Ala Ile Tyr Ile Phe Ser Pro Ser Ala Phe Met Ser Glu Ser 180 185 190 Gln Ile Pro Arg Arg Asp Ile Tyr Pro Tyr Val Met Ser Thr Asn Glu 195 200 205 Ser Glu Asp Ile Asp Ser Lys Ile Asp Leu Ile Arg Val Glu Lys Leu 210 215 220 Leu Arg Gly Ser Phe Tyr Glu 225 230 <210> 57 <211> 346 <212> PRT <213> Campylobacter jejuni <220> <223> NeuB, Neu5 Ac synthase, GenBank ID: AAK91726.1 <400> 57 Met Lys Glu Ile Lys Ile Gln Asn Ile Ile Ile Ser Glu Glu Lys Ala 1 5 10 15 Pro Leu Val Val Pro Glu Ile Gly Ile Asn His Asn Gly Ser Leu Glu 20 25 30 Leu Ala Lys Ile Met Val Asp Ala Ala Phe Ser Ala Gly Ala Lys Ile 35 40 45 Ile Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser Lys Ala Ala 50 55 60 Lys Lys Val Ile Pro Gly Asn Ala Lys Ile Ser Ile Tyr Glu Ile Met 65 70 75 80 Gln Lys Cys Ala Leu Asp Tyr Lys Asp Glu Leu Ala Leu Lys Glu Tyr 85 90 95 Thr Glu Lys Leu Gly Leu Val Tyr Leu Ser Thr Pro Phe Ser Arg Ala 100 105 110 Gly Ala Asn Arg Leu Glu Asp Met Gly Val Ser Ala Phe Lys Ile Gly 115 120 125 Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys His Ile Ala Ala Phe 130 135 140 Lys Lys Pro Met Ile Val Ser Thr Gly Met Asn Ser Ile Glu Ser Ile 145 150 155 160 Lys Pro Thr Val Lys Ile Leu Leu Asp Asn Glu Ile Pro Phe Val Leu 165 170 175 Met His Thr Thr Asn Leu Tyr Pro Thr Pro His Asn Leu Val Arg Leu 180 185 190 Asn Ala Met Leu Glu Leu Lys Lys Glu Phe Ser Cys Met Val Gly Leu 195 200 205 Ser Asp His Thr Thr Asp Asn Leu Ala Cys Leu Gly Ala Val Val Leu 210 215 220 Gly Ala Cys Val Leu Glu Arg His Phe Thr Asp Ser Met His Arg Ser 225 230 235 240 Gly Pro Asp Ile Val Cys Ser Met Asp Thr Lys Ala Leu Lys Glu Leu 245 250 255 Ile Ile Gln Ser Glu Gln Met Ala Ile Ile Arg Gly Asn Asn Glu Ser 260 265 270 Lys Lys Ala Ala Lys Gln Glu Gln Val Thr Ile Asp Phe Ala Phe Ala 275 280 285 Ser Val Val Ser Ile Lys Asp Ile Lys Lys Gly Glu Val Leu Ser Met 290 295 300 Asp Asn Ile Trp Val Lys Arg Pro Gly Leu Gly Gly Ile Ser Ala Ala 305 310 315 320 Glu Phe Glu Asn Ile Leu Gly Lys Lys Ala Leu Arg Asp Ile Glu Asn 325 330 335 Asp Ala Gln Leu Ser Tyr Glu Asp Phe Ala 340 345 <210> 58 <211> 346 <212> PRT <213> Flavobacterium limnosediminis <220> <223> NeuB, Neu5 Ac synthase, GenBank ID: WP_023580510.1 <400> 58 Met Asn Pro Tyr Ile Glu Ile Ala Gly Arg Lys Ile Gly Leu Asp Tyr 1 5 10 15 Pro Pro Leu Val Ile Ala Glu Ile Gly Ile Asn His Glu Gly Ser Leu 20 25 30 Gln Val Ala Lys Glu Ile Val Asp Ala Ala Phe Arg Ala Gly Val Glu 35 40 45 Val Val Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser Gly Ala 50 55 60 Ala Lys Lys Val Ile Pro Gly Asn Ala Asp Val Ser Ile Tyr Glu Ile 65 70 75 80 Met Glu Arg Cys Ala Leu Asp Glu Ser Glu Glu Leu Glu Leu Lys Asn 85 90 95 Tyr Val Glu Ser Lys Gly Met Ile Phe Ile Ser Thr Pro Phe Ser Arg 100 105 110 Ala Ala Ala Glu Arg Leu Lys Lys Phe Asp Ile Pro Ala Tyr Lys Ile 115 120 125 Gly Ser Gly Glu Cys Asn Asn Tyr Pro Leu Leu Glu His Ile Ala Ser 130 135 140 Phe Gly Lys Pro Val Ile Leu Ser Thr Gly Met Asn Thr Val Asp Ser 145 150 155 160 Ile Arg Lys Ala Val Ala Ile Phe Asp Lys His Asn Val Pro Val Ala 165 170 175 Leu Leu His Thr Thr Asn Leu Tyr Pro Thr Pro Ile His Leu Val Arg 180 185 190 Phe Gly Ala Met Met Glu Met His Asn Ala Phe Pro Asp Lys Val Phe 195 200 205 Gly Leu Ser Asp His Thr Leu Asn Asn Asn Ala Cys Leu Gly Ala Val 210 215 220 Ala Leu Gly Ala Ser Ile Leu Glu Arg His Phe Thr Asp His Met Ser 225 230 235 240 Arg Thr Gly Pro Asp Ile Val Cys Ser Met Asp Glu Gln Ala Thr Arg 245 250 255 Glu Leu Ile Val Asn Ser Ala Glu Ile Ala Leu Met Arg Gly Gly Thr 260 265 270 Lys Lys Pro Ala Ala Glu Glu Gln Val Thr Ile Asp Phe Ala Phe Ala 275 280 285 Thr Val Cys Ser Ile Ala Pro Ile Lys Lys Gly Glu Val Phe Thr Lys 290 295 300 Glu Asn Ile Trp Val Lys Arg Pro Gly Thr Gly Lys Ile Leu Ala Glu 305 310 315 320 His Phe Glu Arg Leu Leu Gly Lys Thr Ala Thr Arg Asp Ile Glu Asn 325 330 335 Asp Glu Gln Leu Asp Phe Ser Asp Val Glu 340 345 <210> 59 <211> 372 <212> PRT <213> Campylobacter jejuni <220> <223> NeuC, UDP-GlcNAc 2-epimerase, GenBank AAK91727.1 <400> 59 Met Lys Lys Ile Leu Phe Ile Thr Gly Ser Arg Ala Asp Tyr Ser Lys 1 5 10 15 Ile Lys Ser Leu Met Tyr Arg Val Gln Asn Ser Ser Glu Phe Glu Leu 20 25 30 Tyr Ile Phe Ala Thr Gly Met His Leu Ser Lys Asn Phe Gly Tyr Thr 35 40 45 Val Lys Glu Leu Tyr Lys Asn Gly Phe Lys Asn Ile Tyr Glu Phe Ile 50 55 60 Asn Tyr Asp Lys Tyr Tyr Gln Thr Asp Lys Ala Leu Ala Thr Thr Ile 65 70 75 80 Asp Gly Phe Ser Arg Tyr Ala Asn Glu Leu Lys Pro Asp Leu Ile Val 85 90 95 Val His Gly Asp Arg Ile Glu Pro Leu Ala Ala Ala Ile Val Gly Ala 100 105 110 Leu Asn Asn Ile Leu Val Ala His Ile Glu Gly Gly Glu Ile Ser Gly 115 120 125 Thr Ile Asp Asp Ser Leu Arg His Ala Ile Ser Lys Leu Ala His Ile 130 135 140 His Leu Val Asn Asp Glu Phe Ala Lys Arg Arg Leu Met Gln Leu Gly 145 150 155 160 Glu Asp Glu Lys Ser Ile Phe Ile Ile Gly Ser Pro Asp Leu Glu Leu 165 170 175 Leu Asn Asp Asn Lys Ile Ser Leu Ser Glu Ala Lys Lys Tyr Tyr Asp 180 185 190 Ile Asn Tyr Glu Asn Tyr Ala Leu Leu Met Phe His Pro Val Thr Thr 195 200 205 Glu Ile Thr Ser Ile Lys Asn Gln Ala Asp Asn Leu Val Lys Ala Leu 210 215 220 Ile Gln Ser Asn Lys Asn Tyr Ile Val Ile Tyr Pro Asn Asn Asp Leu 225 230 235 240 Gly Phe Glu Leu Ile Leu Gln Ser Tyr Glu Glu Phe Lys Asn Asn Pro 245 250 255 Arg Phe Lys Leu Phe Pro Ser Leu Arg Phe Glu Tyr Phe Ile Thr Leu 260 265 270 Leu Lys Asn Ala Asp Phe Ile Ile Gly Asn Ser Ser Cys Ile Leu Lys 275 280 285 Glu Ala Leu Tyr Leu Lys Thr Ala Gly Ile Leu Val Gly Ser Arg Gln 290 295 300 Asn Gly Arg Leu Gly Asn Glu Asn Thr Leu Lys Val Asn Ala Asn Ser 305 310 315 320 Asp Glu Ile Leu Lys Ala Ile Asn Thr Ile His Lys Lys Gln Asp Leu 325 330 335 Phe Ser Ala Lys Leu Glu Ile Leu Asp Ser Ser Lys Leu Phe Phe Glu 340 345 350 Tyr Leu Gln Ser Gly Asp Phe Phe Lys Leu Ser Thr Gln Lys Val Phe 355 360 365 Lys Asp Ile Lys 370 <210> 60 <211> 391 <212> PRT <213> Enterobacteriaceae <220> <223> NeuC, UDP-GlcNAc 2-epimerase, GenBank ID: WP_000723250.1 <400> 60 Met Lys Lys Ile Leu Tyr Val Thr Gly Ser Arg Ala Glu Tyr Gly Ile 1 5 10 15 Val Arg Arg Leu Leu Thr Met Leu Arg Glu Thr Pro Glu Ile Gln Leu 20 25 30 Asp Leu Ala Val Thr Gly Met His Cys Asp Asn Ala Tyr Gly Asn Thr 35 40 45 Ile His Ile Ile Glu Gln Asp Asn Phe Asn Ile Ile Lys Val Val Asp 50 55 60 Ile Asn Ile Asn Thr Thr Ser His Thr His Ile Leu His Ser Met Ser 65 70 75 80 Val Cys Leu Asn Ser Phe Gly Asp Phe Phe Ser Asn Asn Thr Tyr Asp 85 90 95 Ala Val Met Val Leu Gly Asp Arg Tyr Glu Ile Phe Ser Val Ala Ile 100 105 110 Ala Ala Ser Met His Asn Ile Pro Leu Ile His Ile His Gly Gly Glu 115 120 125 Lys Thr Leu Ala Asn Tyr Asp Glu Phe Ile Arg His Ser Ile Thr Lys 130 135 140 Met Ser Lys Leu His Leu Thr Ser Thr Glu Glu Tyr Lys Lys Arg Val 145 150 155 160 Ile Gln Leu Gly Glu Lys Pro Gly Ser Val Phe Asn Ile Gly Ser Leu 165 170 175 Gly Ala Glu Asn Ala Leu Ser Leu His Leu Pro Asn Lys Gln Glu Leu 180 185 190 Glu Leu Lys Tyr Gly Ser Leu Leu Lys Arg Tyr Phe Val Val Val Phe 195 200 205 His Pro Glu Thr Leu Ser Thr Gln Ser Val Asn Asp Gln Ile Asp Glu 210 215 220 Leu Leu Ser Ala Ile Ser Phe Phe Lys Asn Thr His Asp Phe Ile Phe 225 230 235 240 Ile Gly Ser Asn Ala Asp Thr Gly Ser Asp Ile Ile Gln Arg Lys Val 245 250 255 Lys Tyr Phe Cys Lys Glu Tyr Lys Phe Arg Tyr Leu Ile Ser Ile Arg 260 265 270 Ser Glu Asp Tyr Leu Ala Met Ile Lys Tyr Ser Cys Gly Leu Ile Gly 275 280 285 Asn Ser Ser Ser Gly Leu Ile Glu Val Pro Ser Leu Lys Val Ala Thr 290 295 300 Ile Asn Ile Gly Asp Arg Gln Lys Gly Arg Val Arg Gly Ala Ser Val 305 310 315 320 Ile Asp Val Pro Val Glu Lys Asn Ala Ile Val Arg Gly Ile Asn Ile 325 330 335 Ser Gln Asp Glu Lys Phe Ile Ser Val Val Gln Ser Ser Ser Asn Pro 340 345 350 Tyr Phe Lys Glu Asn Ala Leu Ile Asn Ala Val Arg Ile Ile Lys Asp 355 360 365 Phe Ile Lys Ser Lys Asn Lys Asp Tyr Lys Asp Phe Tyr Asp Ile Pro 370 375 380 Glu Cys Thr Thr Ser Tyr Asp 385 390 <210> 61 <211> 391 <212> PRT <213> Escherichia coli S88 <220> <223> NeuC, UDP-GlcNAc 2-epimerase, GenBank ID: CAR04561.1 <400> 61 Met Lys Lys Ile Leu Tyr Val Thr Gly Ser Arg Ala Glu Tyr Gly Ile 1 5 10 15 Val Arg Arg Leu Leu Thr Met Leu Arg Glu Thr Pro Glu Ile Gln Leu 20 25 30 Asp Leu Ala Val Thr Gly Met His Cys Asp Asn Ala Tyr Gly Asn Thr 35 40 45 Ile His Ile Ile Glu Gln Asp Asn Phe Asn Ile Ile Lys Val Val Asp 50 55 60 Ile Asn Ile Asn Thr Thr Ser His Thr His Ile Leu His Ser Met Ser 65 70 75 80 Val Cys Leu Asn Ser Phe Gly Asp Phe Phe Ser Asn Asn Thr Tyr Asp 85 90 95 Ala Val Met Val Leu Gly Asp Arg Tyr Glu Ile Phe Ser Val Ala Ile 100 105 110 Ala Ala Ser Met His Asn Ile Pro Leu Ile His Ile His Gly Gly Glu 115 120 125 Lys Thr Leu Ala Asn Tyr Asp Glu Phe Ile Arg His Ser Ile Thr Lys 130 135 140 Met Ser Lys Leu His Leu Thr Ser Thr Glu Glu Tyr Lys Lys Arg Val 145 150 155 160 Ile Gln Leu Gly Glu Lys Pro Gly Ser Val Phe Asn Ile Gly Ser Leu 165 170 175 Gly Ala Glu Asn Ala Leu Ser Leu His Leu Pro Asn Lys Gln Glu Leu 180 185 190 Glu Leu Lys Tyr Gly Ser Leu Leu Lys Arg Tyr Phe Val Val Val Phe 195 200 205 His Pro Glu Thr Leu Ser Thr Gln Ser Val Asn Asp Gln Ile Asp Glu 210 215 220 Leu Leu Ser Ala Ile Ser Phe Phe Lys Asn Thr His Asp Phe Ile Phe 225 230 235 240 Ile Gly Ser Asn Ala Asp Thr Gly Ser Asp Ile Ile Gln Arg Lys Val 245 250 255 Lys Tyr Phe Cys Lys Glu Tyr Lys Phe Arg Tyr Leu Ile Ser Ile Arg 260 265 270 Ser Glu Asp Tyr Leu Ala Met Ile Lys Tyr Ser Cys Gly Leu Ile Gly 275 280 285 Asn Ser Ser Ser Gly Leu Ile Glu Val Pro Ser Leu Lys Val Ala Thr 290 295 300 Ile Asn Ile Gly Asp Arg Gln Lys Gly Arg Val Arg Gly Ala Ser Val 305 310 315 320 Ile Asp Val Pro Val Glu Lys Asn Ala Ile Val Arg Gly Ile Asn Ile 325 330 335 Ser Gln Asp Glu Lys Phe Ile Ser Val Val Gln Ser Ser Ser Asn Pro 340 345 350 Tyr Phe Lys Glu Asn Ala Leu Ile Asn Ala Val Arg Ile Ile Lys Asp 355 360 365 Phe Ile Lys Ser Lys Asn Lys Asp Tyr Lys Asp Phe Tyr Asp Ile Pro 370 375 380 Glu Cys Thr Thr Ser Tyr Asp 385 390 <210> 62 <211> 392 <212> PRT <213> Pantoea vagans <220> <223> Vague MFS transporter, WP_048785139.1 <400> 62 Met Lys Ser Leu Leu Thr Arg Lys Arg Arg Ile Asn Pro Val Phe Leu 1 5 10 15 Ala Phe Met Ala Ala Ser Phe Met Ile Gly Val Ala Gly Ala Leu Gln 20 25 30 Ala Pro Thr Leu Ser Leu Phe Leu Thr Arg Glu Val Gln Ala Arg Pro 35 40 45 Leu Trp Val Gly Leu Phe Phe Thr Val Asn Ala Ile Ala Gly Ile Val 50 55 60 Val Ser Met Leu Val Ala Lys Arg Ser Asp Ser Arg Gly Asp Arg Arg 65 70 75 80 Thr Leu Ile Leu Phe Cys Cys Ala Met Ala Phe Cys Asn Ala Leu Leu 85 90 95 Phe Ala Phe Thr Arg His Tyr Leu Thr Leu Ile Thr Leu Gly Val Leu 100 105 110 Leu Ser Ala Leu Ala Ser Val Ser Met Pro Gln Ile Phe Ala Leu Ala 115 120 125 Arg Glu Tyr Ala Asp Gln Ser Ala Arg Glu Ala Val Met Phe Ser Ser 130 135 140 Val Met Arg Ala Gln Leu Ser Leu Ala Trp Val Ile Gly Pro Pro Leu 145 150 155 160 Ser Phe Ala Leu Ala Leu Asn Phe Gly Phe Val Thr Leu Phe Leu Val 165 170 175 Ala Ala Ala Leu Phe Leu Val Cys Ile Leu Leu Ile Lys Phe Thr Leu 180 185 190 Pro Ser Val Pro Arg Ala Glu Pro Leu Met Arg Ser Gly Gly Met Pro 195 200 205 Leu Ser Gly Trp Arg Asp Arg Asp Val Arg Leu Leu Phe Ile Ala Ser 210 215 220 Val Thr Met Trp Thr Cys Asn Thr Met Tyr Ile Ile Asp Met Pro Leu 225 230 235 240 Tyr Ile Ser Val Thr Leu Gly Leu Pro Glu Lys Leu Ala Gly Leu Leu 245 250 255 Met Gly Thr Ala Ala Gly Leu Glu Ile Pro Val Met Leu Leu Ala Gly 260 265 270 His Tyr Ala Lys Arg Val Gly Lys Arg Asn Leu Met Leu Ile Ala Val 275 280 285 Ala Ala Gly Val Leu Phe Tyr Ala Gly Leu Ala Met Phe Ala Ser Gln 290 295 300 Thr Ala Leu Met Ala Leu Gln Leu Phe Asn Ala Val Phe Ile Gly Ile 305 310 315 320 Ile Ala Gly Ile Gly Met Leu Trp Phe Gln Asp Leu Met Pro Gly Arg 325 330 335 Pro Gly Ala Ala Thr Thr Met Phe Thr Asn Ser Ile Ser Thr Gly Met 340 345 350 Ile Leu Ala Gly Val Ile Gln Gly Thr Leu Ser Glu Arg Phe Gly His 355 360 365 Ile Ala Val Tyr Trp Leu Ala Leu Gly Leu Ala Val Ala Ala Phe Ala 370 375 380 Met Ser Ala Arg Val Lys Asn Val 385 390 <210> 63 <211> 393 <212> PRT <213> Yersinia frederiksenii <220> <223> Fred MFS transporter, WP_087817556.1 <400> 63 Met Lys Ser Ala Leu Thr Phe Ser Arg Arg Ile Asn Pro Val Phe Leu 1 5 10 15 Ala Phe Phe Val Val Ala Phe Leu Ser Gly Ile Ala Gly Ala Leu Gln 20 25 30 Ala Pro Thr Leu Ser Leu Phe Leu Ser Thr Glu Val Lys Val Arg Pro 35 40 45 Leu Trp Val Gly Leu Phe Tyr Thr Val Asn Ala Ile Ala Gly Ile Thr 50 55 60 Val Ser Phe Val Leu Ala Lys Arg Ser Asp Leu Arg Gly Asp Arg Arg 65 70 75 80 Lys Leu Ile Leu Val Cys Tyr Leu Met Ala Val Gly Asn Cys Leu Leu 85 90 95 Phe Ala Phe Asn Arg Asp Tyr Leu Thr Leu Ile Thr Ala Gly Val Leu 100 105 110 Leu Ala Ala Val Ala Asn Thr Ala Met Pro Gln Ile Phe Ala Leu Ala 115 120 125 Arg Glu Tyr Ala Asp Asn Ser Ala Arg Glu Val Val Met Phe Ser Ser 130 135 140 Ile Met Arg Ala Gln Leu Ser Leu Ala Trp Val Ile Gly Pro Pro Leu 145 150 155 160 Ser Phe Met Leu Ala Leu Asn Tyr Gly Phe Thr Leu Met Phe Cys Ile 165 170 175 Ala Ala Gly Ile Phe Val Leu Ser Ala Leu Val Val Trp Phe Ile Leu 180 185 190 Pro Ser Val Gln Arg Ala Glu Pro Val Met Asp Ala Pro Thr Val Ala 195 200 205 Gln Gly Ser Leu Phe Ala Asp Lys Asp Val Leu Leu Leu Phe Ile Ala 210 215 220 Ser Met Leu Met Trp Thr Cys Asn Thr Met Tyr Ile Ile Asp Met Pro 225 230 235 240 Leu Tyr Ile Thr Ala Ser Leu Gly Leu Pro Glu Arg Leu Ala Gly Leu 245 250 255 Leu Met Gly Thr Ala Ala Gly Leu Glu Ile Pro Ile Met Leu Leu Ala 260 265 270 Gly Tyr Ser Val Arg Arg Phe Gly Lys Arg Lys Ile Met Leu Phe Ala 275 280 285 Val Leu Ala Gly Val Leu Phe Tyr Thr Gly Leu Val Leu Phe Lys Phe 290 295 300 Lys Ser Ala Leu Met Leu Leu Gln Ile Phe Asn Ala Ile Phe Ile Gly 305 310 315 320 Ile Val Ala Gly Ile Gly Met Leu Tyr Phe Gln Asp Leu Met Pro Gly 325 330 335 Arg Ala Gly Ala Ala Thr Thr Leu Phe Thr Asn Ser Ile Ser Thr Gly 340 345 350 Val Ile Leu Ala Gly Val Leu Gln Gly Val Leu Thr Glu Thr Trp Gly 355 360 365 His Asn Ser Val Tyr Val Met Ala Met Ile Leu Ala Ile Leu Ser Leu 370 375 380 Ile Ile Cys Ala Arg Val Arg Glu Ala 385 390 <210> 64 <211> 398 <212> PRT <213> Serratia marcescens <220> <223> Mark MFS transporter, WP_060448169.1 <400> 64 Met Gln Arg Leu Ser Arg Leu Ser Leu Arg Ile Asn Pro Ile Phe Ala 1 5 10 15 Ala Phe Leu Leu Ile Ala Phe Leu Ser Gly Ile Ala Gly Ala Leu Leu 20 25 30 Thr Pro Thr Leu Ser Leu Phe Leu Thr Thr Glu Val Lys Val Arg Pro 35 40 45 Leu Trp Val Gly Leu Phe Tyr Thr Ala Asn Ala Val Ala Gly Ile Val 50 55 60 Val Ser Phe Leu Leu Ala Lys Arg Ser Asp Thr Arg Gly Asp Arg Arg 65 70 75 80 Arg Leu Ile Leu Leu Cys Cys Leu Met Ala Val Gly Asn Cys Leu Leu 85 90 95 Phe Ala Phe Asn Arg Asp Tyr Leu Thr Leu Ile Thr Ala Gly Val Leu 100 105 110 Met Ser Ala Val Ala Asn Thr Ala Met Pro Gln Ile Phe Ala Leu Ala 115 120 125 Arg Glu Tyr Ala Asp Ser Glu Ala Arg Glu Val Val Met Phe Ser Ser 130 135 140 Val Met Arg Ala Gln Leu Ser Leu Ala Trp Val Ile Gly Pro Pro Leu 145 150 155 160 Ser Phe Ala Leu Ala Leu Asn Tyr Gly Phe Thr Val Met Phe Leu Ile 165 170 175 Ala Ala Val Thr Phe Ala Val Cys Val Leu Leu Val Gly Phe Met Leu 180 185 190 Pro Ser Val Pro Arg Ala Ala Glu Asn Glu Gly Leu Gln Gly Gly Val 195 200 205 Ser Ala Pro Ile Ala Pro Ala Ser Ala Trp Arg Asn Arg Asp Val Arg 210 215 220 Leu Leu Phe Ile Ala Ser Met Leu Met Trp Thr Cys Asn Thr Leu Tyr 225 230 235 240 Ile Ile Asp Met Pro Leu Tyr Ile Thr Ala Asp Leu Gly Leu Pro Glu 245 250 255 Gly Leu Ala Gly Val Leu Met Gly Thr Ala Ala Gly Leu Glu Ile Pro 260 265 270 Ala Met Leu Leu Ala Gly Tyr Tyr Val Lys Arg Phe Gly Lys Arg Asn 275 280 285 Met Met Leu Leu Ala Val Val Ala Gly Val Leu Phe Tyr Leu Gly Leu 290 295 300 Thr Val Leu Glu Ser Lys Pro Ala Leu Ile Ala Leu Gln Leu Leu Asn 305 310 315 320 Ala Val Phe Ile Gly Ile Val Ala Gly Ile Gly Met Leu Tyr Phe Gln 325 330 335 Asp Leu Met Pro Gly Arg Pro Gly Ala Ala Thr Thr Leu Phe Thr Asn 340 345 350 Ser Ile Ser Thr Gly Val Ile Leu Ala Gly Val Leu Gln Gly Ala Leu 355 360 365 Val Glu Asn Leu Gly His Gly Ser Val Tyr Trp Met Ala Ala Leu Leu 370 375 380 Ala Leu Ala Ala Leu Gly Met Ser Ala Lys Val Arg Glu Val 385 390 395 <210> 65 <211> 387 <212> PRT <213> Rouxiella badensis <220> <223> Bad MFS transporter, WP_017489914.1 <400> 65 Met Ser Ser Arg Arg Leu Ser Ile Ile Phe Ala Thr Phe Leu Leu Val 1 5 10 15 Ser Phe Leu Thr Gly Ile Ala Gly Ala Leu Gln Ala Pro Thr Leu Ser 20 25 30 Leu Phe Leu Thr Asn Glu Val Lys Val Arg Pro Leu Trp Val Gly Leu 35 40 45 Phe Tyr Thr Val Asn Ala Leu Gly Gly Ile Val Ile Ser Phe Leu Leu 50 55 60 Ala Asn Tyr Ser Asp Lys Lys Gly Asp Arg Arg Lys Leu Leu Phe Phe 65 70 75 80 Cys Thr Leu Met Ala Ile Gly Asn Ser Leu Ile Phe Ala Tyr Ser Arg 85 90 95 Asp Tyr Leu Val Leu Ile Ser Val Gly Val Leu Leu Ala Ala Ile Gly 100 105 110 Asn Ala Ser Met Pro Gln Leu Phe Ala Leu Ala Arg Glu Tyr Ala Asp 115 120 125 Arg Ser Ala His Glu Val Val Met Phe Ser Ser Met Met Arg Ala Thr 130 135 140 Leu Ser Leu Ala Trp Val Leu Gly Pro Pro Ile Ser Phe Thr Leu Ala 145 150 155 160 Leu Asn Tyr Gly Phe Thr Leu Met Tyr Leu Cys Ala Ala Gly Val Phe 165 170 175 Ile Phe Ser Ala Leu Met Val Trp Phe Phe Leu Pro Ser Val Gly Arg 180 185 190 Ile Glu Gln Pro Val Asp Lys Val Val Val His Val Ser Ala Trp Lys 195 200 205 Asn Arg Asp Val Arg Leu Leu Phe Phe Ala Ser Leu Leu Met Trp Thr 210 215 220 Cys Asn Ile Met Tyr Ile Ile Asp Met Pro Leu Tyr Ile Thr Ser Asp 225 230 235 240 Leu Gly Leu Pro Glu Gly Leu Ala Gly Leu Leu Met Gly Ala Ala Ala 245 250 255 Gly Leu Glu Ile Pro Val Met Leu Ile Ala Gly Tyr Leu Val Lys Arg 260 265 270 Thr Gly Lys Arg Arg Leu Met Leu Cys Ala Ala Val Phe Gly Ile Leu 275 280 285 Phe Tyr Leu Gly Leu Val Leu Phe Gln Phe Lys Ala Ala Leu Met Ile 290 295 300 Leu Gln Leu Phe Asn Ala Ile Phe Ile Gly Ile Ile Ala Gly Ile Gly 305 310 315 320 Met Leu Tyr Phe Gln Asp Leu Met Pro Gly Arg Ala Gly Ser Ala Thr 325 330 335 Thr Leu Phe Thr Asn Ser Ile Ser Thr Gly Ala Ile Leu Ala Gly Val 340 345 350 Ile Gln Gly Thr Ile Val Gln Asn Phe Gly His Tyr Gln Val Tyr Trp 355 360 365 Met Ala Leu Ala Leu Ala Val Gly Ala Leu Val Leu Met Thr Arg Val 370 375 380 Lys Asn Val 385 <210> 66 <211> 394 <212> PRT <213> Rosenbergiella nectary <220> <223> Nec MFS transporter, WP_092672081.1 <400> 66 Met Gln Ser Phe Thr Pro Pro Ala Pro Lys Gly Gly Asn Pro Val Phe 1 5 10 15 Met Met Phe Met Leu Val Thr Phe Phe Val Ser Ile Ala Gly Ala Leu 20 25 30 Gln Ala Pro Thr Leu Ser Leu Tyr Leu Ser Gln Glu Leu Ala Ala Lys 35 40 45 Pro Phe Met Val Gly Leu Phe Phe Thr Ile Asn Ala Val Thr Gly Ile 50 55 60 Ile Ile Ser Phe Ile Leu Ala Lys Arg Ser Asp Arg Lys Gly Asp Arg 65 70 75 80 Arg Arg Leu Leu Met Phe Cys Cys Ala Met Ala Ile Ala Asn Ala Leu 85 90 95 Met Phe Ala Phe Val Arg Gln Tyr Val Val Leu Ile Thr Leu Gly Leu 100 105 110 Ile Leu Ser Ala Leu Thr Ser Val Val Met Pro Gln Leu Phe Ala Leu 115 120 125 Ala Arg Glu Tyr Ala Asp Arg Thr Gly Arg Glu Val Val Met Phe Ser 130 135 140 Ser Val Met Arg Thr Gln Met Ser Leu Ala Trp Val Ile Gly Pro Pro 145 150 155 160 Ile Ser Phe Ala Leu Ala Leu Asn Tyr Gly Phe Ile Thr Leu Tyr Leu 165 170 175 Val Ala Ala Ala Leu Phe Leu Leu Ser Leu Ile Leu Ile Lys Thr Thr 180 185 190 Leu Pro Ser Val Pro Arg Leu Tyr Pro Ala Glu Asp Leu Ala Lys Ser 195 200 205 Ala Ala Ser Gly Trp Lys Arg Thr Asp Val Arg Phe Leu Phe Ala Ala 210 215 220 Ser Val Leu Met Trp Val Cys Asn Leu Met Tyr Ile Ile Asp Met Pro 225 230 235 240 Leu Tyr Ile Ser Lys Ser Leu Gly Met Pro Glu Ser Phe Ala Gly Val 245 250 255 Leu Met Gly Thr Ala Ala Gly Leu Glu Ile Pro Val Met Leu Leu Ala 260 265 270 Gly Tyr Leu Ala Lys Arg Val Gly Lys Arg Pro Leu Val Ile Val Ala 275 280 285 Ala Val Cys Gly Leu Ala Phe Tyr Pro Ala Met Leu Val Phe His Gln 290 295 300 Gln Thr Gly Leu Leu Ile Ile Gln Leu Leu Asn Ala Val Phe Ile Gly 305 310 315 320 Ile Val Ala Gly Leu Val Met Leu Trp Phe Gln Asp Leu Met Pro Gly 325 330 335 Lys Ala Gly Ala Ala Thr Thr Leu Phe Thr Asn Ser Val Ser Thr Gly 340 345 350 Met Ile Phe Ala Gly Leu Cys Gln Gly Leu Leu Ser Asp Leu Leu Gly 355 360 365 His Gln Ala Ile Tyr Val Leu Ala Thr Val Leu Met Val Ile Ala Leu 370 375 380 Leu Leu Leu Leu Arg Val Lys Glu Gln Ala 385 390 <210> 67 <211> 393 <212> PRT <213> Yersinia bercovieri <220> <223> YberC MFS transporter <400> 67 Met Lys Ser Ala Leu Thr Phe Ser Arg Arg Ile Asn Pro Val Phe Leu 1 5 10 15 Ala Phe Phe Val Val Ala Phe Leu Ser Gly Ile Ala Gly Ala Leu Gln 20 25 30 Ala Pro Thr Leu Ser Leu Phe Leu Ser Thr Glu Val Lys Val Arg Pro 35 40 45 Leu Trp Val Gly Leu Phe Tyr Thr Val Asn Ala Ile Ala Gly Ile Thr 50 55 60 Val Ser Phe Ile Leu Ala Lys Arg Ser Asp Ser Arg Gly Asp Arg Arg 65 70 75 80 Lys Leu Ile Met Val Cys Tyr Leu Met Ala Val Gly Asn Cys Leu Leu 85 90 95 Phe Ala Phe Asn Arg Asp Tyr Leu Thr Leu Ile Thr Ala Gly Val Leu 100 105 110 Leu Ala Ser Val Ala Asn Thr Ala Met Pro Gln Ile Phe Ala Leu Ala 115 120 125 Arg Glu Tyr Ala Asp Ser Ser Ala Arg Glu Val Val Met Phe Ser Ser 130 135 140 Ile Met Arg Ala Gln Leu Ser Leu Ala Trp Val Ile Gly Pro Pro Leu 145 150 155 160 Ser Phe Met Leu Ala Leu Asn Tyr Gly Phe Thr Leu Met Phe Ser Ile 165 170 175 Ala Ala Gly Ile Phe Val Leu Ser Ala Leu Val Val Trp Phe Ile Leu 180 185 190 Pro Ser Val Pro Arg Ala Glu Pro Val Val Asp Ala Pro Val Val Val 195 200 205 Gln Gly Ser Leu Phe Ala Asp Lys Asn Val Leu Leu Leu Phe Ile Ala 210 215 220 Ser Met Leu Met Trp Thr Cys Asn Thr Met Tyr Ile Ile Asp Met Pro 225 230 235 240 Leu Tyr Ile Thr Ala Ser Leu Gly Leu Pro Glu Arg Leu Ala Gly Leu 245 250 255 Leu Met Gly Thr Ala Ala Gly Leu Glu Ile Pro Ile Met Leu Leu Ala 260 265 270 Gly Tyr Ser Val Arg Tyr Phe Gly Lys Arg Lys Ile Met Leu Phe Ala 275 280 285 Val Leu Ala Gly Val Leu Phe Tyr Thr Gly Leu Val Leu Phe Lys Phe 290 295 300 Lys Thr Ala Leu Met Leu Leu Gln Ile Phe Asn Ala Ile Phe Ile Gly 305 310 315 320 Ile Val Ala Gly Ile Gly Met Leu Tyr Phe Gln Asp Leu Met Pro Gly 325 330 335 Arg Ala Gly Ala Ala Thr Thr Leu Phe Thr Asn Ser Ile Ser Thr Gly 340 345 350 Val Ile Leu Ala Gly Val Leu Gln Gly Gly Leu Thr Glu Thr Trp Gly 355 360 365 His Asp Ser Val Tyr Val Met Ala Met Val Leu Ser Ile Leu Ala Leu 370 375 380 Ile Ile Cys Ala Arg Val Arg Glu Ala 385 390 <210> 68 <211> 107 <212> DNA <213> Escherichia coli <220> <223> Plac, lac operon promoter <400> 68 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 60 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagct 107 <210> 69 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> PgatY_70UTR, variant of E. coli promoter for gatYZABCD; tagatose-1,6-bisP aldolase <300> <310> WO / 2020 / 255054 <311> 6 / 19 / 2020 <312> 12 / 24 / 2020 <400> 69 cggcaaccta tgcctgatgc gacgctgaag cgtcttatca tgcctacata gcactgccac 60 gtatgtttac accgcatccg gcataaaaac acgcgcactt tgctacggct tccctatcgg 120 gaggccgttt tttgccttt cactcctcga atattttca tattgtcgtt ttgtgatcg 180 ttatctcgat atttaaaac aaataatttc attatatttt gtgcctacaa gcatcgtgga 240 ggtccgtgac ttcacccat acaaaaca tcaaccagg aggaacagc t 291 <210> 70 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF, E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 12 / 19 / 2018 <312> 27.06.2019 <400> 70 gcggcacgcc ttgcagatta cggtttgcca cactttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aaaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg ttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaac taaccaagga ggaacagct 300 <210> 71 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD1, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 71 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaaatt cgaaacagct 300 <210> 72 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD10, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 72 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaactg agaaacagct 300 <210> 73 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD2, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 73 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagcg caaaacagct 300 <210> 74 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD3 variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 74 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagaa caaaacagct 300 <210> 75 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD4, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 75 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaacta ggaaacagct 300 <210> 76 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD5, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 76 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaaccg agaaacagct 300 <210> 77 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD6, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 77 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagag ctaaacagct 300 <210> 78 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD7, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 78 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagag caaaacagct 300 <210> 79 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD8, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 79 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaagag aaaaacagct 300 <210> 80 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> PglpF_SD9, variant of PglpF E. Coli promoter sequence of glpFKX operon <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 80 gcggcacgcc ttgcagatta cggtttgcca cacttttcat ccttctcctg gtgacataat 60 ccacatcaat cgaaaatgtt aataaatttg ttgcgcgaat gatctaacaa acatgcatca 120 tgtacaatca gatggaataa atggcgcgat aacgctcatt ttatgacgag gcacacacat 180 tttaagttcg atatttctcg tttttgctcg ttaacgataa gtttacagca tgcctacaag 240 catcgtggag gtccgtgact ttcacgcata caacaaacat taaccaaagg aaaaacagct 300 <210> 81 <211> 107 <212> DNA <213> Artificial Sequence <220> <223> Plac_16UTR, variant of E. coli lac operon promoter <300> <310> WO / 2019 / 123324 <311> 19.12.2018 <312> 27.06.2019 <400> 81 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 60 gttgtgtgga attgtgagcg gataacaatt tcaaggagga aacagct 107 <210> 82 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR, variant of E. coli promoter for mglBAC galactose / methyl-galactosity transporter <300> <310> WO / 2020 / 255054 <311> 6 / 19 / 2020 <312> 12 / 24 / 2020 <400> 82 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa ggaggaaaca gct 203 <210> 83 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD4, variant of E. coli promoter for mglBAC; galactose / methylgalactose transporter <300> <310> WO / 2020 / 255054 <311> June 19, 2020 <312> December 24, 2020 <400> 83 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgcataacaaa 180 cattaaccaa ctaggaaaca gct 203 <210> 84 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> CP6 promoter sequence <400> 84 catgtgggag tttattcttg acacagatat ttccggatga tataataact gagtactgtt 60 cacacaggaa acagct 76 <210> 85 <211> 269 <212> DNA <213> Escherichia coli <220> <223> PosmY, promoter for osmY-ytjA; perplasmic chaperone <400> 85 caaaattgtg atctatattt aaaaagtga tgacattctct gacggcgtta ataccgttc 60 aatgcgtaga tatcagtatc taagccgtc gattgtcatt ctaccgatat taatactga 120 ttcagaggct gtaatggtcg ttattcatca ctcatcgctt tgtgatggc gaccattgac 180 ttctgtagag ggtgaagtct ctccctattc agcaatgcaa cctcgtgttg ccaggctcaa 240 attack slow slow 269 <210> 86 <211> 483 <212> PRT <213> Avibacterium gallinarum <220> <223> SacC_Agal, glycoside hydrolase family 32 protein, WP_103853210.1 <400> 86 Meanwhile, the Asn Glu Gly Lys Tyr Lys Is Leu Tyr Ala Ala Glu 1 5 10 15 Gln Gly Glu Leu Glu Lys With Gln Thr Val Ala Gln Asp Gln Asp 20 25 30 Phe Arg Pro Val Tyr His Leu Ala Pro Thr Gly Leu Leu Asn Asp 35 40 45 Pro Asn Gly Leu Ile Phe Asp Gly Glu Lys Tyr His Leu Phe Tyr Gln 50 55 60 Trp Tyr Pro Phe Asp Ala Leu His Gly Met Lys His Trp Gln His Phe 65 70 75 80 Ile Thr Gln Asp Phe Lys Gln Phe Ser Gln Ala Asp Leu Leu Val Pro 85 90 95 Cys Glu Leu Tyr Glu Ser His Gly Cys Tyr Ser Gly Gly Ala Val Lys 100 105 110 Ile Gly Asp Gln Ile Ala Val Phe Tyr Thr Gly Asn Thr Arg Arg Pro 115 120 125 Ser Asp Asn Gln Arg Val Pro Tyr Gln Asn Leu Ala Ile Phe Ser Lys 130 135 140 Asp Gly Lys Leu Leu Ser Lys Arg Pro Leu Ile Glu Gln Ala Pro Gln 145 150 155 160 Gly Tyr Thr Glu His Val Arg Asp Pro Lys Pro Phe Leu Thr Lys Asp 165 170 175 Gly Lys Ile Arg Phe Ile Cys Gly Ala Gln Arg Glu Asn Leu Thr Gly 180 185 190 Thr Ala Leu Val Phe Glu Met Asp Asn Leu Ala Asp Thr Pro Arg Leu 195 200 205 Leu Gly Glu Leu Ala Leu Pro Ala Phe Asp Asn Gln Gly Val Phe Met 210 215 220 Trp Glu Cys Pro Asp Leu Ser Gln Met Gly Asp Lys Ser Leu Phe Ile 225 230 235 240 Trp Ser Pro Gln Gly Lys Ala Arg Glu Leu Glu Gln Tyr Gln Asn Asn 245 250 255 Tyr His Ala Val Tyr Ala Leu Gly Glu Leu Ala Asp Arg Gln Phe His 260 265 270 Ala Glu Gln Ile Ala Glu Leu Asp Gln Gly Phe Asp Phe Tyr Ala Pro 275 280 285 Gln Thr Phe Ser Gly Thr Gln Thr Met Leu Leu Gly Trp Val Gly Leu 290 295 300 Pro Asp Leu Ser Tyr Pro Thr Asp Leu Tyr Lys Trp His Ser Met Leu 305 310 315 320 Ser Met Pro Arg Gln Leu Arg Leu Gln Asp Gly Lys Ile Tyr Gln Gln 325 330 335 Pro Ile Glu Asn Ile Tyr Lys Asn Leu Thr Ala Leu Gln Ser Ile Thr 340 345 350 Val Glu Lys Glu Ala Glu Ile Ala Asp Leu Asp Arg Ala Tyr Leu Lys 355 360 365 Phe Asp Ala Asn Ala Gln Pro Phe Ser Leu Lys Phe Phe Asn Asn Ala 370 375 380 Gln Asn Gln Arg Leu Ile Leu Ser Tyr Asp Gly Glu Met Leu Cys Leu 385 390 395 400 Asp Arg Ser Gln Thr Glu Gln Thr Asp Ser Met Lys Ser Phe Gly Asp 405 410 415 Lys Arg Tyr Cys Arg Ile Glu Asp Leu Arg Gln Val Glu Ile Phe Phe 420 425 430 Asp Arg Ser Val Ala Glu Ile Phe Leu Asn Gln Gly Glu Lys Ala Met 435 440 445 Thr Ser Arg Phe Phe Ile Cys Ala Arg Glu Asn Gln Leu Cys Thr Asp 450 455 460 Lys Pro Leu Thr Leu Gln Val Gly Tyr Pro Lys Lys Ile Glu Val Asp 465 470 475 480 Tyr Thr Lys <210> 87 <211> 548 <212> PRT <213> Arthrobacter globiformis <220> <223> Bff, beta-fructofuranosidase protein, BAD18121.1 <400> 87 Met Glu Arg Thr Cys Ile Thr Val Arg Ala Ile Val Arg Phe His Ile 1 5 10 15 Glu Gln Arg Gln Thr Ile Val Asn Lys Gln Arg Thr Lys Arg Gly Ile 20 25 30 Leu Thr Ala Ala Leu Ser Ile Gly Ala Leu Gly Ala Thr Leu Ile Ser 35 40 45 Gly Pro Ala Val Ala Ala Thr Asp Ala Ala Pro Gly Phe Pro Gln Pro 50 55 60 Thr Glu His Thr Gln Lys Ala Tyr Ser Pro Thr Asp Asn Phe Thr Ser 65 70 75 80 Arg Trp Thr Arg Ala Asp Ala Lys Gln Leu Lys Ala Met Ser Asp Pro 85 90 95 Asp Ala Gly Ser Arg Glu Asn Ser Met Pro Thr Glu Tyr Thr Met Pro 100 105 110 Thr Val Ser Gln Asp Phe Pro Asp Met Ser Asn Glu Lys Val Trp Val 115 120 125 Trp Asp Thr Trp Pro Leu Ile Asp Glu Asn Ala Asn Gln Tyr Ser Val 130 135 140 Asn Gly Gln Glu Ile Ile Phe Ser Leu Val Ala Asp Arg Lys Leu Gly 145 150 155 160 Phe Asp Glu Arg His Gln Tyr Ala Arg Ile Gly Tyr Phe Tyr Arg Pro 165 170 175 Ala Gly Ile Pro Ala Asp Glu Arg Pro Glu Asp Gly Gly Trp Thr Tyr 180 185 190 Gly Gly Gln Val Phe Asp Glu Gly Val Thr Gly Lys Ile Phe Glu Asp 195 200 205 Gln Ser Phe Thr His Gln Thr Gln Trp Ser Gly Ser Ala Arg Val Ser 210 215 220 Lys Asn Gly Glu Ile Lys Leu Phe Phe Thr Asp Val Ala Phe Tyr Arg 225 230 235 240 Asp Lys Asp Gly Gln Asp Val Lys Pro Tyr Asp Ser Arg Ile Ala Leu 245 250 255 Ser Val Gly His Val His Ser Asn Lys Lys Gly Val Lys Leu Thr Gly 260 265 270 Phe Asn Lys Val Lys Glu Leu Leu Gln Ala Asp Gly Lys Asn Tyr Gln 275 280 285 Asn Ala Ala Gln Asn Ser Tyr Tyr Asn Phe Arg Asp Pro Phe Thr Phe 290 295 300 Val Asp Pro Ala His Pro Gly Glu Thr Tyr Met Val Phe Glu Gly Asn 305 310 315 320 Ser Ala Met Asp Arg Asp Glu Ala Lys Cys Thr Ala Glu Asp Leu Gly 325 330 335 Tyr Arg Glu Gly Glu Thr Asn Gly Glu Thr Val Glu Gln Val Asn Asn 340 345 350 Ser Gly Ala Thr Tyr Gln Ile Gly Asn Val Gly Leu Ala Arg Ala Lys 355 360 365 Asn Lys Ala Leu Thr Glu Trp Glu Phe Leu Pro Pro Ile Leu Ser Ala 370 375 380 Asn Cys Val Thr Asp Gln Thr Glu Arg Pro Gln Ile Tyr Met Gln Asp 385 390 395 400 Gly Lys Tyr Tyr Leu Phe Thr Ile Ser His Arg Ser Thr Phe Ala Thr 405 410 415 Gly Ile Asp Gly Pro Glu Gly Val Tyr Gly Phe Val Gly Asn Gly Ile 420 425 430 Arg Ser Asp Tyr Gln Pro Leu Asn Arg Gly Ser Gly Leu Ala Leu Gly 435 440 445 Ser Pro Thr Asn Leu Asn Phe Ala Ala Gly Thr Pro Phe Ala Pro Asp 450 455 460 Tyr Asn Gln His Pro Gly Gln Phe Gln Ala Tyr Ser His Tyr Val Met 465 470 475 480 Pro Gly Gly Leu Val Gln Ser Phe Ile Asp Thr Ile Gly Thr Lys Asp 485 490 495 Asn Phe Val Arg Gly Gly Thr Leu Gly Pro Thr Val Lys Leu Asn Ile 500 505 510 Lys Gly Asp Ser Ala Thr Val Asp Tyr Asn Tyr Gly Asp Asn Gly Leu 515 520 525 Gly Gly Trp Ala Asp Ile Pro Ala Asn Arg Glu Leu Lys Asn Ser Lys 530 535 540 Ala Val Ala Lys 545 <210> 88 <211> 505 <212> PRT <213> Klebsiella pneumoniae <220> <223> ScrY, sucrose porin, GeneBank ID CAA40657.1 <300> <310> WO / 2015 / 197082 <311> 29.06.2015 <312> 30.12.2015 <400> 88 Met Tyr Lys Lys Arg Lys Leu Ala Ile Leu Ile Ala Leu Leu Thr Gly 1 5 10 15 Thr Ala Ala Ala His Gly Gln Thr Asp Leu Asn Ser Ile Glu Ala Arg 20 25 30 Leu Ala Ala Leu Glu Lys Arg Leu Gln Asp Ala Glu Thr Arg Ala Ser 35 40 45 Thr Ala Glu Ser Arg Ala Ala Ser Ala Glu Gln Lys Val Gln Gln Leu 50 55 60 Thr Gln Gln Gln Gln Gln Thr Gln Ala Thr Thr Gln Gln Val Ala Arg 65 70 75 80 Arg Thr Thr Gln Leu Glu Glu Lys Ala Glu Arg Pro Gly Gly Phe Glu 85 90 95 Phe His Gly Tyr Ala Arg Ser Gly Val Ile Met Asn Asp Ser Ala Ala 100 105 110 Ser Thr Lys Ser Gly Ala Tyr Met Thr Pro Ala Gly Glu Thr Gly Gly 115 120 125 Ala Ile Gly Arg Leu Gly Asn Gln Ala Asp Thr Tyr Val Glu Met Asn 130 135 140 Leu Glu His Lys Gln Thr Leu Asp Asn Gly Ala Thr Thr Arg Phe Lys 145 150 155 160 Val Met Val Ala Asp Gly Gln Thr Thr Tyr Asn Asp Trp Thr Ala Ser 165 170 175 Ser Ser Asp Leu Asn Val Arg Gln Ala Phe Val Glu Leu Gly Asn Leu 180 185 190 Pro Thr Phe Glu Gly Pro Phe Lys Gly Ser Thr Leu Trp Ala Gly Lys 195 200 205 Arg Phe Asp Arg Asp Asn Phe Asp Ile His Trp Ile Asp Ser Asp Val 210 215 220 Val Phe Leu Ala Gly Thr Gly Gly Gly Ile Tyr Asp Val Lys Trp Asn 225 230 235 240 Asp Ser Leu Arg Ser Asn Phe Ser Leu Tyr Gly Arg Asn Phe Gly Asp 245 250 255 Ile Ala Asp Ser Ser Asn Ser Val Gln Asn Tyr Ile Val Ser Met Asn 260 265 270 Asn Phe Ala Gly Pro Val Gln Met Met Val Ser Gly Met Arg Ala Lys 275 280 285 Asp Asn Asp Asp Arg Gln Asp Ala Asn Gly Asn Leu Val Lys Gly Asp 290 295 300 Ala Ala Asn Thr Gly Val His Ala Leu Leu Gly Leu His Asn Glu Ser 305 310 315 320 Phe Tyr Gly Leu Arg Asp Gly Thr Ser Lys Thr Ala Leu Leu Tyr Gly 325 330 335 His Gly Leu Gly Ala Glu Val Lys Gly Ile Gly Ser Asp Gly Ala Leu 340 345 350 Arg Pro Gly Ala Asn Thr Trp Arg Phe Ala Ser Tyr Gly Thr Thr Pro 355 360 365 Leu Ser Asp Arg Trp Phe Ile Ala Pro Ala Val Leu Ala Gln Ser Ser 370 375 380 Lys Asp Arg Tyr Val Asp Gly Asp Ser Tyr Gln Trp Ala Thr Leu Asn 385 390 395 400 Leu Arg Leu Ile Gln Glu Val Thr Gln Asn Phe Ala Leu Ala Trp Glu 405 410 415 Gly Ser Tyr Gln Tyr Met Asp Leu Gln Pro Glu Gly Tyr Asn Asp Arg 420 425 430 His Ala Val Asn Gly Ser Phe Tyr Lys Leu Thr Phe Ala Pro Thr Phe 435 440 445 Lys Val Gly Ser Ile Gly Asp Phe Phe Ser Arg Pro Glu Ile Arg Phe 450 455 460 Tyr Thr Ser Trp Met Asp Trp Ser Lys Lys Leu Asp Asn Tyr Ala Asn 465 470 475 480 Asp Asp Ala Leu Gly Ser Asn Gly Phe Lys Ser Gly Gly Glu Trp Ser 485 490 495 Phe Gly Met Gln Met Glu Thr Trp Phe 500 505 <210> 89 <211> 456 <212> PRT <213> Klebsiella pneumoniae <220> <223> ScrA, sucrose-specific enzyme II, GeneBank ID: CAA40658.1 <300> <310> WO / 2015 / 197082 <311> 29.06.2015 <312> 30.12.2015 <400> 89 Met Asp Phe Glu Gln Ile Ser Arg Ser Leu Leu Pro Leu Leu Gly Gly 1 5 10 15 Lys Glu Asn Ile Ala Ser Ala Ala His Cys Ala Thr Arg Leu Arg Leu 20 25 30 Val Leu Val Asp Asp Ala Leu Ala Asp Gln Gln Ala Ile Gly Lys Ile 35 40 45 Asp Gly Val Lys Gly Cys Phe Arg Asn Ala Gly Gln Met Gln Ile Ile 50 55 60 Phe Gly Thr Gly Val Val Asn Lys Val Tyr Ala Ala Phe Ile Gln Ala 65 70 75 80 Ala Gly Ile Ser Glu Ser Ser Lys Ser Glu Ala Ala Asp Leu Ala Ala 85 90 95 Lys Lys Leu Asn Pro Phe Gln Arg Ile Ala Arg Leu Leu Ser Asn Ile 100 105 110 Phe Val Pro Ile Ile Pro Ala Ile Val Ala Ser Gly Leu Leu Met Gly 115 120 125 Leu Leu Gly Met Val Lys Thr Tyr Gly Trp Val Asp Pro Ser Asn Ala 130 135 140 Leu Tyr Ile Met Leu Asp Met Cys Ser Ser Ala Ala Phe Ile Ile Leu 145 150 155 160 Pro Ile Leu Ile Gly Phe Thr Ala Ala Arg Glu Phe Gly Gly Asn Pro 165 170 175 Tyr Leu Gly Ala Thr Leu Gly Gly Ile Leu Thr His Pro Ala Leu Thr 180 185 190 Asn Ala Trp Gly Val Ala Ala Gly Phe His Thr Met Asn Phe Phe Gly 195 200 205 Ile Glu Val Ala Met Ile Gly Tyr Gln Gly Thr Val Phe Pro Val Leu 210 215 220 Leu Ala Val Trp Phe Met Ser Met Val Glu Lys Arg Leu Arg Arg Val 225 230 235 240 Ile Pro Asp Ala Leu Asp Leu Ile Leu Thr Pro Phe Leu Thr Val Ile 245 250 255 Ile Ser Gly Phe Ile Ala Leu Leu Leu Ile Gly Pro Ala Gly Arg Ala 260 265 270 Leu Gly Asp Gly Ile Ser Phe Ile Leu Ser Thr Leu Ile Ser His Ala 275 280 285 Gly Trp Leu Ala Gly Leu Leu Phe Gly Gly Leu Tyr Ser Val Ile Val 290 295 300 Ile Thr Gly Ile His His Ser Phe His Ala Ile Glu Ala Gly Leu Leu 305 310 315 320 Gly Asn Pro Ser Ile Gly Val Asn Phe Leu Leu Pro Ile Trp Ala Met 325 330 335 Ala Asn Val Ala Gln Gly Gly Ala Cys Phe Ala Val Trp Phe Lys Thr 340 345 350 Lys Asp Ala Lys Ile Lys Ala Ile Thr Leu Pro Ser Ala Phe Ser Ala 355 360 365 Met Leu Gly Ile Thr Glu Ala Ala Ile Phe Gly Ile Asn Leu Arg Phe 370 375 380 Val Lys Pro Phe Ile Ala Ala Leu Val Gly Gly Ala Ala Gly Gly Ala 385 390 395 400 Trp Val Val Ser Met His Val Tyr Met Thr Ala Val Gly Leu Thr Ala 405 410 415 Ile Pro Gly Met Ala Ile Val Gln Ala Ser Ser Leu Leu Asn Tyr Ile 420 425 430 Ile Gly Met Ala Ile Ala Phe Ala Val Ala Phe Ala Leu Ser Leu Thr 435 440 445 Leu Lys Tyr Lys Thr Asp Ala Glu 450 455 <210> 90 <211> 466 <212> PRT <213> Salmonella enterica subsp. enterica serovar Typhimurium <220> <223> ScrB, beta-fructofuranosidase, GeneBank ID: CAA47974.1 <300> <310> WO / 2015 / 197082 <311> 29.06.2015 <312> 30.12.2015 <400> 90 Met Ser Leu Pro Ser Arg Leu Pro Ala Ile Leu Gln Ala Val Met Gln 1 5 10 15 Gly Gln Pro Arg Ala Leu Ala Asp Ser His Tyr Pro Arg Trp His His 20 25 30 Ala Pro Val Thr Gly Leu Met Asn Asp Pro Asn Gly Phe Ile Glu Phe 35 40 45 Ala Gly Arg Tyr His Leu Phe Tyr Gln Trp Asn Pro Leu Ala Cys Asp 50 55 60 His Thr Phe Lys Cys Trp Ala His Trp Ser Ser Ile Asp Leu Leu His 65 70 75 80 Trp Gln His Glu Pro Ile Ala Leu Met Pro Asp Glu Glu Tyr Asp Arg 85 90 95 Asn Gly Cys Tyr Ser Gly Ser Ala Val Asp Asn Asn Gly Thr Leu Thr 100 105 110 Leu Cys Tyr Thr Gly Asn Val Lys Phe Ala Glu Gly Gly Arg Thr Ala 115 120 125 Trp Gln Cys Leu Ala Thr Glu Asn Ala Asp Gly Thr Phe Arg Lys Ile 130 135 140 Gly Pro Val Leu Pro Leu Pro Glu Gly Tyr Thr Gly His Val Arg Asp 145 150 155 160 Pro Lys Val Trp Arg His Glu Asp Leu Trp Tyr Met Val Leu Gly Ala 165 170 175 Gln Asp Arg Gln Lys Arg Gly Lys Val Leu Leu Phe Ser Ser Ala Asp 180 185 190 Leu His Gln Trp Thr Ser Met Gly Glu Ile Ala Gly His Gly Ile Asn 195 200 205 Gly Leu Asp Asp Val Gly Tyr Met Trp Glu Cys Pro Asp Leu Phe Pro 210 215 220 Leu Gly Asp Gln His Ile Leu Ile Cys Cys Pro Gln Gly Ile Ala Arg 225 230 235 240 Glu Glu Glu Cys Tyr Leu Asn Thr Tyr Pro Ala Val Trp Met Ala Gly 245 250 255 Glu Phe Asp Tyr Ala Ala Gly Ala Phe Arg His Gly Glu Leu His Glu 260 265 270 Leu Asp Ala Gly Phe Glu Phe Tyr Ala Pro Gln Thr Met Leu Thr Ser 275 280 285 Asp Gly Arg Arg Leu Leu Val Gly Trp Met Gly Val Pro Glu Gly Glu 290 295 300 Glu Met Leu Gln Pro Thr Leu Asn Asn Gly Trp Ile His Gln Met Thr 305 310 315 320 Cys Leu Arg Glu Leu Glu Phe Ile Asn Gly Gln Leu Tyr Gln Arg Pro 325 330 335 Leu Arg Glu Leu Ser Ala Leu Arg Gly Glu Ala Asn Gly Trp Ser Gly 340 345 350 Asn Ala Leu Pro Leu Ala Pro Met Glu Ile Asp Leu Gln Thr Arg Gly 355 360 365 Gly Asp Met Leu Ser Leu Asp Phe Gly Gly Val Leu Thr Leu Glu Cys 370 375 380 Asp Ala Ser Gly Leu Arg Leu Ala Arg Arg Ser Leu Ala Ser Asp Glu 385 390 395 400 Met His Tyr Arg Tyr Trp Arg Gly Asn Val Arg Ser Leu Arg Val Phe 405 410 415 Ile Asp Gln Ser Ser Val Glu Ile Phe Ile Asn Gly Gly Glu Gly Val 420 425 430 Met Ser Ser Arg Tyr Phe Pro Ala Cys Ser Gly Gln Leu Thr Phe Ser 435 440 445 Gly Ile Thr Pro Asp Ala Phe Cys Tyr Trp Pro Leu Arg Thr Cys Met 450 455 460 Val Glu 465 <210> 91 <211> 334 <212> PRT <213> Salmonella enterica subsp. enterica serovar Typhimurium <220> <223> ScrR, sucrose repressor, GeneBank ID: CAA47975.1 <300> <310> WO / 2015 / 197082 <311> 29.06.2015 <312> 30.12.2015 <400> 91 Met Lys Thr Lys Arg Val Thr Ile Lys Asp Ile Ala Glu Gln Ala Gly 1 5 10 15 Val Ser Lys Ala Thr Ala Ser Leu Val Leu Asn Gly Arg Gly Lys Glu 20 25 30 Leu Arg Val Ala Gln Glu Thr Arg Glu Arg Val Leu Ser Ile Ala Arg 35 40 45 Lys His His Tyr Gln Pro Ser Ile His Ala Arg Ser Leu Arg Asn Asn 50 55 60 Arg Ser His Thr Ile Gly Leu Val Val Pro Glu Ile Thr Asn His Gly 65 70 75 80 Phe Ala Val Phe Ala His Glu Leu Glu Met Leu Cys Arg Glu Ala Gly 85 90 95 Val Gln Leu Leu Ile Ser Cys Thr Asp Glu Asn Pro Gly Gln Glu Ser 100 105 110 Val Val Val Asn Asn Met Ile Ala Arg Gln Val Asp Gly Met Ile Val 115 120 125 Ala Ser Cys Met His Asn Asp Ala Asp Tyr Leu Lys Leu Ser Gln Gln 130 135 140 Leu Pro Val Val Leu Phe Asp Arg Cys Pro Asn Glu Ser Ala Leu Pro 145 150 155 160 Leu Val Met Thr Asp Ser Ile Thr Pro Thr Ala Glu Leu Ile Ser Arg 165 170 175 Ile Ala Pro Gln His Ser Asp Glu Phe Trp Phe Leu Gly Gly Gln Ala 180 185 190 Arg Leu Ser Pro Ser Arg Asp Arg Leu Thr Gly Phe Thr Gln Gly Leu 195 200 205 Ala Gln Ala Gly Ile Ala Leu Arg Pro Glu Trp Val Ile Asn Gly Asn 210 215 220 Tyr His Pro Ser Ser Gly Tyr Glu Met Phe Ala Ala Leu Cys Ala Arg 225 230 235 240 Leu Gly Arg Pro Pro Lys Ala Leu Phe Thr Ala Ala Cys Gly Leu Leu 245 250 255 Glu Gly Val Leu Arg Tyr Met Ser Gln His His Leu Leu Asp Ser Asp 260 265 270 Ile His Leu Thr Ser Phe Asp Asp His Tyr Leu Tyr Asp Ser Leu Ser 275 280 285 Leu Arg Ile Asp Thr Val Gln Gln Asp Asn Arg Gln Leu Ala Trp His 290 295 300 Cys Tyr Asp Leu Ile Ser Gln Leu Ile Glu Gly Asp Thr Pro Glu Thr 305 310 315 320 Leu Gln Arg Tyr Leu Pro Ala Thr Leu Gln Phe Arg His Gln 325 330 <210> 92 <211> 730 <212> Ms <213> Escherichia coli <220> <223> glpR coding sequence <400> 92 atgaaacaaa cacaacgtca caacggtatt atcgaactgg ttaaacagca gggttatgtc 60 agtaccgaag agctggtaga gcatttctcc gtcagcccgc agactattcg ccgcgacctc 120 aatgagctgg cggagcaaaa cctgatcctg gccatcatgg cggtgcggcg ctgccttcca 180 gttcggttaa cacgccgtgg cacgatcgca aggccaccca gaccgaagaa aaagagcgca 240 tcgcccgcaa agtggcggag caaatcccca atggctcgac gctgtttatc gatatcggca 300 ccacgccgga agcggtagcg cacgcactgc tcaatcacag caatttgcgc attgtcacca 360 acaatctcaa cgttgctaac acgttgatgg taaaagaaga ttttcgcatc attctcgccg 420 gtggcgaatt acgcagccgc gatggcggga tcattggcga agcgacgctc gattttatct 480 cccagttccg ccttgatttc ggcattctgg ggataagcgg catcgatagc gacggctcgc 540 tgctggagtt cgattaccac gaagttcgca ccaaacgcgc cattattgag aactcgcgcc 600 acgttatgct ggttgtcgat cactcgaaat ttggccgtaa cgcgatggtc aatatgggca 660 gcatcagcat ggtagatgcc gtctacaccg acgccccgcc gccagtaagc gtgatgcagg 720 tgctgacgga 730 <210> 93 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD8 promoter sequence <400> 93 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa gagaaaaaca gct 203 <210> 94 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD10 promoter sequence <400> 94 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa ctgagaaaca gct 203 <210> 95 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB-54UTR promoter sequence <400> 95 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaacaaa aaccggagat acc 203 <210> 96 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> Plac 70_UTR promoter sequence <400> 96 tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 60 gttgtgtgga atgcctacaa gcatcgtgga ggtccgtgac tttcacgcat acaacaaaca 120 ttaaccaagg aggaaacagc t 141 <210> 97 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD9 promoter sequence <400> 97 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa aggaaaaaca gct 203 <210> 98 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD5 promoter sequence <400> 98 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa ccgagaaaca gct 203 <210> 99 <211> 203 <212> DNA <213> Artificial Sequence <220> <223> PmglB_70UTR_SD7 promoter sequence <400> 99 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgtgcctac aagcatcgtg gaggtccgtg actttcacgc atacaacaaa 180 cattaaccaa gagcaaaaca gct 203 <210> 100 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> PglpA_70UTR promoter sequence <400> 100 gaaaacattc ataaattaaa tgtgaattgc cgcacacatt attaaataag atttacaaaa 60 tgttcaaaat gacgcatgaa atcacgtttc actttcgaat tatgagcgaa tatgcgcgat 120 gcctacaagc atcgtggagg tccgtgactt tcacgcatac aacaaacatt aaccaaggag 180 gaaacagct 189 <210> 101 <211> 239 <212> DNA <213> Artificial Sequence <220> <223> PglpT_70UTR promoter sequence <400> 101 ccatttagcc atagtaaaaa catgaattgt ttgatttcgc gcatattcgc tcataattcg 60 aaagtgaaac gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt 120 gtgcggcaat tcacatttaa tttatgaatg ttttcttaac atcgcggcat gcctacaagc 180 atcgtggagg tccgtgactt tcacgcatac aacaaacatt aaccaaggag gaaacagct 239 <210> 102 <211> 350 <212> DNA <213> Artificial Sequence <220> <223> PmglB_16UTR promoter sequence <400> 102 tgcgtcgcca ttctgtcgca acacgccaga atgcggcggc gatcactaac tcaacaaatc 60 aggcgatgta accgctttca atctgtgagt gatttcacag tatcttaaca atgtgatagc 120 tatgattgca ccgttttaac gttgtaaccc gtatgtaaca gtgaataatc acttttgccg 180 aggtaacagc gtcataacaa caattaaagc cgttttctgg agcgttaccg ggcatggaag 240 aacgaatttt aaaaagtgag cttcggcgtt cagtaacact tcattaactc tactgccccg 300 ccgagcattt atctcaagca ctaccctgca taagcaagga ggaaacagct 350 <210> 103 <211> 189 <212> DNA <213> Artificial Sequence <220> <223> PglpA_16UTR promoter sequence <400> 103 gaaaacattc ataaattaaa tgtgaattgc cgcacacatt attaaataag atttacaaaa 60 tgttcaaaat gacgcatgaa atcacgtttc actttcgaat tatgagcgaa tatgcgcgat 120 gcctacaagc atcgtggagg tccgtgactt tcacgcatac aacaaacatt aaccaaggag 180 gaaacagct 189 BRIEF DESCRIPTION OF THE DRAWINGS

[0352] Figure 1

[0353] Total sample analysis revealed the effects of lacY overexpression on the relative titers of LNT-II, LNT, and pLNH2 and the total HMO content in (a) yberC-expressing strains MP1 and MP2 and (b) nec-expressing strains MP3 and MP4.

[0354] Figure 2

[0355] Effect of lacY overexpression on (a) the LNT fraction (%) of the final HMO blend and (b) the final optical density achieved by yberC-expressing strains MP1 and MP2 and nec-expressing strains MP3 and MP4.

[0356] Figure 3

[0357] Analysis of supernatant and cell pellet samples revealed the effect of lacY overexpression on the fractions of LNT-II, LNT, and pLNH2 detected in the culture supernatants of (a) yberC-expressing strains MP1 and MP2 and (b) nec-expressing strains MP3 and MP4.

[0358] Figure 4

[0359] Total sample analysis revealed the effect of lacY overexpression on the relative titers of LNT-II, LNT, pLNnH, and total HMO content in vag-expressing strains MP5 and MP6.

[0360] Figure 5

[0361] Effect of lacY overexpression on (a) the LNnT fraction (%) in the final HMO blend and (b) the final optical density achieved by vag-expressing strains MP5 and MP6.

[0362] Figure 6

[0363] Performance of LNT-producing strains MP7 and MP8 in a fermentation run using a high-lactose process. Shown are time courses of: (a) lactose concentration, (b) biomass concentration, (c) relative cumulative LNT production on sucrose, (d) LNT-II to LNT ratio, and (e) pLNH2 to LNT ratio.

[0364] Figure 7

[0365] Performance of LNnT-producing strains MP5 and MP6 in a fermentation run using a high-lactose process. Shown are time courses of: (a) lactose concentration, (b) biomass concentration, (c) relative cumulative LNnT production on sucrose, (d) ratio of LNT-II to LNnT, and (e) ratio of pLNnH to LNnT.

[0366] Figure 8

[0367] The percentage of 3'SL production in the corresponding MFS transporter strain was set to 100%. The strain containing the MFS transporter nec is represented by a dotted line, the yberC strain by a cross-striped line, and the fred strain by a horizontal striped line.

[0368] Figure 9

[0369] The percentage of 2'FL production over the control nec transporter strain (MP18) was set to 100%.The 2'FL titers of strains containing the MFS transporter nec combined with an additional lacY copy expressed from PglpF_SD7 (MP19) or PglpF (MP20) relative to the nec transporter strain are shown.

[0370] Sequence ID

[0371] This application contains a sequence listing in both text and electronic formats, which is incorporated herein by reference. Table 9 provides a summary of the sequences in this application.

[0372] Table 9. Sequence

[0373]

[0374]

[0375]

[0376] Example

[0377] Example 1 - Improvement of the LNT production system by increasing expression of the lacY gene encoding the lactose permease in cells expressing a heterologous MFS transporter.

[0378] Genotypes of strains MP1, MP2, MP3, and MP4

[0379] The strain (genetically engineered cell) constructed in this application is based on Escherichia coli K-12DH1, with genotypes: Fˉ, gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. The E. coli K-12DH1 strain was additionally modified to generate the platform strain "MDO" with the following modifications: lacZ: 1.5 kbp deletion, lacA: 0.5 kbp deletion, nanKETA: 3.3 kbp deletion, melA: 0.9 kbp deletion, wcaJ: 0.5 kbp deletion, mdoH: 0.5 kbp deletion, and the Plac promoter was inserted upstream of the gmd gene.

[0380] Methods for inserting or deleting a gene of interest into or from the E. coli genome are well known to those skilled in the art. Insertion of a genetic cassette into the E. coli chromosome can be accomplished 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) along with specific selection marker genes and screening methods.

[0381] Based on the platform strain "MDO" (e.g., also reported in WO2020255054A1 or WO2019123324A1), the modifications summarized in the table below were performed to obtain full-chromosome strains MP1, MP2, MP3, and MP4. This strain can produce the tetrasaccharide HMO LNT. The glycosyltransferase LgtA (β-1,3-N-acetylglucosaminyltransferase) from Neisseria meningitidis and the GalTK (β-1,3-galactosyltransferase) from Helicobacter pylori (H. pylori) are present in all four strains. In addition, MP1 and MP2 express the heterologous transporter YberC from Yersinia bercovieri, while strains MP3 and MP4 express the heterologous transporter Nec ...

Claims

1. A method for producing one or more HMOs, comprising: a) providing a genetically engineered cell, i. overexpressing one or more lactose permeases, ii. expressing a heterologous MFS-transporter selected from the group consisting of: I. Amino acid sequence Vag according to SEQ ID NO: 62, II. Amino acid sequence Nec according to SEQ ID NO: 66, III. Amino acid sequence according to Fred of SEQ ID NO:63, IV. Amino acid sequence according to Marc of SEQ ID NO: 64, and V. The amino acid sequence of Bad according to SEQ ID NO: 65, and iii. expressing two or more glycosyltransferases selected from β-1,3-GlcNAc-transferase, β-1,3-Gal-transferase and β-1,4-Gal-transferase, b) culturing the cells in a suitable medium supplemented with lactose, and c) harvesting said one or more HMOs, Wherein the one or more HMOs are LNT and / or LNnT.

2. The method for producing one or more HMOs according to claim 1, wherein the genetically engineered cells express a sucrose utilization system.

3. The method for producing one or more HMOs according to claim 2, wherein the sucrose utilization system is a polypeptide capable of hydrolyzing sucrose into glucose and fructose selected from the group consisting of SEQ ID NO: 86 and SEQ ID NO:

87.

4. The method of any one of the preceding claims, 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, EDI1749185.1, WP_084912833.1, WP_103826752.1, WP_021804673.1, WP_084984472.1, WP_199428647.1, WP_046596210.1 and XP_452193.

1.

5. The method according to any one of the preceding claims, wherein the amino acid sequence of the one or more lactose permeases is identical to GenBank ID NP_414877.

1.

6. The method according to any one of the preceding claims, wherein the MFS-transporter is Nec or Vag.

7. A method according to any one of the preceding claims, wherein a) the β-1,3-Gal-transferase is selected from CvB3galT and GalTK, or b) The β-1,4-Gal-transferase is GalT.

8. The method according to any one of the preceding claims, wherein the β-1,3-GlcNAc-transferase is selected from the group consisting of LgtA, PmnagT and HD0466.

9. The method of any one of the preceding claims, wherein the genetically engineered cell comprises one or more heterologous nucleic acid sequences encoding one or more heterologous polypeptides that enable the utilization of sucrose as the sole carbon and energy source for the genetically engineered cell.

10. The method according to any one of the preceding claims, wherein the genetically engineered cell expresses one or more polypeptides involved in activated sugar nucleotide biosynthesis.

11. The method according to claim 10, wherein the one or more polypeptides involved in activated sugar nucleotide biosynthesis are selected from the group consisting of Pgm, GalU, GalE, GlmM, GlmU and GlmS.

12. The method according to any one of the preceding claims, wherein the genetically engineered cell comprises at least one nucleic acid sequence encoding the lactose permease according to claim 4 or 5.

13. The method according to any one of the preceding claims, wherein the genetically engineered cell comprises more than one nucleic acid sequence encoding one or more lactose permeases according to claim 4 or 5.

14. The method according to any one of the preceding claims, wherein the genetically engineered cell comprises at least one nucleic acid sequence encoding one or more heterologous glycosyltransferases according to any one of claims 7 to 8.

15. The method according to any one of the preceding claims, wherein the genetically engineered cell comprises at least one polypeptide encoding one or more polypeptides involved in activated sugar nucleotide biosynthesis according to claim 10.

16. The method according to any one of claims 12 to 15, wherein the expression of any one or more of the nucleic acid sequences is regulated by one or more regulatory elements.

17. The method of claim 16, wherein the regulatory elements comprise one or more promoter sequences.

18. The method of claim 17, wherein the one or more promoter sequences are selected from Plac, PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, Plac_16UTR, PmglB_70UTR, PmglB_70UTR_SD4, CP6 and PosmY.

19. The method of claim 18, wherein the one or more promoter sequences are selected from PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, Plac_16UTR and Plac.

20. The method according to any one of claims 16 to 19, wherein the regulatory element comprises an endogenous, native, heterologous, synthetic and / or optimized Shine-Dalgarno sequence.

21. The method according to any one of claims 12 to 20, wherein the nucleic acid sequence is integrated into the genome of the genetically engineered cell.

22. The method of any one of claims 12 to 20, wherein the one or more nucleic acid sequences are expressed from a plasmid.

23. The method of claim 22, wherein the plasmid is a low copy number plasmid.

24. The method of claim 22, wherein the plasmid is a medium copy number plasmid.

25. The method of claim 22, wherein the plasmid is a high copy number plasmid.

26. The method of any one of the preceding claims, wherein the genetically engineered cell is Escherichia coli.

27. The method of any one of the preceding claims, wherein the genetically engineered cells are derived from E. coli K-12.

28. A genetically engineered cell that produces LNT and / or LNnT, and wherein the cell a) overexpressing one or more lactose permease genes, b) expressing a heterologous MFS-transporter selected from the group consisting of: i. The amino acid sequence of Vag according to SEQ ID NO: 62, ii. Amino acid sequence according to Nec of SEQ ID NO: 66, iii. Amino acid sequence according to Fred of SEQ ID NO: 63, iv. the amino acid sequence according to Marc of SEQ ID NO: 64, and v. the amino acid sequence of Bad according to SEQ ID NO: 65, and c) expressing two or more glycosyltransferases selected from the group consisting of β-1,3-GlcNAc-transferase, β-1,3-Gal-transferase and β-1,4-Gal-transferase, and optionally, d) expressing a sucrose utilization system, and / or e) expressing one or more polypeptides involved in activated sugar biosynthesis.

29. The genetically engineered cell of claim 28, 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, EDI1749185.1, WP_084912833.1, WP_103826752.1, WP_021804673.1, WP_084984472.1, WP_199428647.1, WP_046596210.1, and XP_452193.1, ii. two or more glycosyltransferases selected from CvB3galT, GalTK, GalT, LgtA, PmnagT and HD0466, and optionally iii. one or more polypeptides involved in activating sugar biosynthesis selected from the group consisting of Pgm, GalU, GalE, GlmM, GlmU and GlmS.

30. Use of a cell according to any one of claims 28 to 29 for the production and / or manufacture of one or more HMOs, wherein the one or more HMOs are LNT and / or LNnT.

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